Anti-ILT4 Compositions and Methods

Abstract

Provided herein is an antibody or antigen-binding fragment thereof that binds to ILT4.Described herein is an antibody or antigen-binding fragment thereof that antagonizes the interaction between ILT4 and HLA-G and / or HLA-A.Also provided herein is a method for treating autoimmune disorder, neoplastic disorder, or inflammatory disorder by administering an antibody or antigen-binding fragment thereof that binds to ILT4 alone or in combination with additional therapeutic agent.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 324,544, filed March 28, 2022, and U.S. Provisional Patent Application No. 63 / 374,250, filed September 1, 2022, the disclosures of which are incorporated by reference in their entireties herein.

[0002] Sequence Listing The Sequence Listing submitted with this application by EFS, entitled "4494-143PCT.xml", created on March 23, 2023, and having a size of 130,000 bytes, is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to the field of biotechnology, and more specifically to anti-ILT4 antibodies and antigen-binding fragments thereof, as well as methods and compositions of anti-ILT4 antibodies and antigen-binding fragments. [Background technology]

[0004] Immunoglobulin-like transcript 4 (ILT4), also known as leukocyte immunoglobulin-like receptor B2 (LILRB2), is an immunosuppressive molecule that is expressed primarily in myeloid cells, including monocytes, macrophages, dendritic cells, and granulocytes, tumor cells, and stromal cells. ILT4 is abundant in tumor and stromal cells in the tumor microenvironment of certain malignancies. ILT4 expression and signaling in myeloid cells creates a tumor-suppressive microenvironment that favors tumor progression. ILT4 has been shown to bind to a variety of ligands, including human leukocyte antigen G (HLA)-G, major histocompatibility complex class I (MHC-I) proteins such as (HLA-A), angiopoietin-like protein (AngptI), Nogo66, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), β-amyloid, semaphorin 4A (Sema4A), CD1c / d, and complement split products (CSPs). Among the endogenous ligands of ILT4, human leukocyte antigen G (HLA)-G, a non-classical MHC class I molecule, is expressed by a wide range of tumors. HLA-G has been shown to mediate immune tolerance in cancer through its interaction with ILT4. HLA-G binding to ILT4 can also directly inhibit the function of monocytes, dendritic cells, and neutrophils. As a result, tumor cells can evade innate and adaptive immune responses by expressing (HLA)-G. Mechanisms of immune tolerance resulting from HLA-G / ILT4 interactions include impaired proliferation, differentiation, cytotoxicity, cytokine secretion, and chemotaxis of immune cells. Interaction between HLA-G and monocytes by ILT4 inhibits maturation of human monocyte-derived antigen-presenting cells (APCs), leading to reduced expression of MHC class II antigens and costimulatory molecules via Stat3 activation. Blocking ILT4 can shift suppressed macrophages (M2) to an activated state (M1-like).

[0005] Immune checkpoint inhibitors have become state-of-the-art treatments for a variety of malignancies. These include programmed cell death protein 1 (PD-1) / programmed cell death ligand 1 (PD-L1) inhibitors, which are used as both first-line and second-line treatments. PD-1, the programmed cell death 1 protein, is a member of the CD28 family and an immunosuppressive receptor expressed on the surface of activated T cells and B cells. The interaction between PD1 and PD-L1 can be blocked to significantly improve the tumor-killing activity of CD8+ cytotoxic T cells. In particular, PD-1 / PD-L1 inhibitors are used to treat patients with non-small cell lung cancer (NSCLC). For example, compared with conventional therapy, PD-1 / PD-L1 inhibitor monotherapy can significantly prolong survival without the levels of side effects observed with previous therapies used to treat advanced NSCLC. However, some patients harbor tumors that are refractory to PD-1 / PD-L1 inhibitors or that become refractory to PD-1 / PD-L1 inhibitors following treatment and may evade T cell-mediated tumor responses.

[0006] Thus, there is an unmet need for inhibitors of ILT4 that can block the HLA-G / ILT4-mediated immunosuppressive effects on myeloid cell populations in the tumor microenvironment. Summary of the Invention [Problem to be solved by the invention]

[0007] The leukocyte immunoglobulin-like receptor (LILR) family contains activating and inhibitory members that can upregulate or downregulate immune cell activity. Inhibitory LILR family members include LILRB1 (CD85j / ILT2), LILRB2 (CD85d / ILT4), LILRB3 (CD85a / ILT5), LILRB4 (CD85K, ILT3), and LILRB5 (CD85C), while activating LILR family members include, but are not limited to, LILRA1 (CD85l), LILRA2 (CD85h / ILT1), LILRA4 (CD85g / ILT7), LILRA5 (CD85f), and LILRA6 (CD85b). LILRA3 (CD85e / ILT6) is exclusively expressed in soluble form. [Means for solving the problem]

[0008] The present disclosure relates to antibodies or antigen-binding fragments thereof that bind to ILT4, i.e., LILRB2. In some embodiments, the antibody or antigen-binding fragment thereof that binds ILT4 comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of DYYMN (SEQ ID NO: 1), GYSVN (SEQ ID NO: 9), DSYMN (SEQ ID NO: 23), GYFMN (SEQ ID NO: 30), SYWMN (SEQ ID NO: 38), DYTIH (SEQ ID NO: 46), DNYLQ (SEQ ID NO: 52), DYGMH (SEQ ID NO: 60), and TYGMS (SEQ ID NO: 68), DINPNNGGTSYNQKFKG (SEQ ID NO: 2), RINPYNGDIFNNQKFKG (SEQ ID NO: 10), RIYPGVYRTHYNEKFKD (SEQ ID NO: 17), YINPDNGVTRYNQKFKG (SEQ ID NO: 24), RINPYNGDIFYNQKFKG (SEQ ID NO: 31), QIYPGHGDTNYNGKFKG (SEQ ID NO: 39), WFYPGTVSIKYNEKFKD (SEQ ID NO: 69), or a CDR-H2 comprising an amino acid sequence selected from the group consisting of: 47), PGSGNTYYSDNFTG (SEQ ID NO:53), YISSDSSTIYYADTVKG (SEQ ID NO:61), and WINTYSGEPTYADEFKG (SEQ ID NO:69), and a CDR-H3 comprising an amino acid sequence selected from the group consisting of GGAELTGTYWYFDV (SEQ ID NO:3), GTTVGGAWFAY (SEQ ID NO:11), SGYYGGTYEEDAMDY (SEQ ID NO:18), EGTITTDLSWFAY (SEQ ID NO:25), GITVAAGSFDV (SEQ ID NO:32), EGSELGRLFAY (SEQ ID NO:40), HEHPHYYGDSYDAMGY (SEQ ID NO:48), STVVYFDV (SEQ ID NO:54), RAAQGYVMDY (SEQ ID NO:62), and RGYDGYYYTMDY (SEQ ID NO:70).In some embodiments, the light chain variable domain comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of RASENIYSNLA (SEQ ID NO: 4), RASESVDSYGYSFLH (SEQ ID NO: 12), RASESVDNYGNTFMH (SEQ ID NO: 33), SASSSVSFMY (SEQ ID NO: 41), SNYAN (SEQ ID NO: 55), and KASQSVSDDVA (SEQ ID NO: 63), GATNLAD (SEQ ID NO: 5), LASNLES (SEQ ID NO: 13), AATSLAD (SEQ ID NO: 19), ASTNLAD (SEQ ID NO: 26), RASNLES (SEQ ID NO: 34), LTSNLAS (SEQ ID NO: 42), (SEQ ID NO: 53), GTNRAP (SEQ ID NO:56), ASNRYT (SEQ ID NO:64), and AATNLAD (SEQ ID NO:71), and a CDR-L3 comprising an amino acid sequence selected from the group consisting of QHFWDSPFT (SEQ ID NO:6), QQSNEDLMYT (SEQ ID NO:14), QNFWDTPYT (SEQ ID NO:20), QHFWDTPYT (SEQ ID NO:27), QQSSDHPLT (SEQ ID NO:35), QQWSSNPPT (SEQ ID NO:43), QHFWGTPYT (SEQ ID NO:49), WYSNHWV (SEQ ID NO:57), QQDYGSPT (SEQ ID NO:65), and QHFFGAPWT (SEQ ID NO:72).

[0009] In some embodiments, an antigen or antigen-binding fragment thereof that binds to ILT4 is selected from the group consisting of CDR-H1 sequences of DYYMN (SEQ ID NO: 1), GYSVN (SEQ ID NO: 9), DSYMN (SEQ ID NO: 23), or GYFMN (SEQ ID NO: 30), CDR-H2 sequences of DINPNNGGTSYNQKFKG (SEQ ID NO: 2), RINPYNGDIFNNQKFKG (SEQ ID NO: 10), RIYPGVYRTHYNEKFKD (SEQ ID NO: 17), YINPDNGVTRYNQKFKG (SEQ ID NO: 24), or RINPYNGDIFYNQKFKG (SEQ ID NO: 31), or CDR-H2 sequences of GGAELTGTYWYFDV (SEQ ID NO: 3), GTTVGGAWFAY (SEQ ID NO: 11), SGYYGGTYEEDAMDY (SEQ ID NO: 18), EGTITTDLSWFAY (SEQ ID NO: 19), or CDR-H3 sequences of GGAELTGTYWYFDV (SEQ ID NO: 3), GTTVGGAWFAY (SEQ ID NO: 11), SGYYGGTYEEDAMDY (SEQ ID NO: 18), EGTITTDLSWFAY (SEQ ID NO: 20), or CDR-H4 sequences of GGAELTGTYWYFDV (SEQ ID NO: 3), GTTVGGAWFAY (SEQ ID NO: 11), SGYYGGTYEEDAMDY (SEQ ID NO: 19), EGTITTDLSWFAY (SEQ ID NO: 21), or CDR-H5 sequences of GGAELTGTYWYFDV (SEQ ID NO: 3), GTTVGGAWFAY (SEQ ID NO: 22), SGYYGGTYEEDAMDY (SEQ ID NO: 23), and the CDR-L1 sequence of RASENIYSNLA (SEQ ID NO: 4), RASESVDSYGYSFLH (SEQ ID NO: 12), or RASESVDNYGNTFMH (SEQ ID NO: 33); the CDR-L2 sequence of GATNLAD (SEQ ID NO: 5), LASNLES (SEQ ID NO: 13), AATSLAD (SEQ ID NO: 19), ASTNLAD (SEQ ID NO: 26), or RASNLES (SEQ ID NO: 34); and the CDR-L3 sequence of QHFWDSPFT (SEQ ID NO: 6), QQSNEDLMYT (SEQ ID NO: 14), QNFWDTPYT (SEQ ID NO: 20), QHFWDTPYT (SEQ ID NO: 27), or QQSSDHPLT (SEQ ID NO: 35).

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable domain CDR comprising DYYMN (SEQ ID NO: 1), DINPNNGGTSYNQKFKG (SEQ ID NO: 2), and GGAELTGTYWYFDV (SEQ ID NO: 3), and a light chain variable domain CDR comprising RASENIYSNLA (SEQ ID NO: 4), GATNLAD (SEQ ID NO: 5), and QHFWDSPFT (SEQ ID NO: 6); (b) a heavy chain variable domain CDR comprising GYSVN (SEQ ID NO: 9), RINPYNGDIFNNQKFKG (SEQ ID NO: 10), and GTTVGGAWFAY (SEQ ID NO: 11), and a light chain variable domain CDR comprising RASESVDSYGYSFLH (SEQ ID NO: 12), LASNLES (SEQ ID NO: 13), and QQSNEDLMYT (SEQ ID NO: 14); (c) a DYYMN (SEQ ID NO: 1), RIYPGVYRTHYNEKFKD (SEQ ID NO: 17), and SGYYGGTYEEDAMDY (SEQ ID NO: 18). (d) heavy chain variable domain CDRs comprising DYYMN (SEQ ID NO: 1), RIYPGVYRTHYNEKFKD (SEQ ID NO: 17), and SGYYGGTYEEDAMDY (SEQ ID NO: 18), and light chain variable domain CDRs comprising RASENIYSNLA (SEQ ID NO: 4), AATSLAD (SEQ ID NO: 19), and QNFWDTPYT (SEQ ID NO: 20); or (e) heavy chain variable domain CDRs comprising DSYMN (SEQ ID NO: 23), YINPDNGVTRYNQKFKG (SEQ ID NO: 24), and EGTITTDLSWFAY (SEQ ID NO: 25), and light chain variable domain CDRs comprising RASENIYSNLA (SEQ ID NO: 4), ASTNLAD (SEQ ID NO: 26), and QHFWDTPYT (SEQ ID NO: 27).

[0011] In some embodiments, the antibody or antigen-binding fragment thereof has the following amino acid sequence: DIQMTQSPASLSISVGETVTITCRASENIYSNLAWYQQKQGKSPQVLVYGATNLADGVPSRFSGSGSGTQYSLKIKSLQSEDFGSYYCQHFWDSPFTFGSGTKLEIK (SEQ ID NO: 8), VIVLTQSPASLAVSLGQRAAISCRASESVDSYGYSFLHWYQQKPGQPPKLLIYLASNLESGIPARFSGSGSGTDFTLTINPVEADDVATYYCQQSNEDLMYTFGGGTKLEIK (SEQ ID NO: 16), DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATSLADGVPSRFRGSGSGTQYSLKISSLQSEDFGNYYCQNFWDTPYTFGGGTKLEIK (SEQ ID NO: 22), DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYASTNLADGAPATFSGSGSGTQYSLKINSLQSVDFGSYYCQHFWDTPYTFGGGTKLEIK (SEQ ID NO: 29), DIVLTQSPASLAVSLGQRATISCRASESVDNYGNTFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSKTDFTLTINPVEADDVATYYCQQSSDHPLTFGAGTKLELS (SEQ ID NO: 37), QIVLTQSPALMSASPGEKVTMTCSASSSVSFMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK (SEQ ID NO: 45), DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFGGSGSGTQYSLKINSLQPEDFGSYYCQHFWGTPYTFGGGTKLEIT (SEQ ID NO:51), QAVVTQESALTTSPGETVTLTCRSSTGTVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDQAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO:59), SIVMTQTPKFLLVSAGDRVTITCKASQSVSDDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYGSPTFGGGTKLEIK (SEQ ID NO: 67), or DIQMTQSPASLSASVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVSAATNLADGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHFFGAPWTFGGGTKLEIK (SEQ ID NO:74), and a light chain variable domain comprising an amino acid sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to: EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGGAELTGTYWYFDVWGTGTTVTVSS (SEQ ID NO: 7), DVQLQQSGPELVKPGNSVKISCKAAGYSFTGYSVNWVKERHGKSLEWIGRINPYNGDIFNNQKFKGKATLTVDKSSSTAHMELRSLTSEDSAVYYCARGTTVGGAWFAYWGQGTLVTVSA (SEQ ID NO: 15), QVQLKQSGAELVRPGASVKLSCRASGYTFTDYYMNWVKQRPGQGLEWIARIYPGVYRTHYNEKFKDKATLTAEKSSSTAYMELSSLTSEDSAVYFCARSGYYGGTYEEDAMDYWGQGTSVTVSS (SEQ ID NO: 21), EVQLQQSGPELVIPGASVKISCKASGYTFTDSYMNWVKQSHGKSLEWIAYINPDNGVTRYNQKFKGKATLTVHKSSSTAYMELRSLTSEDSAVYYCAREGTITTDLSWFAYWGQGTLVTVSA (SEQ ID NO: 28), EVHLQQSGPELVKPGASVKISCKASGYSFIGYFMNWMKQSHGKSLEWIGRINPYNGDIFYNQKFKGKATLTVDKSSTTAHMDLLSLTSEDFAVYYCARGITVAAGSFDVWGTGTTVTVSS (SEQ ID NO: 36), QVQLQQSGAELVKPGASVKISCKASGYAFSSYWMNWVKQRPGKGLEWIGQIYPGHGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAKEGSELGRLFAYWGQGTLVTVSA (SEQ ID NO: 44), QVQLQQSGTELVKPGASVKLSCKASGYIFTDYTIHWVKQRSGQGLEWIGWFYPGTVSIKYNEKFKDKATLTADRSSSIVYMELSRLTSEDSGVYFCARHEHPHYYGDSYDAMGYWGQGTSVTVSS (SEQ ID NO: 50), QVQLQQSGPELVKPGASVKISCKASGYIFTDNYLQWVKQRPGQGLEWIGWIFPGSGNTYYSDNFTGKATLTVDKSSITAYMLLSSLTSEDSAVYFCSRSTVVYFDVWGTGTTVTVSS (SEQ ID NO: 58), EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKRLEWVAYISSDSSTIYYADTVKGRFTISRDNAKNTLFLEMTSLRSEDTAMYYCARRAAQGYVMDYWGQGTSVTVSS (SEQ ID NO: 66), or QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMAWINTYSGEPTYADEFKGRFAFSLETSVSTAYLQINNLKNEDTATYFCARRGYDGYYYTMDYWGQGTSVTVSS (SEQ ID NO: 73), comprising a heavy chain variable domain comprising amino acids that are at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMAWINTYSGEPTYADEFKGRFAFSLETSVSTAYLQINNLKNEDTATYFCARRGYDGYYYTMDYWGQGTSVTVSS (SEQ ID NO: 73).

