Tetravalent anti-psgl-1 antibodies and uses thereof

Abstract

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 (CH 1 ) domain, an antibody hinge region, an second heavy chain constant region (CH 2 ) domain, and a third heavy chain constant region (CH 3 ) 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)