Antibodies to EDIL3 and methods of use thereof

Abstract

The present invention relates to human monoclonal antibodies that bind to epidermal growth factor-like repeats and discoidin I-like domain 3 (EDIL3) and methods of use thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 430,840, filed December 7, 2022, the entire contents of which are incorporated herein by reference.

[0003] Statement on Federally Sponsored Research

[0004] This invention was made with government support under CA143832 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Sequence Listing

[0006] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created at [ ], has the name [ ] and is [ ] bytes in size. [Background technology]

[0007] Tumors create a microenvironment that is continuously angiogenic and immunosuppressive. There remains a need for therapies that can reverse immune exclusion or immune dysfunction in the tumor microenvironment, leading to improved patient outcomes. The present disclosure identifies specific targets and corresponding antibodies that can promote immune cell trafficking in the tumor microenvironment and reverse the immunosuppressive effects in the tumor microenvironment. Summary of the Invention

[0008] Aspects relate to antibodies or antigen-binding fragments or variants thereof that bind to epidermal growth factor-like repeat and discoidin I-like domain 3 (EDIL3) protein. In embodiments, the antibody or antigen-binding fragment may comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR). In embodiments, the HCVR comprises complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprises CDRs LCDR1, LCDR2, and LCDR3. In embodiments, the amino acid sequence of HCDR1 is SYAMS, the amino acid sequence of HCDR2 is AISDSGGSTYYADSVKG, the amino acid sequence of HCDR3 is EGLITFGGVIVIGYFDY, the amino acid sequence of LCDR1 is QASQDISNYLN, the amino acid sequence of LCDR2 is DASNLET, and the amino acid sequence of LCDR3 is QQYDNLPIT. In one embodiment, the amino acid sequence of HCDR1 is SYWMS, the amino acid sequence of HCDR2 is NIKQDGSQKYYVDSVKG, the amino acid sequence of HCDR3 is RGNFFFDN, the amino acid sequence of LCDR1 is RASQYVSSYLA, the amino acid sequence of LCDR2 is DASNRAT, and the amino acid sequence of LCDR3 is QQRNNWPPT. In one embodiment, the amino acid sequence of HCDR1 is NHYWS, the amino acid sequence of HCDR2 is YIYYSGSTNYNPSLKS, and the amino acid sequence of HCDR3 is GFAY, the amino acid sequence of LCDR1 is RASQGITNYLA, the amino acid sequence of LCDR2 is AASTLQS, and the amino acid sequence of LCDR3 is QKYNSAPWT. In embodiments, the amino acid sequence of HCDR1 is SYAMN, the amino acid sequence of HCDR2 is AISGSGDSTYSTDSVKG, the amino acid sequence of HCDR3 is EYYDILTGYWDWYFDL, the amino acid sequence of LCDR1 is RASQSINSNLA, the amino acid sequence of LCDR2 is GASTRAT, and the amino acid sequence of LCDR3 is QQYNNWPLT.In one embodiment, the amino acid sequence of HCDR1 is SYGMH, the amino acid sequence of HCDR2 is VMWYDGSDRYSADSVKG, the amino acid sequence of HCDR3 is GYDILTGPDHFDY, the amino acid sequence of LCDR1 is RASQSISSYLN, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR3 is QQSYSTPLT. In one embodiment, the amino acid sequence of HCDR1 is SYDMN, the amino acid sequence of HCDR2 is TISGSGSHTYYADSVRG, the amino acid sequence of HCDR3 is EGGATAFDI, the amino acid sequence of LCDR1 is RASQGISSYLA, the amino acid sequence of LCDR2 is VASTLQS, and the amino acid sequence of LCDR3 is QQLNNYPT. In one embodiment, the amino acid sequence of HCDR1 is TYGMH, the amino acid sequence of HCDR2 is LIWYDGINKYYADSVKG, the amino acid sequence of HCDR3 is PYYDILTGYFDY, the amino acid sequence of LCDR1 is RASQSDSSSYLA, the amino acid sequence of LCDR2 is GTSSRAT, and the amino acid sequence of LCDR3 is QQYGSSPLT. In one embodiment, the amino acid sequence of HCDR1 is SYGMH, the amino acid sequence of HCDR2 is VIWYDGTNKYYADSVKG, the amino acid sequence of HCDR3 is DPSLWFGEFPHYYGMDV, the amino acid sequence of LCDR1 is QASQDISNYLN, the amino acid sequence of LCDR2 is DASNLET, and the amino acid sequence of LCDR3 is QQYDNLPLT. In embodiments, the amino acid sequence of HCDR1 is GYYWS, the amino acid sequence of HCDR2 is EIQHSGSTNYKPSLKS, the amino acid sequence of HCDR3 is LTGDSLLFEY, the amino acid sequence of LCDR1 is RASQSVSSYLA, the amino acid sequence of LCDR2 is DTSNRAT, and the amino acid sequence of LCDR3 is QQRSNWPIT.In one embodiment, the amino acid sequence of HCDR1 is SYGMH, the amino acid sequence of HCDR2 is VIWYDGSNKYYADSVKG, the amino acid sequence of HCDR3 is DSASDYFDY, the amino acid sequence of LCDR1 is RASQSVSSNLA, the amino acid sequence of LCDR2 is GASTRAT, and the amino acid sequence of LCDR3 is QQYSDWPT. In one embodiment, the amino acid sequence of HCDR1 is GYYWS, the amino acid sequence of HCDR2 is EINHSGSTNYKPSLKS, the amino acid sequence of HCDR3 is LTGDSLLFEY, the amino acid sequence of LCDR1 is RASQSVSSYLA, the amino acid sequence of LCDR2 is DTSNRAT, and the amino acid sequence of LCDR3 is QQRSNWPIT.

[0009] In embodiments, an antibody or antigen-binding fragment or variant thereof may comprise a light chain variable region (LCVR) and a heavy chain variable region (HCVR). In embodiments, the amino acid sequence of the LCVR is DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIK, and the amino acid sequence of the HCVR is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISDSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAQEGLITFGGVIVIGYFDYWGQGTLVTVSS. In embodiments, the amino acid sequence of the LCVR is EIVLTQSPATLSLSPGERATLSCRASQYVSSYLAWYHQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNNWPPTFGQGTKVEIK, and the amino acid sequence of the HCVR is EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSQKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRRGNFFFDNWGQGTLVTVSS. In embodiments, the amino acid sequence of the LCVR is DIQMTQSPSSLSASVGDRVTITCRASQGITNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDVATYYCQKYNSAPWTFGQGTKVEIK, and the amino acid sequence of the HCVR is QVQLQESGPGLVKPSETLSLTCTVSGGSISNHYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGFAYWGQGTLVTVSS.In embodiments, the amino acid sequence of the LCVR is EIVMTLSPATLSVSPGERATLSCRASQSINSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEIK, and the amino acid sequence of the HCVR is EVQLLESGGGLGQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGSGDSTYSTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEYYDILTGYWDWYFDLWGQGTLVTVSS. In embodiments, the amino acid sequence of the LCVR is DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK, and the amino acid sequence of the HCVR is QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSDRYSADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYDILTGPDHFDYWGQGTLVTVSS. In embodiments, the amino acid sequence of the LCVR is DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNNYPTFGGGTKVEIK and the amino acid sequence of the HCVR is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMNWVRQAPGKGPVWVSTISGSGSHTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEGGATAFDIWGQGTMVTVSS.In embodiments, the amino acid sequence of the LCVR is EIVLTQSPGTLSLSPGERATLSCRASQSDSSSYLAWYQQKPGQAPRLLIYGTSSRATGISDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIK, and the amino acid sequence of the HCVR is QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVALIWYDGINKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARPYYDILTGYFDYWGQGTLVTVSS. In embodiments, the amino acid sequence of the LCVR is DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK and the amino acid sequence of the HCVR is QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGTNKYYADSVKGRFTISRDNSKNTLYLQVNSLRAEDTAVYYCARDPSLWFGEFPHYYGMDVWGQGTTVTVSS. In embodiments, the amino acid sequence of the LCVR is EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTVSSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK, and the amino acid sequence of the HCVR is QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIQHSGSTNYKPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAELTGDSLLFEYWGQGTLVTVSS.In embodiments, the amino acid sequence of the LCVR is EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSDWPTFGGGTKVEIR and the amino acid sequence of the HCVR is QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDTSKNTLYLQMNSLRAEDTAVYYCARDSASDYFDYWGQGTLVTVSS. In embodiments, the amino acid sequence of the LCVR is EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTVSSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK, and the amino acid sequence of the HCVR is QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYKPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAELTGDSLLFEYWGQGTLVTVSS.

[0010] In embodiments, an antibody or antigen-binding fragment thereof may comprise a light chain variable region (LCVR) and a heavy chain variable region (HCVR). In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISDSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAQEGLITFGGVIVIGYFDYWGQGTLVTVSS. In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASQYVSSYLAWYHQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNNWPPTFGQGTKVEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSQKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRRGNFFFDNWGQGTLVTVSS.In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQGITNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDVATYYCQKYNSAPWTFGQGTKVEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLQESGPGLVKPSETLSLTCTVSGGSISNHYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGFAYWGQGTLVTVSS. In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVMTLSPATLSVSPGERATLSCRASQSINSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EVQLLESGGGLGQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGSGDSTYSTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEYYDILTGYWDWYFDLWGQGTLVTVSS. In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSDRYSADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYDILTGPDHFDYWGQGTLVTVSS.In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNNYPTFGGGTKVEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMNWVRQAPGKGPVWVSTISGSGSHTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEGGATAFDIWGQGTMVTVSS. In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSDSSSYLAWYQQKPGQAPRLLIYGTSSRATGISDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVALIWYDGINKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARPYYDILTGYFDYWGQGTLVTVSS. In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGTNKYYADSVKGRFTISRDNSKNTLYLQVNSLRAEDTAVYYCARDPSLWFGEFPHYYGMDVWGQGTTVTVSS.In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTVSSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIQHSGSTNYKPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAELTGDSLLFEYWGQGTLVTVSS. In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSDWPTFGGGTKVEIR, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDTSKNTLYLQMNSLRAEDTAVYYCARDSASDYFDYWGQGTLVTVSS. In embodiments, the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTVSSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK, and the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYKPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAELTGDSLLFEYWGQGTLVTVSS.

[0011] In embodiments, the antibody fragment or antigen-binding fragment or variant thereof may comprise a F(ab), Fv, or scFv. In embodiments, the antibody fragment or antigen-binding fragment or variant thereof may comprise a VhH.

[0012] Aspects relate to therapeutic antibodies that bind to epidermal growth factor-like repeats and discoidin I-like domain 3 (EDIL3) protein, comprising a variable domain and a constant domain. In embodiments, the constant domain is an IgG, and the variable domain comprises framework regions and complementarity determinants for binding to epidermal growth factor-like repeats and discoidin I-like domain 3 (EDIL3) protein. In embodiments, the constant region of the therapeutic antibody is an IgG1. In embodiments, the therapeutic antibody may comprise any one of the antibodies set forth in Tables 1-4.

[0013] Aspects relate to pharmaceutical compositions that can include any of the antibodies described herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0014] Aspects relate to methods of treating cancer in a subject, which may include administering to a subject in need thereof an effective amount of any of the antibodies described herein or the pharmaceutical compositions described herein. In embodiments, the method of treating cancer may include administering to the subject an angiogenesis inhibitor, a checkpoint blockade inhibitor, or a combination thereof. In embodiments, the angiogenesis inhibitor comprises bevacizumab. In embodiments, the checkpoint blockade inhibitor comprises ipilimumab.

[0015] Aspects relate to the use of any of the antibodies described herein or any of the pharmaceutical compositions described herein to treat cancer. In some embodiments, any of the antibodies described herein or any of the pharmaceutical compositions described herein can be used to treat cancer.

[0016] Aspects relate to methods of reducing the immunosuppressive effect of cancer-associated fibroblasts in a subject, which can include administering to the subject an effective amount of any of the antibodies described herein, or any of the pharmaceutical compositions described herein.

[0017] Aspects relate to the use of any of the antibodies described herein, or any of the pharmaceutical compositions described herein, to reduce the immunosuppressive effects of cancer-associated fibroblasts.

[0018] Aspects relate to any of the antibodies described herein, or any of the pharmaceutical compositions described herein, for use in reducing the immunosuppressive effects of cancer-associated fibroblasts.

[0019] Aspects relate to nucleic acids encoding any of the antibodies described herein. In embodiments, the nucleic acid sequence encoding HCDR1 is AGCTATGCCATGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GCTATTAGTGATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is GAGGGTTTGATTACGTTTGGGGGAGTTATCGTTATAGGCTACTTTGACTAC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is CAGGCGAGTCAGGACATTAGCAACTATTTAAAT or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATGCATCCAATTTGGAAACA or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAACAGTATGATAATCTCCCGATCACC or a degenerate variant thereof. In embodiments, the nucleic acid sequence encoding HCDR1 is AGCTATTGGATGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is AATATAAAGCAAGATGGAAGTCAGAAATACTATGTGGACTCTGTGAAGGGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is CGTGGGAACTTCTTCTTTGACAAT or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGTATGTTAGCAGCTACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATGCATCCAACAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGCGTAACAACTGGCCTCCGACG or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding HCDR1 is AATCACTACTGGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is TATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGT or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is GGGTTTGCTTAC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is CGGGCGAGTCAGGGCATTACCAATTATTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GCTGCATCCACTTTGCAATCA or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAAAAGTATAACAGTGCCCCGTGGACG or a degenerate variant thereof. In embodiments, the nucleic acid sequence encoding HCDR1 is AGCTATGCCATGAAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GCTATCAGTGGCAGTGGTGATAGCACATACTCCACAGACTCCGTGAAGGGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is GAGTATTACGATATTTTGACTGGTTATTGGGACTGGTACTTCGATCTC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTATTAACAGCAACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GGTGCATCCACCAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGTATAATAACTGGCCGCTCACT or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding HCDR1 is AGCTATGGCATGCAT or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GTTATGGGTATGATGGAAGTGATAGATACTCTGCAGACTCCGTGAAGGGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is GGGTACGATATTTTGACTGGTCCCGACCACTTTGACTAC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is CGGGCAAGTCAGAGCATTAGCAGTTATTTAAAT or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GCTGCATCCAGTTTGCAAAGT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAACAGAGTTACAGTACCCCGCTCACT or a degenerate variant thereof. In embodiments, the nucleic acid sequence encoding HCDR1 is AGCTATGACATGAAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is ACTATTAGTGGTAGTGGTAGTCACACATACTACGCAGACTCCGTGAGGGGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is GAGGGGGGAGCTACTGCTTTTGATATC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is CGGGCCAGTCAGGGCATTAGCAGTTATTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GTTGCATCCACTTTGCAAAGT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAACAGCTTAATAATTACCCCACT or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding HCDR1 is ACCTATGGCATGCAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is CTTATATGGTATGATGGAATTAATAAATACTATGCGGACTCCGTGAAGGGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is CCCTATTACGATATTTTGACTGGTTATTTTGACTAC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTGATAGCAGCAGCTACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GGTACATCCAGTAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGTATGGTAGCTCACCGCTCACT or a degenerate variant thereof. In embodiments, the nucleic acid sequence encoding HCDR1 is AGCTATGGCATGCAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GTTATATGGTATGATGGAACTAATAAATACTATGCAGACTCCGTGAAGGGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is GATCCCTCCTTATGGTTCGGGGAGTTCCCTCATTACTACGGTATGGACGTC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is CAGGCGAGTCAGGACATTAGCAATTATTTAAAT or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATGCATCCAATTTGGAAACA or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAACAGTATGATAATCTCCCGCTCACT or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding HCDR1 is GGTTACTACTGGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GAAATCCAACATAGTGGAAGCACCAACTACAAACCGTCCCTCAAGAGT or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is CTAACTGGGGATTCCCTTTTGTTTGAGTAC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is AGGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATACATCCAACAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGCGTAGCAACTGGCCGATCACC or a degenerate variant thereof. In embodiments, the nucleic acid sequence encoding HCDR1 is AGCTATGGCATGCAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is GATAGCGCCTCGACTACTTTGACTAC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GGTGCATCCACCAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGTATAGTGACTGGCCCACT or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding HCDR1 is GGTTACTACTGGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GAAATCAATCATAGTGGAAGCACCAACTACAAACCGTCCCTCAAGAGT or a degenerate variant thereof, the nucleic acid sequence encoding HCDR3 is CTAACTGGGGATTCCCTTTTGTTTGAGTAC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATACATCCAACAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGCGTAGCAACTGGCCGATCACC or a degenerate variant thereof.

