ANTI-GALECTIN-1 (GAL1) MONOCLONAL ANTIBODIES AND FRAGMENTS THEREOF FOR NEUTRALIZING GAL1
The present invention is based, in part, on the discovery of galectin 1 (Gal1) epitopes against which anti-Gal1 agents can neutralize Gal1 function, as well as anti-Gal1 agents and methods useful for neutralizing Gal1 function.
1 . A recombinant polypeptide of less than or equal to about 60 amino acids in length comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of residues 102-115 of SEQ ID NOs: 1-16.
2 . The recombinant polypeptide of claim 1 , wherein said polypeptide is equal to 14 amino acids in length.
3 . The recombinant polypeptide of claim 1 or 2 , wherein said amino acid sequence is identical to the amino acid sequence of residues 102-115 of any one of SEQ ID NOs: 1-16.
4 . The recombinant polypeptide of any one of claims 1 - 3 , wherein said polypeptide further comprises a heterologous sequence.
5 . The recombinant polypeptide of any one of claims 1 - 4 , wherein the polypeptide is isolated.
6 . The recombinant polypeptide of any one of claims 1 - 5 , wherein said polypeptide is covalently linked to a detectable label.
7 . The recombinant polypeptide of any one of claims 1 - 6 , wherein said polypeptide is covalently bonded to a carrier molecule or immobilized on an object.
8 . The recombinant polypeptide of claim 7 , wherein the object is selected from the group consisting of a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array, and a capillary tube.
9 . A recombinant nucleic acid molecule having a sequence that hybridizes under stringent conditions with the complement of a nucleic acid encoding a polypeptide of any one of claims 1 - 8 or having a sequence with at least about 95% homology to a nucleic acid encoding a polypeptide of any one of claims 1 - 8 , wherein the nucleic acid molecule is only as long as required to encode the polypeptide.
10 . A vector comprising the recombinant nucleic acid of claim 9 , optionally wherein the vector is an expression vector comprising a promoter to which the nucleic acid is operably linked.
11 . A host cell which expresses the polypeptide of any one of claims 1 - 8 , comprises the nucleic acid of claim 9 , or comprises the vector of claim 10 .
12 . An immunogenic composition comprising the polypeptide of any one of claims 1 - 8 , the nucleic acid of claim 9 , the vector of claim 10 , or the host cell of claim 11 ; and a pharmaceutically acceptable carrier.
13 . The immunogenic composition of claim 12 , further comprising at least one additional immunostimulatory agent.
14 . The immunogenic composition of claim 13 , wherein the immunostimulatory agent is an adjuvant and/or an immune checkpoint inhibitor.
15 . The immunogenic composition of claim 14 , wherein the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, LAG-3, TIM-1, CTLA-4, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 284, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-4, BTLA, SIRPalpha (CD47), CD48, 284 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, A2aR, and combinations thereof.
16 . The immunogenic composition of claim any one of claims 12 - 15 , wherein the composition is capable of eliciting neutralizing anti-Gal1 antibodies in mammals.
17 . An isolated neutralizing anti-Gal1 antibody, or antigen binding portion thereof, that specifically binds to the polypeptide of any one of claims 1 - 8 .
18 . The antibody, or antigen binding portion thereof, of claim 17 , which is a monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, single-chain antibody, antibody fragment, composite, murine, human, or is detectably labeled.
19 . The antibody, or antigen-binding fragment thereof, of claim 17 or 18 , which is detectably labeled, comprises an effector domain, comprises an Fc domain, and/or is selected from the group consisting of Fv, Fav, F(ab′)2), Fab′, dsFv, scFv, sc(Fv)2, and diabodies fragments.
20 . A method of identifying a neutralizing anti-Gal1 antibody comprising
(a) administering an effective amount of an agent selected from the group consisting of the polypeptide of any one of claims 1 - 8 , the nucleic acid of claim 9 , the vector of claim 10 , the host cell of claim 1 , or the immunogenic composition of any one of claims 12 - 16 , to a subject to generate antibodies that neutralize Gal1; and
(b) isolating anti-Gal1 antibodies specific for the administered agent.
