High affinity SIRP-alpha reagents and methods of using
High affinity SIRP-α reagent are provided, which (i) comprise at least one amino acid change relative to the wild-type protein; and (ii) have an increased affinity for CD47 relative to the wild-type protein. Compositions and methods are provided for modulating phagocytosis in a mammal by administering a therapeutic dose of a pharmaceutical composition comprising a high affinity SIRPα reagent, which blocks the physiological binding interaction between SIRPα and its ligand CD47.
1. A soluble high affinity SIRPα polypeptide comprising a human SIRPα d1 domain sequence set forth in SEQ ID NO: 1 or a d1 domain of a human SIRPα set forth in SEQ ID NO: 2, and further comprising at least two and no more than 15 amino acid substitutions within the d1 domain of SEQ ID NO: 1 or SEQ ID NO: 2,
wherein two or more of the substitutions are selected from the group consisting of: 31T or 31S or 31F; 53R; 54Q; 56P or 56R; 66T or 66G; and 68R,
wherein the amino acid positions are relative to the d1 domain of SEQ ID NO: 1 or SEQ ID NO: 2,
wherein the substitutions increase the affinity of the high affinity SIRPα polypeptide binding to human CD47 relative to its wild-type SIRPα, and
wherein the high affinity SIRPα polypeptide lacks a SIRPα transmembrane domain.
2. The soluble high affinity SIRPα polypeptide of claim 1 , comprising at least three substitutions, wherein three or more of the substitutions are selected from the group consisting of: 31T or 31S or 31F; 53R; 54Q; 56P or 56R; 66T or 66G; and 68R.
3. The soluble high affinity SIRPα polypeptide of claim 1 , comprising at least four substitutions, wherein four or more of the substitutions are selected from the group consisting of: 31T or 31S or 31F; 53R; 54Q; 56P or 56R; 66T or 66G; and 68R.
4. The soluble high affinity SIRPα polypeptide of claim 1 , comprising at least five substitutions, wherein five or more of the substitutions are selected from the group consisting of: 31T or 31S or 31F; 53R; 54Q; 56P or 56R; 66T or 66G; and 68R.
5. The soluble high affinity SIRPα polypeptide of claim 1 , comprising at least six substitutions, wherein six substitutions are selected from the group consisting of: 31T or 31S or 31F; 53R; 54Q; 56P or 56R; 66T or 66G; and 68R.
6. The soluble high affinity SIRPα polypeptide of claim 1 , comprising:
(i) 27I or 27L; 53R; 66T or 66G; 68R; and 103V;
(ii) 4V or 4I; 27I or 27L; 47V or 47L; 53R; 54Q; 66T or 66G; 68R; and 92I;
(iii) 4V or 4I; 6I or 6L; 21V; 27I or 27L; 31T or 31S or 31F; 47V or 47L; 53R; 56P or 56R; 66T or 66G; 68R; and 94L or 94V;
(iv) 6I or 6L; 27I or 27L; 31T or 31S or 31F; 47V or 47L; 53R; 54Q; 56P or 56R; 66T or 66G; 92I; and 94L or 94V;
(v) 4V or 4I; 21V; 27I or 27L; 31T or 31S or 31F; 47V or 47L; 53R; 54Q; 56P or 56R; 66T or 66G; 94L or 94V; and 103V;
(vi) 4V or 4I; 6I or 6L; 27I or 27L; 31T or 31S or 31F; 47V or 47L; 53R; 56P or 56R; 66T or 66G; 68R; 92I; and 94L or 94V;
(vii) 4V or 4I; 6I or 6L; 31T or 31S or 31F; 47V or 47L; 53R; 56P or 56R; 66T or 66G; 92I; and 103V;
(viii) 6I or 6L; 27I or 27L; 31T or 31S or 31F; 47V or 47L; 53R; 54Q; 56P or 56R; 66T or 66G;
(ix) 4V or 4I; 6I or 6L; 27I or 27L; 31T or 31S or 31F; 47V or 47L; 53R; 54Q; 56P or 56R; 63I; 66T or 66G; 68R; and 92I;
(x) 6I or 6L; 27I or 27L; 31T or 31S or 31F; 47V or 47L; 53R; 54Q; 56P or 56R; 66T or 66G; 68R; 92I; and 103V;
(xi) 6I or 6L; 27I or 27L; 31T or 31S or 31F; 47V or 47L; 53R; 54Q; 56P or 56R; 66T or 66G; and 92I.