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:8, 16, 22, 29, or 37, and a heavy chain variable domain comprising an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:7, 15, 21, 28, or 36.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 8, 16, 22, 29, or 37, and a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 7, 15, 21, 28, or 36.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable domain comprising the amino acid sequence of: EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGGAELTGTYWYFDVWGTGTTVTVSS (SEQ ID NO: 7); and (b) a light chain variable domain comprising the amino acid sequence of QVLVYGATNLADGVPSRFSGSGSGTQYSLKIKSLQSEDFGSYYCQHFWDSPFTFGSGTKLEIK (SEQ ID NO: 8); (c) a light chain variable domain comprising the amino acid sequence of DVQLQQSGPELVKPGNSVKISCKAAGYSFTGYSVNWVKERHGKSLEWIGRINPYNGDIFNNQKFKGKATLTVDKSSSTAHMELRSLTSEDSAVYYCARGTTVGGAWFAYWGQGTLVTVSA (SEQ ID NO: 15); a heavy chain variable domain comprising the amino acid sequence of VIVLTQSPASLAVSLGQRAAISCRASESVDSYGYSFLHWYQQKPGQPPKLLIYLASNLESGIPARFSGSGSGTDFTLTINPVEADDVATYYCQQSNEDLMYTFGGGTKLEIK (SEQ ID NO: 16); A heavy chain variable domain comprising the amino acid sequence of KDKATLTAEKSSSTAYMELSSLTSEDSAVYFCARSGYYGGTYEEDAMDYWGQGTSVTVSS (SEQ ID NO: 21), and a light chain variable domain comprising the amino acid sequence of DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATSLADGVPSRFRGSGSGTQYSLKISSLQSEDFGNYYCQNFWDTPYTFGGGTKLEIK (SEQ ID NO: 22);(d) a heavy chain variable domain comprising the amino acid sequence of EVQLQQSGPELVIPGASVKISCKASGYTFTDSYMNWVKQSHGKSLEWIAYINPDNGVTRYNQKFKGKATLTVHKSSSTAYMELRSLTSEDSAVYYCAREGTITTDLSWFAYWGQGTLVTVSA (SEQ ID NO: 28), and a light chain variable domain comprising the amino acid sequence of DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYASTNLADGAPATFSGSGSGTQYSLKINSLQSVDFGSYYCQHFWDTPYTFGGGTKLEIK (SEQ ID NO: 29); or e) a heavy chain variable domain comprising the amino acid sequence of EVHLQQSGPELVKPGASVKISCKASGYSFIGYFMNWMKQSHGKSLEWIGRINPYNGDIFYNQKFKGKATLTVDKSSTTAHMDLLSLTSEDFAVYYCARGITVAAGSFDVWGTGTTVTVSS (SEQ ID NO: 36), and a light chain variable domain comprising the amino acid sequence of DIVLTQSPASLAVSLGQRATISCRASESVDNYGNTFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSKTDFTLTINPVEADDVATYYCQQSSDHPLTFGAGTKLELS (SEQ ID NO: 37).

[0015] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 comprising an amino acid sequence of X1X2X3X4N, X1IX2PX3X4X5X6X7X8X9NX 10 KFKX 11 (SEQ ID NO: 76) (wherein X1 is R, D or Y, X2 is N or Y, X3 is G, N, D or Y, X4 is V or N, X5 is G or Y, X6 is R, G, V or D, X7 is I or T, X8 is H, S, R or F, X9 is Y or N, and X 10 is E or Q, and X 11X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 (wherein X1 is G, S or E, X2 is G, T or I, X3 is A, T or Y, X4 is E, V, Y or I, X5 is L, G, T or A, X6 is T, G or A, X7 is G, A, T or D, X8 is T, W, Y, L or S, X9 is Y, F, E or S, and X 10 is W, A, E or D, and X 11 is Y, D, F or V, and X 12 is F, A, or absent, and X 13 is D, M, Y, or absent, and X 14 is V, D or absent, and X 15 is Y, or absent).

[0016] In one embodiment, the antibody or antigen-binding fragment thereof is 10 (SEQ ID NO: 78) (wherein X1 is N or S, X2 is I or V, X3 is Y or D, X4 is S or Y, X5 is N or Y, X6 is L or G, X7 is A, Y or N, X8 is S, T or not present, X9 is F or not present, and X 10X1QX2X3X4DX5X6X7T (wherein X1 is absent or Q, X2 is S, H, N or Q, X3 is N, F or S, X4 is E, W or S, X5 is L, S, T or H, X6 is M or P, and X7 is Y, F or L).

[0017] In some embodiments, an antibody or antigen-binding fragment thereof that binds ILT4 comprises heavy chain variable domain CDRs comprising DYYMN (SEQ ID NO: 1), IYPGVYRT (SEQ ID NO: 113), and SGYYGGTYEEDAMDY (SEQ ID NO: 18), and light chain variable domain CDRs comprising ENIYSN (SEQ ID NO: 114), AAT, and QNFWDTPYT (SEQ ID NO: 20). In some embodiments, an antibody or antigen-binding fragment thereof that binds ILT4 comprises heavy chain variable domain CDRs comprising DYYMN (SEQ ID NO: 1), INPNNGGT (SEQ ID NO: 116), and GGAELTGTYWYFDV (SEQ ID NO: 3), and light chain variable domain CDRs comprising ENIYS (SEQ ID NO: 117), GAT, and QHFWDSPFT (SEQ ID NO: 6). In some embodiments, an antibody or antigen-binding fragment thereof that binds ILT4 comprises heavy chain variable domain CDRs comprising GYSVN (SEQ ID NO:9), INPYNGDI (SEQ ID NO:119), and GTTVGGAWFAY (SEQ ID NO:11), and light chain variable domain CDRs comprising ESVDSYGYSF (SEQ ID NO:120), LAS, and QQSNEDLMYT (SEQ ID NO:14). In some embodiments, an antibody or antigen-binding fragment thereof that binds ILT4 comprises heavy chain variable domain CDRs comprising GYFMN (SEQ ID NO:30), INPYNGDI (SEQ ID NO:119), and GITVAAGSFDV (SEQ ID NO:32), and light chain variable domain CDRs comprising ESVDNYGNTF (SEQ ID NO:122), RAS, and QQSSDHPLT (SEQ ID NO:35).

[0018] The present disclosure also relates to a method for treating a subject with an autoimmune disorder, a neoplastic disorder, or an inflammatory disorder, comprising administering an antibody or an antigen-binding fragment that binds to ILT4. In some aspects, the present disclosure relates to a method for treating a subject with cancer. In some embodiments, the cancer is a solid cancer or a liquid cancer. In one aspect, the cancer is a carcinoma or a sarcoma. In particular, the median ILT4 expression is particularly high in non-small cell lung cancer (NSCLC) compared to other cancers, and NSCLC patients with high ILT4 expression have a poor prognosis compared to NSCLC patients with low ILT4 expression.

[0019] In some embodiments, the method includes a) blocking the interaction of ILT4 with HLA-G, b) downregulating ILT4 activity or HLA-G levels, c) removing immune suppression by ILT4 or HLA-G, d) enhancing monocyte activation, or e) any combination of a)-d). In some embodiments, the method includes treating or reducing the severity of cancer by reversing or reducing immune suppression by administering an antibody or antigen-binding fragment thereof that binds ILT4. In some embodiments, reversing or reducing immune suppression includes reprogramming bone marrow cells by administering an antibody or antigen-binding fragment thereof that binds ILT4. In some aspects, the present disclosure includes improving T cell activation in a subject by administering an antibody or antigen-binding fragment thereof of the present disclosure. In some aspects, the present disclosure includes improving antigen presentation for T cell priming in a subject by administering an antibody or antigen-binding fragment thereof of the present disclosure. In some aspects, the present disclosure includes promoting macrophage reprogramming in a subject by administering an antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the present disclosure includes activating dendritic cells in a subject by administering an antibody or antigen-binding fragment of the present disclosure. In some embodiments, the present disclosure includes increasing T cell costimulation in a subject by administering an antibody or antigen-binding fragment of the present disclosure.

[0020] The present disclosure also relates to a method of inducing apoptosis of a cancer cell or a cancer stem cell comprising contacting the cancer cell or a cancer stem cell with an antibody or antigen-binding fragment thereof that binds to ILT4.

[0021] Other features and characteristics of the subject matter of the present disclosure, as well as its method of operation, function of associated elements of construction and combination of parts, and economies of manufacture, will become more apparent from a consideration of the following description and the accompanying drawings, sequence listing, and claims, all of which form a part of this specification. [Brief description of the drawings]

[0022] [Figure 1] 1A and 1B show the levels of TNF-α in the supernatants of monocyte-derived macrophages from (A) donor A and (B) donor B, respectively. Cells were treated with the indicated antibodies at the indicated concentrations (μg / ml).

[0023] [Diagram 2] 2A and 2B show the levels of IL-6 in the supernatants of monocyte-derived macrophages from (A) donor A and (B) donor B, respectively. Cells were treated with the indicated antibodies at the indicated concentrations (μg / ml).

[0024] [Diagram 3] 3A and 3B show the mean fluorescence intensity of CD206 for monocyte-derived macrophages from (A) donor A and (B) donor B, respectively. Cells were treated with the indicated antibodies at the indicated concentrations (μg / ml).

[0025] [Figure 4] Figures 4A and 4B show the mean fluorescence intensity of CD209 for monocyte-derived macrophages from (A) donor A and (B) donor B, respectively. Cells were treated with the indicated antibodies at the indicated concentrations (μg / ml).

[0026] [Diagram 5] FIG. 5 shows cell-associated fluorescence of CHO-S parental and CHO-S / ILT4 cell lines following incubation with PE-conjugated HLA-G.

[0027] [Figure 6] Figure 6 shows cell-associated fluorescence after pre-incubation with the indicated mouse anti-human ILT4 antibodies prior to PE-HLA-G incubation. The results measure HLA-G blockade by candidate mouse anti-human ILT4 antibodies.

[0028] [Figure 7]FIG. 7 shows IC50 curves from an exemplary ILT4 antibody blocking assay using flow cytometry.

[0029] [Figure 8] Figure 8 shows the blocking activity of humanized anti-ILT4 antibodies against HLA-G binding to ILT4-expressing CHO cells. Graph represents n=3. Each data point is the mean of triplicates with SD.

[0030] [Figure 9] Figure 9 shows the blocking activity of humanized anti-ILT4 antibodies against HLA-A binding to ILT4-expressing CHO cells. Graph represents n=3. Each data point is the mean of triplicates with SD.

[0031] [Figure 10] Figure 10 shows the effect of anti-ILT4 mAbs on TNFα production from macrophages. Levels of TNF-α in the supernatants of monocyte-derived macrophages from donor 4 and donor 5, respectively. Cells were treated with the indicated antibodies at 4ug / ml, titrated 1:4 at eight concentration points. Anti-ILT4 antibodies and LPS were added 24 hours prior to evaluation.

[0032] [Figure 11] Figure 11 shows the effect of anti-ILT4 mAbs on IL-6 production from macrophages. Levels of TNF-α in the supernatants of monocyte-derived macrophages from (A) donor 4 and (B) donor 5, respectively. Cells were treated with the indicated antibodies at 4ug / ml, titrated 1:4 at eight concentration points. Anti-ILT4 antibodies and LPS were added 24 hours prior to evaluation.

[0033] [Figure 12]Figure 12 shows the effect of anti-ILT4 mAb on cell surface marker expression on macrophages before and after treatment. Flow cytometry data showing mean fluorescence intensity (MFI) as fold increase over control staining for monocyte-derived macrophages. This data compares expression levels before (media) and after treatment with 4ug / ml of anti-ILT4 antibody against CD206, ILT4, and CD163.

[0034] [Figure 13] Figure 13 shows an MLR assay to assess T cell activation. M2 macrophages and CD4 T cells were plated at a ratio of 1:5 in the presence of anti-ILT4 or anti-ILT4 + anti-PD-1 (Tori). (A) Donor 1 and (B) Donor 2. IgG1 (hIgG1 LALA isotype control).

[0035] [Figure 14] FIG. 14 shows competition of anti-ILT4 mAb binding to ILT4-CHO cells in competition with AF647-Hz45.01-LALA.

[0036] [Figure 15] FIG. 15 plots the data from FIG. 14 on the same graph (left) with Hz45.01-LALA versus 1E1 and HuIgG1-LALA (as controls), and on the same graph (right) with Hz45.01-LALA versus J19h1 and HuIgG1-LALA (as controls).

[0037] [Figure 16] FIG. 16 shows competition of anti-ILT4 mAb binding to ILT4-CHO cells in competition with AF647-Hz156.03-LALA.

[0038] [Figure 17]FIG. 17 plots the data from FIG. 16 on the same graph (left) with Hz45.01-LALA versus 1E1 and HuIgG1-LALA (as controls), and on the same graph (right) with Hz45.01-LALA versus J19h1 and HuIgG1-LALA (as controls).

[0039] [Figure 18] FIG. 18 shows binding of AF647-Hz45.01-LALA and AF647-Hz156.03-LALA to ILT4-CHO cells at various concentrations (0-10 μg / ml) incubated with ILT4-CHO cells and analyzed by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Aspects of the subject matter of the present disclosure may be embodied in a variety of forms, and the following description is intended to disclose only some of these forms as illustrative examples of the subject matter encompassed by the present disclosure, and therefore, the subject matter of the present disclosure is not intended to be limited to the forms or embodiments so described.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other documents mentioned herein are incorporated by reference in their entirety.In case of discrepancy, the present specification, including definitions, will prevail.

