Fc variants with enhanced binding to FcRn and extended half-life
阅读说明：本技术 具有与FcRn增强的结合及延长的半衰期的Fc变体 (Fc variants with enhanced binding to FcRn and extended half-life ) 是由 邱华伟 B·麦肯斯 于 2019-01-25 设计创作，主要内容包括：本公开提供包含经修饰的Fc结构域的结合多肽(例如,抗体和免疫粘附素)。本公开还提供编码该结合多肽的核酸、重组表达载体和用于制备此类结合多肽的宿主细胞。还提供使用本文公开的结合多肽治疗疾病的方法。(The present disclosure provides binding polypeptides (e.g., antibodies and immunoadhesins) comprising a modified Fc domain. The disclosure also provides nucleic acids encoding the binding polypeptides, recombinant expression vectors, and host cells for making such binding polypeptides. Also provided are methods of treating diseases using the binding polypeptides disclosed herein.)
1. An isolated binding polypeptide comprising a modified Fc domain comprising:
aspartic acid (D) or glutamic acid (E) at amino acid position 256, and/or tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprising:
phenylalanine (F) or tyrosine (Y) at amino acid position 434; or
A tyrosine (Y) at amino acid position 252,
wherein the amino acid positions are according to EU numbering.
2. An isolated binding polypeptide comprising a modified Fc domain comprising a combination of amino acid substitutions at positions selected from the group consisting of:
a) tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256;
b) aspartic acid (D) at amino acid position 256 and phenylalanine (F) at amino acid position 434;
c) aspartic acid (D) at amino acid position 256 and tyrosine (Y) at amino acid position 434;
d) tryptophan (W) at amino acid position 307 and phenylalanine (F) at amino acid position 434;
e) tyrosine (Y) at amino acid position 252 and tryptophan (W) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434;
f) aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434;
g) aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307 wherein tyrosine (Y) is not at amino acid position 434;
h) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434; and
i) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, and glutamine (Q) at amino acid position 307, wherein threonine (T) is not at amino acid position 254, histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434; wherein the amino acid substitutions are according to EU numbering.
3. An isolated binding polypeptide comprising a modified Fc domain comprising:
a) a dual amino acid substitution selected from the group consisting of M252Y/T256D, M252Y/T256E, M252Y/T307Q, M252Y/T307W, T256D/T307Q, T256D/T307W, T256E/T307Q, and T256E/T307W, wherein threonine (T) is not at amino acid position 254, histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434; or
b) A triple amino acid substitution selected from the group consisting of M252Y/T256D/T307Q, M252Y/T256D/T307W, M252Y/T256E/T307Q, and M252Y/T256E/T307W, wherein threonine (T) is not at amino acid position 254, histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434;
wherein the amino acid substitutions are according to EU numbering.
4. The isolated binding polypeptide of any one of claims 1-3, wherein the modified Fc domain is a modified human Fc domain.
5. The isolated binding polypeptide of any one of claims 1-4, wherein the modified Fc domain is a modified IgG1 Fc domain.
6. The isolated binding polypeptide of any one of claims 1-5, wherein the binding polypeptide has human FcRn binding affinity.
7. The isolated binding polypeptide of any one of claims 1-5, wherein the binding polypeptide has rat FcRn binding affinity.
8. The isolated binding polypeptide of any one of claims 1-7, wherein the binding polypeptide has human and rat FcRn binding affinity.
9. The isolated binding polypeptide of any one of claims 2-8, wherein the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.
10. The isolated binding polypeptide of claim 9, wherein the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.
11. The isolated binding polypeptide of any one of claims 1-8, wherein the isolated binding polypeptide has altered FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
12. The isolated binding polypeptide of claim 11, wherein the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
13. The isolated binding polypeptide of any one of claims 1-12, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain.
14. The isolated binding polypeptide of any one of claims 1-13, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to FcRn binding affinity of the binding polypeptide at elevated non-acidic pH.
15. The isolated binding polypeptide of any one of claims 12-14, wherein the enhanced FcRn binding affinity comprises a reduced FcRn binding off-rate.
16. The isolated binding polypeptide of any one of claims 13-15, wherein the acidic pH is about 6.0.
17. The isolated binding polypeptide of any one of claims 13-16, wherein the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
18. The isolated binding polypeptide of any one of claims 1-17, wherein the isolated binding polypeptide has altered fcyriiia binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
19. The isolated binding polypeptide of any one of claims 1-18, wherein the isolated binding polypeptide has reduced fcyriiia binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
20. The isolated binding polypeptide of any one of claims 1-18, wherein the isolated binding polypeptide has enhanced fcyriiia binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
21. The isolated binding polypeptide of any one of claims 1-18, wherein the isolated binding polypeptide has about the same fcyriiia binding affinity as a binding polypeptide comprising a wild-type Fc domain.
22. The isolated binding polypeptide of any one of claims 1-21, wherein the isolated binding polypeptide has about the same thermostability as a binding polypeptide comprising a wild-type Fc domain.
23. The isolated binding polypeptide of any one of claims 1-21, wherein the isolated binding polypeptide has about the same thermostability as a binding polypeptide comprising a modified Fc domain having triple amino acid substitutions M252Y/S254T/T256E according to EU numbering.
24. The isolated binding polypeptide of any one of claims 1-23, wherein the isolated binding polypeptide is an antibody.
25. The isolated binding polypeptide of any one of claims 1-24, wherein the isolated binding polypeptide is a monoclonal antibody.
26. The isolated binding polypeptide of any one of claims 24-25, wherein the isolated antibody is a chimeric, humanized, or human antibody.
27. The isolated binding polypeptide of any one of claims 24-26, wherein the isolated antibody is a full length antibody.
28. The isolated binding polypeptide of any one of claims 1-27, wherein the isolated binding polypeptide specifically binds one or more human targets.
29. An isolated nucleic acid molecule comprising a nucleic acid encoding the isolated polypeptide of any one of claims 1-28.
30. A vector comprising the isolated nucleic acid molecule of claim 29.
31. The vector of claim 30, wherein the vector is an expression vector.
32. A host cell comprising the vector of any one of claims 30-31.
33. The host cell according to claim 32, wherein the host cell is of eukaryotic or prokaryotic origin.
34. The host cell according to any one of claims 32-33, wherein the host cell is of mammalian origin.
35. The host cell according to any one of claims 32-33, wherein the host cell is of bacterial origin.
36. A pharmaceutical composition comprising the isolated binding polypeptide of any one of claims 1-28.
37. A pharmaceutical composition comprising the isolated antibody of any one of claims 24-27.
38. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307 according to EU numbering.
39. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307 according to EU numbering.
40. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises a tyrosine (Y) at amino acid position 252 and an aspartic acid (D) at amino acid position 256, according to EU numbering.
41. The isolated binding polypeptide of any one of claims 38-40, wherein the modified Fc domain is a modified human Fc domain.
42. The isolated binding polypeptide of any one of claims 38-41, wherein the modified Fc domain is a modified IgG1 Fc domain.
43. The isolated binding polypeptide of any one of claims 38-42, wherein the binding polypeptide has human FcRn binding affinity.
44. The isolated binding polypeptide of any one of claims 38-42, wherein the binding polypeptide has rat FcRn binding affinity.
45. The isolated binding polypeptide of any one of claims 38-44, wherein the isolated binding polypeptide has increased serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
46. The isolated binding polypeptide of any one of claims 38-44, wherein the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
47. The isolated binding polypeptide of any one of claims 38-44, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain.
48. The isolated binding polypeptide of any one of claims 38-47, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH.
49. The isolated binding polypeptide of any one of claims 46-48, wherein the enhanced FcRn binding affinity comprises a reduced FcRn binding off-rate.
50. The isolated binding polypeptide of claim 47 or 48, wherein the acidic pH is about 6.0.
51. The isolated binding polypeptide of claim 47, wherein the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
52. The isolated binding polypeptide of claim 48, wherein the acidic pH is about 6.0.
53. The isolated binding polypeptide of any one of claims 38-52, wherein the isolated binding polypeptide has altered FcyRIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
54. The isolated binding polypeptide of any one of claims 38-53, wherein the isolated binding polypeptide is a monoclonal antibody.
55. The isolated binding polypeptide of any one of claims 38-54, wherein the antibody is a chimeric, humanized, or human antibody.
56. The isolated binding polypeptide of any one of claims 38-55, wherein the isolated binding polypeptide specifically binds one or more human targets.
57. An isolated nucleic acid molecule comprising a nucleic acid encoding the isolated polypeptide of any one of claims 38-56.
58. An expression vector comprising the isolated nucleic acid molecule of claim 57.
59. A host cell comprising the expression vector of claim 58.
60. A pharmaceutical composition comprising the isolated binding polypeptide of any one of claims 38-58.
61. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises a combination of at least four amino acid substitutions comprising:
aspartic acid (D) or glutamic acid (E) at amino acid position 256 and tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprising:
phenylalanine (F) or tyrosine (Y) at amino acid position 434; and
a tyrosine (Y) at amino acid position 252,
wherein the amino acid positions are according to EU numbering.
62. An isolated binding polypeptide comprising a modified Fc domain having a combination of amino acid substitutions at positions selected from the group consisting of:
a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434;
b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434;
c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434;
d) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434; or
e) Tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434,
wherein the amino acid substitutions are according to EU numbering.
63. An isolated binding polypeptide comprising a modified Fc domain comprising:
a quadruple amino acid substitution selected from the group consisting of M252Y/T256D/T307Q/N434Y, M252Y/T256E/T307W/N434Y, M252Y/T256E/T307Q/N434Y, M252Y/T256D/T307Q/N434F and M252Y/T256D/T307W/N434Y,
wherein the amino acid substitutions are according to EU numbering.
64. The isolated binding polypeptide of any one of claims 61-63, wherein the modified Fc domain is a modified human Fc domain.
65. The isolated binding polypeptide of any one of claims 61-64, wherein the modified Fc domain is a modified IgG1 Fc domain.
66. The isolated binding polypeptide of any one of claims 61-65, wherein the binding polypeptide has human FcRn binding affinity.
67. The isolated binding polypeptide of any one of claims 61-65, wherein the binding polypeptide has rat FcRn binding affinity.
68. The isolated binding polypeptide of any one of claims 61-67, wherein the binding polypeptide has human and rat FcRn binding affinity.
69. The isolated binding polypeptide of any one of claims 61-68, wherein the isolated binding polypeptide has an altered FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
70. The isolated binding polypeptide of claims 61-69, wherein the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
71. The isolated binding polypeptide of any one of claims 61-70, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain.
72. The isolated binding polypeptide of any one of claims 61-71, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
73. The isolated binding polypeptide of any one of claims 61-72, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising a wild-type Fc domain.
74. The isolated binding polypeptide of any one of claims 61-73, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at non-acidic pH as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
75. The isolated binding polypeptide of any one of claims 61-74, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH, as compared to a binding polypeptide comprising a wild-type Fc domain.
76. The isolated binding polypeptide of any one of claims 61-75, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH, as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
77. The isolated binding polypeptide of any one of claims 61-76, wherein the acidic pH is about 6.0.
78. The isolated binding polypeptide of any one of claims 61-77, wherein the non-acidic pH is about 7.4.
79. The isolated binding polypeptide of any one of claims 61-78, wherein the isolated binding polypeptide has an altered serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
80. The isolated binding polypeptide of any one of claims 61-79, wherein the isolated binding polypeptide has a reduced serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
81. The isolated binding polypeptide of any one of claims 61-80, wherein the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
82. The isolated binding polypeptide of any one of claims 61-81, wherein the isolated binding polypeptide has altered FcyRIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
83. The isolated binding polypeptide of any one of claims 61-82, where the isolated binding polypeptide has reduced FcyRIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
84. The isolated binding polypeptide of any one of claims 61-83, wherein the isolated binding polypeptide has reduced fcyriiia binding affinity as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
85. The isolated binding polypeptide of any one of claims 61-84, where the isolated binding polypeptide has reduced thermostability as compared to a binding polypeptide comprising a wild-type Fc domain.
86. The isolated binding polypeptide of any one of claims 61-85, wherein the isolated binding polypeptide has reduced thermostability as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
87. The isolated binding polypeptide of any one of claims 61-86, wherein the isolated binding polypeptide is an antibody.
88. The isolated binding polypeptide of any one of claims 61-87, wherein the isolated binding polypeptide is a monoclonal antibody.
89. The isolated binding polypeptide of any one of claims 61-88, wherein the isolated antibody is a chimeric, humanized, or human antibody.
90. The isolated binding polypeptide of any one of claims 61-89, wherein the isolated antibody is a full length antibody.
91. The isolated binding polypeptide of any one of claims 61-90, wherein the isolated binding polypeptide specifically binds to one or more targets.
92. An isolated nucleic acid molecule comprising a nucleic acid encoding the isolated polypeptide of any one of claims 61-91.
93. A vector comprising the isolated nucleic acid molecule of claim 92.
94. The vector according to claim 93, wherein the vector is an expression vector.
95. A host cell comprising the vector of any one of claims 93-94.
96. The host cell according to claim 95, wherein the host cell is of eukaryotic or prokaryotic origin.
97. The host cell according to any one of claims 95-96, wherein the host cell is of mammalian origin.
98. The host cell according to any one of claims 95-96, wherein the host cell is of bacterial origin.
99. A pharmaceutical composition comprising the isolated binding polypeptide of any one of claims 61-92.
100. A pharmaceutical composition comprising the isolated antibody of any one of claims 87-90.
101. An isolated binding polypeptide comprising a modified Fc domain comprising tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434 according to EU numbering.
