Antibodies against ilt2 and uses thereof

Abstract

The present invention provides monoclonal antibodies or antigen-binding portions thereof directed against ILT2, as well as pharmaceutical compositions comprising the monoclonal antibodies or antigen-binding portions thereof and methods of producing the monoclonal antibodies or antigen-binding portions thereof. Also provided are methods of treating cancer comprising administering the antibodies or compositions of the invention. Methods of treating cancer, combination therapy, and patient selection are also provided.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 034,569, filed June 4, 2020, and U.S. Provisional Patent Application No. 62 / 885,374, filed August 12, 2019, the contents of which are incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present invention belongs to the field of monoclonal antibodies and modulating immune responses against cancer. BACKGROUND

[0004] ILT2, also known as LILRB1, LIR1, and CD85j, is a cell surface protein expressed on immune cells with known functions to suppress immune responses. The protein contains 4 Ig C domains in the extracellular region and 4 intracellular ITIM domains. It is a member of the ILT family, which consists of ILT1, ILT2, ILT3, and ILT4. ILT2 is most similar to ILT4, with about 80% homology. Known ligands of ILT2 include MHC-1 as well as non-classical MHC molecules such as HLA-F, HLA-G, HLA-B27, and UL18 (human CMV). The known strongest interactors of ILT2 in the human genome are HLA-G1.

[0005] HLA-G1 is widely expressed on the surface of a variety of malignancies, including breast, cervical, CRC, lung, gastric, pancreatic, thyroid, and ovarian cancer cells, as well as glioblastoma multiforme, melanoma cells. Its expression is associated with a worse clinical outcome. In addition, ILT2 expression in the tumor microenvironment is associated with a worse clinical response to oncolytic immunotherapy, even in the absence of HLA-G1. Harnessing the immune response as a weapon against cancer and for cancer surveillance is a promising way for cancer prevention and treatment. However, ILT2 appears as an obstacle to effective immunotherapy. There is a great need for therapeutic modalities that can circumvent the ILT2-HLA-G1 axis as well as the non-HLA-G1 dependent functions of ILT2. SUMMARY

[0006] The present invention provides monoclonal antibodies that bind to ILT2 and inhibit ILT2-mediated suppression of immune cells; and pharmaceutical compositions comprising the monoclonal antibodies. Also provided are methods of treating cancer comprising administering the compositions of the present invention, methods of producing the antibodies, binding fragments, and compositions of the present invention, and methods of increasing the efficacy of PD-1 / PD-L1 based therapies.

[0007] According to a first aspect, there is provided a monoclonal antibody or antigen binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0008] a. CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 13 (SGYYWN), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 14 (YISYDGSNNYNPSLKN), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 15 (GYSYYYAMDX), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 16 (RTSQDISNYLN), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 17 (YTSRLHS), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 18 (QQGNTLPT), wherein said X is selected from A, C and S;

[0009] b. CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (DHTIH), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 2 (YIYPRDGSTKYNEKFKG), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 3 (TWDYFDY), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 4 (RASESVDSYGNSFMH), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 5 (RASNLES), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 6 (QQSNEDPYT); or

[0010] c. CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 7 (GYTFTSYGIS), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 8 (EIYPGSGNSYYNEKFKG), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 9 (SNDGYPDY), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 10 (KASDHINNWLA), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 11 (GATSLET), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 12 (QQYWSTPWT).

[0011] According to some embodiments, the antibody or antigen-binding fragment of the application comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARTWDYFDYWGQGTTLTVSS), SEQ ID NO: 21 (QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWVGEIYPGSGNSYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARSNDGYPDYWGQGTTLTVSS) and SEQ ID NO: 23 (DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDXWGQGTSVTVSS), wherein said X is selected from the group consisting of A, C and S.

[0012] According to some embodiments, the antibody or antigen-binding fragment of the present application comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 (EVQLVESGGGLVQPGGSLRLSCAASGFTLSSNAMSWVRQAPGKGLEWVSAISSGSDTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAADGYTSSSSVNTGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYYSYPLTFGGGTKLEIK), SEQ ID NO: 15 (EVQLVESGGGLVQPGGSLRLSCAASGFTLSSNAMSWVRQAPGKGLEWVSAISSGSDTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAADGYTSSSSVNTGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYYSYPLTFGGGTKLEIK), SEQ ID NO: 17 (EVQLVESGGGLVQPGGSLRLSCAASGFTLSSNAMSWVRQAPGKGLEWVSAISSGSDTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAADGYTSSSSVNTGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYYSYPLTFGGGTKLEIK), and SEQ ID NO: 19 (EVQLVESGGGLVQPGGSLRLSCAASGFTLSSNAMSWVRQAPGKGLEWVSAISSGSDTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAADGYTSSSSVNTGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYYSYPLTFGGGTKLEIK).

[0013] According to some embodiments, the antibody or antigen-binding fragment is humanized and X is selected from the group consisting of A and S.

[0014] According to some embodiments, X is A, SEQ ID NO: 15 is GYSYYYAMDA (SEQ ID NO: 25) and SEQ ID NO: 23 is DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDAWGQGTSVTVSS (SEQ ID NO: 28).

[0015] According to another aspect, a monoclonal antibody or antigen-binding fragment is provided that binds to an epitope of human leukocyte immunoglobulin-like receptor subfamily B member 1 (ILT2), said epitope being selected from the human ILT2 sequence: VKKGQFPIPSITWEH (SEQ ID NO:41), LELVVTGAYIKPTLS (SEQ ID NO:42), VILQCDSQVAFDGFS (SEQ ID NO:43), and WYRCYAYDSNSPYEW (SEQ ID NO:44).

[0016] According to some implementations, the epitope is a three-dimensional epitope comprising SEQ ID NO:41, 42, 43 and 44.

[0017] According to some embodiments, a monoclonal antibody or antigen-binding fragment is provided that binds to ILT2 and inhibits the direct interaction between ILT2 and β-2 microglobulin (B2M).

[0018] According to some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and HLA or MHC-I proteins by inhibiting the direct interaction between ILT2 and B2M.

[0019] According to some implementation schemes, the HLA is HLA-G.

[0020] According to another aspect, a monoclonal antibody or antigen-binding fragment is provided that binds to ILT2 and induces at least three of the following in a subject with cancer:

[0021] a. Increased cytotoxicity of natural killer (NK) cells;

[0022] b. Increased T cell cytotoxicity, proliferation, or both;

[0023] c. Increased macrophage phagocytosis, increased production of M1 inflammatory macrophages, decreased production of M2 suppressive macrophages, or a combination thereof; and

[0024] d. Increased dendritic cell homing to cancerous tumors, increased dendritic cell activation, or a combination thereof.

[0025] According to some implementation schemes, the cancer is a cancer that expresses HLA-G or MHC-I.

[0026] According to some embodiments, the antibody or antigen-binding fragment of the present invention is used for at least one of the following: binding to ILT2, inducing / enhancing anti-tumor T cell responses, increasing T cell proliferation, reducing cancer-induced myelosuppressive activity, increasing the cytotoxicity of natural killer cells, increasing macrophage phagocytosis, increasing the generation of M1 inflammatory macrophages, reducing the generation of M2 suppressive macrophages, increasing the number of dendritic cells in the tumor microenvironment, increasing dendritic cell activation, treating cancers expressing HLA-G, and treating cancers expressing MHC-I.

[0027] According to some embodiments, the antibody or antigen-binding fragment of the present invention, in combination with an opsonizer, is used to treat cancers expressing HLA-G or MHC-I.

[0028] According to some embodiments, the antibody or antigen-binding fragment of the present invention is combined with an anti-PD-L1 / PD-1-based therapy for the treatment of cancers expressing HLA-G or MHC-I.

[0029] According to another aspect, a method is provided for treating a subject with cancer expressing HLA-G or MHC-I, the method comprising administering to the subject a pharmaceutical composition comprising an antibody or antigen-binding fragment of the present invention.

[0030] According to some embodiments, the method of the present invention further includes administering a conditioning agent to the subject.

[0031] According to some embodiments, the opsonizer is an EGFR inhibitor, optionally wherein the EGFR inhibitor is cetuximab.

[0032] According to some embodiments, the method of the present invention further includes administering an anti-PD-L1 / PD-1-based immunotherapy to the subject.

[0033] According to another aspect, a method is provided for treating HLA-G or MHC-I-expressing cancers in a subject of need, the method comprising:

[0034] a. Confirm that the expression of ILT2 or soluble HLA-G in the subjects is higher than a predetermined threshold; and

[0035] b. Administer to the subject an agent that inhibits ILT2-based immunosuppression; thereby treating the subject's cancer.

[0036] According to some implementation schemes, the confirmation includes measuring the expression of ILT2 or soluble HLA-G in the subject prior to the administration.

[0037] According to some embodiments, the method of the present invention includes confirming the expression of ILT2, wherein the expression of ILT2 is in the immune cells of the subject.

[0038] According to some implementation schemes, the immune cells are selected from peripheral blood immune cells and tumor-associated immune cells.

[0039] According to some implementation schemes, the immune cells are selected from CD8-positive T cells, macrophages, NK cells, and TEMRA cells.

[0040] According to some implementation schemes, the immune cells are peripheral blood CD8-positive T cells.

[0041] According to some embodiments, the method of the present invention includes confirming the expression of soluble HLA-G.

[0042] According to some embodiments, the method of the present invention further includes administering an anti-PD-L1 / PD-1-based therapy to the subject.

[0043] According to another aspect, a method is provided for treating HLA-G or MHC-I-expressing cancers in a subject of need, the method comprising:

[0044] a. Administering to the subject an agent that inhibits ILT2-based immunosuppression; and

[0045] b. Administer anti-PD-L1 / PD-1-based therapy to the subjects;

[0046] This allows them to treat the cancer in the test subjects.

[0047] According to another aspect, a method is provided to increase the efficacy of anti-PD-L1 / PD-1-based therapies against cancer cells expressing HLA-G, MHC-I, or both, the method comprising contacting the cancer cells with an ILT2 antagonist.

[0048] According to some implementation schemes, the agent that inhibits ILT2-based immunosuppression is an ILT2 antagonist.

[0049] According to some implementation schemes, the ILT2 antagonist is an antibody or antigen-binding fragment that specifically binds to ILT2 and inhibits ILT2-mediated immune cell suppression.