[0042] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

[0043] The present disclosure also relates to a humanized immunoglobulin having binding specificity for ILT4, comprising an antigen-binding region of non-human origin and at least a portion of an immunoglobulin of human origin. In one aspect, the humanized immunoglobulin comprises an antigen-binding region of non-human origin that binds to ILT4 and a constant region derived from a human constant region. In another embodiment, the humanized immunoglobulin that binds to ILT4 comprises a complementarity-determining region of non-human origin and a variable framework region of human origin, and may comprise a constant region of human origin. For example, the humanized immunoglobulin may comprise a heavy chain and a light chain, the light chain comprising a complementarity-determining region derived from an antibody of non-human origin that binds to ILT4 and a framework region derived from a light chain of human origin, and the heavy chain comprising a complementarity-determining region derived from an antibody of non-human origin that binds to ILT4 and a framework region derived from a heavy chain of human origin.

[0044] The present disclosure also relates to a humanized immunoglobulin light chain or a humanized immunoglobulin heavy chain. In one embodiment, the present disclosure relates to a humanized light chain comprising one or more light chain CDRs of non-human origin and a human light chain framework region. In another embodiment, the present disclosure relates to a humanized immunoglobulin heavy chain comprising one or more heavy chain CDRs of non-human origin and a human heavy chain framework region. The CDRs can be derived from a non-human immunoglobulin.

[0045] Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kDa) and two identical heavy chains (about 55 or 70 kDa) form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region, which can be distinguished from the more conserved constant (C) region of the remainder of each chain.

[0046] Human immunoglobulins can be divided into classes and subclasses depending on the isotype of the heavy chain. The classes include IgG, IgM, IgA, IgD and IgE, with the heavy chains being of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) type, respectively. The subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, with the heavy chains being of gamma 1, gamma 2, gamma 3, gamma 4, alpha 1 and alpha 2 type, respectively. Human immunoglobulin molecules of a selected class or subclass may contain either kappa (κ) or lambda (λ) light chains.

[0047] Antibodies can be raised against a suitable immunogen in a suitable mammal (e.g., mouse, rat, rabbit or sheep). Antibody-producing cells (e.g., lymphocytes) can be isolated, for example, from the lymph nodes or spleen of the immunized animal. The cells can then be fused to a suitable immortalized cell (e.g., a myeloma cell line), thereby forming a hybridoma. The fused cells can be isolated using selective culture techniques using conventional methods known to those skilled in the art. Cells producing antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA). Immunoglobulins of non-human origin with binding specificity for ILT4 can also be obtained from antibody libraries (e.g., phage libraries containing non-human Fab molecules).

[0048] In one embodiment, the antigen-binding region of the humanized immunoglobulin comprises CDRs of non-human origin. In this embodiment, the humanized immunoglobulin with binding specificity for ILT4 comprises at least one CDR of non-human origin. For example, the CDRs may be derived from the light and heavy chain variable regions of an immunoglobulin of non-human origin, such that the humanized immunoglobulin substantially comprises heavy chain CDR1, CDR2 and / or CDR3, and / or light chain CDR1, CDR2 and / or CDR3 from one or more immunoglobulins of non-human origin, and the resulting humanized immunoglobulin has binding specificity for ILT4. In some aspects, all three CDRs of the selected chain are substantially identical to the CDRs of the corresponding chain of the donor, and in some aspects, all three CDRs of the light and heavy chains are substantially identical to the CDRs of the corresponding donor chain.

[0049] The humanized immunoglobulin or immunoglobulin chain portion (human portion) of human origin can be derived from any suitable human immunoglobulin or immunoglobulin chain. For example, the human constant region or portion can be derived from the kappa or lambda light chain, and / or g (e.g., gamma 1, gamma 2, gamma 3, gamma 4), mu, alpha (e.g., alpha 1, alpha 2), delta or epsilon heavy chain of a human antibody, including allelic variants, if present. A particular constant region (e.g., IgG1), variant or portion thereof can be selected to modulate effector function. For example, a mutated constant region (variant) can be used to minimize the ability to bind to Fc receptors and / or fix complement.

[0050] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a cell" includes one or more cells.

[0051] As used herein, "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate a reasonable deviation from that numerical value as well as values ​​known to one of ordinary skill in the art. For example, ±20%, ±10%, or ±5%, may be within the intended meaning of the recited value, where appropriate. The numerical values ​​given are approximate, meaning that "around," "about," or "approximately" can be inferred if not explicitly stated.

[0052] Concentrations, amounts, and other numerical data may be expressed or presented in a range format herein. It should be understood that such range formats are used merely for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. As an example, a numerical range of "about 0.01 to 2.0" should be interpreted not only to include the explicitly recited values ​​of about 0.01 to about 2.0, but also to include the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 0.5, 0.7, and 1.5, as well as subranges such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5, etc. Moreover, such interpretation should be applied regardless of the breadth of the range or the characteristics being described. Furthermore, it should be noted that all percentages are by weight unless otherwise specified.

[0053] In understanding the scope of the present disclosure, as used herein, the terms "including" or "comprising" and their derivatives are intended to be open-ended terms that specify the presence of the described features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other undescribed features, elements, components, groups, integers, and / or steps. The above also applies to words having similar meanings, such as the terms "including", "having", and their derivatives. As used herein, the term "consisting of" and its derivatives are intended to be closed terms that specify the presence of the described features, elements, components, groups, integers, and / or steps, but exclude the presence of other undescribed features, elements, components, groups, integers, and / or steps. As used herein, the term "consisting essentially of" is intended to specify the presence of the described features, elements, components, groups, integers, and / or steps, and is also intended to specify the presence of those that do not substantially affect the basic and novel characteristics of the features, elements, components, groups, integers, and / or steps. It is understood that reference to any one of these transition terms (i.e., "comprising," "consisting of," or "consisting essentially of") provides direct support for substitution with any of the other transition terms not specifically used. For example, modifying a term from "comprising" to "consisting essentially of" or "consisting of" would find direct support due to this definition for any element disclosed throughout this disclosure. Based on this definition, any element disclosed or incorporated by reference herein may be included in or excluded from the claimed invention.

[0054] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as if each member of the list were individually identified as a separate and unique member. Thus, the individual members of such lists should not be construed as de facto equivalents of any other members of the same list simply based on their presentation in a common group, unless otherwise indicated.

[0055] Furthermore, a particular molecule, construct, composition, element, moiety, excipient, disorder, condition, property, step, etc. may be discussed in the context of one particular embodiment or aspect, or in a separate paragraph or section of this disclosure. This is merely for convenience and brevity, and it is understood that any such disclosure is equally applicable to, and intended to be combined with, any other embodiment or aspect found anywhere in this disclosure, figures, and claims, all of which form the invention as filed and claimed as of the filing date. For example, a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, composition, or method is intended to directly support, and will be found to be, the embodiment related to the construct, composition, formulation, and method described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.

[0056] Unless otherwise specified, "nucleic acid sequences" that encode a "protein" or "polypeptide" include all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.

[0057] The term "exogenous" refers to any substance introduced into or originating from outside a cell, tissue, or organism that is not produced by or originating from the same cell, tissue, or organism into which it is introduced.

[0058] The terms "transduced," "transfected," or "transformed" refer to the process by which exogenous nucleic acid is introduced or transferred into a cell. A "transduced," "transfected," or "transformed" cell (e.g., a mammalian cell) is one that has been transduced, transfected, or transformed with an exogenous nucleic acid (e.g., a vector) that includes an exogenous nucleic acid encoding any of the molecules described herein.

[0059] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, or a combination thereof. Unless otherwise limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties to the referenced nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.

[0060] Modifications can be introduced into the nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include the aliphatic hydroxyamino acids (e.g., serine and threonine), the amide family (e.g., asparagine and glutamine), the aliphatic family (e.g., alanine, valine, leucine and isoleucine), and the aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).

[0061] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or Fc receptor). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity (microscopic equilibrium dissociation constant) that reflects a 1:1 interaction between members of a binding pair (e.g., antibody / Fc receptor or antibody and antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D) Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in this disclosure and are known to those of skill in the art.

[0062] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0063] As used herein, the terms "cytotoxic" and "cytotoxic agent" refer to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes, such as nucleases and fragments thereof; antibiotics; small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or mutant forms; and various anti-tumor or anti-cancer drugs disclosed below.

[0064] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Non-limiting examples of chemotherapeutic agents include alkalizing or alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; chlorambucil, tetracycline ... Nitrogen mustards such as cyclohexyl, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterin, prednimustine, trofosfamide, and uracil mustard; nitrosuureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, and cactinomycin. , calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens such as calcitonin, dromostanol propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergics such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitine; elliptinium acetate; etoglucinamide; do;gallium nitrate;hydroxyurea;lentinan;lonidamine;mitoguazone;mitoxantrone;mopidanmol;nitracrine;pentostatin;phenamet;pirarubicin;podophyllic acid;2-ethylhydrazide;procarbazine;PSK(R);razoxane;sizofiran;spirogermanium;tenuazonic acid;triaziquone;2,2',2"-trichlorotriethylamine;urethane;vindesine;dacarbazine;manomustine;mitobronitol ;Mitrolactol;Pipobroman;Gacytosine;Arabinoside ("Ara-C");Thiotepa;Taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);Chlorambucil;Gemcitabine;6-Thioguanine;Mercaptopurine;Methotrexate Cert;Platinum analogues such as cisplatin and carboplatin;Platinum;Etoposide (VP-16);Ifosfamide;Mitomycin C;Mitoxantrone;Vincristine;Vinblastine;Vinorelbine;Navelbine;Novantrone;Teniposide;Daunomycin;Aminopterin;Xeloda;Ibandronate;CPT-11;Topoisomerase inhibitors RFS2000;Difluoromethylornithine (DMFO);Retinoic acid;Esperamicin;Capecitabine;and pharma- ceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antihormonal drugs that act to regulate or inhibit hormone action on tumors, such as antiestrogens, including tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and other chemotherapeutic drugs, such as prednisolone. Also included are pharma-ceutically acceptable salts, acids, or derivatives of any of the above.;

[0065] A detectable moiety is a compound or composition that can be directly or indirectly conjugated to an antibody. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0066] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain having a structure substantially similar to a native antibody structure or containing an Fc region as defined herein.

[0067] The term "treating" or "treatment" as used herein and well understood in the art, refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or not, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, reduction in recurrence of disease, and remission (partial or total). "Treat" and "treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. In addition to being useful as a treatment method, the methods described herein can be useful for the prevention or prophylaxis of disease.

[0068] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0069] As used herein, the term "pharmaceutical acceptable" refers to a solvent, co-solvent, surfactant, carrier, diluent, excipient, buffer, salt, solvate, hydrate, and / or other ingredient that is compatible with other ingredients of the formulation and not harmful to the recipient. A "pharmaceutical acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. A pharmaceutical acceptable carrier may include, but is not limited to, one or more of a solvent, co-solvent, surfactant, diluent, buffer, excipient, stabilizer, or preservative.

[0070] As used herein, "buffering agent" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. A buffering agent may be present in the liquid or solid formulation of the invention. The buffering agent adjusts the pH of the formulation to a pH of about 5.0 to about 8.5, about 5.5 to about 7.5, about 6.0 to about 6.5, or about 6.3. In one embodiment, examples of buffering agents that control the pH in the range of 5.0 to 7.5 include acetate, succinate, gluconate, histidine, tartarate, TRIS, citric acid, phosphoric acid, maleate, cacodylate, 2-[N-morpholino]ethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris), N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine, and other organic acid buffers. In another aspect, the buffering agent herein is histidine or citrate.

[0071] As used herein, "saccharides" refer to sugars having the general formula (CHO) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. In one aspect, examples of sugars herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, erythritol, glycerol, arabitol, siritol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, and the like. The sugar may be a lyoprotectant. In another aspect, the sugar herein is a non-reducing disaccharide, such as sucrose.

[0072] "Surfactant" herein refers to an agent that reduces the surface tension of a liquid. Surfactants may be non-ionic surfactants. In one aspect, examples of surfactants herein include polysorbates (polyoxyethylene sorbitan monolaurate, e.g., polysorbate 20 and polysorbate 80); TRITON (t-octylphenoxypolyethoxyethanol, a non-ionic detergent, Union of Dow Chemical Co., Midland Mich.); Carbide Subsidiaries);Sodium Dodecyl Sulfate (SDS);Sodium Lauryl Sulfate;Sodium Octyl Glycoside;Lauryl-, Myristyl-, Linoleyl-, or Stearyl-Sulfobetaine;Lauryl-, Myristyl-, Linoleyl-, or Stearyl-Sarcosine;Linoyl-, Myristyl-, or Cetyl-Betaine;Lauramidopropyl-, Cocamidopropyl-, Linoleamidopropyl-, Myristamidopropyl-, Palmidopropyl-, or Isostearamidopropyl-Betaine (e.g., Lauramidopropyl);Myristamidopropyl-, Palmidopropyl-, or Isostearamidopropyl-Dimethylamine;Sodium Methyl Cocoyl Taurate, or Disodium Methyl Oleyl Taurate;Sorbitan Monopalmitate;and the MONAQUAT Series (Mona Industries, Inc., Paterson, NJ; polyethyl glycol (PEG), polypropylene glycol (PPG), and copolymers of polyoxyethylene and polyoxypropylene glycol (e.g., Pluronic / Poloxamer, PF68, etc.). In other embodiments, the surfactant is polysorbate 80.

[0073] "Lyophilized" or "lyophilization" refers to freeze-drying, which is commonly used to preserve proteins by removing water from protein preparations of interest. Freeze-drying, or lyophilization, is a process in which the material to be dried is first frozen and then the ice or freezing solvent is removed by sublimation in a vacuum environment. One or more excipients can be included in the pre-lyophilized formulation to enhance stability during the freeze-drying process and / or to improve the stability of the freeze-dried product during storage.

[0074] A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein formulation in a carrier or diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration (e.g., parenteral administration) to a patient to be treated.

[0075] A "lyoprotectant" is a molecule that, when combined with a protein of interest, significantly prevents or reduces the chemical and / or physical instability of the protein during lyophilization and subsequent storage. Exemplary lyoprotectants include sugars / saccharides such as sucrose or trehalose; amino acids such as monosodium glutamate or histidine; methylamines such as betaine; lyotropic salts such as magnesium sulfate; trihydric or higher sugar alcohols, e.g., polyols such as glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronics; and combinations thereof. The lyoprotectant may be a non-reducing sugar, e.g., trehalose or sucrose. The lyoprotectant may be added to the formulation prior to lyophilization in a "lyoprotecting amount," meaning that after the protein is lyophilized in the presence of a lyoprotecting amount of the lyoprotectant, the protein essentially retains its physical and chemical stability and integrity during lyophilization and storage.

[0076] The "carrier or diluent" of interest herein is one that is pharma- ceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0077] A "preservative" is a compound that can be added to a diluent to essentially reduce bacterial activity in the reconstituted formulation, thus facilitating the production of, for example, a reconstituted formulation for multiple use. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0078] A "bulking agent" is a compound that adds mass to a lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol.

[0079] As used herein, the term "tonicity adjusting agent" is intended to mean one or more compounds that can be used to adjust the tonicity of liquid formulations.Suitable tonicity adjusting agents include glycerin, lactose, mannitol, dextrose, sodium chloride, magnesium sulfate, magnesium chloride, sodium sulfate, sorbitol, trehalose, sucrose, raffinose, maltose, and others known to those skilled in the art.In one embodiment, the tonicity of the liquid formulation is close to that of blood or plasma.

[0080] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0081] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example, Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting.