102. An isolated binding polypeptide comprising a modified Fc domain comprising tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434 according to EU numbering.
103. An isolated binding polypeptide comprising a modified Fc domain comprising tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434 according to EU numbering.
104. An isolated binding polypeptide comprising a modified Fc domain comprising tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434 according to EU numbering.
105. An isolated binding polypeptide comprising a modified Fc domain comprising tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434 according to EU numbering.
106. The isolated binding polypeptide of any one of claims 101-105, wherein the modified Fc domain is a modified human Fc domain.
107. The isolated binding polypeptide of any one of claims 101-106, wherein the modified Fc domain is a modified IgG1 Fc domain.
108. The isolated binding polypeptide of any one of claims 101-107, wherein the binding polypeptide has human FcRn binding affinity.
109. The isolated binding polypeptide of any one of claims 101-108, wherein the isolated binding polypeptide has a reduced serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
110. The isolated binding polypeptide of any one of claims 101-109, wherein the isolated binding polypeptide has a reduced serum half-life as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
111. The isolated binding polypeptide of any one of claims 101-110, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain.
112. The isolated binding polypeptide of any one of claims 101-111, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
113. The isolated binding polypeptide of claim 111 or 112, wherein the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
114. The isolated binding polypeptide of any one of claims 101-113, wherein the isolated binding polypeptide has reduced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
115. The isolated binding polypeptide of any one of claims 101-114, wherein the isolated binding polypeptide has reduced fcyriiia binding affinity as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
116. The isolated binding polypeptide of any one of claims 101-115, wherein the isolated binding polypeptide has reduced thermostability as compared to a binding polypeptide comprising a wild-type Fc domain.
117. The isolated binding polypeptide of any one of claims 101-116, wherein the isolated binding polypeptide has reduced thermostability as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
118. The isolated binding polypeptide of any one of claims 101-117, wherein the isolated binding polypeptide is a monoclonal antibody.
119. The isolated binding polypeptide of claim 118, wherein the antibody is a chimeric, humanized, or human antibody.
120. The isolated binding polypeptide of any one of claims 101-119, wherein the isolated binding polypeptide specifically binds to one or more targets.
121. An isolated nucleic acid molecule comprising a nucleic acid encoding the isolated polypeptide according to any one of claims 101-120.
122. An expression vector comprising the isolated nucleic acid molecule of claim 121.
123. A host cell comprising the expression vector of claim 122.
124. A pharmaceutical composition comprising an isolated binding polypeptide according to any one of claims 101-120.
125. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide of any one of claims 1-28, 38-58, 61-91, and 101-120, or administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 36-37, 60, 99-100, and 124.
126. The method of claim 125, wherein the disease or disorder is cancer.
127. The method of claim 126, wherein the cancer is a tumor.
128. The method of claim 125, wherein the disease or disorder is an autoimmune disorder.
129. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide of any one of claims 1-28 and 38-58, or administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 36, 37, and 60.
130. A method of treating an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide of any one of claims 61-91 and 101-120, or administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 99, 100, and 124.
Background
The interaction of antibodies with neonatal Fc receptors (FcRn) is a determinant to maintain and prolong the serum half-life of antibodies and other Fc-derived therapeutics. FcRn is a heterodimer of a class I MHC-like a-domain and a β 2-macroglobulin (β 2-m) subunit that recognizes a different region of the antibody Fc heavy chain than other Fc γ receptors (Fc γ rs). Although FcRn is expressed in a variety of tissues, it is thought to act primarily in the vascular endothelium, kidney and at the blood brain barrier, respectively, to prevent IgG degradation, excretion and trigger inflammatory responses.
Antibodies that bind FcRn are highly pH dependent, and this interaction occurs with high affinity (high nanomolar to low micromolar) only at low pH (pH <6.5), but not at physiological pH (pH around 7.4). Upon acidification of the endosomes to pH less than 6.5, the interaction between IgG and FcRn becomes very favorable and is directly responsible for the degradation of the FcRn-bound antibody and promotes the recycling of the FcRn-bound antibody to the cell surface. The increase in pH weakens the interaction and facilitates the release of antibodies into the bloodstream.
Fc engineering using high-throughput mutagenesis methods has been widely used to identify variants that enhance FcRn binding affinity, as enhanced binding will likely result in increased efficacy and decreased dose frequency of therapeutic antibodies as a direct result of the increased serum half-life compared to wild-type IgG antibodies. However, variants that enhance FcRn binding affinity may have unpredictable results. For example, certain IgG variants that show a large increase in FcRn affinity at pH6.0, such as N434W or P257I/Q311I and the like, have wild-type or severely reduced serum half-lives in cynomolgus and human FcRn (hFcRn) transgenic mouse studies (see, e.g., Kuo et al 2011 supra; Datta-Mannan et al 2007, J.biol.Chem.282: 1709-1717; and Datta-Mannan et al 2007, Metab.Dispos.35: 86-94). The T250Q/M428L (QL) variant has shown IgG scaffold-specific results in animal models (see, e.g., Datta-Mannan et al 2007, J.biol. chem.282: 1709-1717; and Hinton et al 2006, J.Immunol.176: 346-356). The M252Y/S254T/T256E (YTE, EU numbering) variant has been shown to enhance 10-fold in vitro, but to exhibit antibody-dependent cell-mediated reduced cellular cytotoxicity (ADCC) in vivo due to a 2-fold reduction in affinity for the Fc γ RIIIa receptor (see, e.g., Dall' Acqua et al 2002 supra).
Thus, there remains a need for alternative Fc variants with enhanced binding to FcRn and extended circulating half-life.
Summary of The Invention
The present invention is based on the discovery of novel IgG antibodies having one or more of the following characteristics: increased serum half-life, enhanced FcRn binding affinity at acidic pH, enhanced fcyriiia binding affinity, and similar thermostability compared to wild-type IgG antibodies.
Thus, in certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising aspartic acid (D) or glutamic acid (E) at amino acid position 256, and/or tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprises phenylalanine (F) or tyrosine (Y) at amino acid position 434 or tyrosine (Y) at amino acid position 252, wherein the amino acid positions are according to EU numbering.
In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.
In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.
In certain exemplary embodiments, the isolated binding polypeptide has an altered serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has altered FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to FcRn binding affinity of the binding polypeptide at elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding off-rate.
In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
In certain exemplary embodiments, the isolated binding polypeptide has altered Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has about the same Fc γ RIIIa binding affinity as a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has about the same thermostability as a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has about the same thermostability as a binding polypeptide comprising a modified Fc domain having triple amino acid substitutions M252Y/S254T/T256E according to EU numbering.
In certain exemplary embodiments, the isolated binding polypeptide is an antibody, e.g., a monoclonal antibody. In certain exemplary embodiments, the isolated antibody is a chimeric, humanized, or human antibody. In certain exemplary embodiments, the isolated antibody is a full-length antibody.
In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.
In other aspects, an isolated binding polypeptide is provided comprising a modified Fc domain comprising a combination of amino acid substitutions at positions selected from the group consisting of: a) tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256, b) aspartic acid (D) at amino acid position 256 and phenylalanine (F) at amino acid position 434, c) aspartic acid (D) at amino acid position 256 and tyrosine (Y) at amino acid position 434, D) tryptophan (W) at amino acid position 307 and phenylalanine (F) at amino acid position 434, e) tyrosine (Y) at amino acid position 252 and tryptophan (W) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434, F) aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434, g) aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434, H) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434, and i) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, and glutamine (Q) at amino acid position 307, wherein threonine (T) is not at amino acid position 254, histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434, wherein the amino acid substitutions are in accordance with EU numbering.
In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.
In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.
In certain exemplary embodiments, the isolated binding polypeptide has an altered serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has altered FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to FcRn binding affinity of the binding polypeptide at elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding off-rate. In certain exemplary embodiments, the isolated binding polypeptide has less FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain having the double amino acid substitution M428L/N434S according to EU numbering.
In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
In certain exemplary embodiments, the isolated binding polypeptide has altered Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has about the same Fc γ RIIIa binding affinity as a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has about the same thermostability as a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has about the same thermostability as a binding polypeptide comprising a modified Fc domain having triple amino acid substitutions M252Y/S254T/T256E according to EU numbering.
In certain exemplary embodiments, the isolated binding polypeptide is an antibody, e.g., a monoclonal antibody. In certain exemplary embodiments, the isolated antibody is a chimeric, humanized, or human antibody. In certain exemplary embodiments, the isolated antibody is a full-length antibody.
In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.
In other aspects, there is provided an isolated binding polypeptide comprising a modified Fc domain comprising a) a dual amino acid substitution selected from the group consisting of M252Y/T256D, M252Y/T256E, M252Y/T307Q, M252Y/T307W, T256D/T307Q, T256D/T307W, T256E/T307Q, and T256E/T307W, wherein threonine (T) is not at amino acid position 254, histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434, or b) an amino acid substitution selected from the group consisting of M252W/T256W/T307W, M252W/T307W, wherein the amino acid substitution is not at amino acid position 434, and the triple amino acid substitution is not at amino acid position 434, wherein the amino acid substitutions are according to EU numbering.
In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.
In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.
In certain exemplary embodiments, the isolated binding polypeptide has an altered serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has altered FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to FcRn binding affinity of the binding polypeptide at elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding off-rate. In certain exemplary embodiments, the isolated binding polypeptide has less FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain having the double amino acid substitution M428L/N434S according to EU numbering.
In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
In certain exemplary embodiments, the isolated binding polypeptide has altered Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has about the same Fc γ RIIIa binding affinity as a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has about the same thermostability as a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has about the same thermostability as a binding polypeptide comprising a modified Fc domain having triple amino acid substitutions M252Y/S254T/T256E according to EU numbering.
In certain exemplary embodiments, the isolated binding polypeptide is an antibody, e.g., a monoclonal antibody. In certain exemplary embodiments, the isolated antibody is a chimeric, humanized, or human antibody. In certain exemplary embodiments, the isolated antibody is a full-length antibody.
In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.
In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307 according to EU numbering.
In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.
In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity or both human and rat FcRn binding affinity.
In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to FcRn binding affinity of the binding polypeptide at elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding off-rate.
In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
In certain exemplary embodiments, the isolated binding polypeptide has altered Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the antibody is a chimeric, humanized, or human antibody.
In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.
In certain aspects, an isolated nucleic acid molecule is provided comprising a nucleic acid encoding the isolated polypeptide.
In certain aspects, a vector comprising the isolated nucleic acid molecule is provided. In certain exemplary embodiments, the vector is an expression vector. In certain aspects, an expression vector comprising the isolated nucleic acid molecule is provided.
In certain aspects, a host cell comprising the vector is provided. In certain aspects, a host cell comprising the expression vector is provided.
In certain exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain exemplary embodiments, the host cell is of mammalian origin. In certain exemplary embodiments, the host cell is of bacterial origin.
In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.
In certain aspects, a pharmaceutical composition comprising the isolated antibody is provided.
In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307, according to EU numbering.
In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.
In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity or both human and rat FcRn binding affinity.
In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to FcRn binding affinity of the binding polypeptide at elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding off-rate.
In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
In certain exemplary embodiments, the isolated binding polypeptide has altered Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the antibody is a chimeric, humanized, or human antibody.
In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.
In certain aspects, an isolated nucleic acid molecule is provided comprising a nucleic acid encoding the isolated polypeptide.
In certain aspects, a vector comprising the isolated nucleic acid molecule is provided. In certain exemplary embodiments, the vector is an expression vector. In certain aspects, an expression vector comprising the isolated nucleic acid molecule is provided.
In certain aspects, a host cell comprising the vector is provided. In certain aspects, a host cell comprising the expression vector is provided.
In certain exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain exemplary embodiments, the host cell is of mammalian origin. In certain exemplary embodiments, the host cell is of bacterial origin.
In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.
In certain aspects, a pharmaceutical composition comprising the isolated antibody is provided.
In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain, wherein the modified Fc domain comprises a tyrosine (Y) at amino acid position 252 and an aspartic acid (D) at amino acid position 256, according to EU numbering.
In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.
In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity or both human and rat FcRn binding affinity.
In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to FcRn binding affinity of the binding polypeptide at elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding off-rate.
In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
In certain exemplary embodiments, the isolated binding polypeptide has altered Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the antibody is a chimeric, humanized, or human antibody.
In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.
In certain aspects, an isolated nucleic acid molecule is provided comprising a nucleic acid encoding the isolated polypeptide.
In certain aspects, a vector comprising the isolated nucleic acid molecule is provided. In certain exemplary embodiments, the vector is an expression vector. In certain aspects, an expression vector comprising the isolated nucleic acid molecule is provided.
In certain aspects, a host cell comprising the vector is provided. In certain aspects, a host cell comprising the expression vector is provided.
In certain exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain exemplary embodiments, the host cell is of mammalian origin. In certain exemplary embodiments, the host cell is of bacterial origin.