7. The soluble high affinity SIRPα polypeptide of claim 1 , comprising:
(i) 27I; 53R; 66T; 68R; F103V;
(ii) 4V; 27L; 47V; 53R; 54Q; 66G; 68R; 92I;
(iii) 4V; 6I; 21V; 27I; 31T; 47L; 53R; 56P; 66T; 68R; 94L;
(iv) 6I; 27I; 31S; 47V; 53R; 54Q; 56P; 66G; 92I; 94L;
(v) 4I; 21V; 27I; 31F; 47V; 53R; 54Q; 56R; 66G; 94V; 103V;
(vi) 4V; 6I; 27I; 31F; 47V; 53R; 56R; 66G; 68R; 92I; 94L;
(vii) 4V; 6L; 31F; 47V; 53R; 56P; 66G; 92I; 103V;
(viii) 6I; 27I; 31F; 47L; 53R; 54Q; 56P; 66T;
(ix) 4V; 6I; 27I; 31F; 47V; 53R; 54Q; 56P; 63I; 66T; 68R; 92I;
(x) 6I; 27I; 31T; 47V; 53R; 54Q; 56P; 66G; 68R; 92I; 103V; or
(xi) 6I; 27I; 31F; 47V; 53R; 54Q; 56P; 66T; 92I.
8. The soluble high affinity SIRPα polypeptide of claim 1 , comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 3-9 and 37-39.
9. The soluble high affinity SIRPα polypeptide of claim 1 , wherein the high affinity SIRPα polypeptide has a K D less than 279 nM for human CD47.
10. The soluble high affinity SIRPα polypeptide of claim 1 , comprises amino acid sequences from SIRPα outside of the d1 domain.
11. The soluble high affinity SIRPα polypeptide of claim 1 , wherein the polypeptide is multimeric.
12. The soluble high affinity SIRPα polypeptide of claim 1 , wherein the polypeptide is monomeric.
13. The soluble high affinity SIRPα polypeptide of claim 1 , which is pegylated.
14. The soluble high affinity SIRPα polypeptide of claim 1 , further comprising one or more substitutions selected from the group consisting of: 4V or 4I; 6I or 6L; 21V; 27I or 27L; 47V or 47L; 63I; 92I; 94L or 94V; and 103V.
15. A composition comprising the soluble high affinity SIRPα polypeptide of claim 1 and a pharmaceutically acceptable excipient.
16. A method of increasing phagocytosis of a cell expressing human CD47 comprising contacting the cell with the composition of claim 15 and a tumor specific antibody.
17. The method of claim 16 , wherein the contacting is in vitro.
18. The method of claim 16 , wherein the contacting is in vivo.
19. The method of claim 16 , wherein the cell expressing human CD47 is a cancer cell.
20. The soluble high affinity SIRPα polypeptide of claim 1 , further comprising a detectable label.
21. A method of imaging a tumor, the method comprising contacting cancer cells with a polypeptide as set forth in claim 20 .
22. A chimeric protein comprising the soluble high affinity SIRPα polypeptide of claim 1 and an immunoglobulin Fc sequence.
23. A composition comprising the chimeric protein of claim 22 and a pharmaceutically acceptable excipient.
24. A method of increasing phagocytosis of a cell expressing human CD47, the method comprising contacting the cell with the composition of claim 23 .
25. The method of claim 24 , further comprising contacting the cell with a tumor specific antibody.
26. The method of claim 24 , wherein the contacting is in vitro.
27. The method of claim 24 , wherein the contacting is in vivo.
28. The method of claim 24 , wherein the cell expressing human CD47 is a cancer cell.