Multivalent and multispecific DR5-binding fusion proteins and methods of modulating immune cells
The disclosure relates generally to molecules that specifically engage death receptor 5 (DR5), a member of the TNF receptor superfamily (TNFRSF). More specifically the disclosure relates to multivalent and multispecific molecules that bind at least DR5.
1. A method of modulating immune cells to enhance tumor destruction comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide that binds death receptor 5 (DR5) and comprises a plurality of DR5 binding domains (DR5BDs), wherein each DR5BD is a VHH comprising an amino acid sequence selected from SEQ ID NOs: 16-20, 87, and 89, wherein adjacent DR5BDs are operably linked by an amino acid linker, and wherein the isolated polypeptide comprises an immunoglobulin Fc region that forms a homodimer.
2. The method of claim 1 , wherein the plurality of DR5BDs is two DR5BDs.
3. The method of claim 1 , wherein the plurality of DR5BDs is four DR5BDs.
4. The method of claim 1 , wherein the plurality of DR5BDs is six DR5BDs.
5. The method of claim 1 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
6. The method of claim 2 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
7. The method of claim 3 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
8. The method of claim 4 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
9. The method of claim 1 , wherein the isolated polypeptide comprises an immunoglobulin hinge region.
10. The method of claim 9 , wherein the immunoglobulin hinge region comprises an amino acid sequence selected from EPKSSDKTHTCPPC (SEQ ID NO: 6), DKTHTCPPC (SEQ ID NO: 7), and ESKYGPPCPPC (SEQ ID NO: 8).
11. The method of claim 9 , wherein the immunoglobulin Fc region comprises an amino acid sequence selected from SEQ ID NOs: 1-5 or 127.
12. The method of claim 1 , wherein the immunoglobulin Fc region is an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG4 Fc region.
13. The method of claim 1 , wherein the polypeptide has the structure: DR5BD-Linker-DR5BD-Linker-Hinge-Fc, where each DR5BD is a humanized VHH sequence, and wherein the polypeptide forms a homodimer that is tetravalent.
14. The method of claim 13 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
15. The method of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 113.
16. The method of claim 15 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 113 fused to an Fc region polypeptide, and wherein the polypeptide forms a homodimer that is tetravalent.
17. The method of claim 1 , wherein each VHH is a humanized VHH.
18. The method of claim 1 , wherein each amino acid linker consists of 5-20 amino acids.
19. The method of claim 18 , wherein each amino acid linker is composed predominantly of glycine and serine.
20. The method of claim 19 , wherein each amino acid linker comprises an amino acid sequence selected from GGSGGS (SEQ ID NO: 11); GGSGGSGGS (SEQ ID NO: 12); GGSGGSGGSGGS (SEQ ID NO: 13); and GGSGGSGGSGGSGGS (SEQ ID NO: 14).
21. The method of claim 14 , wherein each amino acid linker consists of 5-20 amino acids.
22. The method of claim 21 , wherein each amino acid linker is composed predominantly of glycine and serine.
23. The method of claim 22 , wherein each amino acid linker comprises an amino acid sequence selected from GGSGGS (SEQ ID NO: 11); GGSGGSGGS (SEQ ID NO: 12); GGSGGSGGSGGS (SEQ ID NO: 13); and GGSGGSGGSGGSGGS (SEQ ID NO: 14).
24. A method of modulating immune cells to enhance tumor destruction comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide that binds death receptor 5 (DR5), wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 113 fused to an Fc region polypeptide of SEQ ID NO: 2, and wherein the polypeptide forms a homodimer that is tetravalent.