In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.
In certain aspects, a pharmaceutical composition comprising the isolated antibody is provided.
In certain aspects, an isolated binding polypeptide comprising a modified Fc domain is provided, wherein the modified Fc domain comprises a combination of at least four amino acid substitutions comprising: aspartic acid (D) or glutamic acid (E) at amino acid position 256 and tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprising phenylalanine (F) or tyrosine (Y) at amino acid position 434; and tyrosine (Y) at amino acid position 252, wherein amino acid position is according to EU numbering.
In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain having a combination of amino acid substitutions at positions selected from the group consisting of: a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; d) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434; or e) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434, wherein the amino acid substitutions are in accordance with EU numbering.
In certain aspects, there is provided an isolated binding polypeptide comprising a modified Fc domain comprising: a quadruple amino acid substitution selected from the group consisting of M252Y/T256D/T307Q/N434Y, M252Y/T256E/T307W/N434Y, M252Y/T256E/T307Q/N434Y, M252Y/T256D/T307Q/N434F and M252Y/T256D/T307W/N434Y, wherein the amino acid substitution is according to EU numbering.
In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.
In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity. In certain exemplary embodiments, the binding polypeptide has rat FcRn binding affinity. In certain exemplary embodiments, the binding polypeptide has human and rat FcRn binding affinity.
In certain exemplary embodiments, the isolated binding polypeptide has altered FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.
In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at non-acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at non-acidic pH as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the non-acidic pH is about 7.4.
In certain exemplary embodiments, the isolated binding polypeptide has an altered serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the isolated binding polypeptide has altered Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced fcyriiia binding affinity as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the isolated binding polypeptide has reduced thermostability as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced thermostability as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the isolated binding polypeptide is an antibody. In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the isolated antibody is a chimeric, humanized, or human antibody. In certain exemplary embodiments, the isolated antibody is a full-length antibody.
In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more targets.
In certain aspects, an isolated nucleic acid molecule is provided comprising a nucleic acid encoding the isolated polypeptide.
In certain aspects, a vector comprising the isolated nucleic acid molecule is provided.
In certain exemplary embodiments, the vector is an expression vector.
In certain aspects, a host cell comprising the vector is provided.
In certain exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain exemplary embodiments, the host cell is of mammalian origin. In certain exemplary embodiments, the host cell is of bacterial origin.
In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.
In certain aspects, a pharmaceutical composition comprising the isolated antibody is provided.
In certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434, according to EU numbering.
In certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434, according to EU numbering.
In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain comprising tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434, according to EU numbering.
In certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434, according to EU numbering.
In certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434, according to EU numbering.
In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.
In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity.
In certain exemplary embodiments, the isolated binding polypeptide has a reduced serum half-life as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.
In certain exemplary embodiments, the isolated binding polypeptide has reduced Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced fcyriiia binding affinity as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the isolated binding polypeptide has reduced thermostability as compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced thermostability as compared to a binding polypeptide comprising M252Y/S254T/T256E/H433K/N434F.
In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the antibody is a chimeric, humanized, or human antibody.
In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more targets.
In certain aspects, an isolated nucleic acid molecule is provided comprising a nucleic acid encoding the isolated polypeptide.
In certain aspects, an expression vector comprising the isolated nucleic acid molecule is provided.
In certain aspects, a host cell comprising the expression vector is provided.
In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.
In certain aspects, a method of treating a disease or disorder in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide, or administering to the subject a therapeutically effective amount of the pharmaceutical composition.
In certain exemplary embodiments, the disease or disorder is cancer. In certain exemplary embodiments, the cancer is a tumor.
In certain exemplary embodiments, the disease or disorder is an autoimmune disorder.
In certain aspects, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide, or administering to the subject a therapeutically effective amount of the pharmaceutical composition.
In certain aspects, a method of treating an autoimmune disorder in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide, or administering to the subject a therapeutically effective amount of the pharmaceutical composition.
Brief Description of Drawings
The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments taken together with the accompanying drawings.
FIGS. 1A-1B depict the structure of an FcRn interacting with the Fc region of IgG1 FIG. 1A depicts the interaction between hFcRn and IgG1 Fc (pdb: 4n0u) showing one Fc monomer (dark gray band), including glycosylation of the rods shown as "glycan" tags in complexes with the α domain (gray) and β 2-m (light gray) hFcRn subunitsH2-
Fig. 2A-2D depict Octet screening assays and results. Fig. 2A schematically presents an Octet screening assay. The NiNTA biosensor captures histidine-tagged antigen and subsequently antibody variants for rat fcrn (rfcrn) binding kinetics. Figure 2B depicts the binding kinetics curves for rFcRn at pH6.0 for the wild type (solid line), T307A/E380A/N434A (AAA) variants (dashed line), LS (dashed line interspersed with a single dot), YTE (long dashed line), H435A (dashed line interspersed with a single dot) and H310A/H435Q (dashed line interspersed with a two dot) antibodies, which align with the start of the rFcRn association phase. The H435A and H310A/H435Q variants showed little to no FcRn binding. YTE variants were detected in the Octet rFcRn binding assay with the slowest FcRn off-rate. Figure 2C graphically depicts FcRn binding kinetics normalization by Octet screening of the mutant subset at pH 6.0. Most mutants retained significant binding to rFcRn, but several mutants were similar to the mock control (dash-dot line), showing loss of all rFcRn binding (long-dashed line, located below the dash-dot line (mock)). Both variants (solid line) have slower rFcRn off-rates than the wild-type antibody (bold dash). Figure 2D depicts a scatter plot analysis of the rFcRn off-rates of all point mutations, where rFcRn binding kinetics separated by residue positions can be observed. The saturation variants fall within one of the following four rFcRn off-rate schemes: no binding (not shown), faster binding (black), wild-type-like binding (white), slower binding (grey). The 18 mutants showed significantly slower off-rates from rFcRn than the wild-type antibody (black dot and dash).
Figure 3 graphically depicts Biacore kinetics of the baseline and wild-type variants with human and rat FcRn at pH6.0 and pH 7.4. All FcRn binding curves for each human (first and third columns) and rat (second and fourth columns) FcRn at pH6.0 (first and third rows) and pH7.4 (second and fourth rows) are shown for a range of concentrations of wild type (top left), AAA variant (top right), M428/N434S (LS) variant (bottom left) and M252Y/S254T/T256E (YTE) variant (bottom right). AAA, LS and YTE variants showed slower off-rates from FcRn than wild-type antibody. In general, the antibody binds rFcRn with about 10-fold increased affinity compared to wild-type. The LS variant has the closest affinity for hFcRn at pH7.4 and the greatest residue binding at pH7.4, while rFcRn binds the YTE variant most tightly.
Figure 4A graphically depicts Biacore kinetics of the lead-saturated variant with human and rat FcRn at pH 6.0. FcRn binding kinetic traces are shown for a concentration series of 18 lead saturation variants. M252Y, T256D, T256E, N434F, N434P, N434Y, T307A, T307E, T307F, T307Q and T307W have slower off-rates from both human and rat FcRn. The remaining variants are specific only for rat FcRn.
Figure 4B graphically depicts FcRn binding kinetics of WT, baseline and lead single-saturated variants to human FcRn at pH 6.0. FcRn binding sensorgram for human FcRn at pH6.0 for a range of concentrations of WT, LS, YTE and 18 saturated variants. The single saturated variants used in the combinatorial library are underlined and in bold.
Figures 5A to 5D depict data showing multiple variants with slower off-rates from both human and rat FcRn at pH 6.0. Figures 5A and 5B depict Biacore sensorgrams for a number of variations. FIG. 5A depicts the off-rates of human FcRn for YTE variant (long dashed line with a single dot), LS variant (long dashed line with a two dot, wild type (WT; dotted line) and leader saturation variant (leader; solid lines of various shades) at pH6.0 in FIG. 5A normalized sensorgrams are depicted showing improved off-rates of hFcRn compared to WT.5B depicts the off-rates of rat FcRn for AAA variant (dashed line), LS variant (dashed line with two dot), YTE variant (dashed line with a single dot inclusion), wild type (solid line) and leader saturation variant (dashed lines of different frequencies and thicknesses) at pH 6.0. for clarity representative injections of each of the 11 leader antibodies are shown. these leader single variants show improved off-rate kinetics from human and rat FcRn compared to wild type. FIGS. 5C and 5D depict association rates using Biacore kinetics measurements, leader saturation (white circles) and wild type (black circles) antibody variants binding affinity maps to human (figure 5C) and rat (figure 5D) FcRn. The baseline variant is shown: AAA (diagonal facing down right), LS (dotted line), and YTE (diagonal facing down left). Despite the improved FcRn off-rate, most variants do not have tighter affinity for human or rat FcRn due to slower association kinetics. The 11 variants had slower off rates from the FcRn of both species.
Fig. 6A-6D depict data showing that the combination of leading saturation mutations further improves FcRn off-rate and binding affinity. Fig. 6A and 6B depict representative Biacore sensorgrams showing FcRn off-rates for human and rat FcRn, respectively. Figure 6A depicts normalized sensorgrams of human FcRn for representative variants of the single (dashed line), double (solid light grey), triple (solid grey) and quadruple (solid black line) combination variants compared to wild type (dashed line) and LS variants (dashed long line interspersed with two dots). Figure 6B depicts normalized sensorgrams of rat FcRn for representative variants of single (two-dot interspersed dash), double (single-dot interspersed dash), triple (dash-long line), and quadruple (dash-short line) combination variants compared to wild type (dot-dash line) and YTE variants (solid line). Incorporation of multiple mutations reduces the off-rate and enhances the binding affinity for FcRn to a greater extent than the baseline variant. Figures 6C and 6D depict combined saturation variant plots showing association rate as a function of off-rate for human (figure 6C) or rat (figure 6D) FcRn, revealing that most variants have enhanced binding to FcRn at pH6.0 compared to the baseline variant. The most tightly bound variants for human and rat FcRn are the quadruple and double combinations, respectively.
Figure 7D in figure 7A depicts data showing that enhanced FcRn binding at pH6.0 disrupts the pH dependence of the interaction. FIGS. 7A and 7B depict singleness (two-interspersed) at pH7.4 compared to wild type (dot-dash line) and LS (FIG. 7A, solid line) and YTE (FIG. 7B, solid line) variantsRepresentative sensorgrams of Biacore FcRn binding kinetics for long-dashed lines of dots), double (long-dashed lines interspersed with single dots), triple (long-dashed lines), and quadruple (short-dashed lines) combinatorial variants. Increasing the number of mutations that enhance FcRn binding results in greater residue binding at physiological pH, with most double, triple and quadruple variants showing robust binding to both fcrns. Figures 7C and 7D depict steady state RU (r) FcRn for all saturating variants at pH7.4 for human (figure 7C) or rat (figure 7D)
Figures 8A to 8C depict data obtained from FcRn affinity chromatography and differential scanning fluorescence assay (DSF) of the baseline variants. FIG. 8A depicts the normalized elution profiles of WT (solid black line), AAA (dot-dash line), LS (long-dashed line with two dots), YTE (long-dashed line with one dot), H435A (solid light grey line) and H310A/H435Q (AQ; solid dark grey line) variants. The pH values are shown at the top of the graph. The variants that did not bind FcRn (H435A, H310A/H435Q) did not bind to the column and eluted in the effluent (<10 mL). AAA, LS and YTE variants eluted at higher pH than WT antibody. Figure 8B depicts DSF curves for WT (black), LS (grey) and YTE (dark grey) variants. YTE is unstable compared to WT and LS. Figure 8C depicts the FcRn affinity column elution profiles of the 7 lead single variants for the combined variants compared to WT and LS variants (vertical dashed lines). Both variants (N434F/Y) eluted at higher pH than LS, indicating a pH-dependent decrease in the interaction with FcRn for variants containing these mutations.
Fig. 9A to 9D depict data showing that the combination variants significantly perturb pH dependence and thermostability. Figure 9A depicts representative FcRn affinity chromatograms for single (long dashed lines interspersed with two dots), double (long dashed lines interspersed with a single dot), triple (long dashed lines), and quadruple variants (dashed lines). Increasing the number of FcRn binding enhancing mutations shifts the elution to higher pH values; LS variant (small vertical dashed line). Figure 9B depicts a box plot of elution pH for lead saturation and combination variants including single (white circles), double (horizontal lines), triple (vertical lines), and quadruple (checkered) mutants, indicating a trend of higher pH values with increasing number of FcRn-enhancing mutants. FIG. 9C shows a high correlation between elution pH from FcRn affinity chromatography and hFcRn off-rate using Biacore (R)20.94) revealed a pH-dependent loss of antibody-FcRn interaction, which was accompanied by improved FcRn dissociation kinetics. AAA (diagonal to the bottom right), LS (dot-dash line) and YTE (diagonal to the bottom left) variants had similar hFcRn off-rates and elution pH values as the duplex variant. FIG. 9D depicts T obtained from DSF combining saturated variantsmBox plot of (a), which reveals that additional mutations that enhance FcRn binding destabilize the antibody compared to WT, single or baseline variants.