[0020] In embodiments, the nucleic acid sequence encoding an LCVR is GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA or a degenerate variant thereof, and the nucleic acid sequence encoding an HCVR is GAGGTGCAGC TGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGT TCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCAGGAGGGTTTGATTACGTTTGGGGGAGTTATCGTTATAGGCTACTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding the LCVR is GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGTATGTTAGCAGCTACTTAGCCTGGTACCACCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAACAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA or a degenerate variant thereof, and the nucleic acid sequence encoding the HCVR is The acid sequence is GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGTGGCCAATATAAAGCAAGATGGAAGTCAGAAATACTATG TGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTACGAGACGTGGGAACTTCTTCTTTGACAATTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding the LCVR is GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTACCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA, or a degenerate variant thereof; The nucleic acid sequence encoding is CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACTTGCACTGTCTCTGGTGGCTCCATCAGTAATCACTACTGGAGCTGGATTCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGGGGGTTTGCTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCTTCA or a degenerate variant thereof.

[0023] In embodiments, the nucleic acid sequence encoding the LCVR is GAAATAGTGATGACGCTGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAACAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof, and the nucleic acid sequence encoding the HCVR is GAGGTGCA GCTGTTGGAATCTGGGGGAGGCTTGGGACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCAGCTATCAGTGGCAGTGGTGATAGCACATACTCCACAGACTCCGTGAAGGGCC GGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAAAGAGTATTACGATATTTTGACTGGTTATTGGGACTGGTACTTCGATCTCTGGGGCCAAGGCACCCTGGTCACTGTCTCCTCA or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding an LCVR is GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGAGTTACAGTACCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof, and the nucleic acid sequence encoding an HCVR is CAGG TGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCATTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTGGTATGATGGAAGTGATAGATACTCTGCAGACTCCGTGA AGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATTTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGGGTACGATATTTTGACTGGTCCCGACCACTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding an LCVR is GACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGTTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATAATTACCCCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof; and the nucleic acid sequence encoding an HCVR is GAAGTACAGTTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCCGGTGTGGGTCTCAACTATTAGTGGTAGTGGTAGTCACACATACTACGCAG ACTCCGTGAGGGGCCGGTTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGGGAGCTACTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding LCVR is GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGATAGCAGCAGCTACTTAGCCTGGTATCAGCAGAAACCTGGCCAGGCTCC CAGGCTCCTCATATATGGTACATCCAGTAGGGCCACTGGCATCTCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCT CACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof, and the nucleic acid sequence encoding HCVR is CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATGGTATGATGGAATTAATAAATACTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA. ATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGACCCTATTACGATATTTTGACTGGTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof. In embodiments, the nucleic acid sequence encoding the LCVR is GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAATTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof, and the nucleic acid sequence encoding the HCVR is CAGGTGCAGC TGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAACTAATAAATACTATGCAGACTCCGTGAAGGGCCGAT TCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAGTGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCCCTCCTTATGGTTCGGGAGTTCCCTCATTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding an LCVR is GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATACATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCGTCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding an HCVR is GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGAAAGAGCCACCCTCCTGCAGGGCCAGAGTGTTAGCAGCTACTGGAGATTAAA, or a degenerate variant thereof. The column is CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATACGCCAGCCCCAGGAAGGGGCTGGAGTGGATTGGGGAAATCCAACATAGTGGAAGCACCAACTACAAACCGT CCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGGAGCTAACTGGGGATTCCCTTTTTGTTTGAGTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding the LCVR is GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGATTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAGTGACTGGCCCACTTTCGGCGGAGGGACCAAGGTGGAGATCAGA or a degenerate variant thereof; and the nucleic acid sequence encoding the HCVR is CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAATAAATACTATGCAG ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACACTTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGCGCTCCGACTACTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof.In embodiments, the nucleic acid sequence encoding an LCVR is GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATACATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCGTCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding an HCVR is GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGAAAGAGCCACCCTCCTGCAGGGCCAGAGTGTTAGCAGCTACTGGAGATTAAA, or a degenerate variant thereof. The column is CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATACGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAAACCGT CCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGGAGCTAACTGGGGATTCCCTTTTTGTTTGAGTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof.

[0021] Aspects relate to vectors comprising the nucleic acids described herein.

[0022] Furthermore, aspects relate to cells comprising the nucleic acids or vectors described herein.

[0023] Aspects relate to cells that produce the monoclonal antibodies described herein.

[0024] Aspects relate to methods for treating cancer. In embodiments, the methods may include administering a pharmaceutically effective amount of one or more EDIL3 inhibitors to a subject suffering from cancer. In embodiments, the methods for treating cancer may result in the reversal of an immune-negative tumor microenvironment. In embodiments, the methods for treating cancer may result in the reversal of an immune-dysfunctional tumor environment. In embodiments, the methods for treating cancer may reduce the immunosuppressive effect of cancer-associated fibroblasts (CAFs) in a subject suffering from cancer. In some embodiments, the methods for treating cancer may counteract the effect of EDIL3-dependent blockade of LFA-1 binding to endothelial cells. In some embodiments, the EDIL3 inhibitor is an antibody that binds to EDIL3 protein, or an antigen-binding fragment or variant thereof.

[0025] In some embodiments, the method of treating cancer may include one or more additional therapies for treating cancer. In some embodiments, the additional therapies relate to angiogenesis and / or checkpoint blockade. In some embodiments, the method of treating cancer reduces the immunosuppressive effect of cancer-associated fibroblasts (CAFs) in a subject. In some embodiments, the method of treating cancer may counteract the effects of EDIL3-dependent blockade of LFA-1 binding to ICAM-1. In some embodiments, the method of treating cancer may counteract the effects of EDIL3-dependent blockade of LFA-1 binding to endothelial cells. In some embodiments, the EDIL3 inhibitor is an antibody or antigen-binding fragment or variant thereof that specifically binds to EDIL3 protein. In some embodiments, the method of treating cancer may include one or more additional therapies for treating cancer. In some embodiments, the additional therapy for treating cancer is a therapy that can modify angiogenesis. In some embodiments, the additional therapy for treating cancer is a therapy that can modify checkpoint blockade. [Brief explanation of the drawings]

[0026] [Figure 1-1]This shows that humoral immune responses elicited against EDIL3 by ipilimumab plus bevacizumab (Ipi-Bev) were associated with clinical outcomes in patients with metastatic melanoma. Panel a: Fold change in EDIL3 antibodies measured by ELISA in pre- and post-treatment plasma samples from 42 Ipi-Bev patients. Each bar represents a patient, and bar color indicates clinical assessment (CR, complete response (green); PR, partial response (green); SD, unchanged (blue); and PD, progressive disease (red)). EDIL3 antibody titers were considered significant when their fold change was 1.5 or greater. Panel b: The frequency of EDIL3 antibodies increases with clinical response. [Figure 1-2] The humoral immune response elicited against EDIL3 by ipilimumab plus bevacizumab (Ipi-Bev) was associated with clinical outcomes in patients with metastatic melanoma. Panel c, Immunoblot analysis of EDIL3 Ig expression in pre- and post-treatment plasma samples from a representative patient. Panel d, Kaplan-Meier survival curves for patients based on EDIL3 antibody fold change of ≥1.5 or ≤1.5 (P=0.027). Median survival was 70 weeks (95% CI, 47-81) for patients with an EDIL3 antibody fold change <1.5 but was not reached for patients with a fold change of ≥1.5. Panel e, Percentage of patients with an EDIL3 antibody fold change >1.5 in patient cohorts treated with Ipi-Bev (n=42), ipilimumab (n=34), PD-1 blockade (n=25), and nivolumab-ipilimumab (n=41). [Figure 2] TIDE analysis shows that EDIL3 expression is associated with tumor T cell immune exclusion gene signature. Panel a, Volcano plot of EDIL3, MFGE8, and CTNNB1 for dysfunction versus exclusion correlation across TCGA tumor types. Panel b, EDIL3, MFGE8, and CTNNB1 expression and predictive value of T cell exclusion versus immunotherapy response in the TCGA cutaneous melanoma (SKCM) dataset. [Figure 3]We show that EDIL3-mediated T cell elimination is associated with TGFβ signaling, EMT, and angiogenesis signatures. Cancer-associated fibroblast (CAF) FAP signature scores correlated with EDIL3 and MFGE8 expression levels in T cell elimination determined using the TIDE method (heatmap data not shown). [Figure 4] EDIL3 is abundantly expressed in CAFs and is upregulated by TGF-β1-induced EMT. Panel a: ELISA detection of EDIL3 secreted into conditioned medium from normal fibroblasts (NFs) and patient-derived CAFs (P4-CAFs and CAF2). Panels b-c: NFs pretreated with or without LY2109761 (LY) followed by 24 h of TGF-β1 treatment. EDIL3 expression was examined by ELISA of conditioned medium (panel b) and immunoblot analysis of whole-cell lysates (panel c). Panel d: Quantitative RT-PCR analysis of EDIL3 silencing in CAFs and TGF-β1-mediated induction of EDIL3 and target genes (panel e: transgelin (TAGLN) and panel f: α-SMA (ACTA2)) in control versus EDIL3 siRNA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Mean ± SD of three independent experiments is shown. [Figure 5-1] Figure 1 shows that EDIL3 expression correlates with serum VEGF levels and angiogenic signatures in the TCGA SKCM and Checkmate064 databases. Panel a: Correlation of pre-treatment circulating serum levels of EDIL3 versus VEGF-A in Ipi-Bev-treated melanoma patients (n=42). Panel b: EDIL3 expression is significantly correlated with high angiogenic signatures in the TCGA SKCM and Checkmate064 datasets. [Figure 5-2] EDIL3 expression correlates with serum VEGF levels and angiogenic signatures in the TCGA SKCM and Checkmate064 databases. Panel c, EDIL3 promoted the tube-forming ability of patient-derived endothelial cells comparable to VEGF, as assessed by angiogenesis assays. Mean ± SD of three independent experiments is shown. [Figure 6] EDIL3 blocks lymphocyte endothelial adhesion and inhibits T cell migration. Panel a: Immune cells were screened by flow cytometry for expression of the ICAM-1 ligand LFA-1. Panels b-c: Confluent monolayers of patient-derived endothelial cells were left unstimulated or stimulated with TNF-α for 24 hours, then incubated for 45 minutes with THP-1 cells or activated leukocytes pretreated with human recombinant EDIL3 or / and anti-LFA-1 antibody for 1 hour and allowed to adhere at 37°C. (b) EDIL3 dose-dependently inhibits THP-1 cell adhesion to TNFα-activated endothelial cells. Panel c: EDIL3 blocks lymphocyte-endothelial adhesion by disrupting the interaction between LFA-1 and ICAM-1. Panel d: EDIL3-silenced patient-derived endothelial cells were grown in transwells and incubated with T cells in the lower chamber in the presence or absence of the chemoattractant IP-10. Migration across the endothelial monolayer was measured 2–4 hours later. Silencing of EDIL3 in endothelial cells enhanced transwell transendothelial migration of T cells. Panel e: Patient-derived endothelial cells were cultured on transwells and incubated with untreated or EDIL3-pretreated T cells in the lower chamber in the presence or absence of IP-10. Migration across the endothelial monolayer was measured 2–4 hours later. rEDIL3 partially blocked transwell transendothelial migration of T cells. [Figure 7] (Panel a) Pre-treated T cells with EDIL3 and addition of rEDIL3 to the culture medium reduced overall IP-10-induced migration of T cells (p<0.01), and (Panel b) EDIL3 pre-treatment significantly (p<0.05) down-regulated T cell migration across the vascular network. [Figure 8]Humoral responses induced to Ipi-Bev therapy in patients with metastatic melanoma. Panel a, Frequency of MFGE8 antibodies by clinical response (CR, complete response; PR, partial response; PD, progressive disease; SD, unchanged). Panel b, Kaplan-Meier survival analysis of patients based on MFGE8 antibody fold change ≥1.5 or ≤1.5 (P=0.61). Panel c, EDIL3 antibody titers in pre- and post-treatment plasma samples of patients with improved clinical outcomes. [Figure 9] Ipi-Bev therapy altered IgG titers for EDIL3 and MFGE8 antibody responses. Panel a, Pre- and Post-treatment Ig titers in patients with EDIL3 (n=42) and panel b, MFGE8 (n=39). Pre- and post-treatment Ig titers for Ipi-Bev therapy in patients stratified by clinical response: panel c, EDIL3 (CR / PR (n=22), PD (n=13), and SD (n=7)) and panel d, MFGE8 (CR / PR (n=6), PD (n=9), and SD (n=18)). [Figure 10] Figure 1 shows a longitudinal analysis of EDIL3 antibody responses to Ipi-Bev treatment in patients with metastatic melanoma. Optical density (OD) obtained by ELISA of EDIL3 antibody titers for representative clinical responses was plotted against time from the start of Ipi-Bev treatment in patients. Anti-EDIL3 responses over time are shown for panel a P6; panel b P13; panel c P12; panel d P21; panel e P17; and panel f P26. CR, complete response; PR, partial response; PD, disease progression; and SD, unchanged. [Figure 11] TPM (transcripts per million) based on RNA sequencing are shown. Panel a: Overexpression of EDIL3 in CAFs (cancer-associated fibroblasts) versus NFs (normal fibroblasts) and its induction by TGFβ1. Panel b: Overexpression of MFGE8 in CAFs versus NFs (unaltered by TGFβ1). Panels c-d: CAFs showing positive expression of surface markers FAP and LRRC15 under TGFβ1 control. [Figure 12]Molecular validation data are shown. Panel a: Relative expression of EDIL3 in endothelial cells (HDMEC, TEC (tumor endothelial cells), and HUVEC). Panel b: Validation of TNFα-mediated activation of HUVEC and TEC by induction of ICAM1 expression. Panel c: Silencing of EDIL3 in TEC using siRNA. Panel d: Representative image and quantification results of a binding assay showing that activated THP-1 cells bind EDIL3 with higher affinity than ICAM1. Panel e: Representative image of the adhesion assay in Figure 6, panel c. [Figure 13] (Panel a) Western blot and (Panels b and c) graphs showing that combination therapy patients exhibited antibody responses to EDIL3 in response to treatment. [Figure 14] MFGE8-associated clinical outcomes are shown. Panel A shows a graph of MFGE8 Ig frequency by outcome. Panel B shows a graph of overall survival. [Figure 15] Figure 1 shows EDIL3 and MFG-E8 in melanoma and melanoma cell lines. Panel A shows data for 064. Panel B shows data for melanoma cell lines. Panel C shows Western blot. [Figure 16] Panels A and B show graphs of EDIL3, and panel C shows a Western blot. [Figure 17] The expression levels of CAFs are shown. [Figure 18] The relative mRNA expression levels of EDIL3 (Panel A), ACTA2 (Panel B), and TGLN (Panel C) are shown. [Figure 19] Relative mRNA expression data are shown. [Figure 20-1]