[0082] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria in some embodiments, especially when glycosylation and Fc effector function are not required, such as when therapeutic antibodies are conjugated to cytotoxic drugs (e.g., toxins) and the immunoconjugate itself is effective in tumor cell destruction. Full-length antibodies have a longer half-life in circulation. After expression, the antibody can be isolated from E. coli cell paste in a soluble fraction and purified, for example, through a protein A or protein G column, depending on the isotype. Final purification can be carried out similarly to the process for purifying antibodies expressed, for example, in CHO cells.

[0083] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts. Saccharomyces cerevisiae and Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickerhamii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia spp. (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; as well as filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are commonly available and useful herein.

[0084] Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 / 293T or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO); mouse Sertoli cells; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0085] Host cells are transformed with the above-described expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0086] In some aspects, the antibody or antigen-binding fragment thereof antagonizes the interaction between ILT4 and HLA-G or HLA-A. In certain embodiments, the antibody or antigen-binding fragment thereof selectively binds to ILT4 compared to other leukocyte immunoglobulin-like receptor family members, e.g., ILT1, ILT2, ILT3, ILT5, ILT6, and / or ILT7. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human ILT4 with an affinity that is 2-100,000 times greater, or at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 100, 500, 1000, 5000, 10,000, 13,000, 14,000, 15,000, 20,000, 50,000, or 100,000 times greater than the antibody or antigen-binding fragment thereof binds to each of human ILT3 and human ILT2. In some embodiments, there is no cross-reactivity between the antibody or antigen-binding fragment thereof and other ILT (LILR) family members as measured using surface plasmon resonance.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof selectively binds to HLA-G over one or more of MHC-I, AngptI, Nogo66, MAG, OMgp, beta-amyloid, Sema4A, CD1c / d, and CSP, hi some embodiments, the antibody or antigen-binding fragment thereof blocks binding of ILT4 to HLA-A and / or HLA-G.

[0088] In some embodiments, the antibody or antigen-binding fragment thereof has an equilibrium dissociation constant (K D In some embodiments, the antibody or antigen-binding fragment thereof binds to ILT4 with an equilibrium dissociation constant (K D In some embodiments, the antibody or antigen-binding fragment thereof binds to ILT4 with an equilibrium dissociation constant (K DIn some embodiments, the antibody or antigen-binding fragment thereof binds to ILT4 with an equilibrium dissociation constant (K) of 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, or 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, or 0.07 nM or less, 0.07 nM or less, or 0.06 nM or less, 0.05 nM or less, or 0.04 nM or less, 0.03 nM or less, 0.02 nM or less, 0.01 nM or less, or 0.008 nM or less. D In some embodiments, the antibody or antigen-binding fragment thereof binds to ILT4 with an equilibrium dissociation constant (K) of 0.5-10 pM, 1-9 pM, 2-8 pM, 3-7 pM, or about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. D ) binds to ILT4.

[0089] In some embodiments, the antibody or antigen-binding fragment thereof inhibits HLA-G with a potency of about 0.05 to about 0.50 μg / mL, or about 0.10 to about 0.25 μg / mL. In some embodiments, the antibody or antigen-binding fragment thereof inhibits HLA-G with a potency of less than 0.100 μg / ml.

[0090] In some embodiments, the antibody or antigen-binding fragment thereof inhibits HLA-A with a potency of about 0.05 to about 0.50, or about 0.10 to about 0.20 μg / mL. In some embodiments, the antibody or antigen-binding fragment thereof inhibits HLA-A with a potency of less than 0.10 μg / ml.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof activates TNF-α production with a potency of about 7 ng / mL to about 25 ng / mL or about 12 ng / mL to about 20 ng / mL.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof activates IL-6 production with a potency of about 5 ng / mL to about 25 ng / mL, about 6 ng / mL to about 22 ng / mL, or about 8 ng / mL to about 21 ng / mL.

[0093] In some embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, or IgG4, and the antibody comprises a light chain constant region selected from the group consisting of kappa and lambda.

[0094] In another embodiment, the polypeptide may exhibit reduced affinity for at least one receptor, eg, FcγI, FcγIIA, or C1q, compared to a polypeptide comprising a wild-type human IgG Fc region.

[0095] In yet another embodiment, the polypeptide comprises a human IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM Fc region.

[0096] In yet another embodiment, the polypeptide comprises a human IgG1, IgG2, or IgG4 Fc region.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more Fc domains, e.g., a pair of human Fc domains. In some embodiments, the human Fc domains are human IgG1 Fc domains, human IgG2 Fc domains, human IgG3 Fc domains, or human IgG4 Fc domains. In some embodiments, the human Fc domains are human IgG4 Fc domains. In some embodiments, the human Fc domains each comprise a sequence at least 80% identical to SEQ ID NO: 81. In some embodiments, the human Fc domains each comprise a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 81. In some embodiments, the human Fc domains comprise SEQ ID NO: 81. In some embodiments, the human Fc domains comprise mutations to remove glycosylation and / or to reduce Fc-gamma receptor binding. In some embodiments, the human Fc domain comprises the mutations N297Q, N297A, or N297G, in some embodiments the human Fc domain comprises a mutation at position 234 and / or 235, e.g., L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4), in some embodiments the human Fc domain is an IgG2 Fc domain comprising the mutations V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or a combination thereof (all according to Kabat, EU numbering). In some embodiments the human Fc domain comprises the human IgG1 constant region mutations L234A / L235A ("LALA") or the human IgG1 constant region mutations L234A / L235A / P329G ("LALAPG"), respectively. Non-limiting examples of such mutations are provided in SEQ ID NOs:93 and 94.

[0098] Further examples of engineered human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids in which at least one amino acid mutation results in reduced Fc function include, but are not limited to, mutations of amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330, and 331 in the heavy chain constant region (according to Kabat, EU numbering). Examples of combinations of mutated amino acids are also well known in the art, for example, but are not limited to, combinations of mutations at amino acids 234, 235 and 331, such as 234, 235 and 329, such as L234F, L235E and P331S, or combinations of mutations at amino acids 318, 320 and 322, such as E318A, K320A and K322A.

[0099] Further examples of engineered Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1; S239D / I332E IgG1; S239D / I332E / A330L IgG1; S298A / E333A / K334A; in one heavy chain, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1, and in the opposing heavy chain, D270E / K326D, A330M / K334E IgG1; G236A / S239D / I332E IgG1; K326W / E333S IgG1; S267E / H268F / S324T. These include IgG1; E345R / E430G / S440Y IgG1; N297A or N297Q or N297G IgG1; L235E IgG1; L234A / L235A IgG1; F234A / L235A IgG4; H268Q / V309L / A330S / P331S IgG2; V234A / G237A / P238S / H268A / V309L / A330S / P331S IgG2; M252Y / S254T / T256E IgG1; M428L / N434S IgG1; S267E / L328F IgG1; N325S / L328F IgG1, etc. In some embodiments, the engineered Fc domain comprises one or more substitutions of N297A IgG1, N297Q IgG1, and S228P IgG4.

[0100] In one embodiment, polypeptides of the disclosure comprising an Fc variant exhibit reduced affinity for an Fc receptor, e.g., FcγRI, FcγRIIA, FcγRIIIA, as compared to an unmodified antibody. In one embodiment, polypeptides comprising an Fc variant exhibit affinity for an Fc receptor that is at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, or at least 1% lower than the affinity of the wild-type polypeptide.

[0101] In one embodiment, polypeptides comprising an Fc variant of the disclosure exhibit greater than a 700-fold reduction in Fcγ binding, or greater than a 3,500-fold reduction in Fcγ binding.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variant Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM. In certain embodiments, the antibody is an aglycosylated antibody with reduced effector function. In certain embodiments, the variant Fc region of IgG1 comprises (a) an amino acid substitution at Leu234 with alanine, (b) an amino acid substitution at Leu235 with alanine, (c) an amino acid substitution at Pro329 with glycine or arginine, (d) an amino acid substitution at Asn297 with alanine, (e) an amino acid substitution at Asn297 with glutamine, (f) an amino acid substitution at Asn297 with glycine, or (g) any combination of (a)-(f). In certain embodiments, the variant Fc region of IgG2 comprises (g) an amino acid substitution at position Ser228 with proline, (h) an amino acid substitution at position Pro329 with glycine or arginine, or (i) both (g) and (h). In certain embodiments, the variant Fc region of IgG4 comprises (j) an amino acid substitution at position Ser228 with proline, (k) an amino acid substitution at position Leu235 with alanine or glutamic acid, (l) an amino acid substitution at position Pro329 with glycine or arginine, or (m) any combination of (j)-(l).

[0103] The present disclosure also relates to immunoconjugates. In some embodiments, the antibody or antigen-binding fragment thereof is combined with a chemotherapeutic agent, a cytotoxin, a detectable moiety, a diagnostic agent, or a combination thereof. In other embodiments, the antibody or antigen-binding fragment thereof is administered separately from the chemotherapeutic agent, a cytotoxin, a detectable moiety, a diagnostic agent, or a combination thereof.

[0104] The present disclosure also relates to an expression system and a host cell comprising the expression system. The expression system comprises at least one expression vector, for example, including two or more, or three or more expression vectors. Those skilled in the art will readily understand that a single or multiple expression vectors can be incorporated into a suitable host cell using conventional methods, including, but not limited to, transformation, transfection, or viral infection. The expression system may comprise one or more nucleic acid sequences encoding a protein comprising an antibody or antigen-binding fragment thereof that binds to ILT4. Each of the one or more polypeptide chains comprising a fully formed antibody or antigen-binding fragment thereof that binds to ILT4 may be expressed by a single vector in the host cell, or may be expressed separately from two or more vectors. For example, the heavy and light chains that together comprise the antibody or antigen-binding fragment thereof may be expressed from the same vector, or may be expressed from separate vectors.

[0105] The present disclosure also relates to a method of producing an antibody or antigen-binding fragment thereof comprising culturing a host cell comprising an expression system under conditions that allow for the formation of an antibody or antigen-binding fragment thereof that binds to ILT4, and recovering the antibody or antigen-binding fragment thereof that binds to ILT4, in some embodiments, the method further comprises purifying the recovered antibody or antigen-binding fragment thereof that binds to ILT4.

[0106] The present disclosure also relates to compositions or formulations comprising an antibody or antigen-binding fragment thereof that binds ILT4 and a carrier, which in some aspects comprises a pharma- ceutically acceptable carrier or a diagnostic carrier. In some embodiments, the pharmaceutical composition may include an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an antagonist of PD-1 / PD-L1, TIGIT, or CTLA4. In certain embodiments, the PD-L / PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, durvalumab, lambrolizumab, avelumab, and toripalimab. In some aspects, the PD-L / PD-1 antagonist is toripalimab. The additional therapeutic agent may be, for example, a chemotherapeutic agent or a biotherapeutic agent (including, but not limited to, an antibody or antigen-binding fragment thereof that specifically binds to an antigen selected from the group consisting of PD-L1, PD-L2, CTLA4, BTLA, TIM3, HVEM, GITR, CD27, SIRPα, NKG2A, NKG2C, NKG2E, TSLP, IL10, VISTA, VEGF, EGFR, Her2 / neu, VEGF receptor, other growth factor receptors, CD20, CD28, CD40, CD-40L, CD70, CD73, CCR8, OX-40, 4-1BB, and ICOS). The additional therapeutic agent may be selected from the group consisting of STING agonists, poly ADP-ribose polymerase (PARP) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, cyclin-dependent kinase (CDK) inhibitors, indoleamine 2,3-dioxygenase (IDO) inhibitors, tryptophan 2,3-dioxygenase (TDO) selective inhibitors, antiviral compounds, antigens, adjuvants, anticancer drugs, CTLA-4 pathway antagonists, lipids, liposomes, peptides, cytotoxic drugs, chemotherapeutic drugs, immunomodulatory cell lines, checkpoint inhibitors, vascular endothelial growth factor (VEGF) receptor inhibitors, topoisomerase II inhibitors, smoothened inhibitors, alkylating agents, antitumor antibiotics, antimetabolites, retinoids, and immunomodulatory agents including, but not limited to, anticancer vaccines.

[0107] In some embodiments, the pharmaceutical composition or formulation relates to a liquid formulation of an antibody or antigen-binding fragment thereof that binds to ILT4. In yet a further aspect, the present disclosure relates to a lyophilized formulation comprising a mixture of an antibody or antigen-binding fragment thereof that binds to ILT4 and one or more excipients. In certain embodiments, the formulation may further comprise, but is not limited to, one or more of a buffering agent, a tonicity adjusting agent, a surfactant, a lyoprotectant, a preservative, or a bulking agent.

[0108] An antibody or antigen-binding fragment thereof that binds ILT4 in a formulation of the disclosure may have a given concentration, including, for example, a range of 1 mg / mL to 200 mg / mL, or a concentration of at least about 1 mg / mL, at least about 10 mg / mL, at least about 50 mg / mL, at least about 100 mg / mL, at least about 150 mg / mL, at least about 200 mg / mL, or less than 200 mg / mL, or less than 100 mg / mL, or less than 50 mg / mL.

[0109] The present disclosure relates to a method of delivering an antibody or antigen-binding fragment thereof that binds ILT4 to a subject comprising administering to the subject via a route of administration selected from parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intrathecal, intraarachnoid, intraomayal, intravitreal, intraocular, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, intravaginal, intrarectal, buccal, sublingual, intranasal, or transdermal.

[0110] The present disclosure includes a method for administering an antibody or antigen-binding fragment thereof by introducing the antibody or antigen-binding fragment thereof into the body of a subject. For example, the method includes penetrating the body of a subject with the needle of a syringe, patch, pen, on-body injector, or other injection device, and injecting the antibody or fragment into the body of the subject, for example, into the subject's vein, artery, tumor, muscle tissue, or subcutaneous tissue. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, e.g., an autoinjector) that includes, for example, a cylinder or barrel for holding the fluid to be injected (e.g., including an antibody or fragment thereof or a pharmaceutical composition thereof), a needle for puncturing the skin and / or blood vessel for injection of the fluid, and a plunger for pushing the fluid from the cylinder through the hole in the needle. In one embodiment of the present invention, the injection device including the antibody or antigen-binding fragment thereof or pharmaceutical composition of the present invention is an intravenous (IV) injection device. Such devices contain the antibody or fragment thereof, or pharmaceutical composition thereof, within a cannula or trocar / needle that may be attached to a tube that may be attached to a bag or reservoir for holding a fluid (e.g., saline; or lactated Ringer's solution, which contains NaCl, sodium lactate, KCl, CaCl2, and may contain glucose) to be introduced into the subject's body through the cannula or trocar / needle. The invention provides a container (e.g., a plastic or glass vial, e.g., with a cap or with a chromatography column, hollow bore needle, or syringe cylinder) that contains any of the antibodies or antigen-binding fragments described herein, or a pharmaceutical composition thereof, including a pharma- ceutical acceptable carrier.

