COMPOSITIONS AND METHODS FOR MODULATION OF SIRPALPHA-MEDIATED SIGNALING

Abstract

The present disclosure relates generally to compositions and methods for modulating cell surface receptor signaling by specifically recruiting membrane phosphatases, in cis, to a spatial proximity of a signal regulatory protein α (SIRPα) molecule. More particularly, the disclosure provides novel multivalent protein-binding molecules that specifically bind SIRPα and antagonize the SIRPα-mediated signaling through recruitment of a phosphatase activity to dephosphorylate the intracellular domain of SIRPα. Also provided are compositions and methods useful for producing such molecules, methods for promoting maturation dendritic cells and for production of vaccine, as well as methods for the prevention and/or treatment of health conditions associated with the inhibition of signal transduction mediated by SIRPα and/or CD47.

Description

Claims

1. A multivalent polypeptide comprising: a first amino acid sequence comprising a first polypeptide module capable of binding to a signal regulatory protein α (SIRPα); anda second amino acid sequence comprising a second polypeptide module capable of binding to one or more receptor protein-tyrosine phosphatases (RPTP) of R1/R6 subfamily.

2. The multivalent polypeptide of a claim 1, wherein the one or more RPTPs comprises CD45 or a functional variant thereof.

3. The multivalent polypeptide of any one of claims 1-2, wherein at least one of the first and second polypeptide modules comprises an amino acid sequence for a protein-binding ligand or an antigen-binding moiety.

4. The multivalent polypeptide of claim 3, wherein the antigen-binding moiety is selected from the group consisting of a single-chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a VH domain, a VL domain, a single domain antibody (dAb), a VNAR domain, and a VHH domain, a diabody, or a functional fragment of any thereof.

5. The multivalent polypeptide of claim 3, wherein the protein-binding ligand comprises an extracellular domain (ECD) of a cell surface receptor, or an ECD of a RPTP, or a functional variant of any thereof.

6. The multivalent polypeptide of claim 5, wherein the protein-binding ligand comprises an ECD of CD47 or a functional variant thereof.

7. The multivalent polypeptide of any one of claims 1-6, wherein the first polypeptide module is operably linked to the second polypeptide module via a polypeptide linker sequence.

8. The multivalent polypeptide of claim 7, wherein the polypeptide linker sequence comprises a glycine-serine (GS) linker or a 3C linker.

9. The multivalent polypeptide of any one of claims 1-8, comprising: (a) (i) a CD47 ECD, (ii) a polypeptide linker, and (iii) a CD45 scFv;(b) (i) a SIRPα scFv, (ii) a polypeptide linker; and (iii) a CD45 scFv; or(c) (i) a CD45 VHH, (ii) a polypeptide linker, and (iii) a SIRPα scFv.

10. The multivalent polypeptide of claim 9, comprising, in N-terminus to C-terminus direction: (a) (i) a CD47 ECD, (ii) a GS linker, and (iii) a CD45 scFv; or(b) (i) a CD47 ECD, (ii) a C3 linker, and (iii) a CD45 scFv.

11. The multivalent polypeptide of claim 9, comprising, in N-terminus to C-terminus direction: (a) (i) a SIRPα scFv, (ii) a GS linker, and (iii) a CD45 scFv; or(b) (i) a SIRPα scFv, (ii) a C3 linker, and (iii) a CD45 scFv.

12. The multivalent polypeptide of claim 9, comprising, in N-terminus to C-terminus direction: (a) (i) a CD45 VHH, (ii) a GS linker, and (iii) a SIRPα scFv; or(b) (i) a CD45 VHH, (ii) a C3 linker, and (iii) a SIRPα scFv.

13. The multivalent polypeptide of any one of claims 1-12, wherein the multivalent polypeptide comprises an amino acid sequence that has at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-6.

14. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding a multivalent polypeptide according to any one of claims 1-13.

15. The recombinant nucleic acid molecule of claim 14, wherein the nucleotide sequence encodes an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-6.

16. A recombinant cell comprising: (a) a multivalent polypeptide according to any one of claims 1-13, and/or(b) a recombinant nucleic acid molecule of any one of claims 14-15.

17. The recombinant cell of claim 16, wherein the recombinant cell is a phagocytic cell.

18. The recombinant cell of claim 17, wherein the phagocytic cell is a dendritic cell.

19. A method for promoting the maturation of immature dendritic cells (DCs) in vitro, the method comprising: (a) exposing immature DCs to an antigen; and(b) culturing the immature DCs in the presence of a multivalent polypeptide according to any one of claims 1-13 to induce the maturation of immature DCs into mature DCs.