[0039] Figure 1 shows the binding activity of antibodies PR305734, PR305667, PR305633, PR305618, PR305754-p, PR305764, PR305689, PR305684, PR305629, and PR305623 to human EDIL3 protein, cynomolgus monkey EDIL3 protein, and mouse EDIL3 protein. Panel A shows the results of testing antibody binding to EDIL3 at the protein level. Panels B to D show the binding of antibodies PR305618, PR305623, PR305629, and PR305633 to human EDIL3 protein (Panel B), cynomolgus monkey EDIL3 protein (Panel C), and mouse EDIL3 protein (Panel D). [Figure 20-2] Binding activity of antibodies PR305734, PR305667, PR305633, PR305618, PR305754-p, PR305764, PR305689, PR305684, PR305629, and PR305623 to human EDIL3 protein, cynomolgus monkey EDIL3 protein, and mouse EDIL3 protein. Panels E to G show binding of antibodies PR305667, PR305684, and PR305689 to human EDIL3 protein (Panel E), cynomolgus monkey EDIL3 protein (Panel F), and mouse EDIL3 protein (Panel G). Panels HJ show the binding of antibodies PR305734, PR305764, and PR305754-p to human EDIL3 protein (Panel H), cynomolgus monkey EDIL3 protein (Panel I), and mouse EDIL3 protein (Panel J). [Figure 21-1] The blocking activity of antibodies PR305734, PR305667, PR305633, PR305618, PR305754-p, PR305764, PR305689, PR305684, PR305629, and PR305623 is shown. Panel A shows a summary of the blocking activity of purified anti-EDIL3 antibodies. IC50 values ​​are shown in the table. Panels B and C show the blocking activity of PR305618, PR305623, PR305629, and PR305633 against the binding of human EDIL3 to its ligands, LFA-1 protein (Panel B) and αvβ3 protein (Panel C), by ELISA. [Figure 21-2] Panels D-E show the blocking activity of antibodies PR305734, PR305667, PR305633, PR305618, PR305754-p, PR305764, PR305689, PR305684, PR305629, and PR305623. Panels D-E show the blocking activity of PR305667, PR305684, and PR305689 against the binding of the ligands LFA-1 protein (Panel D) and αvβ3 protein (Panel E) to human EDIL3 by ELISA. Panels F-G show the blocking activity of PR305734, PR305754-p, and PR305764 against the binding of the ligands LFA-1 protein (Panel E) and αvβ3 protein (Panel F) to human EDIL3 by ELISA. [Figure 22-1] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-2] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-3] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-4] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-5] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-6] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-7] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-8] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-9] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 22-10] 1 shows the amino acid and nucleic acid sequences of an embodiment of the present invention. [Figure 23] Figure 1 shows that EDIL3 mAb specifically binds to human recombinant EDIL3. The predicted 65 kDa band of EDIL3 under reducing conditions. [Figure 24]1 shows the results of measuring the kinetic and thermodynamic constants of EDIL3 mAb by surface plasma resonance. [Figure 25] Blocking activity of anti-EDIL3 mAb is shown. [Figure 26-1] SPR-based kinetic characterization of anti-EDIL3 mAb shows that the anti-EDIL3 mAb exhibits nM or sub-nM affinity for EDIL3 and a slow dissociation rate (approximately 1 × 10-5 to 6 min-1). [Figure 26-2] SPR-based kinetic characterization of anti-EDIL3 mAb shows that the anti-EDIL3 mAb exhibits nM or sub-nM affinity for EDIL3 and a slow dissociation rate (approximately 1 × 10-5 to 6 min-1). [Figure 27] Figure 1 shows epitope binding analysis of anti-EDIL3 mAbs. Anti-EDIL3 mAbs bind to overlapping but distinct epitopes on EDIL3 depending on antibody display (i.e., solution vs. surface display). None of these antibodies interact with EDIL3 in their own presence (i.e., exhibiting biparatopic activity) or in the presence of a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0027] The aspects described herein can result in the reversal of immune exclusion or immune dysfunction in a tumor microenvironment. In embodiments, the present approach identifies EDIL3 as a specific target that can promote immune cell trafficking in the tumor microenvironment and reverse the immunosuppressive effects of cancer-associated fibroblasts. In embodiments, EDIL3 can be targeted in combination with antiangiogenic therapy and / or immune checkpoint blockade therapy. Without wishing to be bound by theory, a monoclonal antibody against EDIL3 has been developed and validated to reverse immune exclusion.

[0028] The present disclosure provides antibodies, or antigen-binding fragments or variants thereof, that specifically bind to epidermal growth factor-like repeat and discoidin I-like domain 3 (EDIL3) protein.

[0029] The present disclosure further provides a therapeutic antibody or antigen-binding fragment or variant thereof that specifically binds to epidermal growth factor-like repeat and discoidin I-like domain 3 (EDIL3) protein.

[0030] Furthermore, the present disclosure provides a method for treating cancer by administering an effective amount of an anti-EDIL3 antibody or an anti-EDIL3 therapeutic antibody to a subject in need of cancer treatment.

[0031] The present disclosure also provides a method for reducing the immunosuppressive effect of cancer-associated fibroblasts in a subject by administering to the subject an effective amount of an anti-EDIL3 antibody or an anti-EDIL3 therapeutic antibody.

[0032] Additionally, the present disclosure relates to nucleic acids encoding anti-EDIL3 mAbs.

[0033] The present disclosure also provides an antibody of the present disclosure for use in therapy. More specifically, the present disclosure provides an antibody of the present disclosure for use in treating cancer. Furthermore, the present disclosure provides use of an antibody of the present disclosure in the manufacture of a medicament for treating cancer.

[0034] Disclosed herein are isolated recombinant monoclonal antibodies that specifically bind to EDIL3. In some embodiments, the recombinant monoclonal antibodies can bind to recombinant human EDIL3-FC or EDIL3-His.

[0035] As used herein, "specifically binds" or "immunoreacts" can refer to an antibody that reacts with one or more antigenic determinants of EDIL3 and not with other polypeptides.

[0036] As used herein, the term "isolated," when used in reference to a cell, a protein or polypeptide, or a nucleic acid (e.g., DNA or RNA), can refer to a cell, protein or polypeptide, or nucleic acid that has been purified to some extent. The term "isolated" can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. For example, an "isolated nucleic acid" can include a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated" can also refer to a cell or polypeptide that has been separated from other cellular proteins or tissues. An isolated polypeptide can include both purified and recombinant polypeptides.

[0037] The term "recombinant" in reference to a polypeptide (such as an antibody) or polynucleotide can refer to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which is one that can be made by combining polynucleotides or polypeptides that would not normally occur together.

[0038] The EDIL3 antibodies described herein bind to EDIL3. In one embodiment, the EDIL3 antibodies have high affinity and high specificity for EDIL3. In some embodiments, the EDIL3 antibodies can block the binding of EDIL3 to LFA-1. In some embodiments, blocking the binding of EDIL3 to LFA-1 allows immune cells to bind to endothelial cells and cross over into the TME. Without wishing to be bound by theory, the antibodies described herein can counteract the EDIL3-dependent inhibitory effect of LFA-1 binding, thereby increasing the permissiveness of lymphocytes to cross the vascular endothelium and into the tumor microenvironment.

[0039] As used herein, "antibody" may refer to an immunoglobulin molecule comprising two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. The amino-terminal portion of each LC and HC comprises a variable region of approximately 100-120 amino acids that is primarily responsible for antigen recognition via the complementarity-determining regions (CDRs) contained therein. The CDRs are interspersed with regions termed framework regions (FRs), which are well-known and generally conserved between species (e.g., mouse and human). The amino acids comprising the CDRs and framework regions can be readily identified by those skilled in the art, as they have been previously defined for heavy chain variable regions or light chain variable regions, respectively.

[0040] In embodiments, the CDRs are interspersed with FRs. The antibodies disclosed herein have four FRs, designated FR1, FR2, FR3, and FR4. In embodiments, the FRs are human FRs (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3rd ed. 2018)).

[0041] The three CDRs of the light chain (LC) are referred to as "LCDR1, LCDR2, and LCDR3," and the three CDRs of the heavy chain (HC) are referred to as "HCDR1, HCDR2, and HCDR3." The functional ability of an antibody to bind to a specific antigen is primarily determined by the six CDRs. The assignment of amino acids to the CDR domains within the LCVR and HCVR regions of the antibodies of the present disclosure is based on the Kabat numbering convention.

[0042] The constant region of an antibody defines the antibody's isotype. Antibodies of the present disclosure include IgG. IgG antibodies can be further classified into subclasses, such as IgG1, IgG2, IgG3, and IgG4. In certain embodiments, an antibody of the present disclosure is an IgG1. The carboxy-terminal portion of each HC defines a constant region primarily responsible for effector function. In certain embodiments, an antibody of the present disclosure has one or more modifications in the constant region of each HC that reduce effector function.

[0043] EDIL3 binds to LFA-1 integrin, thereby preventing intracellular adhesion molecule-1 (ICAM-1) binding and thus preventing leukocyte trafficking across the endothelium. Therefore, EDIL3 contributes to carcinogenesis by reducing apoptosis of cancer cells and promoting tumor angiogenesis. Therefore, EDIL3 is a target for immunotherapy. As used herein, the term "EDIL3 antibody" can refer to an antibody that binds to EDIL3 and, without wishing to be bound by theory, interferes with EDIL3 binding to LFA-1 integrin. Antibodies within this range include the disclosed antibodies and their functional equivalents. Functionally equivalent antibodies contain different specific amino acid residues but maintain binding activity to EDIL3. Functionally equivalent antibodies may, for example, have slightly different abilities to interfere with EDIL3 binding to LFA-1 integrin and therefore have therapeutic effects.

[0044] The nucleic acid and amino acid sequences of the monoclonal EDIL3 antibody are shown below:

[0045] [Table 1]

[0046] [Table 2]

[0047] [Table 3-1] [Table 3-2] [Table 3-3]

[0048] [Table 4-1] [Table 4-2] [Table 4-3]

[0049] [Table 5]

[0050] [Table 6]

[0051] [Table 7]

[0052] [Table 8]

[0053] Further provided herein are antibody fragments, such as well-characterized Fab (e.g., Fab, Fab', F(ab'), F(ab)), Fv (light chain variable region and heavy chain variable region expressed as two chains), and single-chain fragments (e.g., single-chain variable region fragments, scFv, scFv-Fc, and single-chain Fab (scFab)), which also bind to EDIL3. Antibody fragments can include aptamers, minibodies, and diabodies. Methods for producing these fragments are conventional (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3rd ed. 2018)).

[0054] The antibodies and fragments thereof disclosed herein can also include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies.

[0055] As used herein, the phrase "complementarity determining means" describes six complementarity determining regions (CDRs) that collectively form specific interactions with EDIL3. CDRs within the complementarity determining means are the disclosed CDRs and their functional equivalents. Functionally equivalent CDRs contain different specific amino acid residues but maintain binding to EDIL3. Functionally equivalent CDRs may, for example, have slightly different abilities to interfere with EDIL3 binding to LFA-1 integrin and therefore may have therapeutic effects.

[0056] Conservative variants of the disclosed antibodies and fragments thereof are contemplated herein. Proteins are conservative variants containing conservative amino acid substitutions that do not substantially affect or reduce the affinity of the protein. For example, an antibody that binds to EDIL3 may contain at least 1, 2, 5, 10, or 15 conservative substitutions in the constant domain, for example, and still be able to bind to Edil3. Conservative amino acid substitution tables providing functionally similar amino acids are well known to those skilled in the art. The following groups are examples of amino acids that are considered conservative substitutions for each other: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0057] As used herein, the term "degenerate variant" may refer to a polynucleotide that encodes a polypeptide (such as an antibody or a fragment thereof) that includes a sequence that is degenerate based on the genetic code (i.e., each of the 20 naturally occurring amino acids can be specified by multiple codons). All degenerate nucleotide sequences that encode the disclosed antibodies and polypeptide sequences of fragments are included.

[0058] Also contemplated are variants of the disclosed antibodies and fragments thereof having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the sequences set forth in Tables 1A-4B. As used herein, "sequence identity," "sequence homology," or "sequence similarity" can refer to the similarity between amino acid or nucleic acid sequences, expressed as the similarity between the sequences. Sequence identity can frequently be measured as a percentage of identity, with two sequences being considered more similar the higher the percentage. Homologs or variants of polypeptides or nucleic acid molecules have a relatively high degree of sequence identity when aligned using well-known standard methods. (See Ceslovas Venclovas, Methods for Sequence-Structure Alignment in Homology Modeling: Methods and Protocols, 55-82 (Andrew Orry and Ruben Abagyan, eds., 2012)).

[0059] As used herein, "binding" (or "binding") can refer to the well-understood interaction between an antibody and a target protein, peptide, or polysaccharide. Binding can be measured in a variety of ways (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)). In embodiments, binding (e.g., avidity) is measured by an ELISA assay. See, e.g., Example 2. A particular antibody or protein binds to a particular target protein, peptide, or polysaccharide and does not bind in significant amounts to other proteins or polysaccharides present in a sample or subject as disclosed herein. Binding occurs between the disclosed antibodies and fragments thereof and an epitope of EDIL3. As used herein, "epitope" can refer to a discrete site on an antigen recognized by the disclosed antibodies and fragments thereof. An epitope may be linear or three-dimensional. The strength or affinity of an immunological binding interaction is measured by the equilibrium binding constant (K d ) and can be expressed in terms of smaller K d indicates a greater affinity. Antibodies have a higher K D is 10 -6 Less than molar, e.g., 10 -7 Less than 10 molar -8 Less than 10 molar -9 Less than molar, or 10 -10 It binds to the target protein when it is in submolar concentration.

[0060] The antibodies herein are monoclonal antibodies ("mAbs"). mAbs can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR or specificity-determining residues, SDR, grafting), or a combination of these or other techniques known in the art. mAbs are antibodies obtained from a single copy or clone, including, for example, a eukaryotic, prokaryotic, or phage clone. In some embodiments, EDIL3 antibodies can be generated using single B-cell cloning technology. A variety of well-known methods and tools can be used to produce and purify the mAbs disclosed herein, including vectors (e.g., plasmids, viruses, or other vehicles for insertion or expression of polynucleotides) and hosts (e.g., microbial, yeast, insect, and mammalian organisms) (see, for example, Process Scale Purification of Antibodies (Uwe Gottschalk, ed., 2nd ed. 2017)). For example, vectors for producing and purifying the mAbs disclosed herein can include DNA segments encoding the monoclonal antibodies described herein. In embodiments, the vectors can be adeno-associated viral (AAV), retroviral, lentiviral, or other vectors.

[0061] Additional antibodies with complementary binding means can be prepared and screened by well-known methods, such as hybridomas, transgenic animals, and phage or yeast display (see, e.g., Monoclonal Antibodies: Methods and Protocols (Vincent Ossipow and Nicolas Fischer, eds., 2d ed. 2014)). Antibodies with equivalent complementary binding means, although differing in their amino acid sequences, perform the same function of binding to the target via CDR-target interactions and acting as (inhibitors / agonists / antagonists) to achieve the same result (inhibition of tumor growth). Preferably, the complementary binding means functions via the same epitope as the disclosed antibodies.

[0062] In embodiments, the antibody may comprise an Fc variant comprising amino acid substitutions that alter the antigen-independent effector functions of the antibody, particularly the circulating half-life of the antibody.

[0063] In embodiments, the antibodies disclosed herein may comprise Fc variants that can reduce or eliminate glycosylation (eg, N-linked or O-linked glycosylation).

[0064] In embodiments, the antibodies disclosed herein may comprise Fc variants that have increased or decreased binding to FcRn when compared to antibodies lacking these substitutions, and may therefore have increased or decreased serum half-life, respectively.

[0065] In embodiments, the antibodies disclosed herein may comprise Fc variants in which mutations have been introduced into the constant region of the mAb to alter the antibody-dependent cellular cytotoxicity (ADCC) activity of the mAb, for example, a LALA mutation in the CH2 domain that reduces ADCC activity.

[0066] The antibodies and fragments thereof disclosed herein can be used therapeutically. In embodiments, the antibodies and fragments thereof disclosed herein can be used to treat, prevent (e.g., via prophylactic treatment), or ameliorate cancer or metastasis. In embodiments, the cancer may include melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), renal cell carcinoma (RCC), chronic lymphocytic leukemia (CLL; e.g., B-cell CLL or T-cell CLL), classical Hodgkin lymphoma (cHL), head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC), gastric cancer, hepatocellular carcinoma (HCC), primary mediastinal large B-cell lymphoma (PMLBCL), bladder cancer, urothelial carcinoma, endometrial cancer, cervical cancer, breast cancer (e.g., triple-negative breast cancer), Merkel cell carcinoma (MCC), and microsatellite instability-high (MSI-H) or DNA mismatch repair deficient (dMMR) adult and pediatric solid tumors.

