Canine PD-1-Binding Polypeptides and Uses Thereof
Provided herein are VHH-containing polypeptides that bind canine PD-1. In some embodiments, VHH-containing polypeptides that bind and antagonize canine PD-1 are provided. Uses of the VHH-containing polypeptides are also provided.
1 . A polypeptide comprising at least one caninized VHH domain that binds canine PD-1, wherein the polypeptide comprises a canine Fc region, and wherein at least one VHH domain that binds canine PD-1 comprises:
a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, a CDR3 comprising the amino acid sequence of SEQ ID NO: 8;
b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR2 comprising the amino acid sequence of SEQ ID NO: 23, a CDR3 comprising the amino acid sequence of SEQ ID NO: 24;
c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or
d) a CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a CDR3 comprising the amino acid sequence of SEQ ID NO: 30.
2 . A polypeptide comprising at least one VHH domain that binds canine PD-1, wherein the polypeptide comprises a canine Fc region, and wherein at least one VHH domain that binds canine PD-1 comprises an amino acid sequence selected from SEQ ID NOs: 2-5.
3 . The polypeptide of claim 1 or 2 , comprising one VHH domain.
4 . The polypeptide of claim 1 or 2 , comprising two VHH domains, wherein the two VHH domains are the same or different.
5 . The polypeptide of claim 1 or 2 , comprising three VHH domains, wherein the three VHH domains are the same or different.
6 . The polypeptide of any one of the preceding claims , wherein each VHH domain binds canine PD-1.
7 . The polypeptide of any one of claims 1 to 6 , wherein the canine Fc region is a canine IgG Fc region.
8 . The polypeptide of claim 7 , wherein the canine Fc region is a canine IgGB Fc region.
9 . The polypeptide of claim 7 or claim 8 , wherein amino acids E233, M234, and L235 of the Fc region, as determined by Kabat numbering, are deleted.
10 . The polypeptide of any one of claims 7 to 9 , wherein the Fc region comprises D265A and N297A substitutions, as determined by Kabat numbering.
11 . The polypeptide of any one of claims 1 to 8 , wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 19.
12 . The polypeptide of any one of claims 1 to 10 , wherein the canine Fc region comprises the amino acid sequence of SEQ ID NO: 20.
13 . The polypeptide of any one of claims 1 to 12 , wherein at least one VHH domain that binds canine PD-1 comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.
14 . The polypeptide of any one of claims 1 to 13 , wherein at least one VHH domain that binds canine PD-1 comprises the amino acid sequence of SEQ ID NO: 3.
15 . The polypeptide of any one of claims 1 to 10 or 12 to 14 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 21.
16 . The polypeptide of any one of claims 1 or 3 to 13 , wherein at least one VHH domain that binds canine PD-1 is caninized.
17 . The polypeptide of claim 16 , wherein at least one caninized VHH domain that binds canine PD-1 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity an amino acid sequence selected from SEQ ID NOs: 9-13.
18 . The polypeptide of claim 16 or claim 17 , wherein at least one caninized VHH domain that binds canine PD-1 comprises an amino acid sequence selected from SEQ ID NOs: 9-13.
19 . The polypeptide of any one of claim 16 to 18 , wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 14-18.
20 . The polypeptide of any one of claims 1 to 19 , which forms a dimer under physiological conditions.
21 . The polypeptide of any one of claims 1 to 20 , wherein the polypeptide decreases canine PD-1 binding to canine PD-L1 in vitro and/or in vivo.
22 . The polypeptide of claim 21 , wherein the polypeptide decreases canine PD-1 binding to canine PD-L1 in vitro by at least 50%, 60%, 70%, 80%, or at least 90%.
23 . The polypeptide of any one of claims 1 to 22 , which is an antagonist of canine PD-1 biological activity.
24 . The polypeptide of any one of claims 1 to 23 , wherein the polypeptide binds canine PD-1 with an affinity (K D ) of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM.
25 . A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 24 and a pharmaceutically acceptable carrier.
26 . An isolated nucleic acid that encodes the polypeptide of any one of claims 1 to 24 .
27 . A vector comprising the nucleic acid of claim 26 .
28 . A host cell comprising the nucleic acid of claim 26 or the vector of claim 27 .
29 . A host cell that expresses the polypeptide of any one of claims 1 to 24 .
30 . A method of producing the polypeptide of any one of claims 1 to 24 comprising incubating the host cell of claim 28 or claim 29 under conditions suitable for expression of the polypeptide.
31 . The method of claim 30 , further comprising isolating the polypeptide.
32 . A method of activating canine CD4+ T cells and/or canine CD8+ T cells comprising contacting the T cells with the polypeptide of any one of claims 1 to 24 .
33 . The method of claim 32 , wherein the T cells are in vitro.
34 . The method of claim 32 , wherein the T cells are in vivo.
35 . A method of treating cancer in a canine comprising administering to a canine with cancer a pharmaceutically effective amount of the polypeptide of any one of claims 1 to 24 or the pharmaceutical composition of claim 25 .
36 . The method of claim 35 , wherein the cancer is selected from lymphoma, hemangiosarcoma, mast cell carcinoma, melanoma, osteosarcoma, mammary cancer, renal cell carcinoma, and a non-small cell lung cancer.
37 . The method of claim 35 or 36 , further comprising an additional anti-cancer therapy.
38 . The method of claim 37 , wherein the additional anti-cancer therapy comprises at least one therapy selected from cancer resection, radiation therapy, and administration of an additional anti-cancer agent.