Fig. 10A-10B depict data obtained from FcRn affinity chromatography and DSF of 7 lead variants. Figure 10A depicts FcRn affinity chromatography of M252Y (solid line), T256D (dashed line with single dot inclusion), T256E (long dashed line), T307Q (long dashed line with single dot inclusion), T307W (long dashed line with two dot inclusion), N434F (dashed line) and N434Y (dashed line) variants. The chromatogram revealed a change in elution pH compared to wild type and LS antibodies (vertical dashed line). N434F and N434Y had higher elution pH (pH around 8.3) than the LS variant (vertical dashed line). The pH at certain elution volumes is shown above the chromatogram for reference. Figure 10B depicts DSF curves for 7 lead variants showing that none of the 7 lead single variants destabilized the antibody to the same extent as the YTE variant (vertical dashed line). All variants except T307Q (dashed long line with single dot inclusion) were unstable compared to WT (dashed vertical line).
Fig. 11A-11C depict data showing reduced Fc γ RIIIa binding in the combination variant containing M252Y. Figure 11A shows Fc γ RIIIa binding sensorgrams for WT (black), LS (grey) and YTE (dark grey) variants revealing reduced binding reactions by YTE variants. Figure 11B depicts boxplots of Fc γ RIIIa binding responses for baseline, single and combination variants as shown. The variant with the M252Y mutation contained a reduced binding response to Fc γ RIIIa, including all quadruple variants. The combination with N434F/Y generally showed enhanced reaction with Fc γ RIIIa. Figure 11C depicts Fc γ RIIIa binding responses of 7 lead single variants compared to WT and YTE variants (horizontal dashed lines). The M252Y mutation showed reduced Fc γ RIIIa binding compared to WT, while 6 variants showed similar WT or enhanced binding to this receptor.
Figures 12A to 12D depict data obtained from FcRn affinity chromatography, DSF and Fc γ RIIIa binding of 7 lead combination variants. Figure 12A depicts the FcRn affinity chromatograms of the 7 lead combinatorial variants compared to wild type antibody and LS variants (vertical dashed and solid lines, respectively). The elution pH of each lead variant was close to the LS variant. Figure 12B shows DSF curves for the lead combination variants compared to YTE and wild type variants (vertical dashed lines as shown). Six of the 7 leader variants had similar or less stable T than YTE variantm: MDWN (long dash with two dotted lines); YTWN (long dashed line); YDTN (solid line); YETN (dash line with single dot); YDQN (dashed line); YEQN (dashed line with single dot inclusion). MDQThe N variant has a similar T to the wild type antibody (dashed line)m. Figure 12C depicts Biacore sensorgrams of Fc γ RIIIa binding kinetics for the 7 lead variants compared to wild-type (larger dashed line) and YTE variants (thick long dashed line). Variant containing M252Y:YDTN (solid line),YDQN (dashed lines with single dot),YTWN (long dash line),YETN (double-dot dash line) andYEQN (smaller dot-dash line), each of which has a reduced steady-state RU in a similar manner to YTE. (D) Shown are the steady state RUs of 7 lead variants, wild-type and YTE variants. Only MDWN and MDQN variant pairsHas similar affinity to wild-type antibodies in Fc γ RIIIa.
Fig. 12E-12H depict data showing that 3 lead variants exhibit a range of key antibody attributes. Figure 12E shows FcRn affinity chromatography elution profiles of DQ (solid line), DW (dashed line) and YD (dashed line) variants compared to WT and LS (vertical dashed line). Each double variant showed an elution pH between WT and LS. Fig. 12F depicts DSF fluorescence curves of the three variants compared to YTE and WT variants (vertical dot-dash line) revealing that YD (dash line) and DW (dot-dash line) are slightly unstable compared to YTE, but DQ (solid line) is similar to WT. Figure 12G depicts Fc γ RIIIa binding sensorgram compared to WT and YTE (horizontal dashed line). YD (dashed line) shows similar binding reactions to YTE, while DQ (solid line) and DW (dashed line) show a slight decrease compared to WT. Figure 12H depicts data showing that the same bridging RF ELISA revealed 3 lead variants and YTE showed significantly reduced or WT-like RF binding different from LS. P <0.001, p < 0.01.
Figures 13A to 13D depict data showing a comparison of FcRn binding kinetics for the lead combination variants at pH6.0 and pH 7.4. Figures 13A and 13B show Biacore FcRn binding sensorgrams for the lead combination variants for human FcRn (figure 13A) or rat FcRn (figure 13B) compared to wild type (dashed line) and LS (hFcRn, figure 13A, dashed line) or YTE (rFcRn, figure 13B, dashed line) at pH 6.0. Each combination variant has an overall tighter binding affinity for the corresponding FcRn despite the altered association and dissociation rates. Figures 13C and 13D show Biacore FcRn sensorgrams at pH 7.4. Each hFcRn lead variant has a similar or reduced steady-state FcRn binding response compared to the LS variant. Only MDQN and MDWThe N variants showed less binding of rFcRn than the YTE variants at pH 7.4.
Fig. 14 is a table depicting Octet rfcn binding off-rates for saturated libraries according to certain embodiments. Wild Type (WT) and similar wild type (WT-like) species are indicated by white rectangles; WT classes are shown. Variants with little to no rFcRn binding compared to wild type are indicated by dark grey rectangles. Variants with faster rFcRn off-rate compared to wild type are indicated by light grey rectangles, and variants with slower rFcRn off-rate compared to wild type are indicated by black rectangles.
Fig. 15A-15C depict a new binding assay developed using a CM5 sensor chip. Fig. 15A is a schematic of this assay. Figure 15B shows direct immobilization of FcRn. Figure 15C shows streptavidin capture of biotinylated FcRn.
Fig. 16A-16B depict FcRn binding of antibody 2 at pH 6.0. Figure 16A depicts human FcRn. Figure 16B depicts mouse FcRn.
Fig. 17A-17B depict FcRn binding of antibody 2 at pH 7.4. Figure 17A depicts human FcRn. Figure 17B depicts a mouse FcRn.
Figure 18 graphically depicts the pH dependence of various antibody 2 variants. The leader variant maintained higher binding affinity at pH6 and lower residue binding than LS at pH 7.4.
Figure 19 depicts a comparison of FcRn binding pH dependence using the scaffolds of
Figure 20 depicts a comparison of the thermostability of
Figure 21 depicts a comparison of Fc γ RIIIa binding using the scaffolds of
Fig. 22A-22I depict various graphs showing that DQ, DW, and YD variants can be transferred between IgG1 backbones. Panels a-c depict FcRn binding sensorgrams normalized at pH6.0 in three IgG1 scaffolds, where WT (light grey), LS (dark grey), DQ (solid black), DW (dashed line) and YD (dashed line) variants show similar kinetics at low pH. The three variants DQ, DW and YD have slightly faster rates of association and dissociation than the LS variant, but maintain tighter FcRn binding affinity. Panels d-f depict FcRn binding sensorgrams at pH 7.4; LS baseline variant (solid black). Panels g to i depict a comparison of FcRn binding response at pH7.4 and binding affinity at pH6.0 for each antibody scaffold with WT (grey), LS (dark grey), DQ (solid black), DW (open circles) and YD (open squares) variants. DQ, DW and YD showed improved FcRn characteristics with enhanced binding at pH6.0 and minimal binding at pH 7.4.
Fig. 23A to 23C show that three leader variants in the mAb2 backbone similarly improved binding to cynomolgus monkey FcRn. Fig. 23A depicts normalized fcrn binding sensorgrams for WT (grey), LS (dark grey), DQ (solid black), DW (dashed line) and YD (dashed line) at pH6.0, showing similar binding kinetics and affinity as hFcRn. Figure 23B depicts a significant decrease in the fcrn binding response of the three variants at physiological pH; LS (dark grey) shows stronger binding than WT (grey) in a similar way as hFcRn. Figure 23C depicts a comparison of residue ccfcrn binding responses at pH7.4 for WT (grey), LS (dark grey), DQ (solid black), DW (open circles) and YD (open squares) to ccfcrn binding affinity at pH6.0, revealing that all three variants maintain the improved FcRn binding characteristics observed for hFcRn.
Fig. 24A-24B show that lead variants extend antibody serum half-life. Pharmacokinetic curves of plasma antibody concentrations as a function of time for WT (black circles with black solid lines), LS (white circles with black dashes), DQ (light grey circles with light grey solid lines), DW (dark grey circles with dark grey solid lines) and YD (black circles with black dashed lines) antibodies in cynomolgus monkeys (fig. 24A) and hFcRn transgenic mice (fig. 24B). All three lead variants extended antibody half-life compared to WT.
Figure 25 depicts a plot of the steady state RU for human FcRn for all saturating variants at pH7.4 as a function of binding affinity at pH 6.0. A comparison of residue FcRn binding at pH7.4 and FcRn binding affinity at pH6.0 is shown. The quadruple combination with improved FcRn binding properties at pH6.0 and pH7.4 is shown in box in the upper right quadrant of the figure. Single (white circles), double (light grey circles), triple (dark grey circles) and quadruple (black circles) variants are shown as well as the baseline AAA, LS and YTE variants (as shown).
Figure 26 depicts a schematic of the biotin CAPture method for capturing biotinylated FcRn.
Figure 27 depicts a graph showing human FcRn binding kinetics at pH6.0 for YTEKF baseline and combination variants as shown.
Figures 28A-28B show FcRn binding kinetics of the combination variants compared to YTEKF baseline at pH6.0 (figure 28A) and pH7.4 (figure 28B). Wild type is indicated by solid black line (WT) and YTEKF reference by dashed line.
Figure 29 depicts a graph of the steady state RU for human FcRn for selected variants at pH7.4 as a function of binding affinity at pH6.0 compared to YTEKF baseline. Several variants (lead quadruplets) showed enhanced binding affinity for human FcRn relative to the YTEKF baseline at pH6.0 and pH 7.4.
Detailed Description
The present disclosure provides binding polypeptides (e.g., antibodies) having altered Fc neonatal receptor (FcRn) binding affinity. In certain embodiments, the binding polypeptide comprises a modified Fc domain that enhances FcRn binding affinity as compared to a binding polypeptide comprising a wild-type (e.g., non-modified) Fc domain. The disclosure also provides nucleic acids encoding the binding polypeptides, recombinant expression vectors and host cells for making the binding polypeptides, as well as pharmaceutical compositions comprising the binding polypeptides disclosed herein. Also provided are methods of treating diseases using the binding polypeptides of the disclosure.
The Fc domain of an immunoglobulin is involved in non-antigen binding functions and has several effector functions mediated by binding of effector molecules (e.g., binding of FcRn). As shown in fig. 1A, the Fc domain comprises a CH2 domain and a CH3 domain. Most of the residues involved in the interaction with FcRn are located at CH2-
In certain embodiments, a binding polypeptide can comprise a modified Fc domain comprising amino acid substitutions that alter the antigen-independent effector function of an antibody, in particular, alter the circulating half-life (e.g., serum half-life) of the binding polypeptide. In some embodiments, a binding polypeptide can comprise a modified Fc domain comprising an amino acid substitution that alters the serum half-life of the binding polypeptide compared to a binding polypeptide comprising a wild-type (i.e., unmodified) Fc domain. In some embodiments, a binding polypeptide can comprise a modified Fc domain comprising an amino acid substitution that increases the serum half-life of the binding polypeptide compared to a binding polypeptide comprising a wild-type (i.e., unmodified) Fc domain. In some embodiments, a binding polypeptide can comprise a modified Fc domain comprising an amino acid substitution that reduces the serum half-life of the binding polypeptide compared to a binding polypeptide comprising a wild-type (i.e., unmodified) Fc domain.
In certain embodiments, a binding polypeptide comprising a modified Fc domain that alters (i.e., increases or decreases) circulatory half-life (e.g., serum half-life) further comprises one or more mutations in addition to the one or more mutations that alter circulatory half-life. In certain embodiments, one or more mutations other than one or more mutations that alter circulating half-life provide one or more desired biochemical characteristics, such as one or more of reduced or enhanced effector function, non-covalent dimerization ability, enhanced ability to localize to a tumor site, reduced serum half-life, increased serum half-life, or the like, when compared to an intact, unaltered antibody of about the same immunogenicity.
The binding polypeptides described herein may exhibit increased or decreased binding to neonatal Fc receptor (FcRn) when compared to binding polypeptides lacking these substitutions, and thus have increased or decreased serum half-life, respectively. Fc domains with improved FcRn affinity are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where it is desirable for the administered antibody to have a long half-life, for example to treat chronic diseases or disorders. Conversely, Fc domains with reduced FcRn binding affinity are expected to have shorter serum half-lives, and such molecules may also be useful, for example, for administration to mammals where reduced circulation time may be advantageous, for example, for in vivo diagnostic imaging or in cases where the starting antibody has toxic side effects when present in circulation for a prolonged period of time. Fc domains with reduced FcRn binding affinity are also less likely to cross the placenta and are therefore also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications that may require a reduction in FcRn binding affinity include applications that are limited to brain, kidney and/or liver.
It is to be understood that the methods described in this disclosure are not limited to the particular methods and experimental conditions disclosed herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Furthermore, unless otherwise indicated, the experiments described herein employ conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled person and are explained fully in the literature. See, for example, Ausubel et al, eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987. 2008), including all supplements, Molecular Cloning, A Laboratory Manual (fourth edition), edited by MR Green and J.Sambrook, and Harlow et al Antibodies, Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2 nd edition).