[0067] In some embodiments, methods for treating cancer may involve administering a pharmaceutically effective amount of one or more EDIL3 inhibitors to a subject afflicted with cancer. In some embodiments, methods for treating cancer result in the reversal of an immunocompromised or immunodysfunctional tumor microenvironment. In embodiments, methods for treating cancer reduce the immunosuppressive effects of cancer-associated fibroblasts (CAFs) in a subject. In some embodiments, methods for treating cancer can counteract the effects of EDIL3-dependent blockade of LFA-1 binding to ICAM-1. In some embodiments, methods for treating cancer can counteract the effects of EDIL3-dependent blockade of LFA-1 binding to endothelial cells. In some embodiments, the EDIL3 inhibitor is an antibody or antigen-binding fragment or variant thereof that specifically binds to EDIL3 protein.

[0068] In some embodiments, the method for treating cancer may include one or more additional therapies for treating cancer. In some embodiments, the additional therapies for treating cancer are therapies that can modify angiogenesis. In some embodiments, the additional therapies for treating cancer are therapies that can modify checkpoint blockade.

[0069] As used herein, "preventing" a disease can refer to inhibiting the full progression of a disease, such as cancer. "Treating" can refer to a therapeutic intervention that ameliorates signs or symptoms of a disease or condition after it has begun to progress, such as reducing tumor burden or the number or size of metastases.

[0070] "Ameliorating" can refer to reducing the number or severity of signs or symptoms of a disease, such as cancer. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease, or who exhibits only early signs, for the purpose of reducing the risk of developing a pathology, such as cancer.

[0071] The antibodies and fragments thereof disclosed herein can be administered to a subject or patient. As used herein, "administration" refers to the act of a physician or caregiver prescribing a drug for administration and thereby administering the drug to a subject through ingestion, infusion, injection, or other means (self-administration or administration by a clinician or other qualified caregiver). For example, administration of a mAb disclosed herein can suppress, inhibit, or prevent the activity of EDIL3 protein.

[0072] The term "therapeutic," in conjunction with the antibodies disclosed herein, refers to antibodies suitable for use in human treatment of cancer. In embodiments, such antibodies are -6 Less than molar, e.g., 10 -7 Less than 10 molar -8 Less than 10 molar -9 Less than molar, or 10 -10 Less than molar K D and any toxic or detrimental effects of the antibody are outweighed by the beneficial effects of the treatment.

[0073] As used herein, "subject" includes both human patients and veterinary subjects, including human and non-human mammals. In embodiments, the subject or patient has or is at risk of having cancer.

[0074] Pharmaceutical compositions of the present disclosure contain an "effective" or "therapeutically effective" amount of a monoclonal antibody of the present disclosure, as used interchangeably herein. The dosage and administration regimen to achieve the desired therapeutic effect will depend on the means of administration and may vary depending on factors such as the individual's medical condition, age, sex, and weight, as well as the ability of the monoclonal antibody to elicit a desired response in the individual. The dose of an antigen-binding polypeptide described herein administered to a subject (e.g., a patient) is typically 0.1-100 mg / kg, 0.1-20 mg / kg, or 1-10 mg / kg of patient body weight. An effective amount is also an amount in which any toxic or adverse effects of the monoclonal antibody of the present disclosure are outweighed by the therapeutically beneficial effects.

[0075] Pharmaceutical compositions of the present disclosure may be formulated to be compatible with the intended route of administration, non-limiting examples of which include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.

[0076] The pharmaceutically acceptable carriers used are conventional (e.g., those described in Remington, The Science and Practice of Pharmacy, 22nd Edition, Loyd V., ed., Pharmaceutical Press, 2012). Generally, the nature of the carrier depends on the mode of administration. For example, parenteral formulations usually contain an injectable fluid containing a pharmaceutically and physiologically acceptable fluid such as water, physiological saline, or balanced salt solution as a vehicle. The pharmaceutical composition may further contain small amounts of non-toxic auxiliary substances for stability.

[0077] In embodiments, the carrier may be sterile and / or suspended, or contained in a unit dosage form containing one or more measured doses of a composition suitable for administering an effective amount of the antibodies and fragments thereof disclosed herein to a subject. Embodiments may also include pharmaceutical agents for use in therapy. In embodiments, the unit dosage form may be placed in a sealed vial containing sterile contents, or in a syringe for injection into a subject, lyophilized for subsequent solubilization and administration, or in a solid or controlled-release dose.

[0078] As used herein, an "effective amount" may refer to an amount sufficient to achieve a desired effect in a subject. For example, this may be the amount necessary to prevent, treat, or ameliorate a disease, e.g., inhibit or suppress cancer. In several embodiments, an effective amount is the amount necessary to eliminate a cancer or tumor, reduce its size, or prevent metastasis. Efficacy is first evident in a cellular response, and various in vitro and cellular assays for measuring this are well known. (Kristina V. Kitaeva et al., Cell Culture Based In Vitro Test Systems for Anticancer Drug Screening, 8 Front. Bioeng. Biotechnol. 322 (2020)). In embodiments, an effective amount is the amount necessary to significantly inhibit or reduce cancer cell proliferation or migration, invasion, or adhesion. The cellular response is manifested as a significant reduction in tumor size, a reduction or suppression of disease progression, and improved survival in the subject. More specifically, an effective dose improves the key cancer endpoints of overall survival (OS), disease-free survival (DFS), response rate, complete response rate, or progression-free survival (PFS). See Dept. of Health and Human Services, Food and Drug Administration, Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologics: Guidance for Industry (2018); E.A. Eisenhauer et al., New Response Evaluation Criteria in Solid Tumors: Revised RECIST Guideline (Version 1.1), 45 Eur. J. Cancer 228 (2009).

[0079] The mAbs disclosed herein can be used in conventional methods for localizing and / or quantifying EDIL3 protein (e.g., for use in measuring the level of EDIL3 protein in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging, etc.).

[0080] The mAbs disclosed herein can be used in methods for isolating EDIL3 polypeptides by conventional techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies to EDIL3 protein (or fragments thereof) can be used diagnostically to monitor protein levels in tissues as part of clinical testing procedures (e.g., to determine the effectiveness of a given treatment regimen).

[0081] Cancer-associated fibroblasts (CAFs) are a group of activated fibroblasts with remarkable heterogeneity and plasticity in the tumor microenvironment, secreting various factors that control tumor initiation, progression, metastasis, and therapeutic resistance. Accordingly, one aspect relates to a method for reducing the immunosuppressive effect of cancer-associated fibroblasts (CAFs) in a subject. Reducing, reversing, or modulating immunosuppression can refer to altering, preventing, or reducing the immunosuppressive properties of cancer-associated fibroblasts. In embodiments, the immunosuppressive effect of CAFs can be reduced by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in a mammal compared to a control sample.

[0082] For example, the antibody is an EDIL3 antibody. In an embodiment, the method further comprises measuring the serum level of EDIL3. In an embodiment, the decrease in immunosuppressive CAF activity can be measured through changes in immune infiltration or cytokines in the tumor microenvironment.

[0083] Aspects relate to methods for identifying subjects who are non-responsive to immune checkpoint blockade therapy. For example, embodiments include detecting EDIL3 expression or protein levels in a sample obtained from the subject. "Detecting" may refer to quantitative, semi-quantitative, qualitative, or other methods for measuring an analyte (e.g., EDIL3) in a sample. For example, detecting may include traditional methods, such as immunohistochemistry of the tumor and / or tumor microenvironment, measuring levels of EDIL3 (e.g., circulating EDIL3), and / or measuring levels of EDIL3 antibodies. In embodiments, elevated EDIL3 expression or protein levels may indicate that the subject is non-responsive to immune checkpoint blockade therapy.

[0084] In embodiments, EDIL3 expression or protein levels can be compared to a control sample, and if altered compared to the control sample, the subject can be identified as non-responsive to immune checkpoint blockade therapy or responsive to immune checkpoint blockade therapy. As used herein, a sample or subject "altered compared to a control" means that the detected level of an analyte or diagnostic or therapeutic indicator (e.g., a marker) is statistically different from that of a normal, untreated sample, or a control sample of an abnormal condition. Determining statistical significance, e.g., the number of standard deviations from the mean that constitute a positive or negative result, is within the capabilities of one of ordinary skill in the art.

[0085] The mAbs of the present disclosure can also be used in combination therapy. In embodiments, a subject is treated with the mAbs disclosed herein in combination with one or more additional therapies for treating cancer, such as radiation therapy, surgery, bone marrow transplantation, chemotherapy, immunotherapy, hormonal therapy, or targeted therapy. It is preferred to use the mAbs of the present disclosure in combination with chemotherapy or immunotherapy. Most preferably, the additional therapy is related to angiogenesis and / or checkpoint blockade. Thus, an aspect of the present invention relates to administering the mAbs disclosed herein in combination with antiangiogenic drugs and / or immune checkpoint blockade therapy.

[0086] As used herein, "combination" therapy or combination refers to the administration of a mAb of the present disclosure to a patient in conjunction with (i.e., before, simultaneously with, or after) any number of related treatments, including, but not limited to, agents such as angiogenesis inhibitors and / or immune checkpoint inhibitors.

[0087] "Angiogenesis" can refer to the formation and spreading of blood vessels. For example, "angiogenesis" can refer to a process involving tissue vascularization, such as proliferation, migration, and invasion of vascular endothelial cells, and the development of new capillaries.

[0088] "Angiogenesis inhibitors" or "anti-angiogenic agents" may refer to substances that inhibit the growth of new blood vessels. In embodiments, angiogenesis inhibitors may include cytokines, EDIL-3 antibodies, VEGF inhibitors, tyrosine kinase inhibitors, or combinations thereof. For example, VEGF inhibitors include VEGF antibodies. For example, angiogenesis inhibitors include axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, regorafenib, sofafenib, sunitinib, thalidomide, vandetanib, and ziv-aflibercept.

[0089] Without wishing to be bound by theory, administration of angiogenesis inhibitors can synergistically enhance a patient's response to immunoblockade therapy.

[0090] The term "immune checkpoint inhibitor" or "immune checkpoint blockade therapy" can refer to any compound that inhibits the function of an immune inhibitory checkpoint protein. Inhibition includes reduction and complete blockade of function. In embodiments, an immune checkpoint inhibitor can be an antibody that specifically recognizes an immune checkpoint protein. In embodiments, immune checkpoint inhibitors can include peptides, antibodies, nucleic acid molecules, and small molecules.

[0091] In embodiments, the checkpoint blockade therapy may include a CTLA4 antibody, a PD-L1 antibody, a PD-1 antibody, a LAG-3 antibody, or a combination thereof. For example, the CTLA4 antibody may include ipilimumab, tremelimumab, or a combination thereof. For example, the PD-L1 antibody may include atezolizumab, avelumab, davelumab, or a combination thereof. For example, the PD-1 antibody may include pembrolizumab, nivolumab, cemiplimab, or a combination thereof. For example, the LAG-3 antibody may include leratolimab.

[0092] equivalent

[0093] Various aspects of the present disclosure may be used alone, in combination, or in various arrangements not specifically described in the above embodiments, and therefore, its application is not limited to the details and arrangements of components set forth in the above description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments. [Example]

[0094] To facilitate a more complete understanding, the following examples are provided. The following examples set forth exemplary modes of making and practicing the present invention. However, these examples are for illustrative purposes only, and the scope is not limited to the specific embodiments disclosed in these examples, as alternative methods may be utilized to achieve similar results.

[0095] Example 1

[0096] EDIL3 as an angiogenic target for immune exclusion

[0097] overview

[0098] Immune checkpoint blockade (ICB) has become the standard of care for many solid tumors. Multiple combination approaches are being investigated to improve these clinical outcomes. The combination of antiangiogenic and immune checkpoint blockade has shown efficacy in several cancers. To better understand the mechanisms underlying these therapies' synergistic effects, we performed a human protein array serological screen using serum from patients with long-term responses treated with ipilimumab and bevacizumab (Ipi-Bev). High-titer antibody responses against EDIL3 were identified, which correlated with favorable clinical outcomes. EDIL3 is a secreted extracellular matrix protein involved in carcinogenesis and has been identified as a marker of poor prognosis in various malignancies. EDIL3 may be associated with an immune exclusion signature for cytotoxic immune cell infiltration and non-responsiveness to ICB by Tumor Immune Dysfunction and Exclusion (TIDE) analysis. Among cells involved in immune exclusion, cancer-associated fibroblasts (CAFs) may be a major source of EDIL3. Furthermore, analysis of TCGA and Checkmate 064 expression revealed that high levels of EDIL3 correlated with increased TGF-β signaling, enrichment of angiogenic signatures, and induction of EMT in fibroblasts. EDIL3 overexpression and TGF-β1 regulation were validated in patient-derived CAFs. Silencing EDIL3 in CAFs disrupted TGF-β1-induced EMT. Circulating EDIL3 levels correlated with VEGF in patient serum samples. Similar to VEGF, EDIL3 promoted the migration and tube-forming ability of patient-derived tumor endothelial cells (TECs). Mechanistically, 3-D microfluidic culture and 2D migration assays using TECs supported EDIL3-mediated disruption of LFA-1 and ICAM-1 interactions as a means of T cell elimination. EDIL3 could be targeted to improve immune cell endothelial migration and the efficacy of ICB therapy.

[0099] preface

[0100] Immune checkpoint blockade has become the standard of care for many malignancies, including melanoma, benefiting approximately 50% of patients with metastatic disease. Combination approaches to improve outcomes and overcome treatment resistance are needed for many patient populations with unmet need. The immune system is known to actively interact with the vasculature as it is the gateway to the tumor microenvironment. Angiogenic factors exhibit immunosuppressive capabilities, and tumor vasculature can limit immune effector cell access to the tumor microenvironment. Innovative strategies involving the combination of immune checkpoint blockade (ICB) and antiangiogenic therapy have demonstrated significant efficacy in many tumor types, including renal cell carcinoma, hepatocellular carcinoma, endometrial carcinoma, non-small cell lung cancer, and melanoma. Correlations have revealed that VEGF blockade is associated with improved lymphocyte trafficking across the endothelium and improved outcomes when tumors contain a myeloid expression signature.

[0101] Treatment with ipilimumab and bevacizumab induced humoral immunity against the vascular antigen targets Gal-1 (galectin-1) (1), Gal-3 (galectin-3) (1,2), and ANGPT2 (angiopoietin 2) (3), which was associated with favorable outcomes. To better understand the immunological role of the combination therapy, we screened a human protein array using serum from patients who achieved long-term responses.

[0102] Materials and Methods

[0103] Blood collection and serum from melanoma patients

[0104] The phase I Ipi-Bev trial conducted in patients with advanced melanoma has been previously reported (1, 2). Briefly, heparinized peripheral blood samples were collected from melanoma patients participating in an IRB-approved protocol at Dana-Farber / Harvard Cancer Center. Blood samples were processed by standard gradient centrifugation on the same day using Ficoll-Paque Plus (GE Healthcare Biosciences). The upper phase was then transferred to 1.5 mL tubes and centrifuged at 12,000 g for 10 min at 4°C. The resulting plasma was aliquoted and stored at -80°C until use. The humoral response study was primarily based on four cohorts of patients with advanced melanoma treated with i) ipilimumab alone, ii) ipilimumab and bevacizumab, iii) anti-PD-1 alone, or iv) nivolumab and ipilimumab (Nivo+Ipi) either as standard of care or through participation in an Ipi-Bev clinical trial.

[0105] ELISA experiments

[0106] To detect antibodies in plasma, ELISA was performed as previously described (1). Briefly, 96-well high-binding microtiter plates (Corning) were coated overnight with 100 ng of purified human recombinant proteins (rhEDIL3 and rhMFGE8 in 50 μl of TBS (Tris-buffered saline)). His-tag and no plasma were used as negative controls. After washing three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST), the plates were blocked with blocking buffer (Pierce Protein-Free Blocking Buffer) for 2 h at room temperature. Plasma samples (diluted 1:500–1:1000 in blocking buffer) were incubated for 1.5 h at 4°C. After washing, HRP-conjugated anti-human IgG (Jackson Laboratories) (diluted 1:4000 in 1% BSA (bovine serum albumin)-PBST) was added for 1 h at room temperature. To increase the sensitivity of the immunoassay, the ELAST amplification system (Perkin-Elmer) was used. Signal was developed for 5 minutes using TMB (3,3',5,5'-tetramethylbenzidine, Sigma) substrate. The reaction was stopped with 1N HCl, and absorbance was measured at 450 nm and 570 nm. Antibody titers were calculated by subtracting the OD (optical density) at 570 nm from the OD at 450 nm and subtracting the "His Tag" and "No Plasma" backgrounds from the EDIL3 / MFGE8 readings. Bars represent the average absorbance obtained from duplicate samples.