[0050] According to some implementation schemes, the antibody or antigen-binding fragment of the method is an antibody or antigen-binding fragment as described herein.

[0051] According to some implementation schemes, the anti-PD-L1 / PD-1-based immunotherapy is an anti-PD-1 blocking antibody.

[0052] According to some implementation schemes, the cancer is refractory to anti-PD-L1 / PD-1 therapy.

[0053] According to some embodiments, the method of the present invention further includes administering a conditioning agent to the subject.

[0054] According to some embodiments, the opsonizer is an EGFR inhibitor, optionally wherein the EGFR inhibitor is cetuximab.

[0055] According to another aspect, a pharmaceutical composition comprising an agent that binds to ILT2 and inhibits ILT2-mediated immunosuppression is provided, said pharmaceutical composition being combined with an anti-PD-L1 / PD-1 based therapy for treating a subject with cancer.

[0056] According to another aspect, a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention is provided.

[0057] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0058] Obtain an agent that binds to the extracellular domain of ILT2 or a fragment thereof, and test the agent's ability to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and select at least one agent that induces at least two of the following: the increased phagocytosis, the increased activity, the increased production, the decreased production, the recruitment, the increased activation, the decreased activity, and the increased cytotoxicity; or

[0059] Culture host cells containing one or more vectors that encode a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected in the following manner:

[0060] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0061] ii. Test the ability of the agent to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and

[0062] iii. Select at least one agent that increases at least two of the following: increased phagocytosis, increased activity, increased production, decreased production, recruitment, increased activation, decreased activity, and increased cytotoxicity;

[0063] This produces the medicine.

[0064] According to some embodiments, the method of the present invention includes testing the ability of the agent to induce at least three of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased generation of M1 macrophages, decreased generation of M2 macrophages, increased recruitment of dendritic cells to the tumor microenvironment, increased dendritic cell activation, and increased cytotoxicity against natural killer (NK) cells, and selecting at least one agent that induces at least three of the following.

[0065] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0066] Obtaining an agent that binds to the extracellular domain of ILT2 or a fragment thereof, testing the ability of said agent to enhance the efficacy of anti-PD-L1 / PD-1 based therapies against cancer cells, and selecting at least one agent that enhances the efficacy of anti-PD-L1 / PD-1 based therapies; or culturing host cells comprising one or more vectors containing a nucleic acid sequence encoding an agent, wherein said nucleic acid sequence is a nucleic acid sequence of an agent selected in such a manner as:

[0067] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0068] ii. To test the ability of the said agent to enhance the efficacy of anti-PD-L1 / PD-1 based therapies against cancer cells; and

[0069] iii. Select at least one agent that enhances the efficacy of anti-PD-L1 / PD-1-based therapies against cancer cells;

[0070] This produces the medicine.

[0071] According to some embodiments, the increased efficacy includes a synergistic increase in the secretion of pro-inflammatory cytokines, or the increased cytotoxicity includes an increase in the secretion of pro-inflammatory cytokines.

[0072] According to some implementation schemes, the pro-inflammatory cytokines are selected from GM-CSF, TNFα, and IFNγ.

[0073] According to some implementation schemes, the increased efficacy includes an increase in T cell activation, cytotoxicity, or a synergistic increase in both.

[0074] According to some implementation schemes, the increase in T cell activation, cytotoxicity, or both includes increased membrane CD107a expression.

[0075] According to some implementations, the increased efficacy includes converting cancers refractory to anti-PD-L1 / PD-1-based therapies into cancers responsive to anti-PD-L1 / PD-1-based therapies.

[0076] According to some implementation schemes, the cancer cells are cancers that express HLA-G or MHC-I.

[0077] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0078] Obtain an agent that binds to the extracellular domain of ILT2 or a fragment thereof, test the ability of said agent to inhibit the interaction between ILT2 and B2M, and select at least one agent that inhibits the interaction between ILT2 and B2M; or culture host cells containing one or more vectors encoding a nucleic acid sequence of an agent, wherein said nucleic acid sequence is a nucleic acid sequence of an agent selected in the following manner:

[0079] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0080] ii. Test the ability of the agent to inhibit the interaction between ILT2 and B2M; and

[0081] iii. Select at least one agent that inhibits the interaction between ILT2 and B2M;

[0082] This produces the medicine.

[0083] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0084] Obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO:41, 42, 43 and 44, or culturing host cells containing one or more vectors with a nucleic acid sequence encoding the drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected by obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO:41, 42, 43 and 44; thereby producing a drug.

[0085] According to another aspect, a nucleic acid molecule encoding an antibody or antigen-binding fragment of the present invention is provided.

[0086] According to some implementation schemes, the nucleic acid molecule is an expression vector.

[0087] Other embodiments of the invention and its full scope will become clear from the detailed description given below. However, it should be understood that while the detailed description and specific examples indicate preferred embodiments of the invention, they are given in an illustrative manner, as various variations and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. Attached Figure Description

[0088] Figure 1 Histograms depicting ILT2 expression on lymphocytes were plotted. Commercial antibody #1 was used at a final concentration of 5 μg / mL. Bindings were plotted as black histograms, while isotype controls were stained and displayed as light gray histograms.

[0089] Figure 2 Histograms depicting ILT2 expression on various immune cells were plotted. Commercial antibody #1 was used at a final concentration of 5 μg / mL. Bindings were plotted as black histograms, while isotype controls were stained and displayed as light gray histograms.

[0090] Figures 3A-3C (3A) Table of cancer indications from the TCGA database in which ILT2 RNA is overexpressed. (3B) Dot plot of the association between MDSC enrichment and ILT2 expression in tumors. Also presented is a bar plot depicting the association between M2 enrichment and ILT2 expression. (3C) Scatter plot of the percentage of various immune cells expressing ILT2 in different tumors.

[0091] Figures 4A-4B (4A) Bar graph showing the percentage of HLA-G positive cases for various cancers as determined by immunohistochemistry (IHC). (4B) Scatter plot of HLA-G IHC scores for various cancers.

[0092] Figure 5 Scatter plot of soluble HLA-G levels in various cancers.

[0093] Figure 6 The heavy and light chain sequences of the three anti-ILT2 antibodies. CDRs identified by the KABAT system are underlined or shown in red.

[0094] Figures 7A-7E(7A) Table of antibody binding values ​​to ILT2 and ILT2 family members. (7B) Histogram of antibody binding to ILT2 on the cell surface of BW cells transfected with human ILT2. (7C) Line graph of binding of chimeric and humanized 19E3 (left panel) and chimeric and humanized 15G8 (right panel) to ILT2 expressed on the surface of BW cells transfected with human ILT2. (7D) Immunostaining of gastric cancer samples with 19E3 antibody. (7E) Scatter plot of the percentage of various immune cells expressing ILT2 in PBMC samples from healthy controls and cancer patients using 15G8 humanized antibody.

[0095] Figures 8A-8P(8A) Bar graph of the percentage of blockade for each ILT2 antibody and positive control (PC, GHI / 75 antibody). (8B) Histogram of ILT2-biotin binding to HLA-G-expressing cells in the presence of ILT2 blocking antibodies. ILT2-biotin binding to cells was determined by flow cytometry analysis using streptavidin-PE. No antibody (grey line), 15G8 (light gray line), allotype control (black line). (8C) Line graph of the blocking activity of the 15G8 humanized antibody as determined by the binding of ILT2-biotin to HLA-G-expressing cells. (8D) Line graph of the blocking activities of chimeric and humanized 19E3 (left panel) and chimeric and humanized 15G8 (right panel) as determined by the binding of ILT2-biotin to HLA-G-expressing cells in the presence of antibodies. (8E) Bar graph of mouse IL-2 secretion from cells expressing the ILT2 signaling reporter construct, in the presence of HLA-G-expressing cells and with or without ILT2 blocking antibodies. PC = positive control (GHI / 75 antibody). (8F) Line graph of the blocking activity of the 15G8 humanized antibody as determined by reporter assay. (8G) Bar graph of mouse IL-2 secretion from cells expressing the ILT2 signaling reporter construct, in the presence or absence of ILT2 blocking antibodies and positive control antibodies. (8H-8K) Bar graph of human IL-2 secretion from (8H) ILT2-deficient or (8I-8K) ILT2-expressing Jurkat cells co-cultured with (8I) MHC-I-only or (8J-8K) MHC-I and exogenous HLA-G-expressing A375 cancer cells, in the presence or absence of ILT2 blocking antibodies and positive control (8I-J) pan-HLA antibody or (8K) HLA-G-specific antibody. (8L-8N) Bar graphs of human IL-2 secretion from Jurakt cells expressing ILT2, cultured with HLA-G-expressing A375 cancer cells in the presence or absence of (8L)15G8 antibody, (8M)GHI / 75 antibody, and (8N)HP-F1 antibody. (8O-8P) Dot graphs of expression of activation markers (8O) phosphorylated ZAP70 and (8P) phosphorylated Syk in TIL cells and NK cells, respectively, in the presence or absence of 15G8 antibody.

[0096] Figures 9A-9D (9A) As determined by a FACS-based method, phagocytosis was measured as a bar graph of the percentage of HLA-G-expressing cancer cells co-cultured with macrophages in the presence of ILT2 antibody relative to a control. (9B) As determined by... Line graphs showing the real-time phagocytosis of cancer cells by macrophages in the presence of ILT2 antibody, as determined by the system. (9C) Bar graphs showing the percentage of various HLA-G and MHC-I-expressing cancer cells co-cultured with macrophages in the presence of ILT2 antibody 15G8 relative to controls. (9D) Bar graphs showing the phagocytosis of macrophages co-cultured with A253-HLA-G cells in the presence of ILT2 antibody, Erbitux, hIgG control, or combinations thereof.

[0097] Figures 10A-10B Bar graphs of IFNγ and granzyme B secretion from activated CD8 T cells co-cultured with (10A) wild-type 721.221 cells or HLA-G-expressing 721.221 cells or (10B) HLA-G-expressing A375 cells in the presence of ILT2 antibody.