[0111] In some embodiments, the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, mesothelioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (or cell carcinoma), gastric cancer. cancer), germ cell tumors, childhood sarcomas, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CN S) neoplasms, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphomas, environmentally induced cancers including those induced by asbestos, virus-related cancers or cancers of viral origin (e.g., human papillomavirus (HPV-related or originating tumors)), and hematological tumors derived from either of the two major blood cell lineages, i.e., myeloid cell lineage (producing granulocytes, erythrocytes, platelets, macrophages and mast cells) or lymphoid cell lineage (producing B, T, NK and plasma cells), e.g., all types of leukemias, lymphomas and myelomas, e.g., acute, chronic, lymphocytic and / or myeloid leukemias, e.g., acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CL ... LL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant [M4E] with eosinophilia), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma;Lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological neoplasms, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, hemocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion Lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), lymphoid hematopoietic neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and Waldenström's macroglobulinemia. Lymphoplasmacytic lymphoma (LPL) with lobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; central and peripheral tumors including seminoma, teratocarcinoma, astrocytoma, and schwannoma tumors of nerve; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors including T-cell and B-cell tumors including but not limited to melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular and teratocarcinoma, hematopoietic tumors of lymphoid system, e.g., T-cell disorders such as T-prolymphocytic leukemia (T-PLL) including small cell and large cell types; large granular lymphocytic leukemia (LGL) of T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / metathymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, or combinations or metastases thereof;

[0112] In certain embodiments, the cancer is a solid tumor. In other embodiments, the cancer is a hematological cancer. In certain embodiments, the cancer is metastatic. In some embodiments, the cancer is recurrent. In other embodiments, the cancer is refractory. In still other embodiments, the cancer is recurrent and refractory. In some embodiments, the cancer is anaplastic astrocytoma, astrocytoma, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., characterized by mutations in BRCA1 and / or BRCA2), carcinoid cancer, cervical cancer, chondrosarcoma, choroid plexus papilloma, colorectal cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, embryonal tumors, hepatocellular carcinoma, idiopathic myelofibrosis, kidney cancer, leukemia, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma ... The cancer is selected from the group consisting of myeloma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, multiple myeloma, neuroblastoma, oligodendroglioma, osteosarcoma, ovarian cancer, pancreatic cancer, polycythemia vera, primitive neuroectodermal tumor, prostate cancer, renal cell carcinoma, renal transitional cell carcinoma, retinoblastoma, renal rhabdoid tumor, rhabdomyosarcoma, salivary gland cancer, sarcoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma (e.g., cutaneous squamous cell carcinoma), synovial sarcoma, thrombocythemia, thyroid cancer, uterine cancer, vestibular schwannoma, and Wilms' tumor. In one embodiment of the present invention, the cancer is, for example, a metastatic cancer of the above types.

[0113] In one embodiment of the invention, the cancer is a myeloid-rich tumor (e.g., mesothelioma, renal cancer, lymphoma, sarcoma, melanoma, head and neck cancer, breast cancer, bladder cancer, gastric cancer, ovarian cancer, or thyroid cancer). Because ILT4 is expressed primarily by myeloid cells and granulocytes, and myeloid cell infiltration into tumors is generally associated with poor prognosis due to the immunosuppressive effects of these cells that can antagonize anti-tumor responses by T cells, treatment with an anti-ILT4 antibody or antigen-binding fragment of the present disclosure benefits subjects with high myeloid or immunosuppressive myeloid cell infiltration.

[0114] In certain embodiments, the cancer is lung cancer, liver cancer, NSCLC, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, pancreatic cancer, glioblastoma, glioma, renal cell carcinoma, stomach cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, or head and neck cancer.

[0115] The present invention includes ELISA assays (enzyme-linked immunosorbent assays) incorporating the use of the antibodies or antigen-binding fragments thereof disclosed herein. For example, such a method for determining whether a sample contains ILT4 or a fragment thereof comprises the following steps:

[0116] (a) coating a substrate (e.g., the surface of a microtiter plate well, e.g., a plastic plate) with an anti-ILT4 antibody (e.g., a fully human antibody, such as an antagonist fully human antibody) or an antigen-binding fragment thereof;

[0117] (b) applying a sample to be tested for the presence of ILT4 to a substrate;

[0118] (c) washing the substrate to remove unbound material from the sample;

[0119] (d) applying a detectably labeled antibody (e.g., an enzyme-linked antibody) that is also specific for the ILT4 antigen;

[0120] (e) washing the substrate to remove unbound labeled antibody;

[0121] (f) detecting the label on the antibody, which, if the labeled antibody is enzyme-linked, applies a chemical that is converted by the enzyme into a detectable, e.g., fluorescent, signal; and

[0122] (g) For example, detecting the label bound to the substrate indicates the presence of ILT4 protein.

[0123] The present invention also includes surface plasmon resonance (SPR) assays incorporating the use of the antibodies or antigen-binding fragments thereof disclosed herein. In some embodiments, the disclosure includes the assessment of antibody binding activity, affinity, and cross-reactivity, comprising the following steps:

[0124] (a) preparing one or more capture surfaces by immobilizing an anti-Fc molecule onto the one or more capture surfaces;

[0125] (b) flowing a solution containing anti-ILT4 antibodies over the one or more capture surfaces to immobilize anti-ILT4 on the one or more surfaces;

[0126] (c) flowing the analyte over the capture surface after step (b) and measuring association of the analyte to the anti-ILT4 antibody;

[0127] (d) flowing a buffer over the capture surface after step (c) to measure dissociation of the analyte from the anti-ILT4 antibody;

[0128] (e) flowing a regeneration solution over the capture surface or surfaces in step (d) to remove any remaining analyte / anti-ILT4 complexes or unbound anti-ILT4 antibodies from the surfaces.

[0129] Binding affinity can be determined by dividing the dissociation rate by the association rate. Some embodiments may include flowing one or more additional analytes over the capture surface.

[0130] The present invention also includes a competitive binding assay between an ILT4 antibody and one or more analytes, comprising the steps of:

[0131] (a) preparing one or more capture surfaces by capturing a first ligand on the one or more capture surfaces;

[0132] (b) preparing a solution comprising an anti-ILT4 antibody and a second analyte;

[0133] (c) flowing a solution containing an anti-ILT4 antibody and a second analyte over the capture surface prepared in step (a); and

[0134] (d) measuring the response.

[0135] In some embodiments, the capture surface may be activated according to the manufacturer's instructions, e.g., with EDC / NHS (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare), and deactivated, e.g., using ethanolamine (Biacore GE Healthcare). In some embodiments, the capture surface is coated, e.g., with avidin, streptavidin, neutravidin, or derivatives thereof, to immobilize biotinylated agents.

[0136] In one embodiment of the present invention, the labeled antibody or antigen-binding fragment thereof is labeled with a peroxidase that reacts with ABTS (e.g., 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) or 3,3',5,5'-tetramethylbenzidine to produce a detectable color change. Alternatively, the labeled antibody or fragment may be labeled with a detectable radioisotope (e.g., 1,2'-diamino-3,4'-diphenylphosphine) that can be detected by a scintillation counter in the presence of a scintillant (e.g., 3 H).

[0137] The anti-ILT4 antibodies (e.g. fully human antibodies, such as antagonist fully human antibodies) or antigen-binding fragments thereof of the present invention may be used in Western blots or immunoprotein blotting. Such procedures form part of the present invention and include, for example, (1) providing a membrane or other solid substrate containing a sample to be tested for the presence of ILT4 or a fragment thereof, which may include, for example, transferring proteins from the sample to be tested for the presence of ILT4 (e.g. from PAGE or SDS-PAGE electrophoretic separation of proteins in the sample) onto the membrane or other solid substrate (e.g. using methods known in the art (e.g. semi-dry blotting or tank blotting)) and contacting the membrane or other solid substrate to be tested for the presence of bound ILT4 or a fragment thereof with the anti-ILT4 antibodies or antigen-binding fragments thereof of the present invention, (2) washing the membrane one or more times to remove unbound anti-ILT4 antibodies or fragments and other unbound materials, and (3) detecting the bound anti-ILT4 antibodies or fragments.

[0138] Such membranes may take the form of, for example, nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membranes onto which proteins to be tested for the presence of ILT4 have been transferred (e.g., after electrophoretic separation in the gel) in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or an SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. Prior to contacting the membrane with an anti-ILT4 antibody or fragment, the membrane may be blocked, for example, with non-fat dry milk, to bind non-specific protein binding sites on the membrane.

[0139] Detection of bound anti-ILT4 antibody or fragment indicates that ILT4 protein is present on the membrane or substrate and in the sample. Detection of bound antibody or fragment may be by binding the antibody or fragment with a detectably labeled secondary antibody (anti-immunoglobulin antibody) and then detecting the presence of the secondary antibody label.

[0140] The anti-ILT4 antibodies (e.g. fully human antibodies, such as antagonist fully human antibodies) and antigen-binding fragments thereof disclosed herein may also be used for immunohistochemistry. Such methods form part of the present invention and include, for example, (1) contacting a cell (e.g. a myeloid cell such as a monocyte, macrophage or dendritic cell) to be tested for the presence of ILT4 protein with an anti-ILT4 antibody of the present invention or an antigen-binding fragment thereof, and (2) detecting the antibody or fragment on or within the cell.

[0141] If the antibody or fragment itself is detectably labeled, it can be detected directly. Alternatively, the antibody or fragment can be bound by a detectably labeled secondary antibody, and the label then detected. EXAMPLES

[0142] The following examples are intended to illustrate the disclosure and not to limit the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples form part of the invention.

[0143] [Example 1]

[0144] Generation and selection of mouse anti-human immunoglobulin-like transcript 4 (ILT4) antibodies: Mice were immunized with human ILT4 and screened for high titer anti-ILT4 antibodies. Primary screening was performed using flow cytometry for hybridomas producing antibodies that bind to ILT4 and block ILT4 / HLA-G interaction. Further screening was performed to confirm binding to ILT4 and characterize ILT4 binding specificity. Antibodies selected after secondary screening were purified and further tested for affinity to ILT4 and potency on ILT4 / HLA-G binding. Antibodies from secondary screening with highest ILT4 affinity and ILT4 / HLA-G blocking activity were selected as leads for further characterization and humanization. Antibodies selected from secondary screening were confirmed by flow cytometry and SPR to have the ability to block ILT4 binding to both HLA-G and HLA-A.

[0145] [Example 2]

[0146] Antibody humanization: The design of humanized VH and VL amino acid sequences was performed according to the procedure described by Tsurushita et al. (Tsurushita et al. 2005. Design of humanized antibodies: From anti-Tac to Zenapax. Methods 36:69-83), which is incorporated herein by reference in its entirety, as follows: First, a three-dimensional molecular model of the variable region of a mouse monoclonal antibody (mouse mAb) was constructed. Second, framework amino acid residues important for the formation of CDR structures or required for antigen binding were predicted using the molecular model. In parallel, cDNA-derived human VH and VL amino acid sequences with high homology to the VH and VL amino acid sequences of the mouse mAb, respectively, were selected. Finally, the CDR sequences together with framework amino acid residues important for the formation of the antigen binding site were grafted from the mouse mAb VH and VL to the corresponding selected human framework sequences.

[0147] Human VH sequences homologous to the mouse-mAb VH framework were searched in the GenBank database, and VH sequences encoded without somatic hypermutations in the framework were selected as acceptors for humanization. The CDR sequences of the mouse-mAb VH were first transferred to the corresponding positions of the VH from the GenBank sequence. Then, amino acid residues of the VH from the GenBank sequence were replaced with the corresponding residues of the mouse mAb VH at framework positions where the three-dimensional model of the mouse mAb variable region showed significant contact with the CDRs.

[0148] Next, the sequences of human VL regions homologous to the mouse-mAb VL framework sequences were searched in the GenBank database, and the VL sequences encoded without somatic hypermutations in the framework were selected as the acceptors for humanization. The CDR sequences of the mouse-mAb VL were first transferred to the corresponding positions of the GenBank VL. Then, at the framework positions where the three-dimensional model of the mouse mAb variable region showed significant contact with the CDRs, the amino acid residues of the GenBank VL were replaced with the corresponding residues of the mouse mAb VL, similar to the VH region above.

[0149] [Example 3]

[0150] Macrophage repolarization with mouse anti-human immunoglobulin-like transcript 4 (ILT4) antibodies: Known ILT4 antibodies were tested in a macrophage repolarization assay developed to confirm the activity of known ILT4 antibodies as a control. Human monocytes were differentiated towards M2 macrophages for 6 days, then stimulated with lipopolysaccharide (LPS) and ILT4 antibodies and assayed using ELISA.

[0151] Mouse anti-human immunoglobulin-like transcript 4 (ILT4) antibodies (L45S4, L77S2, L156S3, L161S and L180S4) and control antibodies (anti-KLH IgG4 negative control; and control antibody J19h1 as a positive control) were used in human macrophage repolarization assays. Macrophage repolarization was measured by Meso Scale Discovery (MSD) assay of secreted cytokines using an MSD plate reader (MESO Sector S 600 from Meso Scale Diagnostics LLC) and flow cytometric analysis of cell surface proteins using a flow cytometer (Attune NXT from ThermoFisher). Human monocyte-derived macrophages of suppressive M2 phenotype were generated by induction in the presence of M-CSF. These cells were stimulated with LPS and phenotype was assessed by MSD analysis and flow cytometry.

[0152] Generation of monocyte-derived macrophages: PBMCs were isolated from fresh buffy coats from two different blood donors using standard density centrifugation. Cells were resuspended in buffer and counted. The cell density was then reduced to 5 × 10 in buffer. 7 Monocytes isolated from PBMCs were centrifuged and resuspended in culture medium. The density was adjusted to 0.5×10 cells / ml in 30 ml of differentiation medium. 6 The concentration was adjusted to cells / ml and the cells were plated. After 3 days, half the volume of additional differentiation medium was added. The cells were cultured for an additional 3 days.

[0153] Macrophage stimulation and treatment with antibodies: Cells were plated using growth medium. Serial dilutions of test antibodies were prepared in duplicate wells of the plate to final concentrations ranging from 30 μg / ml to 0.03 ng / ml. LPS was then added to a final concentration of 100 ng / ml.

[0154] Macrophage phenotype analysis: Human macrophages of the suppressive M2 phenotype were generated and then stimulated with LPS in the presence of ILT4 antibody or control antibody. Two days after LPS stimulation in the presence of ILT4 antibody or control, plates were centrifuged and supernatants were collected for quantification of the inflammatory cytokines TNF-α and IL-6 by MSD analysis.

[0155] Mesoscale discovery assay of cytokine secretion: To determine the effect of mouse anti-human ILT4 antibodies on macrophage polarization, supernatants were analyzed by MSD. For both blood donors, all of the ILT4 antibodies increased TNF-α secretion above the control (Figure 1A and 1B). Maximal effects ranged from a 4- to 5-fold increase over the anti-KLH IgG4 negative control. The control antibody increased TNF-α secretion 8-fold over the negative control.

[0156] For both blood donors, all of the ILT4 antibodies tested also increased IL-6 secretion two-fold above the control at the highest concentration (Figures 2A and 2B). The control antibody increased IL-6 secretion three-fold for donor A and two-fold for donor B.

[0157] Flow cytometric analysis of surface marker expression by macrophages: Expression of cell surface proteins indicative of polarity was assessed by flow cytometry. The mean fluorescent intensity (MFI) of staining for CD206 (Figures 3A and 3B), and CD209 (Figures 4A and 4B) was determined. The ILT4 antibody did not induce a shift in macrophage surface protein expression from M1 to M2 proteins. This was observed for both the control and ILT4 antibodies.

[0158] ILT4 antibody, like the control antibody, increased the production of the inflammatory cytokines TNF-α and IL-6 in a dose-responsive manner. Results of macrophage repolarization assays confirm that ILT4 antibody induces repolarization of inhibitory M2-type macrophages toward a proinflammatory M1 phenotype.

[0159] [Example 4]

[0160] Efficacy of mouse anti-human immunoglobulin-like transcript 4 (ILT4) antibodies for blocking the interaction between human ILT4 and HLA-G: Mouse anti-human immunoglobulin-like transcript 4 (ILT4) antibodies were tested for their ability to block the interaction between human ILT4 and HLA-G. A commercially available non-HLA-G blocking antibody was included as a negative control. A control antibody was included as a positive control. Blocking activity was measured in a flow cytometry assay using soluble fluorescently conjugated HLA-G and CHO-S / ILT4 cell lines engineered to express human ILT4 on their surface. Parental CHO-S cells that do not express human ILT4 were used as a control.