[0107] Measurement of circulating EDIL3

[0108] Secreted EDIL3 levels in patient plasma were measured in duplicate using a human EDIL3 ELISA kit (R&D Systems) according to the manufacturer's instructions. A standard curve was generated for each assay.

[0109] Immunoblotting

[0110] To detect EDIL3 antibodies in plasma, equal amounts of human recombinant EDIL3 protein denatured in 2x SDS-containing Laemmli buffer were separated by SDS-PAGE and transferred to a PVDF membrane. Each lane of the membrane was cut and immunoblotted with pre- and post-treatment plasma samples in separate containers. Plasma samples were diluted 1:500 in 1% BSA in TBS containing 0.1% Tween-20 (TBST). Proteins were visualized using an ECL kit (Perkin Elmer) and digitized using an ImageQuant LAS-4000. The membrane was stripped and reprobed with a commercially available EDIL3 antibody (Abcam) for loading controls.

[0111] Bioinformatics Data Analysis

[0112] EDIL3 and MFGE8 were targets identified from a protein array screen using plasma samples from melanoma patients (1). The immunologically important glycoproteins EDIL3 and MFGE8 were subjected to computational signature analysis of T cell dysfunction and T cell exclusion using the TIDE (Tumor Immune Dysfunction and Exclusion) assay (3). CTNNB1 (β-catenin) was included as a reference marker of immune exclusion (4).

[0113] We accessed the Cancer Genome Atlas (TCGA) and BMS CheckMate 064 datasets to analyze gene expression grouped by TGFβ, EMT, and angiogenesis signatures (PMID: 29443960). Rows in the heatmap indicate the expression (z-score) of the gene of interest. Melanoma cases from TCGA [PMID: 26091043] (n=469) and BMS-064 [PMID: 30021886] pretreated patients (n=90) who underwent RNA sequencing are shown ordered by EDIL3 expression from lowest (left) to highest (right). Gene expression data were log2 transformed. Pathway expression was calculated using the GSVA [PMID: 23323831] tool for TGF-β (TGF-beta), pan-fibroblast TGF-β response signature (F-TBRS), EMT, and angiogenesis (Angio) gene sets described by Mariathasan et al. (5).

[0114] cell line

[0115] Primary TECs were isolated from melanoma patients enrolled in the Ipi-Bev trial according to an institutional review board-approved protocol. Human umbilical vascular endothelial cells (HUVECs) were purchased from ATCC. Endothelial cells were maintained in endothelial basal medium (EBM-2) (PromoCell, Heidelberg, Germany) and used within 3–4 passages after thawing. CAFs were isolated from tumor samples (6). Normal fibroblasts (NF) were purchased from ATCC. CAFs and NFs were maintained in DMEM medium (Gibco-BRL) containing 10% FBS and 1% antibiotics.

[0116] Peripheral blood mononuclear cells (PBMCs) were isolated from normal donor blood samples using Ficoll density gradient separation according to an IRB-approved protocol. T cells were isolated from PBMCs using a Pan T Cell Isolation Kit (Miltenyi Biotech). T cell activation was achieved by adding human monoclonal anti-CD3 (Biolegend) and anti-CD28 (Biolegend) antibodies to RPMI medium supplemented with 10% FBS, 50 μg / ml penicillin, 100 μg / ml streptomycin, and 30 U / ml IL-2, as directed by the manufacturer (Life Technologies).

[0117] RNA extraction and real-time quantitative PCR (qPCR)

[0118] RNA was extracted using the RNeasy Mini Kit (Qiagen) and reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions. Gene expression levels were analyzed by qPCR using primer EDIL3(h)-PR (sc-91971-PR). The standard ABI7500 program was used: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. β-actin was used as a reference.

[0119] EDIL3 silencing

[0120] CAFs or TECs were seeded into 6-well plates at 80% confluency one day before transfection. Cells were transfected in MEM (Corning) with either control siRNA-A (sc-37007) or siEDIL3 (sc-91971) using Lipofectamine™ RNAiMAX Transfection Reagent (Thermofisher Scientific) according to the manufacturer's instructions. After 6 hours, the medium was changed to the cells' default growth medium and cultured overnight. EDIL3 silencing was confirmed by qRT-PCR or immunoblot analysis, as described.

[0121] Wound healing assay

[0122] Wound healing assays were performed as previously described (9). Briefly, TECs were seeded onto fibronectin-coated 12-well plates and grown to confluence. At 90% confluency, cells were starved under low-serum conditions for 24 h, then a scratch was made in the center of the well with a sterile 200 μl pipette tip and incubated in medium supplemented with control, VEGF, or EDIL3. Using a phase-contrast microscope, the wound tip was photographed at 0, 24, and 48 h in the same reference area of ​​each well. Three independent experiments were performed, with each treatment performed in triplicate and with three reference areas per replicate. Percentage wound area was calculated using ImageJ software.

[0123] Tube formation assay

[0124] Endothelial tube formation assays were performed using the Cultrex In Vitro Angiogenesis Assay (R&D systems) according to the protocol. Briefly, TECs were plated in Cultrex® RGF BME-coated wells of a 96-well plate at 30 × 10 per well in EBM-2 medium. 3Cells were seeded in triplicate at a cell density of 100 μl per well. Cultrex® RGF BME vials were thawed overnight on ice prior to seeding. 50 μl of Cultrex® RGF BME was added per well to a chilled 96-well plate using a chilled 200 μl pipette tip. The plate was centrifuged at 250 g for 5 minutes at 4°C to evenly spread the BME. The coated plate was then kept at 37°C for 30 minutes to allow the BME to solidify. Meanwhile, cells were harvested and diluted with conditioned medium supplemented with VEGF or EDIL3 as a control. Cells were seeded and incubated at 37°C for 12 hours, followed by imaging at 4x magnification using a Nikon Eclipse TE2000-S microscope.

[0125] Surface flow cytometry analysis of LFA-1

[0126] THP-1 cells grown in suspension in RPMI-1640 were preactivated with 1 ng / mL PMA for 24 hours and then left overnight. Human Jurkat T cells were grown in logarithmic phase in RPMI-1640 medium. Primary human pan T cells were isolated from PBMCs from healthy donors by negative selection using a pan T cell isolation kit (Miltenyi Biotech). Isolated T cells were stimulated with human T-activating factors CD3 / CD28 in RPMI medium supplemented with IL-2 (100 IU / mL). Cells were counted and cultured at 1–2 × 10 cells / mL by adding fresh RPMI medium containing IL-2 every 2–3 days. 6The cells were maintained at 1000 cells / mL. On day 9, the cells were restimulated with human T-activating factors CD3 / CD28. The expanded T cells were used for flow cytometry analysis to verify LFA-1 expression. Briefly, PMA-activated THP-1, Jurkat T cells, and expanded human T cells were stained with FITC-conjugated CD11a (BioLegend) or an isotype control. Cells were washed twice with FACS buffer and resuspended in PBS fixation buffer containing 1.6% PFA. Cells were acquired on a BD LSRII flow cytometer using FACSDiva software (BD Biosciences). Data were analyzed using FlowJo software.

[0127] Adhesion assay

[0128] Adhesion assays were performed as described with modifications (7, 8). 15 × 10 cells per well were cultured. 3 Endothelial cells were grown on fibronectin-coated 96-well plates at a cell seeding density of 100x100. 24 hours after seeding, cells were treated with BSA or stimulated by adding TNFα (5 ng / ml) for an additional 24 hours. T cells / THP-1 cells were labeled with BCECF (1 μM, Life Technologies) for 30 minutes and then incubated with various concentrations of rEDIL3 at 37°C in the dark or under low light conditions for 1 hour. Endothelial monolayers were washed three times with PBS at the end of treatment. Pretreated labeled T cells / THP-1 cells were added to 50x100 cells. 3 Cells / 100 μl were added to all wells and allowed to adhere for 45 minutes at 37°C. Fluorescence was measured at 490 nm using a SpectraMax® M3 multimode microplate reader before and after four washes with warmed RPMI medium to remove non-adherent cells (input). Percent adhesion is defined as the fluorescence of adherent cells divided by the fluorescence of cell input.

[0129] EC migration assay

[0130] For EDIL3 knockdown, TECs were transfected as described 24 hours before seeding into 24-well transwell plates. Endothelial cells were grown for 48 hours to form monolayers on fibronectin-coated polycarbonate membranes with 5.0 μM pore size and 6.5 mm insert diameter in 24-well transwell plates (Costar). Preactivated T cells labeled with BCECF (1 μM, Life Technologies) were pretreated with BSA or rEDIL3 for 1 hour at 37°C. After gently rinsing the endothelial monolayer with warmed RPMI-1640 medium, 1 × 10 pretreated T cells were added. 5 T cells / 100 μl were added to each insert. These inserts were transferred to a new 24-well plate with a lower chamber containing 0.6 ml of RPMI-1640 medium with or without the chemotactic IP-10 and incubated at 37°C for 2–4 hours. Transendothelial migration of T cells was assessed in each well of the transwell plate in the presence and absence of inserts by measuring fluorescence at 490 nm using a SpectraMax® M3 multimode microplate reader.

[0131] 3D vascular model

[0132] To generate tumor vascular models, H226 spheroid mixtures containing a collagen rat tail hydrogel mixture (2.5 mg / ml) as a source of IP-10 were injected into the central gel region of the 3D microfluidic chamber (10–15 μL per microfluidic chamber). After 30 min of incubation at 37 °C in a sterile humidity chamber, all side walls of one adjacent channel (septal channel) were coated with a 150 μg / ml collagen solution in PBS to improve TEC adhesion to the channel. After 15 min, the channel was washed once with medium.

[0133] 3 x 10 cells to create 3-D blood vessels for tumor vascular models or 3-D vascular models. 6A 25 μL cell suspension of patient-derived TECs at 1000 cells / ml was injected into the collagen-coated media channel. The fluidic channel chip was rotated twice to create a confluent hollow lumen 3D vessel. The chip was placed with the media-gel interface facing downwards for 15 minutes to allow the cells to adhere to the media-gel interface and form a monolayer. 50 μL of the cell suspension was then reinjected, and the chip was inverted to cover the top of the 3D vascular channel. After 90 minutes of incubation in a humidity chamber at 37°C, cell culture medium was gently added to both media channels. The chip was then placed in an incubator to allow the formation of a confluent monolayer. After angiogenesis in the tumor vascular model or 3D vascular model, CD3 / CD8+ T cells (labeled with a cell tracker) pretreated with EDIL3 for 1 hour at 37°C and finally loaded in EDIL3-supplemented media were added at a TEC:T ratio of 2:1. In the 3-D vascular model, IP-10-supplemented media was added to fluidic channels positioned on opposite sides of the vascular barrier. Using a microscope, T cell migration across the vessel in the central chamber was quantified over 48–72 hours.

[0134] statistical analysis

[0135] Bioinformatics and Clinical Data

[0136] All data are presented as mean ± SD and analyzed using an unpaired, two-tailed Student's t-test. A P value <0.05 was considered statistically significant. In vitro assay analysis was performed using GraphPad Prism software version 9.3.1.

[0137] result

[0138] Ipi-Bev therapy induced a functional humoral response against EDIL3 and MFGE8.

[0139] Pre- and post-treatment plasma samples from 42 patients with advanced melanoma treated with the Ipi-Bev combination were screened by protein array (data not shown). Proteins with a Z factor of 0.4 or greater were targeted and quantified according to the manufacturer's instructions. Antibody responses to EDIL3 and MFGE8 were detected in a subset of patients (data not shown). To determine whether humoral responses correlated with clinical outcomes, patients were divided into high- and low-titer groups based on antibody titer, with a significance cutoff of 50%, or a fold change (FC) of 1.5 (Figure 1, panel a). Patients with improved clinical responses were found to have significantly higher FC for EDIL3 antibodies compared with non-responders (median FC, responders vs. non-responders: 2.5 vs. 1.0, p = 0.01). Six of the seven responders showed a significant increase in anti-EDIL3 IgG titers (FC ≥ 1.5). All patients who achieved a complete or partial response (CR / PR) by RECIST criteria to Ipi-Bev combination therapy had significantly increased anti-EDIL3 IgG responses, followed by patients with stable disease (SD) and patients with progressive disease (PD), in descending order of anti-EDIL3 IgG titers (CR / PR = 85.71% vs. SD = 31.82%; PD = 15.38%) (Figure 1, panel b). Thus, humoral responses to EDIL3 were significantly correlated with a higher response rate to Ipi-Bev combination therapy. However, humoral responses to MFGE8 did not correlate with clinical outcomes; they were found to be associated with PD (data not shown), and no change in overall survival was observed (data not shown). The expression and increase of anti-EDIL3 IgG antibodies were further confirmed by performing immunoblot assays (Figure 1, panel c) and ELISA (data not shown) using pre- and post-treatment plasma samples from representative responders from the Ipi-Bev patient cohort. Anti-EDIL3 antibody responses were also associated with improved overall survival (OS) in patients in the Ipi-Bev cohort (long-rank p<0.027). The median survival time was 70 weeks (95% CI, 47-81) in patients with EDIL3 antibody FC <1.5, but was not reached in patients with FC ≥1.5 (Figure 1, panel d).

[0140] To further examine the humoral response to EDIL3 as a function of treatment, we analyzed the FC of anti-EDIL3 antibody titers in pre- and posttreatment plasma samples from patient cohorts treated with ipilimumab alone (n = 34), anti-PD1 alone (n = 25), and the combination of nivolumab and ipilimumab (n = 41) (Figure 1, panel e). The Ipi-Bev cohort had the highest proportion of patients (35.7%) with significantly elevated FC of anti-EDIL3 IgG titers (>1.5), whereas patients in the other cohorts had similar FCs (14% for Ipi alone, 12% for anti-PD1, and 12.2% for Ipi + Nivo).

[0141] EDIL3 and MFGE8 expression correlates with tumor immune rejection

[0142] TIDE analysis, a transcriptome biomarker platform for assessing ICB response by inferring gene function in tumor immune regulation (5), predicted a T cell elimination phenotype but not dysfunction mediated by increased expression of EDIL3 and MFGE8. β-catenin, i.e., CTNNB1, was used as the reference gene for TIDE analysis. CTNNB1 is known to mediate T cell elimination and resistance in melanoma and is therefore a biomarker for predicting ICB response in patients (6, 7). Volcano plots of EDIL3 and MFGE8 expression in melanoma patients showed a high association with T cell elimination, while the volcano plot for β-catenin was consistent with an elimination and dysfunction phenotype (Figure 2, panel a). Furthermore, TIDE analysis revealed that high EDIL3 expression was significantly associated with poor ICB outcome in melanoma patients (p = 7.4e-11), whereas MFGE8 and CTNNB1 expression were not associated with ICB response (Figure 2, panel b). Thus, EDIL3 expression correlates with T cell deletion and predicted non-response to ICB therapy in melanoma patients.

[0143] EDIL3-mediated T cell elimination is associated with TGFβ signaling, EMT, and angiogenic signatures

[0144] Using the gene query tool on the TIDE platform, we evaluated its expression in immune-suppressive cell types that drive T cell elimination in the TME. Among all cell types promoting T cell elimination, CAFs were found to have the highest EDIL3 expression levels, followed by tumor-associated macrophages (TAMs) with an M2-like phenotype. However, myeloid-derived suppressor cells (MDSCs) were negatively correlated with EDIL3 expression. Interestingly, MFGE8 expression was found to be strongly associated with CAFs and weakly associated with TAMs and MDSCs with an M2-like phenotype (Figure 3).