Unless defined otherwise, scientific and technical terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The definitions provided herein take precedence over any dictionary or external definition if any potential ambiguity exists. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and/or" unless otherwise specified. The use of the term "including" as well as other forms such as "includes" and "included" is not limiting.
Generally, the terminology used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed in the present specification, unless otherwise indicated. Enzymatic reactions and purification techniques are generally performed according to the manufacturer's instructions as is commonly practiced in the art or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, and the laboratory procedures and techniques, are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.
In order that the disclosure may be more readily understood, selected terms are defined as follows.
The term "polypeptide" refers to any polymeric chain of amino acids and encompasses natural or artificial proteins, polypeptide analogs, or variants of protein sequences or fragments thereof, unless the context otherwise contradicts. The polypeptide may be monomeric or polymeric. For example, a polypeptide fragment comprises at least about 5 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids.
The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by virtue of its origin or derivative source, is not associated with its naturally associated components with which it naturally accompanies it; substantially free of other proteins from the same species; expressed by cells from different species; or do not occur in nature. Thus, a protein or polypeptide that is chemically synthesized or synthesized in a cellular system other than the cell from which it is naturally derived will be "separated" from its naturally associated components. Proteins or polypeptides may also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.
As used herein, the term "binding protein" or "binding polypeptide" shall refer to a protein or polypeptide (e.g., an antibody or immunoadhesin) that contains at least one binding site that is responsible for selectively binding a target antigen of interest (e.g., a human target antigen). Exemplary binding sites include antibody variable domains, ligand binding sites of receptors, or receptor binding sites of ligands. In certain aspects, a binding protein or binding polypeptide includes a plurality (e.g., two, three, four, or more) binding sites. In certain aspects, the binding protein or binding polypeptide is not a therapeutic enzyme.
The term "ligand" refers to any substance capable of binding or being bound to another substance. Similarly, the term "antigen" refers to any substance that can produce an antibody. Although "antigen" is commonly used to refer to an antibody binding substrate, and "ligand" is often used in reference to a receptor binding substrate, these terms are not distinguished from each other and encompass a wide range of overlapping chemical entities. For the avoidance of doubt, antigen and ligand are used interchangeably herein. The antigen/ligand may be a peptide, polypeptide, protein, aptamer, polysaccharide, sugar molecule, carbohydrate, lipid, oligonucleotide, polynucleotide, synthetic molecule, inorganic molecule, organic molecule, and any combination thereof.
The term "specifically binds" as used herein refers to an antibody or immunoadhesin at up to about 1x 10-6M, about 1X 10-7M, about 1X 10-8M, about 1X 10-9M, about 1X 10-10M, about 1X 10-11M, about 1X 10-12M or lower dissociation constant (Kd) to antigen, and/or the ability to bind to antigen with an affinity that is at least about 2-fold greater than the affinity for a non-specific antigen.
As used herein, the term "antibody" refers to an assembly (e.g., an intact antibody molecule, immunoadhesin or variant thereof) having a significantly known specific immunoreactivity for an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins comprise a light chain and a heavy chain with or without an interchain covalent linkage between the light chain and the heavy chain. The basic immunoglobulin structure in vertebrate systems is relatively clear.
As will be discussed in more detail below, the generic term "antibody" includes five different classes of antibodies that can be biochemically distinguished. While all five classes of antibodies are clearly within the scope of the present disclosure, the following discussion will be directed generally to the IgG class of immunoglobulin molecules. With regard to IgG, an immunoglobulin comprises two identical light chains with a molecular weight of about 23,000 daltons and two identical heavy chains with a molecular weight of 53,000-70,000. The four chains are linked via disulfide bonds in a "Y" configuration, with the light chain ranging from the "Y" port next to the heavy chain and continuing to the end of the variable region.
Immunoglobulin light chains are classified as kappa (. kappa.) or lambda (. lamda.). Each heavy chain class may be associated with a kappa or lambda light chain. Typically, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other via a covalent disulfide linkage or a non-covalent linkage. In the heavy chain, the amino acid sequence extends from the N-terminus of the bifurcated end of the Y configuration to the C-terminus of the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as γ (γ), μ (μ), α (α), () or (), with some subclasses (e.g., γ l- γ 4). The nature of this chain determines the "class" of an antibody as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to confer functional specialization. Modifications of each of these classes and isoforms are readily discernible to the skilled artisan as they are disclosed in time and are therefore within the scope of the disclosure.
Both the light and heavy chains are divided into regions of structural and functional homology. The term "region" refers to a portion of an immunoglobulin or antibody chain ("part" or "portion") and includes constant or variable regions, as well as more discrete fragments or portions of such regions. For example, the light chain variable region comprises "complementarity determining regions" or "CDRs" interspersed between "framework regions" or "FRs" as defined herein.
Regions of immunoglobulin heavy or light chains may be defined as "constant" (C) regions, in the case of "constant regions" based on the relative absence of sequence variation within the region of the plurality of class members, or "variable" (V) regions, in the case of "variable regions" based on significant variation within the region of the plurality of class members. The terms "constant region" and "variable region" may also be used with respect to functionality. In this regard, it is understood that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. In contrast, the constant regions of immunoglobulins or antibodies confer important effector functions such as secretion, transplacental movement, Fc receptor binding, complement fixation, and the like. The subunit structures and three-dimensional configurations of constant regions of various immunoglobulin classes are well known.
The constant and variable regions of immunoglobulin heavy and light chains are folded into domains. The term "domain" refers to a globular region of a heavy or light chain that comprises peptide loops (e.g., comprising 3 to 4 peptide loops) that are stabilized, for example, via beta sheet and/or intrachain disulfide bonds. The constant region on the light chain of an immunoglobulin is interchangeably referred to as a "light chain constant region domain", "CL region", or "CL domain". The constant domains on the heavy chain (e.g., the hinge, CH1, CH2, or CH3 domains) are interchangeably referred to as "heavy chain constant region domains", "CH" region domains, or "CH domains". Variable domains on the light chain are interchangeably referred to as "light chain variable region domains", "VL region domains", or "VL domains". The variable domains on the heavy chain are interchangeably referred to as "heavy chain variable region domains", "VH region domains", or "VH domains".
By convention, the amino acid numbering of the variable constant region domains increases as they move away from the antigen binding site or amino terminus of the immunoglobulin or antibody. The N-terminus of each of the heavy and light chain immunoglobulin chains is the variable region, and the C-terminus is the constant region. The CH3 and CL domains comprise the carboxy-terminal ends of the heavy and light chains, respectively. Thus, the domains of the light chain immunoglobulin are aligned in the VL-CL orientation, while the domains of the heavy chain are aligned in the VH-CH 1-hinge-CH 2-CH3 orientation.
The amino acid assignment for each variable region domain is as defined by Kabat, Sequences of Proteins of immunological Interest (National Institutes of Health, Bethesda, MD,1987 and 1991). Kabat alsoA widely used numbering convention (Kabat numbering) is provided in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number.
As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino-terminal variable domain of an immunoglobulin light chain.
As used herein, the term "
As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain with the CH2 domain. The hinge region comprises about 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al J. Immunol.1998,161: 4083).
As used herein, the term "CH 2 domain" includes that portion of a heavy chain immunoglobulin molecule that extends, for example, from about position 244-. The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. In one embodiment, the binding polypeptides of the present disclosure comprise a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).
As used herein, the term "
As used herein, the term "CL domain" includes the constant region domain of an immunoglobulin light chain, which extends, for example, from about Kabat position 107A to about Kabat position 216. The CL domain is adjacent to the VL domain. In one embodiment, a binding polypeptide of the present disclosure comprises a CL domain derived from a kappa light chain (e.g., a human kappa light chain).
As used herein, the term "Fc region" is defined as that portion of the heavy chain constant region that begins at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, the first residue of the heavy chain constant region being taken as 114) and ends at the C-terminus of the antibody. Thus, a complete Fc region comprises at least a hinge domain, a CH2 domain, and a CH3 domain.
As used herein, the term "native Fc" or "wild-type Fc" refers to a molecule comprising the sequence of a non-antigen-binding fragment, in monomeric or multimeric form, resulting from antibody digestion or otherwise produced; and the term may include a hinge region. The original immunoglobulin source of the native Fc is typically of human origin and may be any immunoglobulin, such as IgG1 and IgG 2. Native Fc molecules consist of monomeric polypeptides that can be joined into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA 2). An example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. As used herein, the term "native Fc" is generic to monomeric, dimeric and multimeric forms.
As used herein, the term "Fc variant" or "modified Fc" refers to a molecule or sequence that is modified from a native/wild-type Fc but still comprises a binding site for FcRn. Thus, the term "Fc variant" may include molecules or sequences that are humanized from a non-human native Fc. In addition, native Fc contains regions that can be removed because they provide undesirable structural features or biological activity for the antibody-like binding polypeptides described herein. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or in which one or more Fc sites or residues have been modified, which affect or are involved in: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).
In certain exemplary embodiments, a particular Fc variant herein has one or more of increased serum half-life, enhanced FcRn binding affinity at acidic pH, enhanced fcyriiia binding affinity, and/or similar thermostability, as compared to an IgG antibody comprising a wild-type Fc.
As used herein, the term "Fc domain" encompasses native/wild-type Fc as defined above as well as Fc variants and sequences. As with Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from intact antibodies or otherwise produced.
As described above, the variable region of an antibody allows it to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains of an antibody combine to form a variable region (Fv) that defines a three-dimensional antigen-binding site. The tetrabasic antibody structure forms an antigen binding site at the end of each arm of the Y. More specifically, the antigen binding site is defined by three Complementarity Determining Regions (CDRs) on each of the heavy and light chain variable regions. As used herein, the term "antigen binding site" includes a site that specifically binds (immunoreacts with) an antigen (e.g., a cell surface or soluble antigen). Antigen binding sites include immunoglobulin heavy and light chain variable regions, and the binding sites formed by these variable regions determine the specificity of the antibody. The antigen binding site is formed by variable regions that differ between antibodies. The altered antibodies of the present disclosure comprise at least one antigen binding site.
In certain embodiments, a binding polypeptide of the present disclosure comprises at least two antigen binding domains that provide binding of the binding polypeptide to a selected antigen. The antigen binding domains need not be derived from the same immunoglobulin molecule. In this regard, the variable region may be derived or derived from any type of animal that can be induced to produce a humoral response and produce immunoglobulins against a desired antigen. Thus, the variable region of a binding polypeptide may be, for example, of mammalian origin, e.g., may be human, murine, rat, goat, sheep, non-human primates (e.g., cynomolgus monkey, macaque, etc.), wolfs, or camelids (e.g., from camels, llamas, and related species).
In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous amino acid sequences that are specifically positioned to form an antigen binding site, since the antibody is assumed to assume its three-dimensional configuration in an aqueous environment. The remainder of the heavy and light chain variable domains exhibit little intermolecular variability in amino acid sequence and are referred to as framework regions. The framework regions adopt predominantly a β -sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β -sheet structure. Thus, these framework regions serve to form a scaffold that provides for the positioning of the six CDRs in the correct orientation by interchain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on the immunoreactive antigen. The complementary surface facilitates non-covalent binding of the antibody to the immunoreactive epitope.
Exemplary binding polypeptides include antibody variants. As used herein, the term "antibody variant" includes synthetic and engineered forms of antibodies that are altered such that they do not naturally occur, e.g., antibodies comprising at least two heavy chain portions but not two complete heavy chains (e.g., domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that are altered to bind to two or more different antigens or to bind to different epitopes on a single antigen; heavy chain molecules linked to scFv molecules, and the like. In addition, the term "antibody variant" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind three, four, or more copies of the same antigen).
As used herein, the term "valency" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to a target molecule or specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site may specifically bind to the same or different molecules (e.g., may bind to different ligands or different antigens, or to different epitopes on the same antigen). The subject binding polypeptides typically have at least one binding site specific for a human antigen molecule.
The term "specificity" refers to the ability to specifically bind to (e.g., immunoreact with) a given target antigen (e.g., a human target antigen). A binding polypeptide may be monospecific and contain one or more binding sites that specifically bind to a target, or a polypeptide may be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In certain embodiments, the binding polypeptide is specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, the binding polypeptide is specific for more than one target. Exemplary binding polypeptides (e.g., antibodies) comprising an antigen binding site that binds an antigen expressed on a tumor cell are known in the art, and one or more CDRs from such antibodies can be included in an antibody as described herein.
As used herein, the term "antigen" or "target antigen" refers to a molecule or a portion of a molecule that is capable of being bound by a binding site of a binding polypeptide. The target antigen may have one or more epitopes.
The term "about" means within about 20%, such as within about 10%, within about 5%, or within about 1% or less of a given value or range.
As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., an isolated binding polypeptide provided herein) present in vitro into a patient, such as by, but not limited to, the term, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art. When controlling or treating a disease or a symptom thereof, administration of the substance typically occurs after onset of the disease or symptom thereof. When preventing a disease or symptoms thereof, administration of the substance typically occurs prior to the onset of the disease or symptoms thereof, and may be continued for a prolonged period of time to delay or reduce the occurrence or extent of disease-related symptoms.
