IgG Fc variants for veterinary use
Provided are various embodiments relating to variant IgG Fc polypeptides of companion animals having increased Protein A binding for ease of purification, decreased C1q binding for reduced complement-mediated immune responses, decreased CD16 binding (e.g., for reduced antibody-dependent cellular cytotoxicity (ADCC) induction, increased stability, and/or the ability to form heterodimeric proteins. In addition, various embodiments relating to antibodies and fusion proteins comprising such variant IgG Fc polypeptides are provided. Also provided are various embodiments relating to contiguous polypeptides comprising one or more variant GLP1 polypeptide(s) having improved serum half-life. Further provided are various embodiments relating to contiguous polypeptides or heterodimeric polypeptides comprising a GLP1 polypeptide and a glucagon polypeptide as a dual GLP1 receptor and glucagon receptor agonist. In various embodiments, such polypeptides may be used to treat, for example, diabetes, obesity, or related indications, in companion animals, such as canines, felines, and equines.
1. A variant IgG Fc polypeptide comprising:
(a) the polypeptide of SEQ ID NO: 1 having an amino acid substitution consisting of a threonine at position 21 and optionally at least one amino acid substitution selected from the group consisting of: a leucine at position 23, an alanine at position 25, a glycine at position 80, an alanine at position 205, and a histidine at position 207, or
(b) a polypeptide that is at least 97% identical to SEQ ID NO: 1, wherein the polypeptide has a threonine at the position corresponding to position 21 of SEQ ID NO: 1 and optionally at least one amino acid substitution selected from the group consisting of: a leucine at the position corresponding to position 23 of SEQ ID NO: 1, an alanine at the position corresponding to position 25 of SEQ ID NO: 1, a glycine at the position corresponding to position 80 of SEQ ID NO: 1, an alanine at the position corresponding to position 205 of SEQ ID NO: 1, and a histidine at the position corresponding to position 207 of SEQ ID NO: 1.
2. The polypeptide of claim 1 , wherein the polypeptide is an antibody, an antibody fusion, or a fusion polypeptide.
3. An isolated nucleic acid encoding the variant IgG Fc polypeptide of claim 1 .
4. A host cell comprising the nucleic acid of claim 3 .
5. A method of producing a variant IgG Fc polypeptide comprising providing a host cell comprising a nucleic acid encoding the variant IgG Fc polypeptide of claim 1 , culturing the host cell, and isolating the variant IgG Fc polypeptide produced.
6. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
7. The pharmaceutical composition of claim 6 , wherein the pharmaceutically acceptable carrier comprises one or more of alumina, aluminum stearate, lecithin, one or more serum protein, human serum albumin, canine or other animal albumin, one or more buffer, glycine, sorbic acid, potassium sorbate, one or more partial glyceride mixtures of saturated vegetable fatty acids, water, one or more salt, one or more electrolyte, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, one or more cellulose-based substances, polyethylene glycol, sucrose, mannitol, or one or more amino acids.
8. The pharmaceutical composition of claim 7 , wherein (i) the one or more buffer comprises one or more of phosphate buffer, citrate buffer, tromethamine buffer, or HEPES buffer; (ii) the one or more amino acids comprises arginine; or both (i) and (ii).
9. A method of delivering a polypeptide to a subject comprising administering the variant IgG Fc polypeptide of claim 1 parenterally.
10. The polypeptide of claim 1 , wherein the variant IgG Fc polypeptide binds to Protein A with a dissociation constant (K d ) of less than 5×10 −6 M, less than 1×10 −6 M, less than 5×10 −7 M, less than 1×10 −7 M, less than 5×10 −8 M, less than 1×10 −8 M, less than 5×10 −9 M, less than 1×10 −9 M, less than 5×10 −10 M, less than 1×10 −10 M, less than 5×10 −11 M, less than 1×10 −11 M, less than 5×10 −12 M, or less than 1×10 −12 M, as measured by biolayer interferometry.
11. A method of delivering a polypeptide to a subject comprising administering the variant IgG Fc polypeptide of claim 1 via by an intramuscular, an intraperitoneal, an intracerebrospinal, a subcutaneous, an intra-arterial, an intrasynovial, an intrathecal, or an inhalation route.
12. A method of delivering a polypeptide to a subject comprising administering the variant IgG Fc polypeptide of claim 1 via subcutaneous administration, intravenous infusion, or intramuscular injection.
13. The polypeptide of claim 1 , wherein the polypeptide sequence comprises SEQ ID NO: 5 or SEQ ID NO: 60.
14. The polypeptide of claim 1 , wherein the polypeptide has increased binding affinity to Protein A and/or reduced binding affinity to C1q and CD16 relative to SEQ ID NO: 1.