[0145] The TCGA Cutaneous Melanoma (SKCM) dataset (PMID: 26091043) cohort utilized all 469 primary and / or metastatic melanoma samples with available RNA-seq data. Additionally, the CheckMate 064 dataset analysis utilized all 90 pretreatment samples from both study arms with available RNA-seq data (PMID: 30021886). Significant enrichment of TGFβ signaling, pan-fibroblast TGFβ response signature, EMT phenotype, and angiogenesis signature was observed with increased EDIL3 expression in TCGA SKCM subjects (data not shown). Next, the TCGA SKCM analysis was compared with the CheckMate 064 dataset generated from subjects with advanced or metastatic melanoma undergoing an open-label, randomized, phase 2 trial of nivolumab administered sequentially with ipilimumab. The expression profile of EDIL3, along with the gene sets of pathways selected from the TCGA SKCM analysis, was visualized as a heatmap for 90 samples from the CheckMate064 dataset. The CheckMate064 dataset analysis confirmed the previous observation from the TCGA SKCM analysis, namely, that EDIL3 expression pathway scores for TGFβ signaling, pan-fibroblast TGF-β response signature, EMT phenotype, and angiogenesis signature were positively correlated when arranged in ascending order (data not shown). Thus, our analysis indicated that immunosuppressive CAFs are a source of EDIL3 and play a role in regulating TGF-β signaling, EMT phenotype, and angiogenesis in the TME of patients with advanced melanoma.

[0146] EDIL3 is abundantly expressed in CAFs isolated from tumor biopsies of melanoma patients and is associated with TGF-β1-induced EMT

[0147] Because TIDE gene query analysis demonstrated EDIL3 expression in CAFs, we analyzed EDIL3 expression in fibroblasts. Compared with NFs, patient-derived CAFs (P4-CAFs and CAF2) secreted abundant levels of EDIL3 into the culture medium, as detected by ELISA (P4-CAFs (p<0.0001), CAF2 (p<0.0001)) (Figure 4, Panel a). Next, NFs were pretreated with or without LY2109761 and subsequently induced with TGFβ1 for 24 hours. TGFβ1 treatment activated downstream SMAD signaling in a concentration-dependent manner. Furthermore, upregulation of EDIL3 protein expression in whole cell lysates and its secretion in the conditioned medium were detected by immunoblot assay and ELISA, respectively (Figure 4, Panels b and c). Inhibition of TGFβ1 signaling using LY2109761 inhibited Smad2 activation and downregulated EDIL3 expression in both the cellular and extracellular compartments, establishing TGFβ1-regulated regulation of EDIL3 expression. RNA sequencing also confirmed the overexpression of EDIL3 and MFGE8 in CAFs compared with NFs, demonstrating only TGFβ1-regulated induction of EDIL3 (Figure 11, panels a and b).

[0148] We evaluated the effect of EDIL3 on TGFβ1-induced EMT in CAFs. EDIL3 was silenced using siRNA in patient-derived CAFs and verified by RT-PCR with or without TGFβ1 induction (Figure 4, panel d). Silencing EDIL3 in CAFs downregulated the basal level of transgelin (TGLN), a key EMT marker, and significantly suppressed TGFβ1-induced TGLN and smooth muscle α-actin (ACTA2) expression in CAFs (Figure 4, panels e and f). Thus, EDIL3 is overexpressed in CAFs from patients with advanced melanoma and regulates TGFβ1 signaling and the associated EMT.

[0149] In the TCGA SKCM and Checkmate064 databases, EDIL3 expression correlates with angiogenesis and its signature.

[0150] To assess the angiogenic role of EDIL3, pretreatment plasma from 39 Ipi-Bev-treated patients was analyzed for the interdependence of circulating levels of VEGF and EDIL3. The Spearman rank correlation was 0.44 (p = 0.005) (Figure 5, panel a). Thus, a "moderate" association was observed, indicating that higher pretreatment EDIL-3 levels were associated with higher pretreatment VEGF levels.

[0151] Based on an angiogenesis signature panel consisting of TEK, CDH5, SOX17, and SOX18 (PMID: 29443960 Mariathasan et al.), 469 subjects from the TCGA SKCM dataset were classified into angiogenesis (high) (n=83) and angiogenesis (low) (n=386) based on agglomerative clustering using two clusters of angiogenesis pathway expression. Consistent with our previous analysis, EDIL3 expression was significantly upregulated in patients with angiogenesis (high) phenotype versus angiogenesis (low) phenotype (p=1.3e-07). Analysis of the Checkmate064 dataset, including 90 patients divided into 22 (high) and 68 (low) angiogenesis signatures, also confirmed a significant association (p=0.0002), i.e., EDIL3 is robustly associated with angiogenesis (high) in patients with advanced melanoma (Figure 5, panel b).

[0152] EDIL3 mRNA expression by RT-PCR was found to be increased in endothelial cells with higher angiogenic potential (HMECs, TECs, and HUVECs) (Figure 12, panel a). To further investigate the functional effects of angiogenesis, recombinant EDIL3 treatment of patient-derived TECs was explored, using VEGF A-induced effects as a positive control. Recombinant human EDIL3 significantly promoted TEC migration at 24 and 48 hours compared with untreated controls, and this was comparable to VEGF A treatment (Figure 5, panel c). It was also found to promote the development of denser capillary-like structures compared with untreated controls and similar to VEGF A treatment (data not shown). Significantly increased branching numbers (mean mesh size, p<0.01) and mesh index (p<0.05) were observed with rEDIL3 treatment compared with control TECs and similar to VEGF treatment (Figure 5, panels d and e). Therefore, our findings support EDIL3 as a positive regulator of pathological angiogenesis.

[0153] EDIL3 blocks lymphocyte endothelial adhesion and inhibits T cell migration

[0154] To verify the functional role of EDIL3 in T cell exclusion in the TME, activated immune cells, namely, THP1 cells, Jurkat cells, and T cells, were screened for lymphocyte function-associated antigen 1 (LFA-1) expression by flow cytometry analysis. THP-1 cells were found to express LFA-1 most abundantly among all three cell types screened (Figure 6, panel a). Immune cell adhesion is a prerequisite for transendothelial migration, and the interaction between LFA-1 on immune cells and ICAM1 (intercellular adhesion molecule 1) on endothelial cells is one of the critical steps. EDIL3 is known to antagonize LFA-1 / ICAM1-dependent adhesion. To verify the mechanism of T cell exclusion, adhesion assays were performed on TECs and THP-1 cells from Ipi-Bev-treated patients as described above (data not shown). Patient-derived TECs were either left unstimulated or stimulated with TNF-α for 24 hours as described in the methods. Compared to untreated THP-1 cells, THP-1 cells pretreated with rEDIL3 (1 h at 37°C) inhibited their adhesion on unstimulated TEC monolayers (p<0.001). TNF-α upregulated ICAM-1 expression on TECs (data not shown), resulting in a significant increase in adhesion of untreated THP-1 cells by 59.6% to 90.2% (p<0.0001) compared to unstimulated TEC monolayers. However, rEDIL3 pretreatment inhibited THP-1 cell adhesion to stimulated TEC monolayers in a dose-dependent manner: 20% (p<0.001) at 10 μg / ml rEDIL3, 51.2% (p<0.0001) at 50 μg / ml EDIL3, and 55% (p<0.0001) at 200 μg / ml EDIL3, respectively (showing saturation) (Figure 6, panel b). We next confirmed that rEDIL3-mediated adhesion inhibition is mediated by disruption of the interaction between LFA-1 and ICAM-1 on TECs. Activated THP-1 cells bound rEDIL3 with higher affinity than rICAM-1 in binding assays (data not shown).THP-1 cells treated with rEDIL3 and anti-LFA-1 antibody showed comparable adhesion inhibition (i.e., 67.8% (p<0.01)) on stimulated endothelial cell monolayers compared with THP-1 cells treated with rEDIL3 antibody alone (67% (p<0.01)) or anti-LFA-1 antibody alone (66% (p<0.01)) (Figure 6, panel c). Thus, disruption of the interaction between LFA-1 and ICAM-1 is one of the mechanisms underlying EDIL3-mediated inhibition of immune cell adhesion on TECs.

[0155] Next, we investigated the effect of EDIL3 on transendothelial migration of T cells across patient-derived TEC monolayers. PBMCs were isolated from donor blood samples using the Ficoll-Paque method, and T cells were purified using a pan T cell isolation kit. IP-10, a positive regulator of chemotactic chemokines known to stimulate T cell migration, was used as a chemoattractant. Migration of activated T cells across TEC monolayers grown on fibronectin-coated microporous membranes in transwells with or without IP-10 in the lower chamber was examined using a Boyden chamber assay. IP-10, when added in the lower chamber, enhanced activated T cell migration compared to control. Silencing EDIL3 in TECs increased T cell migration from 38.5% to 52.85% (p<0.05). IP-10-induced migration was also enhanced by 52.8% to 85.7% after EDIL3 silencing (p<0.01) (Figure 6, panel d). On the other hand, when T cells were pretreated with rEDIL-3, the number of T cells migrating across the endothelial monolayer decreased in both the control and IP-10-treated groups (p<0.05) (Figure 6, panel e).

[0156] T cell migration in the presence of rEDIL3 was further investigated in microfluidic 3D coculture. Endothelial vessels were grown using TECs within DAX-1 3D cell culture chips. Immunofluorescence staining verified the presence of perfusable tubule structures with clear lumens. Cell-tracker dye was used to determine the Y-axis location of T cells within the chip. Two coculture models were used to test T cell migration across the endothelium. In the first model, the 3D vascular model, IP-10 was added to the final chamber, while rEDIL3-pretreated labeled T cells were perfused through the vascular network and allowed to migrate for 48 hours. If significant migration was observed in the control versus IP-10 treatment (p<0.001), ROIs were selected, imaged, and quantified. While IP-10 induced migration 2.3-fold, the addition of EDIL3-pretreated T cells and rEDIL3 to the culture medium significantly reduced overall IP-10-induced migration of T cells (p<0.01) (Fig. 7, panel a).

[0157] In the second model, the tumor vascular model, tumor spheroids were added to the central chamber as an IP-10 source along with a vascular network on one side of the chip to establish a chemotactic gradient and promote T cell migration. The vascular network was perfused with untreated or rEDIL3-pretreated T cells and co-cultured for 48 hours, while maintaining rEDIL3 in the medium supplemented through the vascular port. Next, T cell extravasation from TEC vessels was measured. Fluorescent T cells within the central compartment of the spheroids were quantified. EDIL3 pretreatment significantly (p<0.05) downregulated T cell migration across the vascular network (Figure 7, panel b). Therefore, one mechanism by which EDIL3 inhibits T cell migration may be by blocking the interaction between LFA-1 on T cells and ICAM1 on TECs, which is crucial for adhesion, thereby resulting in T cell elimination in the TME.

[0158] Consideration

[0159] Ipi-Bev combination therapy induced functional humoral immune responses against EDIL3, which correlated with clinical efficacy in patients with advanced melanoma. Interestingly, humoral responses against MFGE8 (milk fat globule-EGF factor 8), another glycoprotein secreted similarly to EDIL3, were also observed. EDIL3 and MFGE8 are evolutionarily related and share sequence similarity (24). They are homologous secreted glycoproteins with an evolutionarily conserved RGD motif. These glycoproteins are involved in leukocyte recruitment and the regulation of inflammation (4). However, their role in tumor immunology and therapy is limited. While anti-MFGE8 responses alone have not been associated with improved clinical outcomes, systemic targeting of MFGE8 enhances cross-presentation of immunogenic antigens (b). Therefore, anti-MFGE8 responses may contribute to the tumor immunogenicity observed in patients treated with Ipi-Bev combination therapy. Among the screened cohort, EDIL3 antibody responses were most robust in patients treated with Ipi-Be combination therapy compared with Ipi alone, anti-PD1, and Ipi-Nivo combination therapy. The combination of bevacizumab and Io has been reported to increase CD8 T lymphocyte infiltration in melanoma tumors (a), and EDIL3 was found to be associated with immune exclusion. Therefore, EDIL3 may be an immunomodulatory target of bevacizumab and may serve as a biomarker for response to Ipi-Bev combination therapy.

[0160] Our TIDE correlation study demonstrated a positive correlation between EDIL3 expression and a gene signature of immune evasion via T cell elimination in a large cohort of melanoma patients. High EDIL3 expression in SKCM subjects predicted non-response to ICB therapy, consistent with previous reports identifying EDIL3 as a poor prognostic marker in multiple malignancies (5, 6). Our in vitro adhesion and transendothelial migration studies confirmed recombinant EDIL3-mediated T cell elimination at the vascular-immune interface. EDIL3 acts as an endogenous inhibitor of LFA-1-dependent leukocyte recruitment in inflammatory disease states (1). This finding supports the strong CD8 expression observed in patients who developed humoral immunity against EDIL3 and benefited from treatment. + This may be important for understanding T cell infiltration. v It is a key player in T cell immunity by promoting Treg responses in inflammatory conditions by upregulating FOXP3 expression via β3 integrin signaling ( 2 ).

[0161] Among cell types known to mediate T cell elimination, CAFs were found to be associated with EDIL3 expression. Existing reports investigating the role of EDIL3 in fibroblasts add weight to current knowledge. Liquid biopsy analysis of colorectal cancer identified EDIL3 as a plasma-derived exosomal cargo protein exclusively associated with patient-derived CAFs (4). EDIL3-secreting CAFs have been implicated as a prognostic signature associated with clinical outcomes, tumor progression, and genetic mutations in breast cancer (3). Consistent with the TCGA analysis, our in vitro studies demonstrated that TGFβ1 induced EDIL3 expression, and its silencing inhibited the TGFβ1-induced EMT phenotype in CAFs. This suggests the existence of an EDIL3-mediated molecular feedback loop in regulating TGFβ-induced EMT in CAFs (7). Mariathasan et al. reported that TGFβ signaling in fibroblasts, specifically CD8 +We reported that humoral responses to EDIL3 were associated with a lack of response to ICB in tumors exhibiting T cell exclusion, thus highlighting the importance of normalizing immune-vascular crosstalk to exert antitumor immunity.

[0162] In conclusion, EDIL3 produced by CAFs supports immune evasion in melanoma tumors through the elimination of T cells and modulation of TGF-β-induced EMT. Without wishing to be bound by theory, EDIL3 may be one of the underlying targets of bevacizumab, which leads to the elimination of the inflammatory phenotype observed in Ipi-Bev treatment responders. Without wishing to be bound by theory, EDIL3 not only provides useful mechanistic insights into the antitumor and synergistic effects of the combination of ICB and anti-VEGF drugs, but also further validates it as a therapeutic target.

[0163] Example 2

[0164] Antibody production and purification

[0165] Harbor H2L2 mice (registered trademark) were immunized with human EDIL3-Fc or EDIL3-his (novoprotein) to generate anti-EDIL3 antibodies. Mice with high serum titers and specific immune responses to human EDIL3-his, mouse EDIL3-his, and cynomolgus monkey EDIL3-his were selected and given a final boost with human EDIL3-Fc or EDIL3-his protein 3 days before sacrifice for collection of spleen, bone marrow, and lymph nodes. This study used nanofluidic optoelectronic B lymphocyte screening technology (NanOBlast). The NanoOBlast workflow begins with the preparation of antibody-secreting cells (ASCs). Selectively enriched antigen-experienced mouse ASCs were isolated from lymphocytes collected from the spleen and lymph nodes using magnetic beads coated with mouse CD138 antibody. ASCs were then loaded onto a 14k-sized chip and isolated into individual nanopens via optoelectronic positioning (OEP). ASCs secreting antigen-specific IgG were detected using a protein-based fluorescent binding assay that produces a characteristic fluorescent bloom (human EDIL3-his, mouse EDIL3-his, and cynomolgus monkey EDIL3-his proteins for screening). Individual ASCs of interest were then unpacked using OEP and exported directly from the chip to a 96-well plate containing cell lysis buffer. Antibody heavy chain variable domain (VH) and light chain variable domain (VL) sequences were recovered using single-cell rapid amplification of cDNA ends (RACE), cloned and recombinantly expressed as standard antibodies using standard methods.

[0166] After obtaining the sequences encoding the light and heavy chain variable regions of each screened antibody molecule, the nucleic acid sequences encoding the light and heavy chain variable regions of each antibody molecule were fused with nucleic acid sequences encoding the light and heavy chain constant domains of a human antibody and expressed using recombinant DNA techniques to obtain recombinant antibody molecules. The nucleic acid sequence encoding the antibody heavy chain variable region (VH) was genetically synthesized and cloned into a mammalian cell expression plasmid vector (pTT5 mammalian expression vector) containing a nucleic acid sequence encoding the heavy chain constant domain of a human IgG1 antibody to encode a full-length heavy chain. The nucleic acid sequence encoding the antibody light chain variable domain (VL) was genetically synthesized and cloned into a mammalian cell expression plasmid vector (pTT5 mammalian expression vector) containing a nucleic acid sequence encoding the light chain constant region of a human Igκ antibody to encode a full-length light chain.