[0161] The ability of control and candidate mouse anti-human ILT4 antibodies to block the interaction between human ILT4 and HLA-G was assessed using soluble fluorescently conjugated HLA-G and CHO-S / ILT4 cells. Cells expressing human ILT4 were pre-incubated with ILT4 antibodies and then incubated with HLA-G. HLA-G binding to the cells was then quantified using flow cytometry. The resulting cell-associated fluorescence was quantified and used to calculate the potency of ILT-4 blockade.

[0162] CHO-S and CHO-S / ILT4 culture: CHO-S cells were cultured in CD OptiCHO medium, and CHO-S / ILT4 cells were cultured in OptiCHO medium containing 20 μg / ml puromycin at 37°C with 5% carbon dioxide and shaking at 150 revolutions per minute (RPM).

[0163] HLA-G titration in HLA-G binding assay: CHO-S parental and CHO-S / ILT4 cells were pelleted by centrifugation and washed at 200,000 cells per ml in FACS buffer (PBS + 0.5% bovine serum albumin (BSA)). Cells were resuspended at 1,000,000 cells per ml in fluorescence activated cell sorting (FACS) buffer and transferred to a 96-well round bottom 96-well plate at 50,000 cells per well and placed on ice. All subsequent manipulations were performed on ice.

[0164] Primary tetramer dilutions of 50 μg / ml were made in FACS buffer. In a separate 96-well plate, 1:2 serial dilutions of tetramer were prepared from 50 μg / ml to 0.05 μg / ml. From each dilution, 50 μl was added to the cells in duplicate. Wells were mixed and incubated at 4°C for 30 minutes. Cells were washed twice with FACS buffer and resuspended in 250 μl of FACS buffer containing 0.1 μg / ml propidium iodide.

[0165] Cells were acquired on an Attune flow cytometer, 5000 cells were collected per sample, and the mean fluorescence intensity (MFI) was recorded. The MFI at different antibody concentrations was plotted using Prism software (Graphpad).

[0166] Antibody titration in HLA-G blocking assay: CHO-S / ILT4 cells were pelleted by centrifugation and washed in FACS buffer (PBS+0.5% BSA) at 200,000 cells per ml. Cells were resuspended in FACS buffer at 1,000,000 cells per ml and transferred to a 96-well round-bottom 96-well plate at 50,000 cells per well and placed on ice. All subsequent manipulations were performed on ice.

[0167] In the first blocking experiment, a negative control non-HLA-G blocking antibody 287219R (Lot: CMIF0221051 from R&D Systems, Cat# MAB2078R) and positive control HLA-G blocking control antibodies J19h1 and 1E1 were evaluated. Primary antibody dilutions were made by diluting the antibody to 30 μg / ml in FACS buffer. Ten three-fold serial dilutions were then made in FACS buffer from 30 μg / ml to 0.001 μg / ml and 50 μl from each dilution was added to the cells in duplicate. Control wells received FACS buffer without antibody. Wells were mixed and incubated for 30 minutes at 4°C. Cells were washed twice with FACS buffer.

[0168] In the second blocking experiment, primary antibody dilutions were made by diluting the antibody to 15 μg / ml in FACS buffer. Three independent sets of eleven 2.5-fold serial dilutions were then made from 15 μg / ml to 0.002 μg / ml in FACS buffer. Each dilution set was handled independently and 50 μl from each was added to the cells in duplicate. Control wells received FACS buffer without antibody. Wells were mixed and incubated at 4°C for 30 minutes. Cells were washed twice with FACS buffer.

[0169] In both blocking experiments, phycoerythrin (PE)-conjugated HLA-G tetramers were diluted to 5 μg / ml in FACS buffer and 100 μl per well was added to the cells. Cells were incubated for 30 min at 4° C., washed twice with FACS buffer, and resuspended in 250 μl of FACS buffer containing 0.1 μg / ml propidium iodide.

[0170] Cells were acquired on an Attune flow cytometer, 5000 propidium iodide negative cells were collected per sample, and MFI was recorded. Relative blocking activity was calculated using the following formula: 100-(sample MFI-background MFI) / (top MFI-background MFI)*100. MFI and relative blocking activity were plotted for each sample using Prism software (Graphpad). IC50 The blocking potency of each antibody, expressed as the blocking potency per unit time, was calculated using Prism software.

[0171] HLA-G titration: Binding of PE-conjugated HLA-G to CHO-S parental and CHO-S / ILT4 cells was quantified by flow cytometry in the PE channel. HLA-G bound to CHO-S / ILT4 cells with a maximum MFI 783-fold higher than untreated cells. Binding to parental CHO-S cells was lower with a maximum MFI 43-fold higher than untreated cells (Figure 5). Based on these plots, 5 μg / ml phycoerythrin-conjugated HLA-G was selected as the concentration to evaluate candidate antibodies for their ability to block HLA-G binding to CHO-S / ILT4 cells.

[0172] In the first experiment, preincubation with J19h1 and 1E1 reduced the MFI of PE-HLA-G by up to 22- and 30-fold, respectively, from cells treated with PE-HLA-G alone. Preincubation with the control antibody 287219R did not reduce the MFI of PE-HLA-G. IC for HLA-G blockade with J19h1 and 1E1 50 The IC values ​​for both were 0.13 μg / ml. 50 It was not possible to calculate values ​​(Table 1). [Table 1]

[0173] In a second experiment, ILT4 antibodies blocked HLA-G binding to CHO-S / ILT4 cells, as shown by a reduction in the MFI of PE-HLA-G on CHO-S / ILT4 cells. The results are shown in Figure 6. 50 The extent of reduction was antibody dependent, with L41S5 reducing the expression level by 14-fold and L45S4 reducing the expression level by 69-fold. IC 50 Values ​​ranged from 0.044 to 0.216 μg / ml (Table 2). [Table 2]

[0174] The results show the ability of mouse anti-human ILT4 antibodies to block the interaction between soluble HLA-G and cell-expressed ILT4 in a flow cytometry assay. A non-blocking antibody used as a negative control had no effect on the MFI of HLA-G on CHO-S / ILT4 cells, indicating that there was no blocking of the HLA-G / ILT4 interaction. Two control antibodies used as positive controls caused a reduction in the MFI of HLA-G on CHO-S / ILT4 cells, demonstrating that this assay detects blocking of the HLA-G / ILT4 interaction. Mouse anti-human ILT4 antibodies also blocked the HLA-G / ILT4 interaction. The effect on the MFI of HLA-G ranged from a 14- to 69-fold reduction in MFI. The potency of the mouse anti-human ILT4 antibodies ranged from 0.065 to 0.171 μg / ml. Figure 7 shows the IC from an exemplary ILT4 blockade of HLA-G assay using flow cytometry. 50 Curves are shown. Control antibodies, 1E1 and J19h1, were included for comparison.

[0175] [Example 5]

[0176] Surface Plasmon Resonance (SPR) Analysis of Mouse, Chimeric, and Humanized Anti-ILT4 Antibodies Binding to ILTR4 (LILRB2): As described above, mouse, chimeric, and humanized anti-ILT4 antibodies were characterized for binding affinity and blocking to ILT4 (LILRB2) using SPR.

[0177] Label-free SPR analysis on a Biacore T200 instrument was utilized to measure antibody affinity for ILT4. CM4 sensor chips from Cytiva Inc. were utilized for all medium resolution analyses. Either anti-mouse Fc-specific or anti-human Fc-specific capture surfaces were prepared using standard amine coupling chemistry. Mouse, chimeric or humanized mAbs were captured on their respective surfaces and ILT4 was injected over the surface at 101 nM to 0.79 nM in a two-fold serial dilution series at a flow rate of 100 μL / min. Since medium resolution analyses were performed here, the association phase was followed for 90 s and the dissociation phase was followed for 1200 s. Sensorgrams were double referenced and globally fitted to a 1:1 interaction model with terms included in mass transport to obtain the equilibrium dissociation constant K D =k off / k on The slowest k that can be measured at a 5% decrease in the dissociation phase over 1200 seconds was calculated as off is 4.27 x 10 -5 s -1 Therefore, 4.27×10 -5 s -1 Slower k off If is first calculated from a global nonlinear fit of the data, then k off During the subsequent refitting in the global analysis, -5 s -1 The binding concentration was kept constant at 100 mM NaCl. All kinetic experiments were carried out at 25° C. in a buffer consisting of Hepes-buffered saline, pH 7.4, containing 0.05% polysorbate 20 (HBS-P+) and 100 μg / ml bovine serum albumin (BSA). Table 3 shows the results of all binding analyses performed. [Table 3] Values ​​highlighted in bold and underlined are k off 4.27 × 10 for 20 min dissociation -5 s -1 This shows that the θ was held constant at its theoretical limit.

[0178] High-resolution binding kinetics of ILT4 binding using humanized anti-ILT4-LALA antibody were measured in triplicate and the results are summarized as follows (numbers in brackets are 95% confidence intervals). JPEG2025511146000005.jpg79160

[0179] The anti-ILT4 antibodies Hz45 and Hz156 were evaluated in a series of in vitro assays evaluating 1) binding to recombinant human and cynomolgus ILT4, 2) binding to ILT4-related molecules from the LILRA and LILRB families, and 3) the effect of binding to ILT4 in inhibiting HLA-A and HLA-G binding to recombinant human ILT4.

[0180] Both antibodies bind tightly to ILT4, with Hz45 binding to K D = 154 pM, and Hz156 is K D = 5.7 pM. The binding rate of both antibodies is approximately 3.3 × 10 6 M -1 s -1 Hz45 has a 29-fold faster off-rate of 53.1 × 10 for ILT4 than Hz156. -5 s -1 and therefore the weaker affinity for Hz45 is mainly due to the faster off-rate observed compared to Hz156.

[0181] Both antibody molecules showed no binding to recombinant cynomolgus ILT4 or to recombinant human LILRA1-6 and LILRB1-4 family members except LILRB2 (ILT4). Antibodies Hz45 or Hz156 precomplexed with recombinant human ILT4 block binding of ILT4 to HLA-A and HLA-G.

[0182] All experiments described herein were performed using a Biacore T200 instrument at 25° C. with a running buffer of Hepes-buffered saline, 0.05% polysorbate 20, pH 7.4 (HBS-P+) supplemented with 100 μg / mL bovine serum albumin (BSA). The running buffer used to immobilize antibodies to the surface was HBS-P+ without the addition of BSA.

[0183] Surface plasmon resonance spectroscopy was used to assess the binding between ILT4 and antibodies Hz45 and Hz156. The binding affinity of each antibody to ILT4 was measured, and both interactions showed fast association rate constants, but Hz156 had a 29-fold slower dissociation rate constant than Hz45. Antibodies Hz45 and Hz156 both showed strong affinity for ILT4, 154 pM and 5.7 pM, respectively. Both molecules showed no binding to recombinant cynomolgus ILT4 or recombinant human LILRA1-6 and LILRB1-4 family members, except LILRB2 (ILT4). Hz45 or Hz156 precomplexed with recombinant human ILT4 blocks ILT4 binding to HLA-A and HLA-G.

[0184] Binding equilibrium exclusion method

[0185] To measure the equilibrium binding affinity and kinetics between human ILT4 and the four humanized LALA-Fc engineered antibodies in solution, the equilibrium exclusion method (KinExA®) was performed. For affinity analysis, the equilibrium dissociation constant K D was experimentally determined and reflects the strength of the binding interaction. on was also experimentally determined, and the dissociation rate k off is expressed by the following formula: off =K D ×k on Calculation is based on:

[0186] K DThe analysis requires immobilization of one of the interaction partners (the titrated binding partner) on a solid phase, which is then used as a probe to capture the other interaction partner, the constant binding partner (CBP). For each experiment, one of the binding partners is titrated in a background of CBP and allowed to reach equilibrium. The solution is then briefly exposed to the solid phase, capturing a portion of the free CBP. The captured CBP is then labeled with a fluorescent secondary molecule. Since the contact time with the solid phase is shorter than the time required for dissociation of the preformed complex in solution, competition between the solution and the titrated binding partner on the solid phase is "kinetically eliminated". Since the solid phase is used only as a probe for the free CBP in each sample, the solution equilibrium does not change during the KinExA measurement.

[0187] The signal generated from the captured CBP is directly proportional to the concentration of free CBP in the equilibrated sample and can be used to calculate the K D The KinExA Pro software performed a least-squares analysis on the measured data to determine the K value for a 1:1 reversible bimolecular interaction. D Fit the optimal solution for .

[0188] Determination of binding kinetics is completed in a similar format to equilibrium analysis, except that measurements are taken "pre-equilibrium" and the binding signal is a function of time. This kinetic experiment is called the direct method. This method holds the titrated CBP concentration constant and probes the solution over time. The amount of free CBP in solution decreases as the sample moves toward equilibrium.

[0189] Binding affinity (K D ), association rate constant (k on ), and the dissociation rate constant (k off ) was measured for four different humanized LALA IgG antibodies against ILT4 using KinExA. The K of the interaction was determined using n-curve analysis of at least two different curves performed at two different CBP concentrations. Dwas determined. ILT4 was used as the concentration reference for all curves. All binding systems have low error and well-defined 95% confidence intervals, providing confidence in the measurements. JPEG2025511146000006.jpg29161

[0190] Competitive binding study of ILT4 between HLA-A and HLA-G and anti-ILT4 antibodies probed by SPR: Using SPR spectroscopy on a Biacore T200 instrument, we investigated whether four chimeric and humanized anti-ILT4 mAbs could block LILRB2 binding to HLA-A (A*02:01; Immunitrack, Denmark) and HLA-G (monomeric HLA-G was prepared at Fred Hutchinson Cancer Center, Seattle, WA). NeutrAvidin was immobilized on all four flows of a CM4 chip using standard amine coupling chemistry, biotinylated HLA-G was captured on flow cell 2, biotinylated HLA-A was captured on flow cell 4, and both flow cells 1 and 3 were used as references, respectively. These competitive experiments were performed using a "premix" format, where 514 nM of ILT4 was mixed with approximately 600 nM of mAb binding sites. At these concentrations, approximately 0.1% of the 514 nM ILT4 in solution is free at equilibrium for most of the antibodies tested. ILT4 / mAb complexes were injected for 60 seconds at 30 μL / min across all four sensor surfaces in HBS-P+ buffer containing 100 μg / mL BSA. The dissociation phase was followed for 60 seconds and all sensorgrams were double referenced. If a binding signal for any given ILT4 / mAb complex was observed, that mAb was designated as a non-competitive binder for ILT4 binding. Conversely, if no significant binding signal was observed for binding of a particular ILT4 / mAb complex to captured HLA-A or HLA-G, that mAb was designated as a competitive binder for ILT4. All four chimeric and humanized mAbs investigated were competitive binders of ILT4 with HLA-A and HLA-G.

[0191] SPR and flow-based characterization of blocking activity for humanized (Hz) versions of L45S4, L77S2, L156S3, and L180S4 between ILT4 and HLA-G. Table 4 below shows the characterization of humanized ILT4 antibodies (numbers in brackets are 95% confidence intervals). [Table 4]

[0192] SPR cross-reactivity study of four humanized anti-ILT4 antibodies with nine LILRA and LILRb homologs: As shown in Table 5, many LILRA and LILRB family members share high homology with LILRB2 (ILT4). Therefore, determining the specificity and cross-reactivity profile of humanized anti-ILT4 antibodies to other family members becomes paramount in characterizing the binding and functional properties of humanized anti-ILT4 mAbs.