As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients (e.g., an isolated binding polypeptide provided herein) in the amounts optionally specified, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the amounts optionally specified.
An "effective amount" refers to an amount of an active agent (e.g., an isolated binding polypeptide of the present disclosure) sufficient to achieve a desired physiological result in an individual in need of the active agent. The effective amount may vary from individual to individual depending on the health and physical condition of the individual to be treated, the classification group of the individual to be treated, the formulation of the composition, the assessment of the medical condition of the individual, and other relevant factors.
As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In certain embodiments, the term "subject" as used herein refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, non-domesticated animals, farm animals, sport animals, and pets.
As used herein, the term "therapy" refers to any regimen, method and/or agent that can be used to prevent, manage, treat and/or ameliorate a disease or a symptom associated therewith. In some embodiments, the term "therapy" refers to any regimen, method, and/or agent that can be used to modulate an immune response to an infection or symptoms associated therewith in a subject. In some embodiments, the terms "plurality of therapies" and "therapy" refer to biological, supportive, and/or other therapies known to those of skill in the art, such as medical personnel, that can be used to prevent, manage, and/or ameliorate a disease or a symptom associated therewith. In other embodiments, the terms "plurality of therapies" and "therapy" refer to biological, supportive, and/or other therapies known to those of skill in the art, such as medical personnel, that can be used to modulate an immune response to an infection or a symptom associated therewith in a subject.
As used herein, the terms "treatment" and "treating" refer to a reduction or improvement in the progression, severity, and/or duration of a disease or symptom associated therewith resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents such as the isolated binding polypeptides provided herein). The term "treating" as used herein may also refer to altering the disease course of the subject being treated. Therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating one or more symptoms, reducing the direct or indirect pathological consequences of a disease, reducing the rate of disease progression, ameliorating or slowing the disease state, and alleviating or improving prognosis.
Binding polypeptides
In one aspect, the present disclosure provides binding polypeptides (e.g., antibodies, immunoadhesins, antibody variants, and fusion proteins) comprising a modified Fc domain. The binding polypeptides disclosed herein encompass any binding polypeptide comprising a modified Fc domain. In certain embodiments, the binding polypeptide is an antibody or immunoadhesin or derivatives thereof. Any antibody from any source or species can be used in the binding polypeptides disclosed herein. Suitable antibodies include, but are not limited to, human, humanized or chimeric antibodies. Suitable antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, full length antibodies, or single chain antibodies.
Fc domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA, and IgE) and species can be used in the binding polypeptides disclosed herein. Chimeric Fc domains comprising partial Fc domains from different species or Ig classes may also be employed. In certain embodiments, the Fc domain is a human Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In other embodiments, the Fc domain is an IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG1 or IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG1 Fc domain. In the case of Fc domains of other species and/or Ig classes or isotypes, the skilled person will appreciate that any amino acid substitutions described herein may be adjusted accordingly. In some embodiments, the modified Fc domain may comprise amino acid substitutions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, or Y436, and any combination thereof, according to EU numbering. In some embodiments, the modified Fc domain may comprise a double amino acid substitution at any two amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 according to EU numbering. In some embodiments, the modified Fc domain may comprise a triple amino acid substitution at any three amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 according to EU numbering. In some embodiments, the modified Fc domain may comprise a quadruple amino acid substitution at any four amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 according to EU numbering. In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution at any amino acid position selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, or Y436, and any combination thereof according to EU numbering, wherein amino acid position N434 is unsubstituted (i.e., amino acid position N434 is wild-type).
In some embodiments, the modified Fc domain may comprise an amino acid substitution selected from M252Y (i.e., tyrosine at amino acid position 252), T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, N434F, N434P, N434Y, Y436H, Y436N, or Y436W, and any combination thereof, according to EU numbering. In some embodiments, the modified Fc domain may comprise a double amino acid substitution according to EU numbering selected from the group consisting of: m252, wherein the substitution is M252Y; t256, wherein the substitution is T256D or T256E; k288, wherein the substitution is K288D or K288N; t307, wherein the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W; e380, wherein the substitution is E380C; n434, wherein the substitution is N434F, N434P, or N434Y; y436, wherein the substitution is Y436H, Y436N or Y436W. In some embodiments, the modified Fc domain may comprise triple amino acid substitutions according to EU numbering selected from the group consisting of: m252, wherein the substitution is M252Y; t256, wherein the substitution is T256D or T256E; k288, wherein the substitution is K288D or K288N; t307, wherein the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W; e380, wherein the substitution is E380C; n434, wherein the substitution is N434F, N434P, or N434Y; y436, wherein the substitution is Y436H, Y436N or Y436W. In some embodiments, the modified Fc domain may comprise a quadruple of amino acid substitutions according to EU numbering selected from the group consisting of: m252, wherein the substitution is M252Y; t256, wherein the substitution is T256D or T256E; k288, wherein the substitution is K288D or K288N; t307, wherein the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W; e380, wherein the substitution is E380C; n434, wherein the substitution is N434F, N434P, or N434Y; y436, wherein the substitution is Y436H, Y436N or Y436W. In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, according to EU numbering, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, according to EU numbering, wherein amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, according to EU numbering, wherein amino acid position N434 is unsubstituted (i.e., amino acid position N434 is wild-type).
In certain embodiments, the modified Fc domain may comprise amino acid substitutions selected from M252, T256, T307, or N434, and any combination thereof, according to EU numbering. In certain embodiments, the modified Fc domain may comprise a double amino acid substitution at any two amino acid positions selected from M252, T256, T307, and N434 according to EU numbering. In certain embodiments, the modified Fc domain may comprise triple amino acid substitutions at any three amino acid positions selected from M252, T256, T307, and N434 according to EU numbering. In certain embodiments, the modified Fc domain may comprise quadruple amino acid substitutions at amino acid positions M252, T256, T307, and N434 according to EU numbering. In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from M252, T256, or T307, and any combination thereof according to EU numbering, wherein amino acid position N434 is unsubstituted (i.e., amino acid position N434 is wild-type).
In exemplary embodiments, the modified Fc domain may comprise amino acid substitutions according to EU numbering selected from the group consisting of: m252, wherein the substitution is M252Y; t256, wherein the substitution is T256D or T256E; t307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y, and any combination thereof. In certain embodiments, the modified Fc domain may comprise a double amino acid substitution at any two amino acid positions selected from the group consisting of: m252, wherein the substitution is M252Y; t256, wherein the substitution is T256D or T256E; t307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y. In certain embodiments, the modified Fc domain may comprise a triple amino acid substitution at any three amino acid positions selected from the group consisting of: m252, wherein the substitution is M252Y; t256, wherein the substitution is T256D or T256E; t307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y. In certain embodiments, the modified Fc domain may comprise quadruple amino acid substitutions at amino acid positions selected from the group consisting of: m252, wherein the substitution is M252Y; t256, wherein the substitution is T256D or T256E; t307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y. In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, according to EU numbering, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, according to EU numbering, wherein amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, according to EU numbering, wherein amino acid position N434 is unsubstituted (i.e., amino acid position N434 is wild-type).
In certain embodiments, the modified Fc domain may comprise amino acid substitutions selected from T256D or T256E and/or T307W or T307Q according to EU numbering, and further comprise amino acid substitutions selected from N434F or N434Y or M252Y. In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from T256D or T256E and/or T307W or T307Q according to EU numbering, and further comprising amino acid substitution M252Y, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from T256D or T256E and/or T307W or T307Q according to EU numbering, and further comprising amino acid substitution M252Y, wherein amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from T256D or T256E and/or T307W or T307Q according to EU numbering, and further comprising amino acid substitution M252Y, wherein amino acid position N434 is unsubstituted (i.e., amino acid position N434 is wild-type).
In some embodiments, the modified Fc domain may comprise a double amino acid substitution selected from M252Y/T256D, M252Y/T256E, M252Y/T307Q, M252Y/T307W, M252Y/N434F, M252Y/N434Y, T256D/T307Q, T256D/T307W, T256D/N434F, T256D/N434Y, T256E/T307Q, T256E/T307W, T256E/N434F, T256E/N434Y, T307Q/N434F, T307Q/N434Y, T307W/N434F, and T307W/N434Y according to EU numbering. In some embodiments, the modified Fc domain may comprise a triple amino acid substitution selected from M252/T256/T307, M252/T256/N434, M252/T256/T307, M252/T256/N434, M252/T307/N434, T256/307Q/N434, T256/307W/N434, T256/307Q/N434, T307/W/N434, T256/Q/N434 and T256/W/N434 according to EU numbering.
In some embodiments, the modified Fc domain may comprise a quadruple amino acid substitution selected from M252Y/T256D/T307Q/N434F, M252Y/T256E/T307Q/N434F, M252Y/T256D/T307W/N434F, M252Y/T256E/T307W/N434F, M252Y/T256D/T307Q/N434Y, M252Y/T256E/T307Q/N434Y, M252Y/T256D/T307W/N434Y, and M252Y/T256E/T307W/N434Y, according to EU numbering.
In some embodiments, it may be desirable for the modified Fc domain to comprise a wild-type amino acid at amino acid position N434 according to EU numbering. In some embodiments, it may be desirable for the Fc domain to not comprise phenylalanine (F) or tyrosine (Y) at amino acid position N434 according to EU numbering. In some embodiments, it may be desirable for the Fc domain to not comprise tyrosine (Y) at amino acid position N434 according to EU numbering. In some embodiments, the modified Fc domain may comprise a double amino acid substitution selected from M252Y/T256D, M252Y/T256E, M252Y/T307Q, M252Y/T307W, T256D/T307Q, T256D/T307W, T256E/T307Q, and T256E/T307W, according to EU numbering. In some embodiments, the modified Fc domain may comprise a triple amino acid substitution selected from M252Y/T256D/T307Q, M252Y/T256D/T307W, M252Y/T256E/T307Q, and M252Y/T256E/T307W, according to EU numbering.
In one embodiment, a binding polypeptide with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions as disclosed herein. In one embodiment, a binding polypeptide having enhanced FcRn binding affinity comprises an Fc domain having one or more amino acid substitutions as disclosed herein. In one embodiment, a binding polypeptide having enhanced FcRn binding affinity comprises an Fc domain having two or more amino acid substitutions as disclosed herein. In one embodiment, a binding polypeptide having enhanced FcRn binding affinity comprises an Fc domain having three or more amino acid substitutions as disclosed herein.
In some embodiments, the binding polypeptide may exhibit species-specific FcRn binding affinity. In one embodiment, the binding polypeptide may exhibit human FcRn binding affinity. In one embodiment, the binding polypeptide can exhibit rat FcRn binding affinity. In some embodiments, the binding polypeptide may exhibit cross-species FcRn binding affinity. Such binding polypeptides are believed to be cross-reactive between one or more different species. In one embodiment, the binding polypeptide may exhibit both human and rat FcRn binding affinity.
Neonatal Fc receptor (FcRn) interacts with the Fc region of antibodies to promote circulation by rescuing normal lysosomal degradation. This process is a pH-dependent process that occurs in endosomes at acidic pH (e.g., pH less than 6.5) rather than under physiological pH conditions of the blood stream (e.g., non-acidic pH). In some embodiments, a binding polypeptide of the present disclosure comprising a modified Fc domain has enhanced FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising a modified Fc domain has enhanced FcRn binding affinity at a pH of less than 7, e.g., at about pH 6.5, about pH6.0, about pH5.5, about pH 5.0, as compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, the binding polypeptide comprising the modified Fc domain has enhanced FcRn binding affinity at a pH of less than 7, e.g., at about pH 6.5, about pH6.0, about pH5.5, about pH 5.0, as compared to the FcRn binding affinity of the binding polypeptide at an elevated, non-acidic pH. The elevated non-acidic pH can be, for example, a pH greater than 7, about pH7, about pH7.4, about pH 7.6, about pH 7.8, about pH 8.0, about pH8.5, and about pH 9.0.
In certain embodiments, it may be desirable for a binding polypeptide comprising a modified Fc domain to exhibit about the same FcRn binding affinity at non-acidic pH as a binding polypeptide comprising a wild-type Fc domain. In some embodiments, it may be desirable for a binding polypeptide comprising a modified Fc domain to exhibit less FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain having the double amino acid substitution M428L/N434S according to EU numbering. Thus, it may be desirable for binding polypeptides comprising a modified Fc domain to exhibit minimal perturbation to pH-dependent FcRn binding.
In some embodiments, a binding polypeptide comprising a modified Fc domain having enhanced FcRn binding affinity at acidic pH has a reduced (i.e., slower) FcRn off-rate compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising a modified Fc domain (the binding polypeptide having enhanced FcRn binding affinity at acidic pH compared to FcRn binding affinity of the binding polypeptide at elevated non-acidic pH) has a slower FcRn off-rate at acidic pH compared to the FcRn off-rate of the binding polypeptide at elevated non-acidic pH.
In some embodiments, binding polypeptides comprising a modified Fc domain that exhibit higher FcRn binding affinity at non-acidic pH as compared to binding polypeptides comprising a wild-type Fc are provided. In some embodiments, a binding polypeptide comprising a modified Fc domain is provided that exhibits a higher FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising a modified Fc domain is provided that exhibits a higher FcRn binding affinity at non-acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain, and exhibits a higher FcRn binding affinity at acidic pH as compared to a binding polypeptide comprising a wild-type Fc domain. Thus, in certain embodiments, a binding polypeptide comprising a modified Fc domain is provided that exhibits a loss of pH-dependent FcRn binding.