20. A mature dendritic cell prepared by the method of claim 19.

21. A method for manufacturing a vaccine, the method comprising: (a) exposing immature DCs to an antigen in vitro to produce a sufficient number of antigen-presenting immature DCs; and(b) promoting the maturation of the antigen-presenting immature DCs in the presence of a multivalent polypeptide according to any one of claims 1-13 to produce mature antigen-presenting DCs.

22. A vaccine manufactured by the method of claim 21.

23. The vaccine of claim 22, further comprises a diluent, an excipient, an auxiliary adjuvant, a bacterial adjuvant, and/or a systemic adjuvant.

24. A pharmaceutical composition comprising a pharmaceutical acceptable excipient, and: (a) a multivalent polypeptide according to any one of claims 1-13;(b) a recombinant nucleic acid molecule according to any one of claims 14-15; and/or(c) a recombinant cell according to any one of claims 16-17;(d) a mature dendritic cell according to claim 20; and /or(e) a vaccine according to any one of claims 22-23.

25. A method for modulating cell signaling mediated by CD47 and/or SIRPα in a subject, the method comprising administering to the subject a composition comprising: (a) a multivalent polypeptide according to any one of claims 1-13;(b) a recombinant nucleic acid molecule according to any one of claims 14-15;(c) a recombinant cell according to any one of claims 16-17;(d) a mature dendritic cell according to claim 20;(e) a vaccine according to any one of claims 22-23; and/or(f) a pharmaceutical composition of claim 24.

26. A method for preventing or treating a health condition in a subject in need thereof, the method comprising administering to the subject a composition comprising: (a) a multivalent polypeptide according to any one of claims 1-13;(b) a recombinant nucleic acid molecule according to any one of claims 14-15;(c) a recombinant cell according to any one of claims 16-17;(d) a mature dendritic cell according to claim 20;(e) a vaccine according to any one of claims 22-23; and/or(f) a pharmaceutical composition of claim 24.

27. The method of any one of claims 25-26, wherein the administered composition recruits the RPTP activity to a spatial proximity of SIRPα, potentiates dephosphorylation of SIRPα, reduces SIRPα-mediated signaling, promotes dendritic cell maturation, and/or promotes macrophage phagocytosis.

28. The method of any one of claims 25-27, wherein the administered composition confers an enhancement in macrophage-mediated phagocytosis.

29. The method of any one of claims 25-28, wherein the subject has or is suspected of having a health condition associated with CD47 and/or SIRPα.

30. The method of any one of claims 25-29, wherein the health condition is a cancer or a chronic infection.

31. A method for preventing or treating a cancer in a subject in need thereof, the method comprising: culturing immature DCs to an antigen in vitro with a cancer-associated antigen or infection-associated antigen to produce antigen-presenting immature DCs;promoting the maturation of antigen-presenting immature DCs in the presence of a multivalent polypeptide according to any one of claims 1-13 to produce mature antigen-presenting DCs; andadministering the subject with the produced mature antigen-presenting DCs.

32. A method of preventing or treating a subject infected or suspected of being suspected with a parasite, a virus, a microfungus, or a bacterium, the method comprising: culturing immature DCs with an antigen derived from a parasite, a virus, a microfungus, or a bacterium to produce antigen-presenting dendritic cells;promoting the maturation of dendritic cells in the presence of a multivalent polypeptide according to any one of claims 1-13 to produce mature antigen-presenting DCs; andadministering the subject with the produced mature antigen-presenting DCs.

33. The method of any one of claims 25-32, wherein the subject is a mammalian subject.

34. The method of claim 33, the mammalian subject is a human.

35. The method of any one of claim 26-34, the composition is administered to the subject individually or as a first therapy in combination with a second therapy.

36. The method of claim 35, wherein the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, or surgery further comprising administering to the subject a second therapy.

37. A kit for modulating cell signaling in a subject or for preventing or for treating a health condition in a subject in need thereof, the kit comprising instructions for use thereof and one or more of the following: (a) a multivalent polypeptide according to any one of claims 1-13;(b) a recombinant nucleic acid molecule according to any one of claims 14-15;(c) a recombinant cell according to any one of claims 16-17;(d) a mature dendritic cell according to claim 20;(e) a vaccine according to any one of claims 22-23; and(f) a pharmaceutical composition of claim 24.