[0098] Figures 11A-11H (11A-11B) Bar graphs showing the percentage of cytotoxicity in NK cell lines co-cultured with various cancer cell lines expressing (11A) HLA-G and (11B) MHC-I in the presence of ILT2 antibody. (11C-11D) Bar graphs showing the secretion of granzyme B and (11D) IFNγ in NK cell lines co-cultured with H&N cancer cells and melanoma cells, respectively, in the presence of 15G8 ILT2 antibody. (11E-11F) Bar graphs showing the expression of (11E) IFNγ and (11F) CD107A in ILT2-positive primary NK cells co-incubated with target cancer cells in the presence of ILT2 antibody. (11G-11H) Scatter plots showing the association between ILT2-positive cells and the expression of (11G) IFNγ and (11H) CD107A in response to ILT2 antibody.

[0099] Figure 12 Linear graphs of HLA-DR and CD80 expression (MFI) in macrophages as determined by flow cytometry, where the macrophages differentiated into M0, M1, or M2 macrophages from monocytes isolated from healthy donors in the presence of IgG or anti-ILT2 antibody. The number of patients with increased expression of the specified marker compared to control IgG is shown for each condition tested.

[0100] Figures 13A-13C (13A) Bar graph showing the phagocytic activity of macrophages co-cultured with various primary tumor cells. (13B-13C) Bar graph showing the dose-dependent phagocytic activity of autologous macrophages on primary tumor cells isolated from (13B) RCC patients and (13C) H&N patients in the presence of the humanized antibody of the present invention.

[0101] Figures 14A-14L (14A) Dot plot of ILT2 and PD-1 expression in tumor cells (left panel) and PBMCs (right panel) from RCC and esophageal cancer patients. (14B-14C) Box plot of (14B) PD-1 and (14C) ILT2 RNA expression in CD8 T cell populations in TME of CRC patients. (14D-14E) (14D) Dot plot of ILT2 expression in CD8 T cells from peripheral blood of healthy donors and (14E) ILT2 and PD-1 expression in TILs from esophageal cancer. (14F) Scatter plot of increased membrane CD107a expression on PBMCs from 10 healthy donors activated with staphylococcal enterotoxin B (SEB) in the presence of 15G8, anti-PD-1 antibody, or a combination of both. (14G) Bar plot of increased CD107a expression in exemplary PBMCs from 3 donors. (14H-14J) Bar graph showing the secretion levels of inflammatory cytokines (14H)IFNγ, (14I)TNFα, and (14J)GM-CSF from activated PBMCs co-cultured with various primary cancer cells in the presence of anti-PD-1 antibody, humanized anti-ILT2 antibody, or both. (14K-14L) Bar graph showing the secretion levels of IFNγ from T cells co-cultured with (14K) dendritic cells or (14L) macrophages in mixed lymphocyte reactions.

[0102] Figures 15A-15F (15A) Line graph of tumor volume of HLA-G and MHC-I-expressing tumors growing in immunocompromised mice supplemented with human macrophages and anti-ILT2 antibodies. (15B) Graph illustration of a mouse treatment schedule for the prevention of lung tumors. (15C) Photograph of the lungs of immunocompromised mice inoculated with HLA-G-positive cancer cells, with or without human PBMCs and ILT2 antibodies. (15D) Scatter plot summarizing data from 15C. (15E) Graph illustration of a mouse treatment schedule for the treatment of established lung tumors. (15F) Box plot of tumor weight.

[0103] Figures 16A-16F Box plots below (16A-16F): (16A) CD107A expression in total CD8 T cells, (16B) T... EMRA CD107A expression in (16C) NK cells, CD69 expression in (16D) total CD8 T cells, and CD69 expression in (16D) total CD8 T cells were observed in mice receiving PBMCs from donors with low or high ILT2 levels, respectively. EMRA In cells or NK cells, (16E)T EMRACD107 expression in cells and CD69 expression in NK cells treated with (16F) combination. * indicates P < 0.005. ** indicates P < 0.0005. *** indicates P < 0.0001.

[0104] Figures 17A-17F (17A) Schematic illustration of the treatment timeline for humanized NSG mice inoculated with H&N cancer and treated with anti-ILT2 or control antibodies. (17B) Line graph of tumor weight from mice treated with IgG and anti-ILT2. (17C-17E) (17C) Bar graph of baseline ILT2 levels in peripheral CD8 T cells in mice that responded (R) or did not respond (NR) to BND-22 treatment. (17D) CD107A expression, (17E) M1 / M2 ratio, and (17F) total number of CD80-positive dendritic cells after intratumoral treatment in four mice treated with anti-ILT2 antibodies.

[0105] Figures 18A-18F (18A) A partial sequence of ILT2, showing residues with significant predicted binding. These residues are categorized into four classes based on their original probability of belonging to epitopes, from purple (highest probability) to light cyan (lowest probability, but still significant). An asterisk indicates the location of the selected mutation. (18B-18C) 3D rendering of the ILT2 surface structure, showing the locations of residues from 18A in (18B) and the four major interacting regions on ILT2 in (18C). 3D banding or surface plot of (18D-18F)ILT2 showing the epitope (yellow / pink) of the (18D)15G8 antibody and the epitopes (red) of the 3H5, 12D12 and 27H5 antibodies from WO2020 / 136145, as well as the secondary epitope (dark blue) of the 3H5 antibody, and the interaction of the (18E-18F) and 15G8 epitopes (pink) on ILT2 with B2M (light purple) in complexes having (18E)HLA-A (blue) or (18F)HLA-G (blue).

[0106] Figures 19A-19C (19A-19B) Bar graphs showing the increased phagocytic percentage of (19A) A375-HLA-G and (19B) SKMEL28-HLA-G cancer cells co-cultured with macrophages in the presence of various anti-ILT2 antibodies, compared to the IgG control. (19C) Line graphs of competitive ILT2 binding ELISA using biotinylated 15G8 antibody in the presence of competitive unbiotinylated GHI / 75, HP-F1, and 15G8 antibodies. Detailed Implementation

[0107] This invention relates to monoclonal antibodies or antigen-binding fragments that bind to ILT2 and inhibit ILT2-mediated immunosuppression, and pharmaceutical compositions. Methods for treating cancer and enhancing PD-1 / PD-L1 immunotherapy are also provided.

[0108] This invention is based, at least in part, on the surprising discovery that ILT2 antagonism synergizes with PD-1 and PD-L1-based immunotherapies to combat cancer cells. Specifically, it was found that the combination of ILT2-blocking antibodies and anti-PD-1 antibodies increases the secretion of pro-inflammatory cytokines by immune cells. This increase is not only additive but also exceeds the sum of the effects of each agent alone. In fact, a de novo increase was observed for at least one cytokine, where the individual agents had no effect. This combination therapy allows for the transformation of PD-1 / PD-L1-refractory cancers into reactive ones.

[0109] Surprisingly, it was also found that ILT2 expression levels in patients' immune cells were correlated with the effectiveness of ILT2 blockade therapy. Responders to the therapy had high ILT2 levels, while non-responders had low ILT2 levels. Specifically, circulating CD8-positive T cells predicted treatment outcomes.

[0110] Finally, a unique epitope was discovered in the ILT2 interdomain between the D1 and D2 domains of the antibody of the present invention. This region is known to be the interaction domain between ILT2 and B2M, and the antibody of the present invention is the first known antibody to directly block this interaction. Furthermore, the antibody of the present invention was found to have immunostimulatory effects not reported with other anti-ILT2 antibodies. The antibody is able to modulate immune surveillance of T cells, NK cells, dendritic cells, and macrophages against cancer cells expressing HLA-G and MHC-I. Specifically, for the first time, it was discovered that the anti-ILT2 antibody used as a monotherapy can enhance phagocytosis of cancer cells.

[0111] Antibodies

[0112] In a first aspect, an antibody or antigen-binding fragment is provided, the antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:1 (DHTIH), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:2 (YIYPRDGSTKYNEKFKG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:3 (TWDYFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:4 (RASESVDSYGNSFMH), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:5 (RASNLES), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:6 (QQSNEDPYT).

[0113] In another aspect, an antibody or antigen-binding fragment is provided, the antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:7 (GYTFTSYGIS), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:8 (EIYPGSGNSYYNEKFKG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:9 (SNDGYPDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:10 (KASDHINNWLA), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:11 (GATSLET), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:12 (QQYWSTPWT).

[0114] In another aspect, an antibody or antigen-binding fragment is provided, the antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:13 (SGYYWN), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO:14 (YISYDGSNNYNPSLKN), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO:15 (GYSYYYAMDX), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:16 (RTSQDISNYLN), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:17 (YTSRLHS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:18 (QQGNTLPT), wherein X is selected from A, C, and S.

[0115] In some embodiments, SEQ ID NO:16 is GYSYYYAMDA (SEQ ID NO:25). In some embodiments, SEQ ID NO:16 is SEQ ID NO:25, and the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, SEQ ID NO:16 is GYSYYYAMDS (SEQ ID NO:26). In some embodiments, SEQ ID NO:16 is SEQ ID NO:26, and the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, SEQ ID NO:16 is GYSYYYAMDC (SEQ ID NO:27). In some embodiments, SEQ ID NO:16 is SEQ ID NO:27, and the antibody or antigen-binding fragment is a mouse antibody.

[0116] In another aspect, an antibody or antigen-binding fragment is provided that binds to the interdomain of human leukocyte immunoglobulin-like receptor superfamily B member 1 (ILT2) between domains D1 and D2.

[0117] On the other hand, an antibody or antigen-binding fragment that binds to an ILT2 epitope is provided, the epitope being selected from the following ILT2 sequences: VKKGQFPIPSITWEH (SEQ ID NO:41), LELVVTGAYIKPTLS (SEQ ID NO:42), VILQCDSQVAFDGFS (SEQ ID NO:43), and WYRCYAYDSNSPYEW (SEQ ID NO:44).

[0118] In another aspect, an antibody or antigen-binding fragment is provided that binds to ILT2 and inhibits the interaction between ILT2 and β-2-microglobulin (B2M).

[0119] On the other hand, an antibody or antigen-binding fragment is provided that binds to ILT2 and induces at least one of the following in a subject with cancer:

[0120] a. Increased cytotoxicity of natural killer (NK) cells;

[0121] b. Increased T cell cytotoxicity, proliferation, or both;

[0122] c. Increased macrophage phagocytosis, increased production of M1 inflammatory macrophages, decreased production of M2 suppressive macrophages, or a combination thereof; and

[0123] d. Increased dendritic cell homing to the tumor of the cancer, increased dendritic cell activation, or a combination thereof.

