Multivalent and multispecific DR5-binding fusion proteins
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. 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 a CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence of SEQ ID NO: 131, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130, and wherein adjacent DR5BDs are operably linked by an amino acid linker.
2. The isolated polypeptide of claim 1 , wherein the plurality of DR5BDs is two DR5BDs.
3. The isolated polypeptide of claim 1 , wherein the plurality of DR5BDs is four DR5BDs.
4. The isolated polypeptide of claim 1 , wherein the plurality of DR5BDs is six DR5BDs.
5. The isolated polypeptide of claim 1 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
6. The isolated polypeptide of claim 2 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
7. The isolated polypeptide of claim 3 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
8. The isolated polypeptide of claim 4 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
9. The isolated polypeptide of claim 3 , wherein the polypeptide is a homodimer of the structure: DR5BD-Linker-DR5BD-Linker-Hinge-Fc, where each DR5BD is a humanized VHH sequence.
10. The isolated polypeptide of claim 9 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
11. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises an immunoglobulin hinge region and an immunoglobulin Fc region.
12. The isolated polypeptide of claim 11 , wherein the immunoglobulin hinge region comprises an amino acid sequence selected from EPKSSDKTHTCPPC (SEQ ID NO: 6), DKTHTCPPC (SEQ ID NO: 7), ESKYGPPCPPC (SEQ ID NO: 8).
13. The isolated polypeptide of claim 11 , wherein the immunoglobulin Fc region is an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG1 Fc region.
14. The isolated polypeptide of claim 11 , wherein the immunoglobulin Fc region comprises an amino acid sequence selected from SEQ ID NOs: 1-5 or 127.
15. The isolated polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 113.
16. The isolated polypeptide of claim 15 , wherein the polypeptide is a homodimer of the amino acid sequence of SEQ ID NO: 113 fused to an Fc region polypeptide.
17. The isolated polypeptide of claim 1 , wherein each amino acid linker consists of 5-20 amino acids.
18. The isolated polypeptide of claim 17 , wherein each amino acid linker is composed predominantly of glycine and serine.
19. The isolated polypeptide of claim 18 , 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).
20. The isolated polypeptide of claim 10 , wherein each amino acid linker consists of 5-20 amino acids.
21. The isolated polypeptide of claim 20 , wherein each amino acid linker is composed predominantly of glycine and serine.
22. The isolated polypeptide of claim 21 , 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).
23. The isolated polypeptide of claim 1 , wherein each VHH is a humanized VHH.
24. An isolated polypeptide that binds death receptor 5 (DR5), wherein the polypeptide is a homodimer of the amino acid sequence of SEQ ID NO: 113 fused to an Fc region polypeptide of SEQ ID NO: 2.