15. The polypeptide of claim 1 , wherein the polypeptide of (a) further comprises an amino acid substitution consisting of a leucine at position 23.
16. The polypeptide of claim 1 , wherein the polypeptide of (a) further comprises an amino acid substitution consisting of a glycine at position 80.
17. The polypeptide of claim 1 , wherein the polypeptide of (a) further comprises an amino acid substitution consisting of an alanine at position 25.
18. The polypeptide of claim 1 , wherein the polypeptide of (a) further comprises an amino acid substitution consisting of a histidine at position 207.
19. The polypeptide of claim 1 , wherein the polypeptide of (a) has at least one amino acid substitution selected from the group consisting of: a leucine at position 23, an alanine at position 25, a glycine at position 80, an alanine at position 205, and a histidine at position 207.
20. The polypeptide of claim 1 , wherein the polypeptide of (b) has at least one amino acid substitution selected from the group consisting of: a leucine at the position corresponding to position 23 of SEQ ID NO: 1, an alanine at the position corresponding to position 25 of SEQ ID NO: 1, a glycine at the position corresponding to position 80 of SEQ ID NO: 1, an alanine at the position corresponding to position 205 of SEQ ID NO: 1, and a histidine at the position corresponding to position 207 of SEQ ID NO: 1.
21. The polypeptide of claim 1 , wherein the polypeptide of (a) further comprises an amino acid substitution consisting of an alanine at position 205.
22. A variant IgG Fc polypeptide comprising:
(a) an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 3, wherein the polypeptide comprises a threonine at the position corresponding to position 21 of SEQ ID NO: 3 and optionally at least one amino acid substitution selected from the group consisting of a leucine at the position corresponding to position 23 of SEQ ID NO: 3 and an isoleucine at the position corresponding to position 24 of SEQ ID NO: 3; or
(b) the polypeptide of SEQ ID NO: 3 having an amino acid substitution consisting of a threonine at position 21 of SEQ ID NO: 3 and optionally at least one amino acid substitution selected from the group consisting of a leucine at position 23 and an isoleucine at position 24.
23. The polypeptide of claim 22 , wherein the polypeptide sequence comprises SEQ ID NO: 6, SEQ ID NO: 61, or SEQ ID NO: 84.
24. The polypeptide of claim 22 , wherein the polypeptide has increased binding affinity to Protein A and/or reduced binding affinity to C1q and CD16 relative to SEQ ID NO: 3.
25. The polypeptide of claim 22 , wherein the polypeptide of (a) has at least one amino acid substitution selected from the group consisting of a leucine at the position corresponding to position 23 of SEQ ID NO: 3 and an isoleucine at the position corresponding to position 24 of SEQ ID NO: 3.
26. The polypeptide of claim 22 , wherein the polypeptide of (b) has at least one amino acid substitution selected from the group consisting of a leucine at position 23 and an isoleucine at position 24.
27. A variant IgG Fc polypeptide comprising:
(a) an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 4, wherein the polypeptide comprises a threonine at the position corresponding to position 21 of SEQ ID NO: 4 and optionally at least one amino acid substitution selected from the group consisting of: a leucine at the position corresponding to position 23 of SEQ ID NO: 4, an alanine at the position corresponding to position 25 of SEQ ID NO: 4, a glycine at the position corresponding to position 80 of SEQ ID NO: 4, and a histidine at the position corresponding to position 207 of SEQ ID NO: 4; or
(b) the polypeptide of SEQ ID NO: 4 having an amino acid substitution consisting of a threonine at position 21 of SEQ ID NO: 4 and optionally at least one amino acid substitution selected from the group consisting of: a leucine at position 23, an alanine at position 25, a glycine at position 80, and a histidine at position 207.
28. The polypeptide of claim 27 , wherein the polypeptide sequence comprises SEQ ID NO: 7 or SEQ ID NO: 62.
29. The polypeptide of claim 27 , wherein the polypeptide has increased binding affinity to Protein A and/or reduced binding affinity to C1q and CD16 relative to SEQ ID NO: 4.
30. The polypeptide of claim 27 , wherein the polypeptide of (a) has at least one amino acid substitution selected from the group consisting of: a leucine at the position corresponding to position 23 of SEQ ID NO: 4, an alanine at the position corresponding to position 25 of SEQ ID NO: 4, a glycine at the position corresponding to position 80 of SEQ ID NO: 4, and a histidine at the position corresponding to position 207 of SEQ ID NO: 4.
31. The polypeptide of claim 27 , wherein the polypeptide of (b) has at least one amino acid substitution selected from the group consisting of: a leucine at position 23, an alanine at position 25, a glycine at position 80, and a histidine at position 207.