[0124] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a mouse antibody. In some embodiments, the antibody is a humanized antibody. As used herein, a "humanized" antibody means an antibody having a human backbone but having a CDR derived from or derived from a non-human antibody. In some embodiments, the CDR may be modified during humanization, but generally still be derived from a non-human antibody's CDR. In some embodiments, the antigen-binding fragment is a single-chain antibody. In some embodiments, the antigen-binding fragment is a single-domain antibody.

[0125] In some embodiments, the antibody or antigen-binding fragment binds to leukocyte immunoglobulin-like receptor subfamily B member 1 (ILT2). In some embodiments, ILT2 is human ILT2. In some embodiments, ILT2 is mammalian ILT2. In some embodiments, ILT2 is primate ILT2. In some embodiments, ILT2 is mouse ILT2. In some embodiments, the antibody or antigen-binding fragment binds to the extracellular domain of ILT2. In some embodiments, the antibody or antigen-binding fragment binds to the ligand pocket of ILT2. In some embodiments, the ligand is B2M. In some embodiments, the ligand is not HLA. In some embodiments, the ligand is HLA. In some embodiments, the HLA is HLA-G. In some embodiments, the ligand is not MHC. In some embodiments, the ligand is MHC. In some embodiments, the MHC is MHC class I (MHC-I). In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 interdomain. In some embodiments, the interdomain is the interface between the D1 and D2 domains. In some embodiments, the intercalation domain is a hinge domain between domains D1 and D2. In some embodiments, the intercalation domain does not contain the N-terminal domain of D1. In some embodiments, the intercalation domain is amino acids 54-184 of SEQ ID NO:31. In some embodiments, amino acids 54-184 of SEQ ID NO:31 contain the intercalation domain. In some embodiments, the intercalation domain is amino acids 90-184 of SEQ ID NO:31. In some embodiments, amino acids 90-184 contain the intercalation domain. In some embodiments, an antibody or antigen-binding fragment binds to an epitope within the intercalation domain. In some embodiments, the epitope comprises at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the intercalation domain. Each possibility represents a separate embodiment of the invention. In some embodiments, the epitope is within D2. In some embodiments, the antibody or antigen-binding domain binds to an epitope in D2. In some embodiments, the epitope is at least partially located in D2. In some embodiments, the antibody or antigen-binding domain binds to an epitope at least partially located in D2. In some embodiments, the epitope spans D1 and D2. In some embodiments, the antibody or antigen-binding fragment does not bind to the ILT2 domain that interacts with the α3 domain of HLA-G.

[0126] In some embodiments, ILT2 is mammalian ILT2. In some embodiments, ILT2 is human ILT2. In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_006660.4. In some embodiments, ILT2 has the following amino acid sequence: (SEQ ID NO:31).

[0127] In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_001075106.2. In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_001075107.2. In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_001075108.2. In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_001265328.2.

[0128] In some embodiments, the D1 domain of ILT2 comprises or consists of the following amino acid sequence: GHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGA (SEQ ID NO:46). In some embodiments, the D1 domain of ILT2 comprises or consists of amino acids 24-121 of SEQ ID NO:31. In some embodiments, the D2 domain of ILT2 comprises or consists of the following amino acid sequence: YIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGV (SEQ ID NO:47). In some embodiments, the D2 domain of ILT2 comprises or consists of amino acids 122-222 of SEQ ID NO:31. In some embodiments, the interstitial domain of ILT2 comprises the amino acids Gln41, Lys65, Trp90, Gly120, Ala121, Val122, Ile123, Gln148, Val149, Ala150, Phe151, Asp201, Asn203, and Glu207 of SEQ ID NO:31. In some embodiments, the epitope comprises the amino acids Gln41, Lys65, Trp90, Gly120, Ala121, Val122, Ile123, Gln148, Val149, Ala150, Phe151, Asp201, Asn203, and Glu207 of SEQ ID NO:31. In some embodiments, the epitope comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids selected from the amino acids Gln41, Lys65, Trp90, Gly120, Ala121, Val122, Ile123, Gln148, Val149, Ala150, Phe151, Asp201, Asn203, and Glu207 of SEQ ID NO:31. In some embodiments, the epitope comprises at least 10 amino acids selected from the amino acids Gln41, Lys65, Trp90, Gly120, Ala121, Val122, Ile123, Gln148, Val149, Ala150, Phe151, Asp201, Asn203, and Glu207 of SEQ ID NO:31. In some implementations, the antibody or antigen-binding fragment binds to the ILT2 epitope provided in SEQ ID NO:41.In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 epitope provided in SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 epitope provided in SEQ ID NO:43. In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 epitope provided in SEQ ID NO:44. In some embodiments, the antibody or antigen-binding fragment binds to a three-dimensional epitope comprising at least two of SEQ ID NO:41, 42, 43, and 44. In some embodiments, the three-dimensional epitope comprises at least three of SEQ ID NO:41, 42, 43, and 44. In some embodiments, the three-dimensional epitope comprises SEQ ID NO:41, 42, 43, and 44.

[0129] In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope comprising an ILT2 residue selected from Q18, G19, K42, L45, S64, I65, T66, W67, E68, G97, A98, Y99, I100, Q125, V126, A127, F128, D178, N180, S181, and E184. In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope comprising an ILT2 residue selected from G97, A98, Y99, I100, Q125, and V126. In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope comprising a plurality of ILT2 residues selected from Q18, G19, K42, L45, S64, I65, T66, W67, E68, G97, A98, Y99, I100, Q125, V126, A127, F128, D178, N180, S181, and E184. In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope comprising a plurality of ILT2 residues selected from G97, A98, Y99, I100, Q125, and V126. In some embodiments, the antibody or antigen-binding fragment binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 residues selected from Q18, G19, K42, L45, S64, I65, T66, W67, E68, G97, A98, Y99, I100, Q125, V126, A127, F128, D178, N180, S181, and E184. Each possibility represents a separate embodiment of the invention. In some embodiments, the antibody or antigen-binding fragment binds to at least 1, 2, 3, 4, 5, or 6 residues selected from G97, A98, Y99, I100, Q125, and V126. Each possibility represents a separate embodiment of the invention. In some implementations, the antibody or antigen-binding fragment binds to G97, A98, Y99, I100, Q125, and V126. It should be understood that the designations used herein refer to SEQ ID NO:31.

[0130] In some embodiments, the antibody or antigen-binding fragment is an ILT2 antagonist. In some embodiments, the antibody or antigen-binding fragment is not an ILT2 agonist. In some embodiments, the antagonism is part of ILT2-mediated immunosuppression. In some embodiments, the antibody or antigen-binding fragment inhibits ILT2-mediated immunosuppression. In some embodiments, the antibody or antigen-binding fragment inhibits ILT2 signaling.

[0131] In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and B2M. In some embodiments, the interaction is a direct interaction. In some embodiments, the antibody or antigen-binding fragment inhibits the contact between ILT2 and B2M. In some embodiments, the contact is a direct contact. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and HLA, MHC, or both. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and B2M, thereby inhibiting the interaction between ILT2 and HLA, MHC, or both. In some embodiments, the interaction is mediated by B2M. In some embodiments, the antibody indirectly inhibits the interaction with HLA, MHC, or both by inhibiting the interaction with B2M. In some embodiments, the interaction is a B2M-mediated interaction. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and the B2M / HLA complex. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and the B2M / MHC complex. In some embodiments, the complex comprises a B2M monomer. In some embodiments, the complex comprises an HLA or MHC monomer. In some embodiments, the complex comprises a B2M dimer. In some embodiments, the complex comprises an HLA or MHC dimer.

[0132] In some embodiments, ILT2-mediated immunosuppression is the suppression of immune cells. In some embodiments, the immune cells are selected from T cells, macrophages, dendritic cells, and natural killer (NK) cells. In some embodiments, ILT2-mediated immunosuppression is the suppression of T cells, macrophages, dendritic cells, and NK cells. In some embodiments, ILT2-mediated immunosuppression is the suppression of T cells, macrophages, and NK cells. In some embodiments, the T cells are CD8-positive T cells. In some embodiments, the T cells are T... EMRA Cells (terminally differentiated effector memory cells reexpressing CD45RA). In some implementations, immune cells are selected from CD8-positive T cells, T cells... EMRACells, dendritic cells, macrophages, and natural killer (NK) cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are macrophages. In some embodiments, the macrophages are tumor-associated macrophages (TAMs). In some embodiments, the immune cells are dendritic cells. In some embodiments, the dendritic cells are tolerogenic dendritic cells. In some embodiments, the immune cells are peripheral blood immune cells. In some embodiments, the immune cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the immune cells are intratumoral immune cells. In some embodiments, the immune cells are immune cells in the tumor microenvironment (TME). In some embodiments, ILT2-mediated immunosuppression is inhibition of macrophage phagocytosis. In some embodiments, ILT2-mediated immunosuppression is inhibition of NK cell cytotoxicity. In some embodiments, ILT2-mediated immunosuppression is inhibition of T cell cytotoxicity. In some embodiments, ILT2-mediated immunosuppression is inhibition of T cell proliferation. In some implementations, ILT2-mediated immunosuppression is the inhibition of immune cell proliferation.

[0133] In some embodiments, the antibody or antigen-binding fragment does not bind to members of the leukocyte immunoglobulin-like receptor superfamily B other than ILT2. In some embodiments, the antibody or antigen-binding fragment is specific for ILT2. In some embodiments, the antibody or antigen-binding fragment preferentially binds to ILT2. In some embodiments, the antibody or antigen-binding fragment does not inhibit members of the leukocyte immunoglobulin-like receptor superfamily B other than ILT2.

[0134] As used herein, “increased binding power” means a greater specific binding to a target or antigen than the binding of an isotype control. In some embodiments, increased binding is an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% in binding power. Each possibility represents a separate embodiment of the invention. In some embodiments, increased binding is the presence of binding as compared to an isotype control that does not bind. Binding of an antibody to a specific domain will be well known to those skilled in the art. Antibody binding can be determined in any manner known to those skilled in the art, including but not limited to: X-ray crystallography, immunoprecipitation, Western blotting, competitive assays, and kinetic repulsion assays. In some embodiments, increased binding power is specific binding.