[0193] SPR was used to determine the specificity of four humanized anti-ILT4 mAbs for binding to LILRB2 and any cross-reactivity with nine other LILRA and LILRB family members. LILR proteins were purchased from Acro Biosystems or R&D Systems and sequences are provided below. Tests were performed on a Biacore 8K instrument using a CM4 sensor chip with a running buffer of HBS-P+, 100 μg / mL BSA. Anti-human Fc specific capture surfaces were prepared using standard amine coupling chemistry. MAbs were captured on flow cell 2 and flow cell 1 was used as the reference flow cell. Each of the nine family members and LILRB2 were injected at 100 nM at 30 μL / min for 90 seconds and allowed to dissociate for 240 seconds. In addition to showing the shape of the true molecular binding interaction, cross-reactivity was determined by examining sensorgrams with significant binding signals. All sensorgrams were double-referenced during processing of the data. Table 5 shows the results from the cross-reactivity studies. mAbs 45_01, 77_01, and 156_03 show no cross-reactivity to any family members and bind only to LILRB2. MAb 180_04 shows some cross-reactivity to family members LILRA1, LILRA3, and LILRB1. Results indicate that greater than 79% homology with LILRB2 was required for cross-reactivity with mAb 180_04 alone. The strongest cross-reactive binding interaction observed with mAb 180_04 was with LILRA3, with a K in the range of 600-700 nM. D It is believed to have the following.

[0194] The above method was performed on a Biacore T200 to determine whether any of these four mAbs cross-react with cynomolgus LILRB2. No cross-reactivity was observed for any of these mAbs (Table 5). [Table 5]

[0195] [Example 6]

[0196] Blocking activity of humanized anti-ILT4 antibodies To demonstrate the ability of anti-ILT4 to block binding of ILT4 to its ligands HLA-A and HLA-G, humanized antibodies Hz45.01-LALA, Hz156.03-LALA, and 1E1, as well as a human IgG1-LALA control, were tested in functional blocking assays to block the interaction between R-phycoerythrin (PE)-HLA-G tetramers and ILT4-CHO cells (Figure 8), or 5ug / ml of PE-HLA-A tetramers (Figure 9) using serial titrations (0-15ug / mL) in triplicate. The data are summarized in Tables 6 and 7. [Table 6] [Table 7]

[0197] Anti-ILT4 antibodies target both HLA-G and HLA-A with similar IC 50 Effectively blocks attacks at range.

[0198] [Example 7]

[0199] Macrophage polarization assay The ability of the anti-ILT4 construct (final format, humanized Ab with LALA mutation on the Fc receptor) to polarize M2 macrophages into M1 macrophages and produce the proinflammatory cytokines TNF-α and IL-6 on in vitro differentiated macrophages was assessed.

[0200] Human monocyte isolation and macrophage differentiation:

[0201] PBMCs were isolated from four healthy human donors by Ficoll-Paque gradient centrifugation. Untreated human monocytes were then purified from PBMCs using the Dynabeads Untouched Human Mononocyte Kit according to the manufacturer's instructions (ThermoFisher Scientific). Monocytes were cultured in RPMI containing 10% FBS and 50 ng / mL M-CSF for 7 days to generate macrophages.

[0202] Processing of macrophages and measurement of cytokine production

[0203] Macrophages were harvested and plated in a 96-well plate at 1 x 10 cells per well. 5 Macrophages were plated at 1000x1000 cells. Macrophages were incubated with humanized LALA anti-ILT 4 antibody or human IgG1-LALA control in the presence of 10ng / mL LPS in a serial titration from 4ug / ml in 1:4 dilutions for 24 hours. All samples were tested in triplicate. Conditioned media was then collected for analysis of IL-6 and TNFα production using ELISA assay kits according to the manufacturer's instructions (R&D Systems) as shown in Figures 10 and 11. ELISA data was analyzed and EC50 values ​​were determined using nonlinear regression equations with Prism GraphPad software as shown in Tables 8 and 9. [Table 8]

[0204] [Table 9]

[0205] Cell surface markers (ILT4, CD163, CD206) on macrophages before and after treatment were analyzed by flow cytometry as shown in FIG.

[0206] Macrophages from two different donors treated with anti-ILT4 antibody produced TNF-α and IL-6 inflammatory cytokines with EC50 values ​​in the ng / ml range. Macrophages before and after antibody treatment expressed ILT4. CD163 surface expression was reduced on macrophages from both donors, whereas anti-ILT4 antibody had a minimal effect on reducing CD206 expression. CD163 is considered an M2 marker. Collectively, these data indicate that anti-ILT4 antibody treatment reprograms M2 macrophages to M1 macrophages by reducing CD163 expression and induces TNF-α and IL-6 inflammatory cytokine production.

[0207] [Example 8] Macrophage mixed leukocyte reaction (MLR) assay

[0208] To evaluate the effect of humanized LALA anti-ILT 4 antibody treatment on macrophages to enhance CD4 T cell activation, as a single or combination treatment with anti-PD1 antibody, as shown in Figure 13.

[0209] Human monocyte isolation and macrophage differentiation:

[0210] PBMCs were isolated from healthy human donors by Ficoll-Paque gradient centrifugation. Human monocytes were then purified from the PBMCs using the EasySep Human Monocyte isolation kit (StemCell) according to the manufacturer's instructions.

[0211] Human monocyte differentiation and culture assays

[0212] The cell density was adjusted to 0.5x10e6 cells / ml in fresh differentiation medium (STEMCELL) and 10ml / dish of monocytes were plated per donor in 5 10cm dishes + 50ng / ml M-CSF (5ul stem cells 100ug / ml human M-CSF added to 10ml ImmunoCult™-SF macrophage medium). After 4 days, half an additional amount of differentiation medium was added (5ml medium per dish). After another 2 days, the cells were detached using 5ml of Versene solution per dish, spun down and the cells were resuspended in RPMI+10%FBS+1%P / S.

[0213] Cells were counted and prepared at a density of 0.5x10e6 cells / ml in RPMI+10%FBS+1%P / S media. 100μl of cell suspension was added to the designated wells of two 96-well U-bottom plates according to the plate map. Anti-ILT4 antibody (humanized LALA) was added to the designated wells (96-well plate) at a final concentration of 1ug / ml.

[0214] CD4+ T cells were isolated from different donors, CD4+ T cells were prepared at a density of 5x10e6 cells / ml, and the cell suspension was added to the designed wells. The plates were incubated at 37°C, 5% CO2 for 5 days. On the sixth day, pipette the solution up and down and transfer all the solution to a V-bottom plate. Centrifuge and collect 100μl of the supernatant from the 96-well plate for IFN-g cytokine analysis.

[0215] Using IFN-g as a readout for T cell activation, IFN-g production was observed as a single agent when compared to isotype control. As shown in Figure 13, increased IFN-g production was observed when combined with anti-PD1 (Toriparimab).

[0216] [Example 9] Competition for anti-ILT4 mAb binding to ILT4-CHO

[0217] Competitive binding assays were performed. The assays involved testing the competition of Hz156.03-LALA (SEQ ID NO: 124 (HC), 125 (LC)), Hz45.01-LALA (SEQ ID NO: 128 (HC), 129 (LC)), 1E1, J19h1, R&D Systems MAB2078R, and human IgG1-LALA in competitive binding assays with Alexa Fluor 647 (AF647)-Hz54.01-LALA or AF647-Hz156.03-LALA for binding to ILT4-CHO cells using serial titrations (0-15 μg / mL). Binding activity was measured by flow cytometry.

[0218] Anti-human ILT4 mAbs at various concentrations (0-15 μg / ml) were incubated with ILT4-CHO cells. Cells were washed and stained with AF647-Hz45.01-LALA. Cells were analyzed by flow cytometry. Figure 14 shows the competition of anti-ILT4 mAbs binding to ILT4-CHO cells in competition with AF647-Hz45.01-LALA. Figure 15 shows the competitive binding of Hz45.01-LALA vs. 1E1 and HuIgG1-LALA (as a control) and Hz45.01-LALA vs. J19h1 and HuIgG1-LALA (as a control).

[0219] Anti-human ILT4 mAbs at various concentrations (0-15 μg / ml) were incubated with ILT4-CHO cells. Cells were washed and stained with AF647-Hz156.03-LALA. Cells were analyzed by flow cytometry. Figure 16 shows the competition of anti-ILT4 mAbs binding to ILT4-CHO cells in competition with AF647-Hz156.03-LALA. Figure 17 shows the competitive binding of Hz45.01-LALA versus 1E1 and HuIgG1-LALA (as a control) and Hz45.01-LALA versus J19h1 and HuIgG1-LALA (as a control).

[0220] All antibodies tested showed dose-dependent blocking of binding to ILT4-CHO cells, with IC 50 The values ​​are summarized in the table below. JPEG2025511146000013.jpg19142

[0221] The binding of AF647-anti-ILT4 mAb to ILT4-CHO cells was measured as shown in Figure 18. Various concentrations (0-10 μg / ml) of AF647-Hz45.01-LALA and AF647-Hz156.03-LALA were incubated with ILT4-CHO cells and analyzed by flow cytometry. The tested antibodies showed dose-dependent binding to ILT4-CHO cells and EC 50 and E.C. 90 The values ​​are summarized in the table below. JPEG2025511146000014.jpg19169

[0222] Hz45.01-LALA, Hz156.03-LALA, 1E1, and J19h1 competed with the binding of AF647-Hz45.01 to ILT4-CHO cells.

[0223] Hz45.01-LALA, Hz156.03-LALA, 1E1, and J19h1 also competed with the binding of AF647-Hz156.03-LALA to ILT4-CHO cells.

[0224] R&D MAB2078R and HuIgG1-LALA did not compete with the binding of AF647-Hz45.01-LALA or AF647-Hz156.03-LALA to ILT4-CHO cells.

[0225] Hz45.01-LALA and Hz156.03-LALA completely competed with the binding of AF647-Hz45.01-LALA and AF647-Hz156.03-LALA.

[0226] Some degree of AF647-Hz45.01-LALA and AF647-Hz156.03-LALA binding activity was maintained at high doses of 1E1 and J19h1 competition. The data indicate that Hz45.01-LALA and Hz156.03-LALA have similar binding properties to each other and somewhat different binding properties compared to 1E1 and J19h1.

[0227] JPEG2025511146000015.jpg251157 JPEG2025511146000016.jpg251158 JPEG2025511146000017.jpg240158 JPEG2025511146000018.jpg226158 JPEG2025511146000019.jpg234159 JPEG2025511146000020.jpg241160 JPEG2025511146000021.jpg207161 JPEG2025511146000022.jpg228162 JPEG2025511146000023.jpg238161 JPEG2025511146000024.jpg235162 JPEG2025511146000025.jpg254161 JPEG2025511146000026.jpg247162 JPEG2025511146000027.jpg230163 JPEG2025511146000028.jpg116162 Amino acids in bold represent the extracellular domain.

[0228] Any of the above protocols or similar variations thereof may be described in various documents related to pharmaceutical products. These documents may include, but are not limited to, protocols, statistical analysis plans, researcher brochures, clinical guidelines, medication guides, risk assessment and intervention programs, prescription information, and other documents that may be related to pharmaceutical products. It is specifically contemplated that such documents may be physically packaged together with pharmaceutical products according to the present disclosure as kits, as may be useful or as indicated by regulatory authorities.

[0229] Although the subject matter of the present disclosure has been described and illustrated in some detail with reference to certain exemplary embodiments, including various combinations and subcombinations of features, those skilled in the art will readily appreciate other embodiments, as well as variations and modifications thereof, that are encompassed within the scope of the present disclosure. Moreover, the description of such embodiments, combinations, and subcombinations is not intended to convey that the claimed subject matter requires any features or combinations of features other than those expressly recited in the claims. Accordingly, the scope of the present disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the following appended claims.

Claims

1. An antibody or antigen-binding fragment that binds to ILT4, CDR-H1 containing an amino acid sequence selected from the group consisting of DYYMN (SEQ ID NO: 1), GYSVN (SEQ ID NO: 9), DSYMN (SEQ ID NO: 23), GYFMN (SEQ ID NO: 30), SYWMN (SEQ ID NO: 38), DYTIH (SEQ ID NO: 46), DNYLQ (SEQ ID NO: 52), DYGMH (SEQ ID NO: 60), and TYGMS (SEQ ID NO: 68), CDR-H2 containing an amino acid sequence selected from the group consisting of DINPNNGGTSYNQKFKG (SEQ ID NO: 2), RINPYNGDIFNNQKFKG (SEQ ID NO: 10), RIYPGVYRTHYNEKFKD (SEQ ID NO: 17), YINPDNGVTRYNQKFKG (SEQ ID NO: 24), RINPYNGDIFYNQKFKG (SEQ ID NO: 31), QIYPGHGDTNYNGKFKG (SEQ ID NO: 39), WFYPGTVSIKYNEKFKD (SEQ ID NO: 47), PGSGNTYYSDNFTG (SEQ ID NO: 53), YISSDSSTIYYADTVKG (SEQ ID NO: 61), and WINTYSGEPTYADEFKG (SEQ ID NO: 69), CDR-H3 containing an amino acid sequence selected from the group consisting of GGAELTGTYWYFDV (SEQ ID NO: 3), GTTVGGAWFAY (SEQ ID NO: 11), SGYYGGTYEEDAMDY (SEQ ID NO: 18), EGTITTDLSWFAY (SEQ ID NO: 25), GITVAAGSFDV (SEQ ID NO: 32), EGSELGRLFAY (SEQ ID NO: 40), HEHPHYYYGDSYDAMGY (SEQ ID NO: 48), STVVYFDV (SEQ ID NO: 54), RAAQGYVMDY (SEQ ID NO: 62), and RGYDGYYYTMDY (SEQ ID NO: 70) Heavy chain variable domains including, CDR-L1 containing an amino acid sequence selected from the group consisting of RASENIYSNLA (SEQ ID NO: 4), RASESVDSYGYSFLH (SEQ ID NO: 12), RASESVDNYGNTFMH (SEQ ID NO: 33), SASSSVSFMY (SEQ ID NO: 41), SNYAN (SEQ ID NO: 55), and KASQSVSDDVA (SEQ ID NO: 63), CDR-L2 containing an amino acid sequence selected from the group consisting of GATNLAD (SEQ ID NO: 5), LASNLES (SEQ ID NO: 13), AATSLAD (SEQ ID NO: 19), ASTNLAD (SEQ ID NO: 26), RASNLES (SEQ ID NO: 34), LTSNLAS (SEQ ID NO: 42), GTNNRAP (SEQ ID NO: 56), ASNRYT (SEQ ID NO: 64), and AATNLAD (SEQ ID NO: 71), CDR-L3 containing an amino acid sequence selected from the group consisting of QHFWDSPFT (SEQ ID NO: 6), QQSNEDLMYT (SEQ ID NO: 14), QNFWDTPYT (SEQ ID NO: 20), QHFWDTPYT (SEQ ID NO: 27), QQSSDHPLT (SEQ ID NO: 35), QQWSSNPPT (SEQ ID NO: 43), QHFWGTPYT (SEQ ID NO: 49), WYSNHWV (SEQ ID NO: 57), QQDYGSPT (SEQ ID NO: 65), and QHFFGAPWT (SEQ ID NO: 72) Light chain variable domains including An antibody or its antigen-binding fragment, including an antibody.

