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 . A method of reducing or depleting the number of T regulatory cells in a tumor in a subject, 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 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 .- 7 . (canceled)
8 . The method of claim 1 , wherein the plurality of DR5BDs is two DR5BDs.
9 . The method of claim 1 , wherein the plurality of DR5BDs is four DR5BDs.
10 . The method of claim 1 , wherein the plurality of DR5BDs is six DR5BDs.
11 . The method of claim 1 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
12 . The method of claim 8 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
13 . The method of claim 9 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
14 . The method of claim 10 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
15 .- 18 . (canceled)
19 . The method of claim 1 , wherein the isolated polypeptide comprises an immunoglobulin hinge region and an immunoglobulin Fc region.
20 . The method of claim 19 , wherein the immunoglobulin Fc region is an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG4 Fc region.
21 . The method of claim 19 , wherein the immunoglobulin Fc region comprises an amino acid sequence selected from SEQ ID NOs: 1-5 or 127.
22 .- 25 . (canceled)
26 . The method of claim 1 , wherein each VHH is a humanized VHH.
27 .- 32 . (canceled)
33 . The method of claim 9 , wherein the polypeptide is a homodimer of the structure: DR5BD-Linker-DR5BD-Linker-Hinge-Fc, where each the DR5BD is a humanized VHH sequence.
34 .- 46 . (canceled)
47 . The method of claim 33 , wherein each DR5BD comprises the amino acid sequence of SEQ ID NO: 87.
48 . The method of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 113.
49 . The method of claim 48 , wherein the polypeptide is a homodimer of the amino acid sequence of SEQ ID NO: 113 fused to an Fc region.
50 . A method of reducing or depleting the number of T regulatory cells in a tumor in a subject, 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 is a homodimer of the amino acid sequence of SEQ ID NO: 113 fused to an Fc region of SEQ ID NO: 2.
51 . The method of claim 1 , wherein each amino acid linker consists of 5-20 amino acids.
52 . The method of claim 51 , wherein each amino acid linker is composed predominantly of glycine and serine.
53 . The method of claim 52 , 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).
54 . The method of claim 19 , 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)
55 . The method of claim 47 , wherein each amino acid linker consists of 5-20 amino acids.
56 . The method of claim 55 , wherein each amino acid linker is composed predominantly of glycine and serine.
57 . The method of claim 56 , 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).
58 . A method of treating, alleviating a symptom of, ameliorating and/or delaying the progression of a viral, bacterial, or parasitic infection, 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 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.
59 . A method of treating, alleviating a symptom of, ameliorating and/or delaying the progression of an autoimmune disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide that binds at least 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.