[0135] The antibodies or antigen-binding fragments, variants, or derivatives disclosed herein may be referred to as having a value greater than or equal to 10. 3 M" 1 sec" 1 5X10 3 M" 1 sec" 1 10 4 M" 1 sec" 1 Or 5x10 4 M" 1 The association rate (k(on)) at which the target antigen (e.g., ILT2) binds is measured in sec. Each possibility represents a separate embodiment of the invention. Antibodies or antigen-binding fragments, variants, or derivatives disclosed herein may be referred to in terms of 10... -6 M or stronger affinity binds to the target antigen, while the typical affinity of most antibodies is 10. -9 M.

[0136] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO:19 (QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARTWDYFDYWGQGTTLTVSS). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO:21 (QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWVGEIYPGSGNSYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARSNDGYPDYWGQGTTLTVSS). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO:23 (DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDXWGQGTSVTVSS), where X is selected from A, C, and S.

[0137] In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO:20 (DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIK). In some embodiments, the antibody or antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO:24 (DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTFGQGTKLEIK). In some embodiments, the antibody or antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO:45 (DIQMTQTTSSLSASLGDRVTISCRTSQDISNYLNWYQQKPDGTVKLLISYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPTFGSGTKLEIK).

[0138] In some embodiments, SEQ ID NO:23 is DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDAWGQGTSVTVSS (SEQ ID NO:28). In some embodiments, SEQ ID NO:23 is SEQ ID NO:28, and the antibody or antigen-binding fragment is humanized. In some embodiments, SEQ ID NO:23 is DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDSWGQGTSVTVSS (SEQ ID NO:29). In some embodiments, SEQ ID NO:23 is SEQ ID NO:29, and the antibody or antigen-binding fragment is humanized. In some embodiments, SEQ ID NO:23 is DVQLQGSGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTSEDTATYYCAHGYSYYYAMDCWGQGTSVTVSS (SEQ ID NO:30). In some embodiments, SEQ ID NO:23 is SEQ ID NO:30, and the antibody or antigen-binding fragment is mouse-derived.

[0139] In some embodiments, the antibody or antigen-binding fragment of the present invention is used to treat or improve cancer in a subject of need. In some embodiments, the cancer is HLA-G positive cancer. In some embodiments, the cancer is MHC-I positive cancer. In some embodiments, the cancer expresses HLA-G. In some embodiments, the cancer expresses MHC-I. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to transform the tumor microenvironment from immunosuppressive to immunostimulatory. In some embodiments, the transformation of the tumor microenvironment includes one or more of the following: inducing / enhancing anti-tumor T cell responses, increasing T cell proliferation, reducing cancer-induced myelosuppressive activity, increasing dendritic cell (DC) activation, increasing dendritic cell homing to the tumor, increasing macrophage phagocytosis, increasing M1 macrophage production, decreasing M2 macrophage production, and increasing NK cell activity. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase T cell responses against cancer cells. In some embodiments, the T cell response includes increased secretion of pro-inflammatory cytokines. In some embodiments, the T cell response includes increased cytotoxicity. In some embodiments, the T cell response includes increased T cell proliferation. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase macrophage phagocytosis of cancer cells. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase dendritic cell homing to a tumor or cancer. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase macrophage phagocytosis. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase macrophage phagocytosis of cancer. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the production of M1 macrophages. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce the production of M2 macrophages. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase NK cell cytotoxicity against cancer cells. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce cancer-induced myelosuppressive activity. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce the activity of tolerant dendritic cells (DCs). In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the activity or number of M1 monocytes. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce the activity or number of M2 monocytes. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the production of M1 macrophages. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce the production of M2 macrophages. In some embodiments, the M1 monocytes / macrophages are inflammatory macrophages / monocytes. In some embodiments, the M2 monocytes / macrophages are suppressive macrophages / monocytes.In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the number of dendritic cells (DCs) in a tumor. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the recruitment of DCs to the tumor. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase DC recruitment to the tumor. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase DC activation. In some embodiments, increasing DC activation includes reducing the activity of toxic dendritic cells. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase antigen presentation. In some embodiments, recruitment to the tumor is recruitment to the tumor microenvironment (TME).

[0140] In some embodiments, the antibody or antigen-binding fragment induces at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 anticancer effects in a subject. Each possibility represents a separate embodiment of the invention. In some embodiments, the antibody or antigen-binding fragment induces at least 2 effects in a subject. In some embodiments, the antibody or antigen-binding fragment induces at least 3 effects in a subject. In some embodiments, the antibody or antigen-binding fragment induces at least 4 effects in a subject. In some embodiments, the effects are selected from: increased NK cell cytotoxicity, increased T cell cytotoxicity, increased T cell proliferation, increased macrophage phagocytosis, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell homing to cancerous tumors, and increased dendritic cell activation. In some embodiments, the effect is selected from: a) increased NK cell cytotoxicity; b) increased T cell cytotoxicity, proliferation, or both; c) increased macrophage phagocytosis, increased M1 macrophage production, decreased M2 macrophage production, or combinations thereof; and d) increased dendritic cell homing to cancerous tumors, increased dendritic cell activation, or combinations thereof. In some embodiments, cytotoxicity is cancer-specific cytotoxicity. In some embodiments, phagocytosis is phagocytosis by cancer cells or cancer cells. In some embodiments, the antibody or antigen-binding fragment induces anticancer activity on T cells, NK cells, dendritic cells, and macrophages in a subject. In some embodiments, the antibody or antigen-binding fragment induces anticancer activity on at least three of the T cells, NK cells, dendritic cells, and macrophages in a subject. In some embodiments, the antibody or antigen-binding fragment induces the effect as a single therapy. In some embodiments, the antibody or antigen-binding fragment induces the effect without combination.

[0141] In some embodiments, the increased cytotoxicity includes increased secretion of pro-inflammatory cytokines. Pro-inflammatory cytokines are well known in the art and include, but are not limited to: IL-1, IL-1β, IL-6, TNFα, IFNγ, MCP-1, IL-12, IL-18, IL-2, IL-15, IL-17, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the pro-inflammatory cytokine is selected from IL-6, interferon-γ (IFNγ), and GM-CSF. In some embodiments, the pro-inflammatory cytokine is GM-CSF.

[0142] "Anti-ILT2 antibody," "antibody that recognizes ILT2," or "antibody against ILT2" is an antibody that binds to ILT2 with sufficient affinity and specificity. In some embodiments, the anti-ILT2 antibody has ILT2 as the antigen it binds to.

[0143] An "antigen" is a molecule or part of a molecule that can trigger antibody formation and be bound by the antibody. An antigen may have one or more epitopes. The specificity mentioned above is intended to indicate that an antigen will react with its corresponding antibody in a highly selective manner, rather than with a variety of other antibodies that may be induced by other antigens.

[0144] The term "antigenic determinant" or "epitope" according to the present invention refers to a region in an antigen molecule that specifically reacts with a particular antibody. Using methods known in the art, peptide sequences derived from epitopes can be used alone or in combination with a vector moiety to immunize animals and generate additional polyclonal or monoclonal antibodies. The IMGT information system (www.imgt.cines.fr / ) can also be used. / V-Quest) analyzes immunoglobulin variable domains to identify variable regions (including CDRs). See, for example, Brochet, X. et al., Nucl. Acids Res. J6: W503-508 (2008).

[0145] Kabat et al. also defined a numbering system for variable domain sequences, applicable to any antibody. Those skilled in the art can explicitly assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described in Kabat et al., U.S. Department of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0146] In some embodiments, the antibody or antigen-binding fragment is used in combination with another agent. In some embodiments, the combination with another agent is for treating cancers expressing HLA-G and / or MHC-I. In some embodiments, the agent is an opsonizer. In some embodiments, the agent is an anti-PD-1 and / or anti-PD-L1 agent. In some embodiments, the antibody or antigen-binding fragment is used in combination with an anti-PD-1 / PD-L1 based therapy.

[0147] As used herein, an opsonizer is any agent that can bind to and opsonize target cells (e.g., cancer cells, cells with intracellular pathogens, etc.). For example, any antibody believed to bind to target cells is an opsonizer, wherein said antibody has an Fc region. In some embodiments, the opsonizer is an antibody that induces antibody-dependent phagocytosis (ADCP). Examples of opsonizers include, but are not limited to, anti-CD47 antibodies, anti-CD20 antibodies, anti-HER2 antibodies, anti-EGFR antibodies, anti-CD52 antibodies, and anti-CD30 antibodies. In some embodiments, the opsonizer is selected from rituximab, trastuzumab, pertuzumab, Herceptin, cetuximab, panitumumab, and Erbitux. In some embodiments, the opsonizer is an anti-EGFR antibody. In some embodiments, the opsonizer is Erbitux.

[0148] As used herein, “anti-PD-1 / PD-L1 therapy” and “PD-1 / PD-L1 therapy” are synonymous and used interchangeably, and refer to a treatment regimen that includes blocking the PD-1 and PD-L1 signaling axes. In some embodiments, the cancer is PD-L1 positive cancer. In some embodiments, PD-1 / PD-L1 therapy is PD-1 / PD-L1 immunotherapy. In some embodiments, PD-1 / PD-L1 therapy is PD-1 / PD-L1 blockade. In some embodiments, PD-1 / PD-L1 therapy is an agent that blocks PD-1-based immunosuppression. In some embodiments, PD-1 / PD-L1 therapy comprises an anti-PD-1 blocking antibody. In some embodiments, PD-1 / PD-L1 therapy comprises an anti-PD-L1 blocking antibody. In some embodiments, PD-1 / PD-L1 therapy enhances immune surveillance. In some embodiments, PD-1 / PD-L1 therapy is an anticancer therapy. In some embodiments, PD-1 / PD-L1 therapy enhances tumor immune surveillance. The term "antibody" (also known as "immunoglobulin") is used in the broadest sense and specifically covers monoclonal antibodies and antibody fragments, provided they exhibit the desired biological activity. In some embodiments, the invention also covers the use of chimeric or humanized antibodies.

[0149] The basic building block of naturally occurring antibody structures is a heterotetrameric protein complex of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains linked together by non-covalent association and disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Five classes of human antibodies exist (IgG, IgA, IgM, IgD, and IgE), and within these classes, multiple subclasses are identified based on structural differences such as the number of immunoglobulin units in a single antibody molecule, the disulfide bridge structure of individual units, and differences in chain length and sequence. Antibody classes and subclasses are their isotypes.