2. a) A heavy chain variable domain CDR comprising DYYMN (SEQ ID NO: 1), DINPNNGGTSYNQKFKG (SEQ ID NO: 2), and GGAELTGTYWYFDV (SEQ ID NO: 3), A light chain variable domain CDR including RASENIYSNLA (SEQ ID NO: 4), GATNLAD (SEQ ID NO: 5), and QHFWDSPFT (SEQ ID NO: 6), or b) A heavy chain variable domain CDR comprising GYSVN (SEQ ID NO: 9), RINPYNGDIFNNQKFKG (SEQ ID NO: 10), and GTTVGGAWFAY (SEQ ID NO: 11), Light chain variable domain CDRs including RASESVDSYGYSFLH (SEQ ID NO: 12), LASNLES (SEQ ID NO: 13), and QQSNEDLMYT (SEQ ID NO: 14), or c) A heavy chain variable domain CDR comprising DYYMN (SEQ ID NO: 1), RIYPGVYRTHYNEKFKD (SEQ ID NO: 17), and SGYYGGTYEEDAMDY (SEQ ID NO: 18), A light chain variable domain CDR containing RASENIYSNLA (SEQ ID NO: 4), AATSLAD (SEQ ID NO: 19), and QNFWDTPYT (SEQ ID NO: 20), or d) A heavy chain variable domain CDR comprising DSYMN (SEQ ID NO: 23), YINPDNGVTRYNQKFKG (SEQ ID NO: 24), and EGTITTDLSWFAY (SEQ ID NO: 25), A light chain variable domain CDR including RASENIYSNLA (SEQ ID NO: 4), ASTNLAD (SEQ ID NO: 26), and QHFWDTPYT (SEQ ID NO: 27), or e) A heavy chain variable domain CDR comprising GYFMN (SEQ ID NO: 30), RINPYNGDIFYNQKFKG (SEQ ID NO: 31), and GITVAAGSFDV (SEQ ID NO: 32), Light chain variable domain CDRs including RASESVDNYGNTFMH (SEQ ID NO: 33), RASNLES (SEQ ID NO: 34), and QQSSDHPLT (SEQ ID NO: 35) The antibody or antigen-binding fragment according to claim 1, comprising:

3. The aforementioned light chain variable domain has the following amino acid sequence: DIQMTQSPASLSISVGETVTITCRASENIYSNLAWYQQKQGKSPQVLVYGATNLADGVPSRFSGSGSGTQYSLKIKSLQSEDFGSYYCQHFWDSPFTFGSGTKLEIK (Sequence ID 8), VIVLTQSPASLAVSLGQRAAISCRASESVDSYGYSFLHWYQQKPGQPPKLLLIYLASNLESGIPARFSGSGSGTDFTLTINPVEADDVATYYCQQSNEDLMYTFGGGTKLEIK (Sequence ID 16), DIQMTQSPASLSVSVGETVTITCCRASENIYSNLAWYQQKQGKSPQLLLVYAATSLADGVPSRGSGSGTQYSLKISSLQSEDFGNYYCQNFWDTPYTFGGGTKLEIK (Sequence ID 22), DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLLVYASTNLADGAPATSGSGSGTQYSLKINSLQSVDFGSYYCQHFWDTPYTFGGGTKLEIK (Sequence ID 29), DIVLTQSPASLAVSLGQRATISCRASESVDNYGNTFMHWYQQKPGQPPKLLLIYRASNLESGIPARFSGSGSSKTDFTLTINPVEADDVATYYCQQSSDHPLTFGAGTKLELS (Sequence ID 37), QIVLTQSPALMSASPGEKVTMTCSASSSVSFMYWYQQKPRSSPKPWIYLTSNLASGVPPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK (Sequence ID 45), DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLLVYGATNLADGVPSRFGGGSGSGTQYSLKINSLQPEDFGSYYCQHFWGTPYTFGGGTKLEIT (Sequence ID 51), QAVVTQESALTTSPGETVTLTCRSSSTGTVTTSNYANWVQEKPDHLFTGLIGGTNNRAAPGVPARFSGSLIGDQAALTITGAGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL (Sequence ID 59), SIVMTQTPKFLLVSAGDRVTITCKASQSVSDDVAAWYQQKPGQSPKLLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYGSPTFGGGTKLEIK (Sequence ID 67), DIQMTQSPASLSASVGETVTITCRASENIYSNLAWYQQKQGKSPQLLLVSAATNLADGVPPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHFFGAPWTFGGGTKLEIK (Sequence ID 74), or DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAAPKVLVYGATNLADGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCQHFWDSPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEEC (Sequence ID 125) It contains an amino acid sequence that is at least 80% identical to, The heavy chain variable domain has the following amino acid sequence: EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTSYNQKFKGKAATLTTVDKSSSTAYMELRSLTSEDSAVYYCARGGAELTGTYWYFDVWGTGTTVTVSS (Sequence No. 7), DVQLQQSGPELVKPGNSVKISCKAAAGYSFTGYSVNWVKERHGKSLEWIGRINPYNGDIFNNQKFKGKAATLTTVDKSSSSTAHMELRSLTSEDSAVYYCARGTTVGGGAWFAYWGQGTLVTVSA (Sequence No. 15), QVQLKQSGAELVRRPGGASVKLSCRASGYTFTDYYYMNWVKQRPGQGLEWIARIYPGVYRTHYNEKFKDKATLTAEKSSSTAYMELSSLTSEDSAVYFCARSGYYGGTYEEDAMDYWGQGTSVTVSS (Sequence No. 21), EVQLQQSGPELVIPGASVKISCKASGYTFTDSYMNWVKQSHGKSLEWIAYINPDNGVTRYNQKFKGKAATLTTVHKSSSSTAYMELRSLTSEDSAVYYCAREGTITTDLSWFAYWGQGTLVTVSA (Sequence No. 28), EVHLQQSGPELVKPGASVKISCKASGYSFIGYFMNWMKQSHGKSLEWIGRINPYNGDIFYNQKFKGKAATLTTVDKSSTTAHMDLLLSLTSEDFAVYYCARGITVAAGSFDVWGTGTTVTVSS (Sequence No. 36), QVQLQQSGAELVKPGASVKISCKASGYAAFSSYWMNWVKQRPGKGLEWIGQIYPGHGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAKEGSELGRLFAYWGQGTLVTVSA (Sequence No. 44), QVQLQQSGTTELVKPGASVKLSCKASGYIFTDYTIHWVKQRSGQGLEWIGWFYPGTVSIKYNEKFKDKATLTADRSSSIVYMELSRLTSEDSGVYFCCARHEHPHYYYGDSYDAMGYWGQGTSVTVSS (Sequence No. 50), QVQLQQSGPELVKPGASVKISCKASGYIFTDNYLQWVKQRPGQGLEWIGWIFPPGSGNTYYSDNFTGKATLTTVDKSSITAYMLLSSLTSEDSAVYFCSRSTVVYFDVWGTGTTVTVSS (Sequence No. 58), EVQLVESGGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKRLEWVAYISSDSSTIYYADTVKGRFTISRDNAKNTLFLEMTSLRRSEDTAMYYYCARRAAQGYVMDYWGQGTSVTVSS (Sequence No. 66), QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMAWINTYSGEPTYADEFKGRFAFSLETSVSSTAYLQINNLKNEDTATYFCCARRGYDGYYYTMDYWGQGTSVTVSS (Sequence ID 73), or QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQRLEWIGDINPNNGGTSYNQKFKGRATITVDTSASTAYMELSSLRSEDTAVYYCARGGAELTGTYWYFDVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 124) The antibody or antigen-binding fragment according to claim 1, comprising an amino acid sequence that is at least 80% identical to that of the antibody or antigen-binding fragment according to claim 1.

4. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment antagonizes the interaction between ILT4 and HLA-G or HLA-A.

5. The antibody or its antigen-binding fragment a) A heavy chain constant region selected from the group consisting of IgG1, IgG2, or IgG4, b) A light chain constant region selected from the group consisting of kappa and lambda, or c) Both (a) and (b) The antibody or antigen-binding fragment according to claim 1, comprising:

6. It contains a variant Fc region of IgG1, IgG2, or IgG4, The mutant Fc region of IgG1 is a) Amino acid substitution by alanine at the Leu234 position, b) Amino acid substitution by alanine at the Leu235 position, c) Amino acid substitution with glycine or arginine at position 329 of Pro, d) Amino acid substitution by alanine at Asn position 297, e) Amino acid substitution by glutamine at Asn position 297, f) Amino acid substitution by glycine at Asn position 297, g) including any combination of (a) to (f), The variant Fc region of IgG2 is h) Amino acid substitution by proline at Ser position 228, i) Amino acid substitution with glycine or arginine at position 329, or j) including both (h) and (i), The mutant Fc region of IgG4 is k) Amino acid substitution by proline at Ser position 228, l) Amino acid substitution with alanine or glutamic acid at the Leu235 position, m) Amino acid substitution with glycine or arginine at position 329 of Pro, n) includes any combination of (k) to (m), The antibody or antigen-binding fragment according to claim 1, wherein the residues are numbered according to the EU index of Kabat.

7. The antibody or its antigen-binding fragment a) Specifically binds to human ILT4 with at least 100 times higher affinity than it binds to human ILT3 and human ILT2, respectively. b) Equilibrium dissociation constant (K) of 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, or 0.08 nM or less Ｄ ) connects to ILT4, or (a) and (b) are both, The antibody or antigen-binding fragment according to claim 1.

8. The antibody or its antigen-binding fragment a) Equilibrium dissociation constants (K) of approximately 0.5 to 10 pM, 1 to 9 pM, 2 to 8 pM, 3 to 7 pM, or 4 to 6 pM. Ｄ ) connects to ILT4, b) IC50 at approximately 0.1 to 0.25 μg / mL to block HLA-G binding to ILT4 ５０ Having a value, c) IC50 at approximately 0.1 to 0.2 μg / mL to block HLA-A binding to ILT4 ５０ Having a value, d) EC production of TNF-α at approximately 7 ng / mL to 25 ng / mL or approximately 12 ng / mL to 20 ng / mL ５０ Having a value, e) EC producing IL-6 at approximately 5 ng / mL to 25 ng / mL, approximately 6 ng / mL to 22 ng / mL, or approximately 8 ng / mL to 21 ng / mL ５０ Having a value, or f) Any combination of (a) to (e), The antibody or antigen-binding fragment according to claim 1.

9. An immunoconjugate comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8, conjugated to a chemotherapeutic agent, a cytotoxin, a detectable moiety, a diagnostic agent, or a combination thereof.

10. An expression system comprising at least one expression vector, The expression system comprises one or more nucleic acid sequences encoding a protein containing the antibody or antigen-binding fragment described in any one of claims 1 to 8.

11. A host cell comprising the expression system described in claim 10.

12. The host cell according to claim 11, wherein the host cell is a mammalian cell.

13. The host cell according to claim 11, wherein the host cell is a Chinese hamster ovary cell.

14. A method for producing an antibody or its antigen-binding fragment, A method comprising culturing the host cells described in claim 11 under conditions for expressing the antibody or its antigen-binding fragment, and recovering the antibody or its antigen-binding fragment.

15. A pharmaceutical composition comprising an immunoconjugate containing an antibody or antigen-binding fragment according to any one of claims 1 to 8, conjugated to an antibody or antigen-binding fragment according to any one of claims 1 to 8, or to a chemotherapeutic agent, cytotoxin, detectable moiety, diagnostic agent, or a combination thereof, and a pharmaceutically acceptable carrier.

16. Including additional medications, The pharmaceutical composition according to claim 15, wherein the additional therapeutic agent is a PD-1 / PD-L1, TIGIT, or CTLA4 antagonist.

17. The pharmaceutical composition according to claim 16, wherein the PD-L / PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, durvalumab, lambrolizumab, avelumab, and tripalimab.

18. For treating subjects with cancer, autoimmune diseases, or inflammatory diseases, The pharmaceutical composition according to claim 15.

19. a) The cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, Endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer (stomach). cancer), bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, head and neck cancer (or cell carcinoma), gastric cancer Cancer, germ cell tumors, pediatric sarcomas, sinus natural killer tumors, melanoma (e.g., metastatic melanoma such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcomas, urethral cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumors, brain cancer, brain Stem nerve glioma, pituitary adenoma, Ewing's sarcoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, mesothelioma, meningioma, chondrosarcoma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocytemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, and carcinoid cancer, leukemia, lymphoma, myeloma, Wilms' cancer, or a combination or metastasis thereof. b) The autoimmune diseases include Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteritis-associated arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behçet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain symptoms associated with systemic inflammatory diseases, systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile diabetes mellitus (also known as type 1 diabetes mellitus), and Wegener's granuloma. Myasthenia gravis, dermatomyositis, inclusion body myositis, polyendocrine insufficiency, Schmidt syndrome, autoimmune uveitis, Addison's disease, Graves' disease, Hashimoto's disease, autoimmune thyroid disease, pernicious anemia, gastric mucosal atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, herpetiform dermatitis, alopecia, scleroderma, progressive systemic hardening of the skin Chromosomal disorders, CREST syndrome (calcification, Raynaud's phenomenon, esophageal motility reduction, cystitis, and telangiectasia), adult-onset diabetes (also known as type II diabetes), mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture syndrome, Chagas disease, sarcomatoidosis, rheumatic fever, asthma, antiphospholipid syndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic diseases, allergic encephalomyelitis, transfusion reactions, leprosy, malaria, and Liegia. Schmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, eczema, lymphomatous granulomatosis, Kawasaki disease, endophthalmitis, psoriasis, erythroblastosis fetus, eosinophilic fasciitis, Schulman syndrome, Felty syndrome, Fuchs cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, transplant rejection, tularemia, periodic fever syndromes, suppurative arthritis, familial Mediterranean fever, TNF receptor-associated periodic syndromes (TRAPS), Mackle-Wells syndrome, or hyper-IgD syndrome, or c) The cancer is acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant [with eosinophilia] M4E), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroplasma; lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, undifferentiated (e.g., Ki 1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, vascular central lymphoma, intestinal T-cell lymphoma, mediastinal primary B-cell lymphoma, progenitor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), lymphoid hematopoietic malignancies, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell Lymphomas of the first type, progenitor B lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma (LPL) with Waldenström macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as painless myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; mesenchymal tumors including myeloid hematopoietic malignancies, fibrosarcomas, and rhabdomyosarcomas; central and peripheral nerve tumors including seminomas, teratomas, astrocytomas, and schwannomas; mesenchymal tumors including fibrosarcomas, rhabdomyosarcomas, and osteosarcomas;Furthermore, melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular and teratogenic carcinomas of the thyroid gland, hematopoietic malignancies of the lymphatic system, other tumors including T-cell disorders such as T-prelymphocytic leukemia (T-PLL), including small cell and cerebral cell types, but not limited to T-cell and B-cell tumors; large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); vascular centripetal (nasal) T-cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myelolynphomas, or combinations thereof or metastases; d) The pharmaceutical composition according to claim 18, wherein the cancer is lung cancer, liver cancer, ovarian cancer, pancreatic cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, glioblastoma, renal cell carcinoma, stomach cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, or head and neck cancer.

20. A heavy chain variable domain CDR containing DYYMN (SEQ ID NO: 1), INPNNNGGT (SEQ ID NO: 116), and GGAELTGTYWYFDV (SEQ ID NO: 3), Light chain variable domain CDRs including ENIYS (SEQ ID NO: 117), GAT, and QHFWDSPFT (SEQ ID NO: 6) An antibody or its antigen-binding fragment that binds to ILT4, including the above.

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