Certain embodiments include the following antibodies: in addition to the Fc mutations described herein that exhibit altered FcRn binding affinity, the antibodies comprise at least one amino acid in one or more constant region domains that has been deleted or otherwise altered to provide a desired biochemical characteristic, e.g., reduced or enhanced effector function, non-covalent dimerization capability, enhanced ability to localize to a tumor site, reduced serum half-life, increased serum half-life, etc., when compared to an intact, unaltered antibody of about the same immunogenicity.
In certain other embodiments, the binding polypeptide comprises constant regions derived from different antibody isotypes (e.g., constant regions from two or more of human IgG1, IgG2, IgG3, or IgG 4). In other embodiments, the binding polypeptide comprises a chimeric hinge (i.e., a hinge comprising a hinged portion derived from hinge domains of different antibody isotypes, e.g., an upper hinge domain from an IgG4 molecule and an IgG1 middle hinge domain).
In certain embodiments, the Fc domain may be mutated to increase or decrease effector function using techniques known in the art. In some embodiments, a binding polypeptide of the present disclosure comprising a modified Fc domain has an altered binding affinity for an Fc receptor. There are several different types of Fc receptors, which are classified based on the type of antibody they recognize. For example, Fc-gamma receptors (Fc γ R) bind to IgG class antibodies, Fc-alpha receptors (Fc α R) bind to IgA class antibodies, and Fc-receptors (FcR) bind to IgE class antibodies. The term Fc γ R encompasses several families of members such as Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa and Fc γ RIIIb. In some embodiments, a binding polypeptide comprising a modified Fc domain has altered Fc γ RIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising a modified Fc domain has reduced binding affinity for Fc γ RIIIa as compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising a modified Fc domain has enhanced binding affinity for fcyriiia as compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising a modified Fc domain has about the same binding affinity for fcyriiia as a binding polypeptide comprising a wild-type Fc domain.
In other embodiments, the binding polypeptides used in the diagnostic and therapeutic methods described herein have a constant region, such as the IgG1 heavy chain constant region, that is altered to reduce or eliminate glycosylation. For example, a binding polypeptide (e.g., an antibody or immunoadhesin) comprising a modified Fc domain can further comprise amino acid substitutions that alter glycosylation of the antibody Fc. For example, the modified Fc domain can have reduced glycosylation (e.g., N or O linked glycosylation).
Exemplary amino acid substitutions that confer reduced or altered glycosylation are disclosed in International PCT publication No. WO05/018572, which is incorporated herein by reference in its entirety. In some embodiments, the binding polypeptide is modified to eliminate glycosylation. Such binding polypeptides may be referred to as "agly" binding polypeptides (e.g., "agly" antibodies). While not being bound by theory, it is believed that "agly" binding polypeptides may have improved in vivo safety and stability. The agly binding polypeptide may have any isotype or subclass thereof, for example IgG1, IgG2, IgG3 or IgG 4. Many art-recognized methods are available for making "agly" antibodies or antibodies with altered glycans. For example, genetically engineered host cells (e.g., modified yeast such as pichia pastoris, or CHO cells) with modified glycosylation pathways (e.g., glycosyltransferase deletions) can be used to produce such antibodies.
In certain embodiments, a binding polypeptide can comprise an antibody constant region (e.g., an IgG constant region, e.g., a human IgG1 constant region) that mediates one or more effector functions. For example, binding of the C1 complex to the constant region of an antibody activates the complement system. Activation of the complement system is important in opsonization and lysis of cellular pathogens. Activation of the complement system also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity reactions. In addition, antibodies bind to receptors on a variety of cells via the Fc domain (the Fc receptor binding site on the Fc region of an antibody binds to an Fc receptor (FcR) on a cell). There are many Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptor), IgE (receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of antibodies to Fc receptors on cell surfaces triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, killing of cells to lyse antibody-coated target cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the binding polypeptide (e.g., an antibody or immunoadhesin) binds to an Fc γ receptor. In alternative embodiments, the binding polypeptide may comprise a constant region that lacks one or more effector functions (e.g., ADCC activity) and/or is incapable of binding to an Fc γ receptor.
Proteins with low thermodynamic stability, including antibodies, have an increased propensity for misfolding and aggregation, and will limit or hinder the activity, efficacy and potential of the protein as a useful therapeutic agent. In certain embodiments, a binding polypeptide comprising a modified Fc domain has about the same thermostability as a binding polypeptide comprising a wild-type Fc domain. In some embodiments, the binding polypeptide comprising the modified Fc domain has about the same thermostability as a binding polypeptide comprising a modified Fc domain having the triple amino acid substitution M252Y/S254T/T256E (YTE).
The resulting physiological characteristics, bioavailability, and other biochemical effects of the modification (e.g., tumor localization, biodistribution, and serum half-life) can be readily measured and quantified using well-known immunological techniques without undue experimentation.
In certain embodiments, a binding polypeptide of the present disclosure can comprise an antigen-binding fragment of an antibody. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with (i.e., specifically binds to) an intact antibody (i.e., the intact antibody from which the polypeptide fragment of the immunoglobulin or antibody is derived). Antigen-binding fragments can be produced by recombinant or biochemical methods well known in the art. Exemplary antigen binding fragments include Fv, Fab 'and (Fab') 2. In exemplary embodiments, the binding polypeptides of the present disclosure comprise an antigen binding fragment and a modified Fc domain.
In some embodiments, the binding polypeptide comprises a single chain variable region sequence (ScFv). The single chain variable region sequence comprises a single polypeptide having one or more antigen binding sites, for example a VL domain linked to a VH domain by a flexible linker. ScFv molecules can be constructed in either the VH-linker-VL orientation or the VL-linker-VH orientation. The flexible hinge connecting the VL and VH domains that make up the antigen binding site comprises from about 10 to about 50 amino acid residues. Linking peptides are known in the art. The binding polypeptide may comprise at least one scFv and/or at least one constant region. In one embodiment, a binding polypeptide of the present disclosure may comprise at least one scFv linked or fused to a modified Fc domain.
In some embodiments, the binding polypeptides of the present disclosure are multivalent (e.g., tetravalent) antibodies produced by fusing a DNA sequence encoding the antibody to an ScFv molecule (e.g., an altered ScFv molecule). For example, in one embodiment, the sequences are combined such that the ScFv molecule (e.g., an altered ScFv molecule) is linked at its N-terminus or C-terminus to the Fc fragment of the antibody via a flexible linker (e.g., gly/ser linker). In another embodiment, the tetravalent antibodies of the present disclosure can be prepared by the following method: the ScFv molecule was fused to a linker peptide fused to the modified Fc domain to construct an ScFv-Fab tetravalent molecule.
In another embodiment, a binding polypeptide of the present disclosure is an altered minibody. The altered minibody of the present disclosure is a dimeric molecule consisting of two polypeptide chains, each polypeptide chain comprising an ScFv molecule fused to a modified Fc domain via a linker peptide. Minibodies can be prepared by constructing the ScFv component and the linker component using methods described in the art (see, e.g., U.S. Pat. No. 5,837,821 or WO94/09817 Al). In another embodiment, tetravalent minibodies may be constructed. Tetravalent minibodies can be constructed in the same manner as minibodies, except that a flexible linker is used to link the two ScFv molecules. The linked scFv-scFv construct is then linked to the modified Fc domain.
In another embodiment, a binding polypeptide of the present disclosure comprises a diabody. Diabodies are dimeric tetravalent molecules each having a polypeptide similar to an scFv molecule, but typically having a short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting the two variable domains, such that the VL and VH domains cannot interact on the same polypeptide chain. In contrast, the VL and VH domains of one polypeptide chain interact (respectively) with the VH and VL domains on a second polypeptide chain (see, e.g., WO 02/02781). Diabodies of the present disclosure comprise scFv-like molecules fused to a modified Fc domain.
In other embodiments, the binding polypeptide comprises a multispecific or multivalent antibody comprising one or more variable domains, e.g., a Tandem Variable Domain (TVD) polypeptide, in tandem on the same polypeptide chain. Exemplary TVD polypeptides include the "double-headed" or "double Fv" configuration described in U.S. patent No. 5,989,830. In a double Fv configuration, the variable domains of two different antibodies are represented in a tandem orientation on two separate chains (one heavy chain and one light chain), with one polypeptide chain having two tandem VH domains separated by a peptide linker (VH 1-linker-VH 2), and the other polypeptide chain consisting of complementary VL domains connected in series by a peptide linker (VL 1-linker-VL 2). In the crossed double-headed configuration, the variable domains of two different antibodies are represented in a tandem orientation on two separate polypeptide chains (one heavy chain and one light chain), one of which has two VH domains in tandem separated by a peptide linker (VH 1-linker-VH 2), and the other polypeptide chain consists of complementary VL domains connected in series in opposite directions by a peptide linker (VL 2-linker-VL 1). Additional antibody variants based on the "double Fv" format include double variable domain IgG (DVD-IgG) bispecific antibodies (see US patent No. 7,612,181) and TBTI format (see US 2010/0226923a 1). In some embodiments, the binding polypeptide comprises a multispecific or multivalent antibody comprising one or more variable domains in tandem on the same polypeptide chain fused to a modified Fc domain.
In another exemplary embodiment, the binding polypeptide comprises a crossed double variable domain IgG (CODV-IgG) bispecific antibody based on a "bipitch" configuration (see US 20120251541a1, which is incorporated herein by reference in its entirety).
In another exemplary embodiment, the binding polypeptide is an immunoadhesin. As used herein, "immunoadhesins"Reference is made to binding polypeptides comprising one or more binding domains (e.g., from a receptor, ligand, or cell adhesion molecule) linked to an immunoglobulin constant domain (i.e., Fc region) (see, e.g., Ashkenazi et al 1995, Methods 8(2): 104-115, and Isaacs (1997) Brit. J. Rheum. 36:305, which are incorporated herein in their entirety by reference
In certain embodiments, the binding polypeptide comprises an immunoglobulin-like domain. Suitable immunoglobulin-like domains include, but are not limited to, fibronectin domains (see, e.g., Koide et al (2007), MethodsMol. biol.352: 95-109, which are incorporated herein by reference in their entirety), DARPin (see, e.g., Stumpp et al (2008) Drug Discov. Today13 (15-16): 695-701, which is incorporated herein by reference in their entirety), Z domains of protein A (see, e.g., Nygren et al (2008) FEBS J.275(11): 2668-76, which is incorporated herein by reference in their entirety), lipocalins (see, e.g., Skerra et al (2008) FEBS J.275(11): 2677-83, which is incorporated herein by reference in their entirety), Affiln (see, e.g., Ebersbach et al (2007) J. mol. biol.372(1): 85, which is incorporated herein by reference in their entirety), Affiln (see, e.g., Effenblack et al. J. 10568, which is incorporated herein by reference in their entirety), Affil. Biolin (1058. 383, which is incorporated herein by reference in their entirety), Avimer (see, e.g., Silverman et al (2005) nat. Biotechnol.23(12): 1556-61, which is incorporated herein by reference in its entirety), Fynomer (see, e.g., Graulovski et al (2007) J Biol Chem282(5): 3196-3204, which is incorporated herein by reference in its entirety), and Kunitz domain peptides (see, e.g., Nixon et al (2006) Curr Opin Drug discovery Devel9(2): 261-8, which is incorporated herein by reference in its entirety).
For the binding polypeptides and immunoadhesins of the present disclosure, virtually any antigen can be targeted by the binding polypeptide, including but not limited to proteins, subunits, domains, motifs, and/or epitopes of target antigens, including both soluble factors (such as cytokines and membrane-bound factors) and transmembrane receptors.
Binding polypeptides of the present disclosure comprising a modified Fc domain described herein may comprise CDR sequences or variable domain sequences of known "parent" antibodies. In some embodiments, the parent antibody and the antibody of the present disclosure may share similar or identical sequences, except for modifications to the Fc domain disclosed herein.
Nucleic acids and expression vectors
In one aspect, the invention provides polynucleotides encoding the binding polypeptides disclosed herein. Also provided are methods of making the binding polypeptides, comprising expressing these polynucleotides.
Polynucleotides encoding the binding polypeptides disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce the desired amount of the claimed antibodies or immunoadhesins. Thus, in certain aspects, the invention provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.
For the purposes of the specification and claims, the term "vector" or "expression vector" is used herein to mean a vector for introducing and expressing a desired gene in a cell. Such vectors can be readily selected from plasmids, phages, viruses and retroviruses, as known to those skilled in the art. Typically, the vector will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
Many expression vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus. Other vector classes involve the use of polycistronic systems with internal ribosome binding sites. In addition, cells that have integrated DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. The marker may provide prototrophy to an auxotrophic subject, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required to optimally synthesize mRNA. These elements may include signal sequences, splicing signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (e.g., human genes) synthesized as described above.