[0167] A plasmid encoding the antibody heavy chain and a plasmid encoding the antibody light chain were co-transfected into human embryonic kidney cells HEK293. Purified recombinant anti-EDIL3 antibody with correctly paired light and heavy chains could be obtained from these HEK293 cells by conventional recombinant protein expression and purification techniques.

[0168] HEK293 cells were grown in FreeStyle™ F17 Expression Medium (Thermo, #A1383504). Prior to transient transfection, cells were cultured at 6–8 × 10 5 The cells were cultured in a shaker at 37°C under 8% CO2 for 24 hours to a concentration of approximately 1.2 × 10 6The concentration was determined as cells / mL. 30 mL of cultured cells was harvested. A plasmid containing a nucleic acid sequence encoding the antibody heavy chain and a plasmid containing a nucleic acid sequence encoding the antibody light chain were mixed at a 2:3 ratio. A total of 30 μg of the plasmid was dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo, 31985088) and sterilized by filtration through a 0.22 μm filter. Next, 1.5 mL of Opti-MEM was mixed with 120 μL of 1 mg / mL PEI (Polysciences, Inc. #23966-2) and allowed to stand for 5 minutes. The PEI was slowly added to the plasmid and incubated at room temperature for 10 minutes. The plasmid and PEI mixture was slowly added dropwise to a culture flask with shaking and cultured at 37°C under 8% CO2 for 5 days. Cell viability was measured after 5 days. The culture was harvested and centrifuged at 3300 g for 10 minutes. The supernatant was then collected and centrifuged at high speed to remove impurities. A gravity column (Bio-Rad, #7311550) containing MabSelect™ (GE Healthcare Life Science, #71-5020-91 AE) was equilibrated with PBS (pH 7.4) and rinsed with 2–5 column volumes of PBS. The supernatant sample was loaded onto the column and rinsed with 5–10 column volumes of PBS, followed by 0.1 M glycine (pH 3.5) to elute the target protein. The eluate was adjusted to neutral with Tris-HCl, pH 8.0, concentrated, and buffer-exchanged into PBS buffer using an ultrafiltration tube (Millipore, UFC901024) to obtain a purified solution of anti-EDIL3 antibody. Finally, the concentration of the purified solution was determined using NanoDrop (Thermo Scientific™ NanoDrop™ One), and the purified solution of anti-EDIL3 antibody was divided into aliquots and stored for later use.

[0169] Detection of antibody binding activity against EDIL3 protein.

[0170] The binding activity of 10 antibodies obtained using the above protocol to EDIL3 protein was determined using an ELISA assay. Human EDIL3 (novoprotein, #C601), cynomolgus monkey EDIL3 (novoprotein, #C18M), and mouse EDIL3 (novoprotein, #C18P) proteins were diluted to a concentration of 1 μg / mL, added to a 96-well plate (100 μL per well), and incubated at 4°C overnight. The 96-well plate was washed three times with PBST solution and then incubated at 37°C for 1 hour with the addition of 2% BSA in PBS. Serial dilutions of the antibodies (starting at 100 nM, 1:10 dilutions, 8 points) were prepared, added to the 96-well plate, and incubated at 37°C for 1 hour. After washing the wells three times with PBST solution, 100 μL of anti-human IgG Fc-HRP secondary antibody solution (5000-fold dilution) was added per well and incubated at 37°C for 50-60 minutes. After washing the wells three times with PBST solution, TMB color development solution was added, and after 5-15 minutes, reaction stop solution was added to stop the reaction.

[0171] The test results are shown in Figures 20A to 20J. Antibodies PR305734, PR305667, PR305633, PR305618, PR305754-p, PR305764, PR305689, PR305684, PR305629, and PR305623 showed EC50 binding activity of single- to double-digit picomolar concentrations against human EDIL3 protein, cynomolgus monkey EDIL3 protein, and mouse EDIL3 protein.

[0172] ELISA-based blocking assay of anti-human EDIL3 monoclonal antibodies against the binding of human EDIL3 to its ligands LFA-1 and αvβ3 proteins

[0173] The blocking activity of anti-human EDIL3 monoclonal antibodies against the binding of human EDIL3 (Novoprotein, C601, 1 mg) to the ligands LFA-1 protein (ACRO, IT2-H53W3, 250 μg) and αvβ3 protein (ACRO, IT3-H52E3, 500 μg) was evaluated. EDIL3 protein was biotinylated using a biotinylation kit (ThermoFisher, A39257, EZ-Link Sulfo-NHS-LC-Biotin) according to the manufacturer's instructions. A 96-well plate (Corning, catalog number: 9018) was coated overnight with 2 μg / mL LFA-1 protein (ACRO, IT2-H53W3-250 μg) or αvβ3 protein (ACRO, IT3-H52E3-500 μg), washed three times with PBST, and then 200 μL of 2% BSA in PBS was added per well and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and 50 μL of test antibody gradient dilutions (starting at 100 nM, 1:3 dilutions, 8 points) and 50 μL of EDIL3-his-biotin (final concentration: 2 μg / ml) were added per well. The plate was incubated at room temperature for 1 hour, after which the supernatant was discarded. 100 μL of a 1:5000 dilution of streptavidin-conjugated HRP (Sigma, catalog number: S2438) was then added per well. The plate was incubated at 37°C for 1 hour and washed three times with PBST. 100 μL of TMB was added per well for color development, and after 15 minutes, the reaction was stopped by adding 50 μL of stop solution. OD450 values ​​were measured using a Spetra max 384 plus (Molecular Devices).

[0174] As shown in Figures 21A-G, PR305618, PR305633, PR305667, and PR305734 are the antibodies with the best blocking activity, with specific IC50 values ​​shown in Figure 21A.

[0175] EDIL3 mAb specifically binds to human recombinant EDIL3

[0176] Binding of EDIL3 mAbs to human recombinant EDIL3 was examined by Western blot analysis and conditioned medium analysis via 10% reducing SDS-PAGE. Human recombinant EDIL3 was transferred to a membrane and probed with EDIL3 polyclonal antibody (Product No. PA5-27994) or EDIL3 mAbs PR305734, PR305754, PR305764, PR305618, PR305633, PR305667, and PR305689 at serial dilutions. Primary antibodies were detected using an HRP-conjugated anti-rabbit IgG antibody. As shown in Figures 23A-B, the EDIL3 protein is the predicted band of approximately 65 kDa under reducing conditions, and the EDIL3 mAbs PR305734, PR305754, PR305764, PR305618, PR305633, PR305667, and PR305689 can bind to human recombinant EDIL3 as well as the EDIL3 polyclonal antibody (Product No. PA5-27994).

[0177] Determination of kinetic and thermodynamic constants of EDIL3 mAbs by surface plasma resonance

[0178] Surface plasmon resonance (SPR), known in the art, was used to determine the binding affinity of the EDIL3 antibodies PR305734, PR305754, PR305764, PR305633, PR305667, PR305618, and PR305689. The SPR-based binding method involves immobilizing a ligand (antibody) on the surface of a sensor chip. The binding partner of interest, i.e., the analyte, flows through a flow channel. Different concentrations of analyte flow over the ligand, allowing the ligand-analyte interaction to be characterized. The SPR signal arises from a change in the refractive index of the light source at the surface of the sensor chip. The increase in mass associated with a binding event causes a proportional increase in the refractive index, which is observed as a change in the response-resonance signal.

[0179] Briefly, for experiments using the antibodies described herein, SPR assays were performed using immobilized antibody (used as analyte) at concentrations ranging from 1 to 1000 nM and flow-through EDIL3 antigen (used as ligand). Kinetic data for the antibody interaction with EDIL3 antigen were fitted to a two-state and 1-1 binding model using Biocore software. Mean and standard deviation K values ​​are derived from at least three independent runs.

[0180] Figures 24 and 26 show kinetic measurements of seven EDIL3 antibodies binding to EDIL3. All seven anti-EDIL3 antibodies exhibit nM or sub-nM affinity for EDIL3, high fidelity, and slow dissociation rates (approximately 1 x 10-5 to 6 min-1). The rankings are in good agreement with previous kinetic evaluations using other SPR instruments.

[0181] Testing the blocking activity of EDIL3 mAb

[0182] The adhesion assay scheme was performed as described in Figure 25. Briefly, TECs were grown on fibronectin-coated 96-well or 24-well black-walled plates. 24 hours after seeding, cells were treated with BSA or stimulated with TNFα (BioLegend; #570102; 5 ng / mL) for an additional 24 hours. Activated T cells were labeled with BCECF (1 mmol / L, Life Technologies, #B1170) for 30 minutes, followed by incubation with the indicated concentrations of rEDIL3 (50 ng / mL) for 1 hour at 37°C in the dark or under low-light conditions. Endothelial monolayers were washed three times with PBS at the end of treatment. Pretreated BCECF-labeled activated T cells were added to all wells and allowed to adhere for 45 minutes at 37°C. Then, specific concentrations of EDIL3, anti-EDIL3, anti-LFA-1, anti-a5b3, EDIL3 antibodies PR305734, PR305754, PR305764, PR305618, PR305633, PR305667, PR305689 are added.

[0183] Fluorescence intensity was measured at 485 nm (excitation) and 530 nm (emission) using a SpectraMax M3 multimode microplate reader before (input) and after washing four times with warm RPMI 1640 medium to remove nonadherent cells. Adhesion data were plotted as a percentage of the untreated control (100%). Representative images were acquired for each treatment group. Figure 25 shows the activity of EDIL3 mAb in blocking LFA-1 binding to ICAM-1.

[0184] Epitope binning analysis of EDIL3 antibody

[0185] To determine whether the EDIL3 antibody binds to a different or similar binding epitope on EDIL3, an epitope competition experiment using the ForteBio Octet® RED96e platform was performed. EDIL3 protein was diluted to 3 μg / ml in kinetic buffer (10x kinetic buffer (catalog no. 18-1105, ForteBio)) and then loaded onto an anti-Penta HIS biosensor (catalog no. 18-5120, ForteBio) to reach a capture level of 0.3 nm. An in-tandem competition assay format was applied, which involved two association steps. First, the antigen-loaded biosensor was allowed to bind to each antibody (i.e., first antibody, first Ab) at a saturating concentration of 400 nM for 300 seconds to reach equilibrium, and then the competing antibody (i.e., second Ab) was allowed to bind at 400 nM for 300 seconds. The second binding signal is recorded as 100% of the signal for each antibody when the first antibody is replaced with kinetic buffer. All binding data were analyzed using ForteBio Data Analysis 11.0 software. The results in Figure 27 show that the seven EDIL3 antibodies, PR305734, PR305754, PR305764, PR305618, PR305633, PR305667, and PR305689, bind to overlapping but distinct epitopes on EDIL3 and that their binding is antibody display (i.e., solution vs. surface display) dependent. None of these antibodies interact with EDIL3 in their own presence (i.e., exhibit biparatopic activity) or in the presence of a negative control.

[0186] *****

[0187] equivalent

[0188] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. An antibody or antigen-binding fragment or variant thereof that specifically binds to epidermal growth factor-like repeat and discoidin I-like domain 3 (EDIL3) protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the HCVR comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprising CDRs LCDR1, LCDR2, and LCDR3; a) the amino acid sequence of HCDR1 is SYAMS, the amino acid sequence of HCDR2 is AISDSGGSTYYADSVKG, the amino acid sequence of HCDR3 is EGLITFGGVIVIGYFDY, the amino acid sequence of LCDR1 is QASQDISNYLN, the amino acid sequence of LCDR2 is DASNLET, and the amino acid sequence of LCDR3 is QQYDNLPIT, or b) the amino acid sequence of HCDR1 is SYWMS, the amino acid sequence of HCDR2 is NIKQDGSQKYYVDSVKG, the amino acid sequence of HCDR3 is RGNFFFDN, the amino acid sequence of LCDR1 is RASQYVSSYLA, the amino acid sequence of LCDR2 is DASNRAT, and the amino acid sequence of LCDR3 is QQRNNWPPT, or c) the amino acid sequence of HCDR1 is NHYWS, the amino acid sequence of HCDR2 is YIYYSGSTNYNPSLKS, the amino acid sequence of HCDR3 is GFAY, the amino acid sequence of LCDR1 is RASQGITNYLA, the amino acid sequence of LCDR2 is AASTLQS, and the amino acid sequence of LCDR3 is QKYNSAPWT; or d) the amino acid sequence of HCDR1 is SYAMN, the amino acid sequence of HCDR2 is AISGSGDSTYSTDSVKG, the amino acid sequence of HCDR3 is EYYDILTGYWDWYFDL, the amino acid sequence of LCDR1 is RASQSINSNLA, the amino acid sequence of LCDR2 is GASTRAT, and the amino acid sequence of LCDR3 is QQYNNWPLT, or e) the amino acid sequence of HCDR1 is SYGMH, the amino acid sequence of HCDR2 is VMWYDGSDRYSADSVKG, the amino acid sequence of HCDR3 is GYDILTGPDHFDY, the amino acid sequence of LCDR1 is RASQSISSYLN, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR3 is QQSYSTPLT, or f) the amino acid sequence of HCDR1 is SYDMN, the amino acid sequence of HCDR2 is TISGSGSHTYYADSVRG, the amino acid sequence of HCDR3 is EGGATAFDI, the amino acid sequence of LCDR1 is RASQGISSYLA, the amino acid sequence of LCDR2 is VASTLQS, and the amino acid sequence of LCDR3 is QQLNNYPT; or g) the amino acid sequence of HCDR1 is TYGMH, the amino acid sequence of HCDR2 is LIWYDGINKYYADSVKG, the amino acid sequence of HCDR3 is PYYDILTGYFDY, the amino acid sequence of LCDR1 is RASQSDSSSYLA, the amino acid sequence of LCDR2 is GTSSRAT, and the amino acid sequence of LCDR3 is QQYGSSPLT, or h) the amino acid sequence of HCDR1 is SYGMH, the amino acid sequence of HCDR2 is VIWYDGTNKYYADSVKG, the amino acid sequence of HCDR3 is DPSLWFGEFPHYYGMDV, the amino acid sequence of LCDR1 is QASQDISNYLN, the amino acid sequence of LCDR2 is DASNLET, and the amino acid sequence of LCDR3 is QQYDNLPLT, or i) the amino acid sequence of HCDR1 is GYYWS, the amino acid sequence of HCDR2 is EIQHSGSTNYKPSLKS, the amino acid sequence of HCDR3 is LTGDSLLFEY, the amino acid sequence of LCDR1 is RASQSVSSYLA, the amino acid sequence of LCDR2 is DTSNRAT, and the amino acid sequence of LCDR3 is QQRSNWPIT, or j) the amino acid sequence of HCDR1 is SYGMH, the amino acid sequence of HCDR2 is VIWYDGSNKYYADSVKG, the amino acid sequence of HCDR3 is DSASDYFDY, the amino acid sequence of LCDR1 is RASQSVSSNLA, the amino acid sequence of LCDR2 is GASTRAT, and the amino acid sequence of LCDR3 is QQYSDWPT; or k) The antibody or antigen-binding fragment or variant thereof, wherein the amino acid sequence of HCDR1 is GYYWS, the amino acid sequence of HCDR2 is EINHSGSTNYKPSLKS, the amino acid sequence of HCDR3 is LTGDSLLFEY, the amino acid sequence of LCDR1 is RASQSVSSYLA, the amino acid sequence of LCDR2 is DTSNRAT, and the amino acid sequence of LCDR3 is QQRSNWPIT.

2. comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); a) the amino acid sequence of the LCVR is DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIK; the amino acid sequence of the HCVR is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISDSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAQEGLITFGGVIVIGYFDYWGQGTLVTVSS; or b) the amino acid sequence of the LCVR is EIVLTQSPATHLSPGERATLSCRASQYVSSYLAWYHQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNNWPPTFGQGTKVEIK; the amino acid sequence of the HCVR is EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSQKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRRGNFFFDNWGQGTLVTVSS; or c) the amino acid sequence of the LCVR is DIQMTQSPSSLSASVGDRVTITCRASQGITNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDVATYYCQKYNSAPWTFGQGTKVEIK; the amino acid sequence of the HCVR is QVQLQESGPGLVKPSETLSLTCTVSGGSISNHYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGFAYWGQGTLVTVSS; or d) the amino acid sequence of the LCVR is EIVMTLSPATHSVSPGERATLSCRASQSINSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEIK; the amino acid sequence of the HCVR is EVQLLESGGGLGQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGSGDSTYSTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEYYDILTGYWDWYFDLWGQGTLVTVSS; or e) the amino acid sequence of the LCVR is DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK; the amino acid sequence of the HCVR is QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSDRYSADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYDILTGPDHFDYWGQGTLVTVSS; or f) the amino acid sequence of the LCVR is DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNNYPTFGGGTKVEIK; the amino acid sequence of the HCVR is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMNWVRQAPGKGPVWVSTISGSGSHTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEGGATAFDIWGQGTMVTVSS; or g) the amino acid sequence of the LCVR is EIVLTQSPGTLSLSPGERATLSCRASQSDSSSYLAWYQQKPGQAPRLLIYGTSSRATGISDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIK; the amino acid sequence of the HCVR is QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVALIWYDGINKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARPYYDILTGYFDYWGQGTLVTVSS; or h) the amino acid sequence of the LCVR is DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK; the amino acid sequence of the HCVR is QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGTNKYYADSVKGRFTISRDNSKNTLYLQVNSLRAEDTAVYYCARDPSLWFGEFPHYYGMDVWGQGTTVTVSS; or i) the amino acid sequence of the LCVR is EIVLTQSPATHLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTVSSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK the amino acid sequence of the HCVR is QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIQHSGSTNYKPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAELTGDSLLLFEYWGQGTLVTVSS; or j) the amino acid sequence of the LCVR is EIVMTQSPATTLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSDWPTFGGGTKVERI R; the amino acid sequence of the HCVR is QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDTSKNTLYLQMNSLRAEDTAVYYCARDSASDYFDYWGQGTLVTVSS; k) the amino acid sequence of the LCVR is EIVLTQSPATHLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTVSSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK; The antibody or antigen-binding fragment or variant thereof of claim 1, wherein the amino acid sequence of the HCVR is QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYKPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAELTGDSLLLFEYWGQGTLVTVSS.

3. comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); a) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISDSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAQEGLITFGGVIVIGYFDYWGQGTLVTVSS; or b) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVLTQSPATHLSPGERATLSCRASQYVSSYLAWYHQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNNWPPTFGQGTKVEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSQKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRRGNFFFDNWGQGTLVTVSS; or c) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQGITNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDVATYYCQKYNSAPWTFGQGTKVEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLQESGPGLVKPSETLSLTCTVSGGSISNHYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGFAYWGQGTLVTVSS; or d) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVMTLSPATHVSPGERATLSCRASQSINSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EVQLLESGGGLGQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGSGDSTYSTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEYYDILTGYWDWYFDLWGQGTLVTVSS; or e) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSDRYSADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYDILTGPDHFDYWGQGTLVTVSS; or f) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNNYPTFGGGTKVEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMNWVRQAPGKGPVWVSTISGSGSHTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEGGATAFDIWGQGTMVTVSS; or g) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVLTQSPGTLSLSPGERATTLSCRASQSDSSSYLAWYQQKPGQAPRLLIYGTSSRATGISDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVALIWYDGINKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARPYYDILTGYFDYWGQGTLVTVSS; or h) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGTNKYYADSVKGRFTISRDNSKNTLYLQVNSLRAEDTAVYYCARDPSLWFGEFPHYYGMDVWGQGTTVTVSS; or i) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVLTQSPATHLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTVSSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIQHSGSTNYKPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAELTGDSLLLFEYWGQGTLVTVSS; or j) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVMTQSPATTLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSDWPTFGGGTKVEIR; the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDTSKNTLYLQMNSLRAEDTAVYYCARDSASDYFDYWGQGTLVTVSS; or k) the VL region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence EIVLTQSPATHLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTVSSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK; The antibody or antigen-binding fragment thereof of claim 1, wherein the VH region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYKPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAELTGDSLLLFEYWGQGTLVTVSS.

4. The antibody or antigen-binding fragment or variant thereof according to any one of claims 1 to 3, wherein the fragment comprises F(ab), Fv, or scFv.

5. The antibody or antigen-binding fragment or variant thereof according to any one of claims 1 to 3, wherein the fragment comprises a VhH.

6. A therapeutic antibody that binds to epidermal growth factor-like repeats and discoidin I-like domain 3 (EDIL3) protein, comprising a variable domain and a constant domain, wherein the constant domain is IgG and the variable domain comprises framework regions and complementarity determining means for binding to the epidermal growth factor-like repeats and discoidin I-like domain 3 (EDIL3) protein.

7. The therapeutic antibody of claim 6, wherein the constant region is IgG4.

8. The therapeutic antibody of any one of claims 6 to 7, comprising any one of the antibodies listed in Tables 1 to 4.

9. A pharmaceutical composition comprising the antibody of any one of claims 1 to 8 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

10. 10. A method of treating cancer in a subject, said method comprising administering to a subject in need thereof an antibody according to any one of claims 1 to 8, or an effective amount of the pharmaceutical composition according to claim 9.

11. 11. The method of claim 10, further comprising administering to the subject an angiogenesis inhibitor, a checkpoint blockade inhibitor, or a combination thereof.

12. 12. The method of claim 11, wherein the angiogenesis inhibitor comprises bevacizumab.

13. 12. The method of claim 11, wherein the checkpoint blockade inhibitor comprises ipilimumab.

14. Use of the antibody according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 for treating cancer.

15. The antibody according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 for use in treating cancer.

16. A method for reducing the immunosuppressive effect of cancer-associated fibroblasts in a subject, comprising administering to the subject an antibody described in any one of claims 1 to 8, or an effective amount of the pharmaceutical composition described in claim 9.

17. Use of the antibody according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 for reducing the immunosuppressive effect of cancer-associated fibroblasts.

18. The antibody according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9, for use in reducing the immunosuppressive effect of cancer-associated fibroblasts.

19. A nucleic acid encoding the antibody of claim 1 or claim 2.

20. a) the nucleic acid sequence encoding HCDR1 is AGCTATGCCATGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GCTATTAGTGATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is GAGGGTTTGATTACGTTTGGGGAGTTATCGTTTATAGGCTACTTTGACTAC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is CAGGCGAGTCAGGACATTAGCAACTATTTAAAT or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATGCATCCAATTTGGAAACA or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAACAGTATGATAATCTCCCGATCACC or a degenerate variant thereof; or b) the nucleic acid sequence encoding HCDR1 is AGCTATTGGATGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is AATATAAAGCAAGATGGAAGTCAGAAATACTATGTGGACTCTGTGAAGGGC or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is CGTGGGAACTTCTTCTTTGACAAT or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGTATGTTAGCAGCTACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATGCATCCAACAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGCGTAACAACTGGCCTCCGACG or a degenerate variant thereof; or c) the nucleic acid sequence encoding HCDR1 is AATCACTACTGGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is TATATCTATTACAGTGGGAGCACCAACTACAACCCCCTCCCTCAAGAGT or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is GGGTTTGCTTAC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is CGGGCGAGTCAGGGCATTACCAATTATTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GCTGCATCCACTTTGCAATCA or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAAAAGTATAACAGTGCCCCGTGGACG or a degenerate variant thereof; or d) the nucleic acid sequence encoding HCDR1 is AGCTATGCCATGAAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GCTATCAGTGGCAGTGGTGATAGCACATACTCCACAGACTCCGTGAAGGGC or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is GAGTATTACGATATTTTGACTGGTTATTGGGACTGGTACTTCGATCTC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTATTAACAGCAACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GGTGCATCCACCAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGTATAATAACTGGCCGCTCACT or a degenerate variant thereof; or e) the nucleic acid sequence encoding HCDR1 is AGCTATGGCATGCAT or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GTTATGTGGTATGATGGAAGTGATAGATACTCTGCAGACTCCGTGAAGGGC or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is GGGTACGATATTTTGACTGGTCCCGACCACTTTGACTAC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is CGGGCAAGTCAGAGCATTAGCAGTTATTTAAAT or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GCTGCATCCAGTTTGCAAAGT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAACAGAGTTACAGTACCCCGCTCACT or a degenerate variant thereof; or f) the nucleic acid sequence encoding HCDR1 is AGCTATGACATGAAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is ACTATTAGTGGTAGTGGTAGTCACACATACTACGCAGACTCCGTGAGGGC or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is GAGGGGGGAGCTACTGCTTTTGATATC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is CGGGCCAGTCAGGGCATTAGCAGTTATTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GTTGCATCCACTTTGCAAAGT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAACAGCTTAATAATTACCCCACT or a degenerate variant thereof; or g) the nucleic acid sequence encoding HCDR1 is ACCTATGGCATGCAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is CTTATATGGTATGATGGAATTAATAAATACTATGCGGACTCCGTGAAGGGC or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is CCCTATTACGATATTTTGACTGGTTATTTTGACTAC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTGATAGCAGCAGCTACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GGTACATCCAGTAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGTATGGTAGCTCACCGCTCACT or a degenerate variant thereof; or h) the nucleic acid sequence encoding HCDR1 is AGCTATGGCATGCAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GTTATATGGTATGATGGAACTAATAAATACTATGCAGACTCCGTGAAGGGC or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is GATCCCTCCTTATGGTTCGGGGAGTTCCCTCATTACTACGGTATGGACGTC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is CAGGCGAGTCAGGACATTAGCAATTATTTAAAT or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATGCATCCAATTTGGAAACA or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAACAGTATGATAATCTCCCGCTCACT or a degenerate variant thereof; or i) the nucleic acid sequence encoding HCDR1 is GGTTACTACTGGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GAAATCCAACATAGTGGAAGCACCAACTACAAACCGTCCCTCAAGAGT or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is CTAACTGGGGATTCCCTTTTGTTTGAGTAC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATACATCCAACAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGCGTAGCAACTGGCCGATCACC or a degenerate variant thereof; or j) the nucleic acid sequence encoding HCDR1 is AGCTATGGCATGCAC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGC or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is GATAGCGCCTCCGACTACTTTGACTAC or a degenerate variant thereof; the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GGTGCATCCACCAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGTATAGTGACTGGCCCACT or a degenerate variant thereof; or k) the nucleic acid sequence encoding HCDR1 is GGTTACTACTGGAGC or a degenerate variant thereof, the nucleic acid sequence encoding HCDR2 is GAAATCAATCATAGTGGAAGCACCAACTACAAACCGTCCCTCAAGAGT or a degenerate variant thereof, and the nucleic acid sequence encoding HCDR3 is CTAACTGGGGATTCCCTTTTGTTTGAGTAC or a degenerate variant thereof; 20. The nucleic acid of claim 19, wherein the nucleic acid sequence encoding LCDR1 is AGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCC or a degenerate variant thereof, the nucleic acid sequence encoding LCDR2 is GATACATCCAACAGGGCCACT or a degenerate variant thereof, and the nucleic acid sequence encoding LCDR3 is CAGCAGCGTAGCAACTGGCCGATCACC or a degenerate variant thereof.

21. a) the nucleic acid sequence encoding LCVR is: GACATCCAGATGACCCAGTCTCCATCCTCCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTT AGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGGCTGGAGTGGGTCTCAGCTATTAGTGATAGTGGTGGTAGCACATACTACGCAGACTCCGT GAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAATCAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCAGGAGGGTTTGATTACGTTTGGGGAGTTATCGTTATAGGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCCA or a degenerate variant thereof; or b) the nucleic acid sequence encoding the LCVR is GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGGAAAGAGCCACCCTCTCTCCTGCAGGGCCAGTCAGTATGTTAGCAGCTACTTAGCCTGGTACCACCAGAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGATGCATCCAACA GGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAACAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC ACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTGGCCAATATAAAGCAAGATGGAAGTCAGAAATACT ATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTACGAGACGTGGGAACTTCTTCTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof; or c) the nucleic acid sequence encoding LCVR is selected from the group consisting of: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTACCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTT TGCAATCAGGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAT or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACTTGCACTGTCTCTGG TGGCTCCATCAGTAATCACTACTGGAGCTGGATTCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCAC CAACTACAACCCCTCCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGGGGGTTTGCTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCTTCA or a degenerate variant thereof; or d) the nucleic acid sequence encoding the LCVR is GAAATAGTGATGACGCTGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGGAAAGAGCCACCCTCTCTCCTGCAGGGCCAGTCAGAGTATTAACAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCA GGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGATTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is GAGGTGCAGCTGTTGGAATCTGGGGGAGGCTTGGGACAGCCTGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTT AGCAGCTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGGAAGGGGCTGGAGTGGGTCTCAGCTATCAGTGGCAGTGGTGATAGCACATACTCCACAGACTCCG TGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAAAGAGTATTACGATATTTTGACTGGTTATTGGGACTGGTACTTCGATCTCTGGGGCCAAGGCACCCTGGTCACTGTCTCCTCA or a degenerate variant thereof; or e) the nucleic acid sequence encoding LCVR is TGCAAAGTGGGGTCCCATCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGAGTTACAGTACCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACC TTCAGTAGCTATGGCATGCATTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTGGTATGATGGAAGTGATAGATACTCTGCAGA CTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATTGCAAATGAAATCAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGGGGTACGATATTTTGACTGGTCCCCGACCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof; or f) the nucleic acid sequence encoding the LCVR is selected from the group consisting of GACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTATCAGCAAAACCAGGGAAAGCCCCCTAAGCTCCTGATCTATGTTGCATCCAC TTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATAATTACCCCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is GAAGTACAGTTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC ACCTTTAGCAGCTATGACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCCGGTGTGGGTCCAACTATTAGTGGTAGTGGTAGTCACACATACTA CGCAGACTCCGTGAGGGGCCGGTTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTGCAAATGAAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGGGAGCTACTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTTCA or a degenerate variant thereof; or g) the nucleic acid sequence encoding LCVR is GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGGAAAGAGCCACCCTCTCTCCTGCAGGGCCAGTCAGAGTGATAGCAGCAGCTACTTAGCCTGGTATCAGCAGAAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTACATCCAG TAGGGCCACTGGCATCTCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACC TTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATGGTATGATGGAATTAATAAATAACTATGCGG ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGACCCTATTACGATATTTGACTGGTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof; or h) the nucleic acid sequence encoding LCVR is selected from the group consisting of GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAATTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTC AGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAACTAATAAAATACTATGCAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAGTGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCCCTCCTTATGGTTCGGGGAGTTCCCTCATTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCCA or a degenerate variant thereof; or i) the nucleic acid sequence encoding LCVR is GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGGAAAGAGCCACCCTCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATACATCCAACA GGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCGTCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGG TCCTTCAGTGGTTACTACTGGAGCTGGATACGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCCAACATAGTGGAAGCACCAACTACAA ACCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGGAGCTAACTGGGGATTCCCTTTTGTTTGAGTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCCA or a degenerate variant thereof; or j) the nucleic acid sequence encoding LCVR is GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGGAAAGAGCCACCCTCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAC CAGGGCCACTGGTATCCCAGCCAGATTCAGTGGCAGTGGGTCTGGGACAGAGATTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAGTGACTGGCCCACTTTCGGCGGAGGGACCAAGGTGGAGATCAGA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTC ACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAAATACTA TGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACACTTCCAAGAACACACTGTATCTGCAAATGAAATACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGCGCCTCCGACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA or a degenerate variant thereof; or k) the nucleic acid sequence encoding LCVR is GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGGAAAGAGCCACCCTCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATACATCCAACA GGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCGTCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA or a degenerate variant thereof; The nucleic acid sequence encoding HCVR is CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTC CTTCAGTGGTTACTACTGGAGCTGGATACGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAAAACCG 20. The nucleic acid of claim 19, which is TCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGGAGCTAACTGGGGATTCCCTTTTGTTTGAGTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCCA or a degenerate variant thereof.

22. A vector comprising the nucleic acid according to any one of claims 19 to 21.

23. A cell comprising the nucleic acid according to any one of claims 19 to 21 or the vector according to claim 22.

Images