TETRAVALENT ANTI-PSGL-1 ANTIBODIES AND USES THEREOF
Provided herein are tetravalent antibodies that specifically bind to human PSGL-1. Unlike bivalent antibodies, these tetravalent antibodies contain a dimer of two monomers, with each monomer comprising two light chain variable (VL) domains and two heavy chain variable (VH) domains. This format allows for cross-linker/FcR-expressing cell-independent tetravalent antibodies against PSGL-1 that show enhanced efficacy as compared to bivalent PSGL-1 antibodies. These tetravalent antibodies can be used in a variety of diagnostic and therapeutic methods, including without limitation treating T-cell mediated inflammatory diseases, transplantations, and transfusions.
1 . A tetravalent antibody that specifically binds to human PSGL-1, the tetravalent antibody comprising a dimer of two monomers, wherein each monomer of the dimer comprises a single-chain polypeptide comprising, from N-terminus to C-terminus:
(a) a first light chain variable (VL) domain;
(b) a first linker sequence;
(c) a first heavy chain variable (VH) domain;
(d) a second linker sequence;
(e) a second VL domain;
(f) a third linker sequence;
(g) a second VH domain;
(h) a fourth linker sequence; and
(i) an antibody Fc domain,
wherein each of the first and the second VL domains comprises a CDR-L1, a CDR-L2, and a CDR-L3; wherein each of the first and the second VH domains comprises a CDR-H1, a CDR-H2, and a CDR-H3; and wherein each of the first and the second VL domains forms a VH-VL binding unit with a corresponding VH domain of the first and the second VH domains, and wherein each of the two VH-VL binding units is specific for human PSGL-1.
2 . The tetravalent antibody of claim 1 , wherein at least one of the two VH domains comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:19; and/or
wherein at least one of the two VL domains comprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:22.
3 . (canceled)
4 . The tetravalent antibody of claim 2 , wherein each of the two VH domains comprises the amino acid sequence of SEQ ID NO:23; an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:23; the amino acid sequence of SEQ ID NO:29; or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:29; and/or
wherein each of the two VL domains comprises the amino acid sequence of SEQ ID NO:24; an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:24; the amino acid sequence of SEQ ID NO:30; or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:30.
5 - 9 . (canceled)
10 . The tetravalent antibody of claim 1 , wherein the first, second and third linker sequences each comprise two or more repeats of the amino acid sequence of SEQ ID NO:25, or the first, second or third linker sequence comprises the amino acid sequence of SEQ ID NO:33, 34, 35, or 36.
11 . (canceled)
12 . (canceled)
13 . The tetravalent antibody of claim 1 , wherein the fourth linker sequence comprises the amino acid sequence of SEQ ID NO:26.
14 . The tetravalent antibody of claim 1 , wherein each of the two single-chain polypeptides comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:1.
15 . (canceled)
16 . A tetravalent antibody that specifically binds to human PSGL-1, the tetravalent antibody comprising a dimer of two monomers, wherein each monomer of the dimer comprises a single-chain polypeptide comprising, from N-terminus to C-terminus:
(a) a first heavy chain variable (VH) domain;
(b) a first linker sequence;
(c) a first light chain variable (VL) domain;
(d) a second linker sequence;
(e) a second VL domain;
(f) a third linker sequence;
(g) a second VH domain;
(h) a fourth linker sequence; and
(i) an antibody Fc domain,
wherein each of the first and the second VL domains comprises a CDR-L1, a CDR-L2, and a CDR-L3; wherein each of the first and the second VH domains comprises a CDR-H1, a CDR-H2, and a CDR-H3; and wherein each of the first and the second VL domains forms a VH-VL binding unit with a corresponding VH domain of the first and the second VH domains, and wherein each of the two VH-VL binding units is specific for human PSGL-1.
17 . The tetravalent antibody of claim 16 , wherein at least one of the two VH domains comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:19; and/or
wherein at least one of the two VL domains comprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:22.