21 . A method of identifying a neutralizing anti-Gal1 antibody comprising
(a) administering an effective amount of an agent selected from the group consisting of the polypeptide of any one of claims 1 - 8 , the nucleic acid of claim 9 , the vector of claim 10 , the host cell of claim 11 , or the immunogenic composition of any one of claims 12 - 16 , to B cells in an in vitro cell culture system to generate antibodies that neutralize Gal1; and
(b) isolating anti-Gal1 antibodies specific for the administered agent.
22 . A method of making an isolated hybridoma which produces a neutralizing anti-Gal1 antibody that specifically binds to Gal1 comprising:
a) immunizing a mammal with or contacting B cells with an effective amount of an agent selected from the group consisting of the polypeptide of any one of claims 1 - 8 , the nucleic acid of claim 9 , the vector of claim 10 , the host cell of claim 11 , or the immunogenic composition of any one of claims 12 - 16 ;
b) optionally isolating splenocytes from the immunized mammal;
c) fusing splenocytes from the immunized mammal or B cells with an immortalized cell line to form hybridomas; and
d) screening individual hybridomas for production of an anti-Gal1 antibody which specifically binds with the agent.
23 . An antibody produced by the method of any one of claims 20 - 22 .
24 . A method of eliciting an anti-Gal1 immune response in a subject comprising administering to the subject a prophylactically or therapeutically effective amount of an agent selected from the group consisting of the polypeptide of any one of claims 1 - 8 , the nucleic acid of claim 9 , the vector of claim 10 , the host cell of claim 11 , the immunogenic composition of any one of claims 12 - 16 , or the antibody of any one of claims 17 - 19 and 23 , to thereby elicit the immune response.
25 . The method of claim 24 , wherein agent is administered in a single dose, administered in multiple doses, or is administered as part of a heterologous prime-boost regimen.
26 . A method of inhibiting Gal1 activity in a subject comprising administering to the subject a prophylactically or therapeutically effective amount of an agent selected from the group consisting of the polypeptide of any one of claims 1 - 8 , the nucleic acid of claim 9 , the vector of claim 10 , the host cell of claim 11 , the immunogenic composition of any one of claims 12 - 16 , or the antibody of any one of claims 17 - 19 and 23 , thereby inhibiting Gal1 activity in the subject.
27 . A method for preventing or delaying the onset of, or slowing the rate of progression of, a disease in a subject mediated by Gal1 activity, comprising administering to the subject a prophylactically or therapeutically effective amount of an agent selected from the group consisting of the polypeptide of any one of claims 1 - 8 , the nucleic acid of claim 9 , the vector of claim 10 , the host cell of claim 1 , the immunogenic composition of any one of claims 12 - 16 , or the antibody of any one of claims 17 - 19 and 23 , thereby preventing or delaying the onset of, or slowing the rate of progression of, the Gal1-mediated disease in the subject.
28 . The method of any one of claims 24 - 27 , further comprising administering at least one additional agent that upregulates an immune response.
29 . The method of claim 28 , wherein the at least one additional agent comprises an inhibitor of an immune checkpoint.
30 . The method of claim 29 , wherein the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, LAG-3, TIM-1, CTLA-4, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-4, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, A2aR, and combinations thereof.
31 . The method of any one of claims 27 - 30 , wherein the Gal1-mediated disease is a Gal1-positive cancer, Gal1-mediated angiogenesis disorder, AP1-dependent lymphoid malignancies, MLL-rearranged ALL, EBV+ post-transplant lymphoproliferative disorder (PTDL), nasopharyngeal carcinoma, Kaposi's sarcoma, breast cancer, prostate cancer, lung cancer, pancreatic cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, and melanoma.
32 . The method of any one of claims 24 - 31 , wherein the subject is a mammal.
33 . The method of claim 32 , wherein the mammal is a human.