[0150] The sequences of the amino-terminal regions of the heavy and light chains are more diverse than those of the carboxyl-terminal regions, and are therefore called variable domains. This part of the antibody structure confers antigen-binding specificity to the antibody. The heavy chain variable (VH) domain and the light chain variable (VL) domain together form a single antigen-binding site; therefore, the basic immunoglobulin unit has two antigen-binding sites. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0151] The carboxyl-terminal portions of the heavy and light chains form constant domains, namely CH1, CH2, CH3, and CL. Although the diversity within these domains is significantly low, differences exist between animal species, and furthermore, several different antibody isotypes exist within the same organism, each with different functions.

[0152] The term "frame region" or "FR" refers to amino acid residues in the variable domains of an antibody that are distinct from the amino acid residues in the hypervariable region as defined herein. The term "hypervariable region," as used herein, refers to amino acid residues in the variable domains of an antibody responsible for antigen binding. The hypervariable region contains amino acid residues from the "complementarity-determining region" or "CDR." The CDR is primarily responsible for binding to the antigen epitope. The ranges of FR and CDR have been precisely defined (see, Kabat et al.). In some embodiments, the CDR is determined using the KABAT system. In some embodiments, the CDR is determined using the Clothia system. In some embodiments, the Clothia system is an enhanced Clothia system (Martin system).

[0153] Monoclonal antibodies as used herein explicitly include “chimeric” antibodies, wherein a portion of the heavy and / or light chains is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984)). Additionally, complementarity-determining region (CDR) transplantation can be performed to alter certain properties of the antibody molecule, including affinity or specificity. Non-limiting examples of CDR transplantation are disclosed in US Patent 5,225,539.

[0154] Chimeric antibodies are molecules whose different parts are derived from different animal species, such as those having a variable region derived from mouse mAbs and a constant region of human immunoglobulins. Antibodies having a variable region framework residue that is essentially derived from human antibodies (called receptor antibodies) and a complementarity-determining region that is essentially derived from mouse antibodies (called donor antibodies) are also called humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in applications and to increase yield in production, for example, in cases where mouse mAbs have higher hybridoma yields but higher human immunogenicity, thus using human / mouse chimeric mAbs. Chimeric antibodies and methods of their production are known in the art (e.g., PCT patent applications WO 86 / 01533, WO 97 / 02671, WO 90 / 07861, WO 92 / 22653 and U.S. patents 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539). As used herein, the term "humanized antibody" refers to an antibody comprising a framework region derived from a human antibody and one or more core-drug receptors (CDRs) derived from a non-human (typically mouse or rat) immunoglobulin. The portion of the humanized immunoglobulin (potentially other than the CDR) is substantially identical to the corresponding portion of the native human immunoglobulin sequence. However, in some cases, such as specific amino acid residues in the framework region, modifications can be made to optimize the performance of the humanized antibody. Importantly, the humanized antibody is expected to bind to the same antigen as the donor antibody providing the CDR. Further details can be found, for example, in U.S. Patent No. 5,225,539 assigned to the Medical Research Council, UK. The terms "frame region derived from recipient human immunoglobulin" and "frame region derived from recipient human immunoglobulin" and similar grammatical expressions are used interchangeably herein to refer to a framework region or a portion thereof having the same amino acid sequence as the recipient human immunoglobulin.

[0155] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies constituting said group are identical and / or bind to the same epitopes, except for possible variants that may occur during the production of the monoclonal antibody, which are typically present in small amounts. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as meaning that the antibody used according to the methods provided herein can be prepared by the hybridoma method first described in Kohler et al., Nature 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described in the following literature: Clackson et al., Nature 352:624-628 (1991) and Marks et al., J.Mol.Biol.222:581-597 (1991).

[0156] The mAbs of this invention can belong to any immunoglobulin class, including IgG, IgM, IgE, or IgA. Hybridomas that produce mAbs can be cultured in vitro or in vivo. High-titer mAbs can be obtained in vivo by intraperitoneal injection of cells from a single hybridoma into primitively sensitized Balb / c mice to produce ascites containing high concentrations of the desired mAb. Isotype IgM or IgG mAbs can be purified from such ascites using column chromatography methods well known to those skilled in the art, or from culture supernatants.

[0157] The terms “antibody fragment” or “antigen-binding fragment” are used synonymously and include, preferably, a portion of the antigen-binding region of a complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; tandem biantibodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062(1995)); monoarmed antibodies, single variable domain antibodies, microantibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, di-scFv, or tandem (di, tri)-scFv); and bispecific T-cell binders (BiTE).

[0158] Papain digestion of the antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site; and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still able to cross-link the antigen.

[0159] The “Fv” is the smallest antibody fragment containing both a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. The three surfaces of the VH-VL dimer are in this configuration. In summary, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three hypervariable regions specific to the antigen) has the ability to recognize and bind to the antigen, but its affinity is lower than that of the entire binding site.

[0160] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name for Fab' in this paper where one or more cysteine ​​residues in the constant domain have at least one free thiol group. The F(ab')2 antibody fragment was initially generated as a Fab' fragment pair with a hinge cysteine ​​residue between the Fab' fragments. Other chemical conjugates of antibody fragments are also known.

[0161] The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be designated as one of two distinct types (called κ and λ) based on the amino acid sequence of its constant domain.

[0162] Antibodies can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, e, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well known.

[0163] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, wherein these domains are contained within a single polypeptide chain. In some embodiments, the Fv polypeptide also contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0164] The term "biantibody" refers to a small antibody fragment having two antigen-binding sites, wherein the fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, resulting in two antigen-binding sites. Biantibodies are described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0165] The term "multispecific antibody" is used in the broadest sense and explicitly covers antibodies that exhibit multi-epitope specificity. Such multispecific antibodies include, but are not limited to, antibodies containing a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VHVL unit has multi-epitope specificity; antibodies having two or more VL and VH domains, wherein each VHVL unit binds to a different epitope; antibodies having two or more single variable domains, wherein each single variable domain binds to a different epitope; full-length antibodies; antibody fragments such as Fab, Fv, dsFv, scFv, biantibodies, bispecific biantibodies, triantibodies, trifunctional antibodies, and antibody fragments that are covalently or non-covalently linked. "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on one or more of the same or different targets.

[0166] The monoclonal antibodies of the present invention can be prepared using methods well known in the art. Examples include a variety of techniques, such as those in the following literature: Kohler, G. and Milstein, C., Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96, MONCLONAL ANTIBODIES ANDCANCER THERAPY, Alan R. Liss, Inc. (1985).

[0167] In addition to conventional methods for antibody production in vivo, phage display technology can be used to generate antibodies in vitro. This generation of recombinant antibodies is significantly faster than conventional antibody production and can generate recombinant antibodies against a very large number of antigens. Furthermore, many antigens have been shown to be non-immunogenic or highly toxic when using conventional methods, making them unsuitable for antibody generation in animals. Additionally, affinity maturation (i.e., increasing affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, a large number of different antibodies against a specific antigen can be generated in a single selection process. To generate recombinant monoclonal antibodies, large libraries of antibodies with different antigen recognition sites can be generated using a variety of methods, all based on display libraries. This library can be prepared in several ways: a synthetic library can be generated by cloning the synthetic CDR3 region from a heavy chain germline gene library, from which recombinant antibody fragments with various specificities can be selected. Human lymphocyte libraries can be used as starting materials for constructing antibody libraries. An initial library of human IgM antibodies can be constructed, resulting in a highly diverse human library. This method has been successfully and widely used to select a large number of antibodies against different antigens. Protocols for phage library construction and recombinant antibody selection are provided in well-known reference texts: Current Protocols in Immunology, Colligan et al. (eds.), John Wiley & Sons, Inc. (1992–2000), Chapter 17, Section 17.1.

[0168] Nonhuman antibodies can be humanized using any method known in the art. In one approach, a nonhuman complementarity-determining region (CDR) is inserted into the frame sequence of a human antibody or a shared antibody. Other variations can then be introduced into the antibody frame to modulate affinity or immunogenicity.

[0169] In some embodiments, the antibodies described herein are neutralizing antibodies. As discussed herein, “neutralization” is defined as the reduction of protein function by the antibodies of the present invention. In one embodiment, as discussed herein, “neutralization” is the binding of an antibody to the surface of immune cells, preferably immature and mature myeloid-derived cells, T cells, and NK cells, thereby blocking the propagation of inhibitory signals within these cells and conferring a less inhibitory phenotype and function.

[0170] In some embodiments, the present invention provides nucleic acid sequences encoding the antibodies of the present invention. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 75% identity with the DNA sequence described herein. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 80% identity with the DNA sequence described herein. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 85% identity with the DNA sequence described herein. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 90% identity with the DNA sequence described herein. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 95% identity with the DNA sequence described herein.

[0171] According to another aspect, a nucleic acid sequence encoding the antibody or antigen-binding fragment of the present invention is provided.

[0172] According to another aspect, a nucleic acid molecule encoding an antibody or antigen-binding fragment of the present invention is provided.

[0173] In some embodiments, the nucleic acid sequence encoding the heavy chain of the antibody or antigen-binding fragment of the present invention is selected from CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGGCGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTATGGTATAAGCTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGGTTGGAGAGATTTATCCTGGAAGTGGTAATTCTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCGTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGAATGATGGTTACCCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:32), GATGTACAGCTTCAGGGGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGATGGTAGCAATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTTCTGAGGACACAGCCACATATTACTGTGCCCATGGTTACTCATATTACTATGCTATGGACTGCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ IDNO: 33), GATGTCCAGCTGCAAGGCTCTGGCCCTGGACTGGTTAAGCCTTCCGAGACACTGTCCCTGACCTGCTCTGTGACCGGCTACTCTATCACCTCCGGCTACTACTGGAACTGGATCAGACAGTTCCCCGGCAAGAAACTGGAATGGATGGGCTACATCTCCTACGACGGCTCCAACAACTACAACCCCAGCCTGAAGAACCGGATCACCATCTCTCGGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAACTCCGTGACCGCTGCCGATACCGCTACCTACTACTGTGCTCACGGCTACTCCTACTACTACGCCATGGATGCTTGGGGCCAGGGCACATCTGTGACAGTGTCCTCT (SEQ ID NO: 34) and CAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGAGCTTCAGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCACTGACCATACTATTCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTAGAGATGGTAGTACTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAAGAACCTGGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 35).