18 . (canceled)
19 . The tetravalent antibody of claim 17 , wherein each of the two VH domains comprises the amino acid sequence of SEQ ID NO:23; an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:23; the amino acid sequence of SEQ ID NO:29; or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:29; and/or
wherein each of the two VL domains comprises the amino acid sequence of SEQ ID NO:24; an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:24; the amino acid sequence of SEQ ID NO:30; or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:30.
20 - 24 . (canceled)
25 . The tetravalent antibody of claim 16 , wherein the first and the third linker sequences have the same sequence comprising five repeats of SEQ ID NO:25.
26 . (canceled)
27 . The tetravalent antibody of claim 16 , wherein the fourth linker sequence comprises the amino acid sequence of SEQ ID NO:26.
28 . The tetravalent antibody of claim 16 , wherein each of the two single-chain polypeptides comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:3.
29 . (canceled)
30 . A tetravalent antibody that specifically binds to human PSGL-1, the tetravalent antibody comprising a dimer of two monomers, wherein each monomer of the dimer comprises an antibody heavy chain and an antibody light chain;
wherein the antibody light chain comprises, from N-terminus to C-terminus:
(i) a first heavy chain variable (VH) domain,
(ii) a first linker sequence,
(iii) a first light chain variable (VL) domain,
(iv) a second linker sequence,
(v) a second VL domain, and
(vi) a light chain constant (CL) domain;
wherein the antibody heavy chain comprises:
(i) a second VH domain, and
(ii) a heavy chain constant region comprising a first heavy chain constant region (CH1) domain, an antibody hinge region, an second heavy chain constant region (CH2) domain, and a third heavy chain constant region (CH3) domain;
wherein each of the first and the second VL domains comprises a CDR-L1, a CDR-L2, and a CDR-L3; wherein each of the first and the second VH domains comprises a CDR-H1, a CDR-H2, and a CDR-H3; and wherein each of the first and the second VL domains forms a VH-VL binding unit with a corresponding VH domain of the first and the second VH domains, and wherein each of the two VH-VL binding units is specific for human PSGL-1.
31 . The tetravalent antibody of claim 30 , wherein at least one of the first and the second VH domains comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:19; and/or
wherein at least one of the first and the second VL domains comprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:22.
32 . (canceled)
33 . The tetravalent antibody of claim 31 , wherein the first and the second VH domains each comprise the amino acid sequence of SEQ ID NO:23; an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:23; the amino acid sequence of SEQ ID NO:29; or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:29; and/or
wherein each of the two VL domains comprises the amino acid sequence of SEQ ID NO:24; an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:24; the amino acid sequence of SEQ ID NO:30; or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:30.
34 - 38 . (canceled)
39 . The tetravalent antibody of claim 30 , wherein the CL domain is a kappa CL domain.
40 . The tetravalent antibody of claim 30 , wherein the first linker sequence comprises five repeats of SEQ ID NO:25; and/or
wherein the second linker sequence comprises the amino acid sequence of SEQ ID NO:28.
41 . (canceled)
42 . The tetravalent antibody of claim 30 , wherein the antibody light chain comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:7; and/or
wherein the antibody heavy chain comprises the amino acid sequence of SEQ ID NO:11, or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:11.
43 - 45 . (canceled)
46 . The tetravalent antibody of claim 1 , wherein the antibody Fc domain is a human antibody Fc domain.
47 - 49 . (canceled)
50 . An isolated polynucleotide encoding the tetravalent antibody of claim 1 .
51 . (canceled)
52 . A vector comprising the isolated polynucleotide of claim 50 .
53 . A host cell comprising the polynucleotide of claim 50 .
54 . A method of producing a tetravalent antibody comprising culturing the host cell of claim 53 so that the tetravalent antibody is produced.
55 . (canceled)
56 . A pharmaceutical composition comprising the tetravalent antibody of claim 1 and a pharmaceutically acceptable carrier.
57 - 63 . (canceled)
64 . A method of treating a T-cell mediated inflammatory disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the tetravalent antibody of claim 1 .
65 . A method for treating an individual in need of a transfusion or transplantation, comprising administering to the individual a therapeutically effective amount of the tetravalent antibody of claim 1 before, concurrently with, and/or after the transfusion or transplantation.
66 - 70 . (canceled)