In other embodiments, a polycistronic construct may be used to express a binding polypeptide as described herein. In such expression systems, multiple gene products of interest, such as the heavy and light chains of an antibody, can be produced from a single polycistronic construct. These systems advantageously use an Internal Ribosome Entry Site (IRES) to provide relatively high levels of polypeptide in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. patent No. 6,193,980, which is incorporated herein by reference. One skilled in the art will appreciate that such expression systems can be used to efficiently produce the full range of polypeptides disclosed in the present application.
More generally, once a vector or DNA sequence encoding a binding polypeptide of the disclosure has been prepared, the expression vector can be introduced into an appropriate host cell. That is, the host cell may be transformed. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and whole virus infection. See, e.g., Ridgway, A.A. G. "Mammalian Expression Vectors," Chapter 24.2, pages 470-472 Vectors, Rodriguez and Denhardt, eds (Butterworth, Boston, MA 1988). The transformed cells are grown under conditions suitable for the production of light and heavy chains, and the heavy and/or light chain protein synthesis is assayed. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA) or fluorescence activated cell sorter analysis (FACS), immunohistochemistry, and the like.
As used herein, the term "transformation" is used in a broad sense to refer to the introduction of DNA into a recipient host cell that alters the genotype and thus results in a change in the recipient cell.
Along the same lines, "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In describing processes for isolating polypeptides from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to refer to a source of antibody unless specifically indicated otherwise. In other words, recovery of the polypeptide from "cells" can mean recovery from whole cells pelleted by centrifugation, or from cell cultures containing both media and suspension cells.
In one embodiment, the host cell line used for expression of the binding polypeptide is of eukaryotic or prokaryotic origin. In one embodiment, the host cell line used for expression of the binding polypeptide is of bacterial origin. In one embodiment, the host cell line used for expression of the binding polypeptide is of mammalian origin; one skilled in the art can determine the particular host cell line best suited for expression of the desired gene product therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR-), HELA (human cervical cancer), CVI (monkey kidney line), COS (with SV 40)Derivative of CVI of T antigen), R1610 (chinese hamster fibroblasts), BALBC/3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney). In one embodiment, the cell line provides altered glycosylation of the antibody expressed thereby, such as nonfucosylation (e.g., per. c6.rtm. (Crucell) or FUT8 knockout CHO cell line (potellligent)TMCells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells may be used. Host cell lines are generally available from commercial services, the American Tissue Culture Collection (American Tissue Culture Collection) or published literature.
In vitro production allows scaling up to give large amounts of the desired binding polypeptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include homogeneous suspension culture (e.g., in an airlift reactor or a continuous stirred reactor), or immobilized or embedded cell culture on beads on agarose or ceramic cartridges (e.g., in hollow fibers, microcapsules). If necessary and/or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, for example gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose and/or (immuno) affinity chromatography.
One or more genes encoding binding polypeptides may also be expressed in non-mammalian cells such as bacterial or yeast or plant cells. In this regard, it is to be understood that a variety of single-cell non-mammalian microorganisms, such as bacteria, i.e., those capable of growing in culture or fermentation, may also be transformed. Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of escherichia coli or salmonella; bacillaceae, such as bacillus subtilis; the genus pneumococcus; streptococcus and Haemophilus influenzae. It is also understood that the polypeptide may be part of an inclusion body when expressed in bacteria. The polypeptide must be isolated, purified and then assembled into a functional molecule.
In addition to prokaryotes, eukaryotic microorganisms may also be used. Saccharomyces cerevisiae or common baker's yeast are the most commonly used among eukaryotic microorganisms, although many other strains are commonly available. For expression in Saccharomyces, for example, the plasmid YRp7(Stinchcomb et al, Nature,282:39 (1979); Kingsman et al, Gene,7:141 (1979); Tschemper et al, Gene,10:157(1980)) is commonly used. This plasmid already contains the TRP1 gene, which provides a selectable marker for mutant strains of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, Genetics,85:12 (1977)). The presence of the trpl lesion then serves as a genomic feature of the yeast host cell to provide an effective environment for detecting transformation by growth in the absence of tryptophan.
Method of treatment
In one aspect, the invention provides a method of treating or diagnosing a patient in need thereof comprising administering an effective amount of a binding polypeptide disclosed herein. In certain embodiments, the present disclosure provides kits and methods for diagnosing and/or treating disorders (e.g., neoplastic disorders in a mammalian subject in need of such treatment). In certain exemplary embodiments, the subject is a human.
The binding polypeptides of the present disclosure can be used in many different applications. For example, in one embodiment, the subject binding polypeptides can be used to reduce or eliminate cells bearing an epitope recognized by the binding domain of the binding polypeptide. In another embodiment, the subject binding polypeptides are effective to reduce the concentration of or eliminate soluble antigen in circulation. In another embodiment, the subject binding polypeptides are effective as T cell conjugates. In one embodiment, the binding polypeptide can reduce tumor size, inhibit tumor growth, and/or prolong the survival time of a tumor-bearing animal. Thus, the disclosure also relates to methods of treating tumors in humans or other animals by administering an effective non-toxic amount of the modified antibodies to such humans or animals.
In one embodiment, the subject binding polypeptides are useful for treating a disease or disorder. For example, the subject binding polypeptides can be used to treat an antibody-related disorder or an antibody-reactive disorder, condition, or disease. As used herein, the term "antibody-related disorder" or "antibody-reactive disorder" or "condition" or "disease" refers to or describes a disease or disorder that can be ameliorated by the administration of a pharmaceutical composition comprising an antibody or binding polypeptide of the disclosure.
In one embodiment, the subject binding polypeptides are useful for treating cancer. As used herein, the term "cancer" or "cancerous" refers to or describes a physiological condition that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomachcancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (kidney or renal cancer), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract, and head and neck cancer.
In another embodiment, the subject binding polypeptides may be used to treat other disorders, including but not limited to infectious diseases, autoimmune disorders, inflammatory disorders, pulmonary diseases, neuronal or neurodegenerative diseases, liver diseases, spinal diseases, uterine diseases, depression, and the like. Non-limiting examples of infectious diseases include infectious diseases caused by RNA viruses (e.g., orthomyxoviruses (e.g., influenza), paramyxoviruses (e.g., respiratory syncytial virus, parainfluenza virus, metapneumovirus), rhabdoviruses (e.g., rabies virus), coronaviruses, alphaviruses (e.g., chikungunya virus), lentiviruses (e.g., HIV), etc.), or DNA viruses. Examples of infectious diseases also include, but are not limited to, bacterial infectious diseases caused by, for example, staphylococcus aureus, staphylococcus epidermidis, enterococcus, streptococcus, escherichia coli, and other infectious diseases, including, for example, infectious diseases caused by candida albicans. Other infectious diseases include, but are not limited to, malaria, SARS, yellow fever, lyme borreliosis, leishmaniasis, anthrax, and meningitis. Exemplary autoimmune disorders include, but are not limited to, psoriasis, rheumatoid arthritis, Sjogren's Syndrome, transplant rejection, Grave's disease, myasthenia gravis, and lupus (e.g., systemic lupus erythematosus). Accordingly, the present disclosure relates to a method of treating a variety of conditions that would benefit from the use of a subject binding polypeptide having, for example, increased half-life.
By routine experimentation, one skilled in the art will be able to determine that an effective, non-toxic amount of the modified binding polypeptide can be used for the purpose of treating a malignant tumor. For example, a therapeutically active amount of a binding polypeptide of the present disclosure may vary depending on factors such as the disease stage (e.g., stage I versus stage IV), age, sex, medical complications (e.g., an immunosuppressive condition or disease) and body weight of the subject, and the ability of the modified antibody to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
In general, the compositions provided in the present disclosure can be used to prophylactically or therapeutically treat any tumor comprising an antigenic marker that allows for targeting of cancer cells by the modified antibody.
Pharmaceutical compositions and their administration
Methods of making and administering the binding polypeptides of the disclosure to a subject are well known or readily determinable to those of skill in the art. The route of administration of the binding polypeptides of the present disclosure may be oral, parenteral, by inhalation, or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. While administration of all of these forms is expressly contemplated as being within the scope of the present disclosure, the form of administration will be a solution for injection, particularly for intravenous or intra-arterial injection or instillation. Generally, suitable pharmaceutical compositions for injection may comprise buffers (e.g., acetate, phosphate or citrate buffers), surfactants (e.g., polysorbates), optional stabilizers (e.g., human albumin), and the like. In some embodiments, the binding polypeptide can be delivered directly to the site of the undesirable cell population, thereby enhancing exposure of the diseased tissue to the therapeutic agent.
Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M, e.g., 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions that may be suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In this case, the composition must be sterile and should be fluid to the extent that easy injection is possible. It should be stable under the conditions of manufacture and storage and generally protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
In any event, sterile injectable solutions can be prepared by incorporating the active compound (e.g., the modified binding polypeptide by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The formulations for injection are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under sterile conditions according to methods known in the art. In addition, the formulations may be packaged and sold in the form of a kit. Such articles typically have a label or package insert indicating that the relevant composition can be used to treat a subject suffering from or susceptible to an autoimmune or oncological disorder.
Effective dosages of the compositions of the present disclosure for treating the above conditions will vary depending upon a number of different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Therapeutic doses can be titrated to optimize safety and efficacy using routine methods known to those skilled in the art.
The binding polypeptides of the present disclosure can be administered multiple times. The interval between single doses may be weekly, monthly or yearly. Intervals may also be irregular, as indicated by measuring blood levels of the modified binding polypeptide or antigen in the patient. In some methods, the dose is adjusted to achieve a plasma modified binding polypeptide concentration of about 1-1000 μ g/ml, and in some methods the concentration is about 25-300 μ g/ml. Alternatively, the binding polypeptide may be administered as a sustained release formulation, in which case less frequent administration is required. For antibodies, the dose and frequency will vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric and non-human antibodies.
The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the antibodies of the invention or mixtures thereof are administered to a patient who is not already in a disease state in order to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose". The precise amount in this use will also depend on the health status and overall immunity of the patient, but will generally be in the range of from about 0.1 to about 25mg per dose, especially from about 0.5 to about 2.5mg per dose. Relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, it is sometimes desirable to administer relatively high doses (e.g., about 1 to 400 mg/kg antibody per dose, with doses of about 5 to 25mg more commonly used for radioimmunoconjugates and higher doses for cytotoxic-drug-modified antibodies) at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient.
The binding polypeptides of the present disclosure can optionally be administered in combination with other agents effective to treat a disorder or condition in need of treatment (e.g., prophylactic or therapeutic). The disclosure is90An effective single therapeutic dose (i.e., a therapeutically effective amount) of the Y-labeled modified antibody ranges between about 5 and about 75mCi, such as between about 10 and about 40 mCi.131An effective single treatment non-bone marrow ablative dose range of the I-modified antibody is between about 5 and about 70mCi or between about 5 and about 40 mCi.131The effective single therapeutic ablative dose of the I-labeled antibody (i.e., possibly requiring autologous bone marrow transplantation) ranges from
Although the binding polypeptide may be administered as described above, it must be emphasized that in other embodiments, the binding polypeptide may be administered to other healthy patients as a first line therapy. In such embodiments, the binding polypeptide can be administered to a patient having normal or average red bone marrow reserve and/or to a patient that has not been treated and is not undergoing treatment. As used herein, administration of a modified antibody or immunoadhesin together or in combination with an adjuvant therapy means sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed antibody. One skilled in the art will appreciate that the various components of the combined therapeutic regimen may be administered or applied periodically to enhance the overall effectiveness of the treatment.
As previously described, the binding polypeptides, immunoadhesins or recombinant thereof of the present disclosure can be administered in a pharmaceutically effective amount for the in vivo treatment of a mammalian disorder. In this regard, it is understood that the disclosed binding polypeptides will be formulated to facilitate administration of the active agent and to enhance the stability of the active agent.
The pharmaceutical composition according to the present disclosure may comprise pharmaceutically acceptable non-toxic sterile carriers such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of this application, a pharmaceutically effective amount of binding polypeptide, immunoadhesin or recombinant thereof, conjugated or unconjugated to the therapeutic agent, should be maintained, which means an amount sufficient to achieve effective binding to the antigen and to obtain a benefit (e.g., sufficient to ameliorate the symptoms of a disease or disorder or to detect a substance or cell). In the case of tumor cells, the modified binding polypeptide may interact with selected immunoreactive antigens on tumor cells or immunoreactive cells and provide an increase in death of these cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in a single dose or in multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.
In keeping with the scope of the disclosure, the binding polypeptides of the disclosure may be administered to a human or other animal in an amount sufficient to produce a therapeutic or prophylactic effect in accordance with the treatment methods described above. The binding polypeptides of the present disclosure may be administered to such humans or other animals in conventional dosage forms prepared by combining the antibodies of the present disclosure with conventional pharmaceutically acceptable carriers or diluents according to known techniques. One skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent will depend on the amount of active ingredient combined therewith, the route of administration, and other well known variables. One skilled in the art will further appreciate that mixtures comprising one or more binding polypeptides described in the present disclosure may prove particularly effective.
The contents of the articles, patents and patent applications, and all other documents and electronically available information mentioned or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The applicant reserves the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.
While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims. Having now described certain embodiments in detail, they will be more clearly understood by reference to the following examples, which are given for purposes of illustration only and are not intended to be limiting.
Examples
The invention is further illustrated by the following examples, which should not be construed as further limiting.
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