[0174] In some embodiments, the nucleic acid sequence encoding the light chain of the antibody or antigen-binding fragment of the present invention is selected from GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO:36), GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGACAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTCCTACACATCAAGATTGCACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA(SEQ IDNO:37),GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGACCTCTCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAACCCGGCAAGGCCGTGAAGCTGCTGATCTCCTACACCTCCA GACTGCACTCTGGCGTGCCCTCCAGATTTTCTGGCTCTGGATCTGGCACCGACTACACCCTGACCATCAGTTCTCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCAGCAGGGCAACACCCTGCCTACCTTTGGCCAGGGCACCAAGCTGGAAATCAAG(SEQ ID NO:38) and GACATCCAGATGACACAATCTTCATCCTACTTGTCTGTATCTCTAGGAGGCAGAGTCACCATTACTTGCAAGGCAAGTGACCACATTAATAATTGGTTAGCCTGGTATCAGCAGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAACCAGT TTGGAAACTGGGGTTCCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:39).

[0175] In some embodiments, the antibody or antigen-binding fragment is mouse-derived, and the sequence encoding the heavy chain is selected from SEQ ID NO:32, 33, and 35. In some embodiments, the antibody or antigen-binding fragment is mouse-derived, and the sequence encoding the light chain is selected from SEQ ID NO:36, 37, and 39. In some embodiments, the antibody or antigen-binding fragment is humanized, and the sequence encoding the heavy chain is SEQ ID NO:34. In some embodiments, the antibody or antigen-binding fragment is humanized, and the sequence encoding the light chain is SEQ ID NO:38.

[0176] As used interchangeably in this article, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA.

[0177] Polynucleotides encoding polypeptides can be obtained from any source, including but not limited to cDNA libraries prepared from tissues believed to possess polypeptide mRNA and express said polypeptide mRNA at detectable levels. Therefore, polynucleotides encoding polypeptides can be readily obtained from cDNA libraries prepared from human tissues. Genes encoding polypeptides can also be obtained from genomic libraries or through known synthetic procedures (e.g., automated nucleic acid synthesis).

[0178] For example, polynucleotides can encode the entire immunoglobulin molecular chain, such as the light chain or the heavy chain. A complete heavy chain includes not only the variable region (VH) but also the constant region (CH), which typically contains three constant domains: CH1, CH2, and CH3; and a "hinge" region. In some cases, the presence of a constant region is desirable.

[0179] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments such as single-domain antibodies (“dAb”), Fv, scFv, Fab', and CHI, with the CK or CL domains cleaved. Because microantibodies are smaller than conventional antibodies, they can achieve better tissue penetration in clinical / diagnostic applications, and in the case of being bivalent, they can retain a higher binding affinity than monovalent antibody fragments (such as dAb). Therefore, unless the context requires otherwise, the term “antibody” as used herein encompasses not only complete antibody molecules but also antigen-binding antibody fragments of the types discussed above. Each frame region present in the encoded polypeptide may contain at least one amino acid substitution relative to the corresponding human receptor frame. Thus, for example, a frame region may contain a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions relative to the receptor frame region. Given the characteristics of the individual amino acids constituting the disclosed protein product, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions can be made, for example, based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved; this is known as "conservative substitution."

[0180] Suitablely, the polynucleotides described herein can be isolated and / or purified. In some embodiments, the polynucleotides are isolated polynucleotides.

[0181] As used herein, the term “non-naturally existing” for a substance, composition, entity, and / or any combination of such substance, composition, or entity, or any grammatical variation thereof, is a conditional term that expressly excludes (but only excludes) the following forms of the substance, composition, entity, and / or any combination of such substance, composition, or entity: which are well known to those skilled in the art as “naturally existing”, or which are determined or interpreted as “naturally existing” by (or may at any time by) a judge, administrative or judicial authority.

[0182] Therapeutic and diagnostic methods

[0183] According to another aspect, a method is provided for treating a subject with cancer expressing HLA, MHC-I, or both, the method comprising administering an antibody or antigen-binding fragment of the present invention to the subject.

[0184] According to another aspect, a method for treating cancer in a subject in need is provided, the method comprising confirming that ILT2 expression in the subject is above a predetermined threshold, and administering to the subject an agent that inhibits ILT2-based immunosuppression, thereby treating the subject's cancer.

[0185] According to another aspect, a method for treating cancer in a subject in need is provided, the method comprising: administering to the subject an agent that inhibits ILT2-mediated immunosuppression; and administering to the subject a PD-1 / PD-L1-based therapy; thereby treating the subject's cancer.

[0186] According to another aspect, a method is provided to increase the efficacy of PD-1 / PD-L1-based therapies against cancer cells, the method comprising contacting the cancer cells with an agent that inhibits ILT2-mediated immunosuppression.

[0187] According to another aspect, an agent that binds to ILT2 and inhibits ILT2-mediated immune cell suppression is provided, which is used in combination with anti-PD-L1 / PD-1 based therapies for the treatment of subjects with cancer.

[0188] As used herein, the terms "treatment" or "treating" a disease, disorder, or condition encompass the reduction of at least one symptom, decrease in severity, or inhibition of progression. Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. For effective treatment, the useful compositions described herein need only reduce the severity of the disease, disorder, or condition, decrease the severity of associated symptoms, or provide an improvement in the quality of life for the patient or subject.

[0189] As used herein, the term "treatment" refers to a clinical intervention that attempts to alter the course of disease in the individual being treated, and can be performed for prevention or during a clinicopathological process. The desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, reducing the pathological consequences of disease, slowing the rate of disease progression, improving disease status, and mitigating or improving prognosis. The term "treatment" can also encompass in vitro procedures that affect cells or tissues in culture.

[0190] In some embodiments, the antibody or antigen-binding fragment is administered as a single therapy. In some embodiments, the antibody or antigen-binding fragment is administered in combination with PD-1 / PD-L1 therapy. In some embodiments, the antibody or antigen-binding fragment is administered in combination with an opsonizer. In some embodiments, the opsonizer is not an anti-CD47 agent. In some embodiments, the anti-CD47 agent is an anti-CD47 antibody. In some embodiments, the antibody or antigen-binding fragment is not administered in combination with an anti-CD47 agent or therapy. In some embodiments, the antibody or antigen-binding fragment is not combined with an anti-CD47 agent or therapy.

[0191] In some embodiments, the treatment includes increasing immune surveillance. In some embodiments, the treatment includes increasing the immune response. In some embodiments, the treatment includes reducing tumor burden. In some embodiments, the treatment includes reducing cancer metastasis. In some embodiments, the treatment includes increasing cytotoxicity against the cancer. In some embodiments, the treatment includes increasing the inflammatory response against the cancer. In some embodiments, the treatment includes increasing phagocytosis by the cancer.

[0192] As used herein, the term "subject" refers to an individual or patient who is a vertebrate, such as a mammal, and particularly includes a human. In some embodiments, the subject is a human. In some embodiments, the subject is a mammal. In some embodiments, the subject has cancer.

[0193] In some embodiments, the cancer is an HLA-expressing cancer. In some embodiments, the HLA is HLA-G. In some embodiments, the cancer is an MHC-I-expressing cancer. In some embodiments, the cancer is a PD-1-expressing cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is refractory to PD-1 and / or PD-L1-based therapies. In some embodiments, the cancer has never responded to PD-1 and / or PD-L1-based therapies. In some embodiments, the cancer has responded to PD-1 and / or PD-L1-based therapies but has become refractory. In some embodiments, the method of the present invention converts refractory cancer into reactive cancer.

[0194] In some embodiments, the method includes confirming that the cancer expresses HLA, MHC-I, or both. In some embodiments, the method includes confirming that the cancer expresses HLA. In some embodiments, the method includes confirming that the cancer expresses MHC-I. In some embodiments, the method includes confirming that the cancer expresses both HLA and MHC-I. In some embodiments, confirmation includes measuring expression in the cancer. In some embodiments, confirmation includes measuring expression on the surface of the cancer. In some embodiments, in the cancer and / or on the cancer is in and / or on cancer cells. In some embodiments, confirmation includes measuring HLA-G secreted by the cancer. In some embodiments, confirmation includes measuring soluble HLA-G. In some embodiments, soluble HLA-G is in body fluids. In some embodiments, the body fluid is blood.

[0195] In some embodiments, the method includes confirming ILT2 expression in the subject. In some embodiments, the method includes confirming that ILT2 expression in the subject is above a predetermined threshold. In some embodiments, confirmation includes measuring ILT2 expression in the subject. In some embodiments, confirmation is performed before administration. In some embodiments, measurement is performed before administration. In some embodiments, ILT2 expression is in immune cells. In some embodiments, ILT2 expression is in the subject's immune cells. In some embodiments, the immune cells are peripheral blood immune cells. In some embodiments, the immune cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the immune cells are intratumoral immune cells. In some embodiments, the immune cells are immune cells in the tumor microenvironment (TME). In some embodiments, the immune cells are selected from CD8-positive T cells, macrophages, NK cells, and T cells. EMRA Cells. In some embodiments, the immune cells are CD8-positive T cells. In some embodiments, the immune cells are peripheral blood CD8-positive T cells.

[0196] In some embodiments, administering the antibody or antigen-binding fragment of the present invention comprises administering a pharmaceutical composition containing the antibody or antigen-binding fragment of the present invention. In some embodiments, a therapeutically effective amount of the antibody or antigen-binding fragment is administered. In some embodiments, the pharmaceutical composition further comprises a carrier, excipient, or adjuvant. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0197] As used herein, the terms “carrier,” “excipient,” or “adjuvant” refer to any component in a pharmaceutical composition that is not an active agent. As used herein, the term “pharmaceuticalally acceptable carrier” refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered astragalus gum; malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline, Ringer's solution; ethanol and phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can be used as carriers herein include sugars, starches, cellulose and their derivatives, powdered astragalus gum, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifiers, and other non-toxic, pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants (such as sodium dodecyl sulfate), as well as colorants, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions considered herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those skilled in the art, such as those described in the following literature: Merck Index, 13th edition, eds., Budavari et al., Merck & Co., Inc., Ravi, NJ (2001); CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th edition (2004); and “Inactive Ingredient Guide,” US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, all contents of which are hereby incorporated by reference in their entirety.Examples of pharmaceutically acceptable excipients, carriers, and diluents that can be used in the compositions of the present invention include distilled water, physiological saline, Ringer's solution, dextran solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th ed., Gilman et al., Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990); and Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, PA (2005), each of which is incorporated herein by reference in its entirety. The compositions described in this invention may also be contained in artificially generated structures such as liposomes, ISCOMS, slow-release particles, and other mediators that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. Liposomes used with the peptides described in this invention are formed from standard vesicle-forming lipids, which typically comprise neutral and negatively charged phospholipids and sterols (such as cholesterol). The choice of lipids is generally determined by considerations such as liposome size and stability in the blood. Various methods can be used to prepare liposomes, as reviewed, for example, in *Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York*, and also see U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0198] The carrier may contain a total of about 0.1% to about 99.99999% by weight of the pharmaceutical composition presented herein.

[0199] The term "therapeutic effective dose" refers to the amount of a drug that is effective in treating a disease or disorder in a mammal. It also refers to the amount that, at the required dose and time period, effectively achieves the desired therapeutic or preventative outcome. The exact dosage form and regimen will be determined by the physician based on the patient's condition.

[0200] In some embodiments, the method further includes administering an opsonizing agent to the subject. In some embodiments, the method further includes contacting cells with the opsonizing agent. In some embodiments, the opsonizing agent is an epidermal growth factor receptor (EGFR) inhibitor. In some embodiments, the EGFR inhibitor is cetuximab. In some embodiments, the opsonizing agent is not an anti-CD47 agent. In some embodiments, the method further includes administering a PD-1 / PD-L1-based therapy to the subject. In some embodiments, the method further includes contacting cells with a PD-1 / PD-L1-based therapy. In some embodiments, the method further includes growing cells in the presence of a PD-1 / PD-L1-based therapy. In some embodiments, the PD-1 / PD-L1-based therapy is a PD-1 or PD-L1 blocking antibody. In some embodiments, the method does not include administering an anti-CD47 agent or therapy. In some embodiments, the method does not administer an anti-CD47 agent or therapy. In some embodiments, the method further includes administering an anti-CD47 agent or therapy.

[0201] In some embodiments, an agent that inhibits ILT2-based immunosuppression binds to ILT2. In some embodiments, the agent binds to the extracellular domain of ILT2. In some embodiments, the agent is an ILT2 antagonist. In some embodiments, the agent is an ILT2 blocking antibody. In some embodiments, the agent inhibits the interaction between ILT2 and B2M. In some embodiments, the agent is the antibody of the present invention.

[0202] In some embodiments, the agent inhibiting ILT2-based immunosuppression is administered before, after, or simultaneously with the opsonizer. In some embodiments, the agent inhibiting ILT2-based immunosuppression and the opsonizer are administered in a single composition. In some embodiments, the agent inhibiting ILT2-based immunosuppression and the opsonizer are administered in separate compositions.

[0203] In some embodiments, the agent inhibiting ILT2-based immunosuppression is administered before, after, or simultaneously with PD-1 / PD-L1 therapy. In some embodiments, the agent inhibiting ILT2-based immunosuppression and PD-1 / PD-L1 therapy are administered in a single composition. In some embodiments, the agent inhibiting ILT2-based immunosuppression and PD-1 / PD-L1 therapy are administered in separate compositions. In some embodiments, at least one of the agents or therapies is suitable for co-administration.

[0204] As used herein, the term "suitable for co-administration" means that the antibody is present in a form that allows it to be safely and readily administered to a subject. In some non-limiting embodiments, co-administration may be performed orally, by injection, or by inhalation. In some embodiments, the antibody will be contained within a pharmaceutical composition that can be safely and readily administered to a subject. In some embodiments, the pharmaceutical composition comprises an antibody and a pharmaceutically acceptable carrier or excipient.

[0205] In some embodiments, the HLA is HLA-G. In some embodiments, the HLA is atypical HLA. In some embodiments, the HLA is canonical HLA. In some embodiments, mRNA expression is confirmed. In some embodiments, protein expression is confirmed. In some embodiments, surface expression of the protein is confirmed. Methods for measuring expression are well known in the art and include PCR, Q-PCR, RNA blotting, immunoblotting, in situ hybridization, immunostaining, and FACS. In some embodiments, the methods include FACS analysis of cancer to confirm surface expression.

[0206] Preparations

[0207] The present invention also contemplates pharmaceutical formulations for human medical use comprising at least one antibody recognizing ILT2 as an active agent, said pharmaceutical formulations for the manufacture of therapeutic compositions for the treatment, diagnosis or prevention of conditions described herein in various ways.

[0208] In such pharmaceutical preparations, the active agent is preferably used in conjunction with one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredient. The one or more carriers must be pharmaceutically acceptable in the sense of compatibility with other ingredients in the preparation and without undue harm to the recipient. The active agent is provided in an amount sufficient to effectively achieve the desired pharmacological action as described above and in an amount suitable for achieving the desired daily dose.

[0209] Typically, the molecules of the present invention, containing the antigen-binding portion of an antibody, are suspended in a sterile saline solution for therapeutic use. Pharmaceutical compositions can alternatively be formulated to control the release of the active ingredient (the molecule containing the antigen-binding portion of an antibody) or to prolong its presence in the patient system. A variety of suitable drug delivery systems are known, including, for example, implantable drug delivery systems, hydrogels, hydroxymethyl cellulose, microcapsules, liposomes, microemulsions, microspheres, etc. Controlled-release formulations can be prepared by using polymer complexes or adsorption of molecules according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of stearic acid dimer and polyanhydride copolymers of sebacic acid. The release rate of the molecules according to the present invention (i.e., antibodies or antibody fragments) from such matrices depends on the molecular weight of the molecules, the amount of the molecules within the matrices, and the size of the dispersed particles.

[0210] The pharmaceutical compositions of the present invention can be administered by any suitable means, such as oral, topical, intranasal, subcutaneous, intramuscular, intravenous, intra-articular, intra-articular, intralesional, or parenteral administration. Generally, intravenous (iv), intra-articular, topical, or parenteral administration is preferred.

[0211] It will be clear to those skilled in the art that the therapeutically effective amount of the molecule according to the invention will depend in particular on the administration schedule, the unit dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, the patient's immune status and health, the therapeutic activity of the molecule administered, and the judgment of the treating physician.

[0212] Although the appropriate dosage of the molecules (antibodies or fragments thereof) of the present invention varies depending on the route of administration, molecular type (peptide, polynucleotide, organic molecule, etc.), and the patient's age, weight, sex, or condition, and should ultimately be determined by a physician, in the case of oral administration, the daily dose is generally between about 0.01 mg and about 500 mg per kg of body weight, preferably about 0.01 mg to about 50 mg, more preferably about 0.1 mg to about 10 mg. In the case of parenteral administration, the daily dose is generally between about 0.001 mg and about 100 mg per kg of body weight, preferably about 0.001 mg to about 10 mg, more preferably about 0.01 mg to about 1 mg. The daily dose can be administered, for example, in a regimen typically administered 1-4 times daily alone. Other preferred methods of administration include intra-articular administration of about 0.01 mg to about 100 mg per kg of body weight. Various considerations for achieving effective doses are described in, for example, the following literature: Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th edition, Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pennsylvania, 1990.

[0213] Suitable dosing regimens for combination chemotherapy are known in the art and described, for example, in Saltz et al., Proc ASCO 1999, 18, 233a and Douillard et al., Lancet 2000, 355, 1041-7.

[0214] The molecules of the present invention, which are the active ingredients, are dissolved, dispersed, or mixed in a well-known pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients are, for example, water, saline, phosphate-buffered saline (PBS), dextran, glycerol, ethanol, and combinations thereof. Other suitable carriers are well known to those skilled in the art. Additionally, the composition may contain small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffers, if desired.

[0215] Generation method

[0216] According to another aspect, a method for producing a drug is provided, the method comprising: obtaining a drug that binds to the extracellular domain of ILT2 or a fragment thereof; testing the ability of the drug to increase at least one of: macrophage inflammatory activity, T cell activity against cancer cells, dendritic cell activity, and natural killer (NK) cell cytotoxicity against cancer cells; and selecting at least one drug that increases at least one of macrophage activity, T cell activity, dendritic cell activity, and cytotoxicity; thereby producing the drug.

[0217] According to another aspect, a method for producing a drug is provided, the method comprising: culturing a host cell containing one or more vectors comprising a nucleic acid sequence encoding the drug, wherein the nucleic acid sequence is a nucleic acid sequence of the drug selected in such a manner as:

[0218] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0219] ii. Test the ability of the agent to increase at least one of the following: macrophage inflammatory activity, T cell activity against cancer cells, dendritic cell activity, and NK cell cytotoxicity against cancer cells; and

[0220] iii. Select at least one agent that increases at least one of phagocytosis, activity, and cytotoxicity;

[0221] This produces the medicine.

[0222] According to another aspect, a method for producing a drug is provided, the method comprising: obtaining a drug that binds to the extracellular domain of ILT2 or a fragment thereof; testing the ability of the drug to increase the efficacy of anti-PD-L1 / PD-1-based therapy against cancer cells; and selecting at least one drug that increases the efficacy of anti-PD-L1 / PD-1-based therapy; thereby producing the drug.

[0223] According to another aspect, a method for producing a drug is provided, the method comprising: culturing a host cell containing one or more vectors comprising a nucleic acid sequence encoding the drug, wherein the nucleic acid sequence is a nucleic acid sequence of the drug selected in such a manner as:

[0224] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0225] ii. To test the ability of the said agent to enhance the efficacy of anti-PD-L1 / PD-1 based therapies against cancer cells; and

[0226] iii. Select at least one agent that enhances the efficacy of anti-PD-L1 / PD-1-based therapies against cancer cells;

[0227] This produces the medicine.

[0228] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0229] An agent is obtained that binds to the extracellular domain of ILT2 or a fragment thereof, and the agent is tested for its ability to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and at least one agent is selected to induce at least two of the following: said increased phagocytosis, said increased activity, said increased production, said decreased production, said recruitment, said increased activation, said decreased activity, and said increased cytotoxicity; thereby producing the agent.

[0230] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0231] Culture host cells containing one or more vectors that encode a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected in the following manner:

[0232] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0233] ii. Test the ability of the agent to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and

[0234] iii. Select at least one agent that increases at least two of the following: increased phagocytosis, increased activity, increased production, decreased production, recruitment, increased activation, decreased activity, and increased cytotoxicity;

[0235] This produces the medicine.

[0236] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0237] A drug that binds to the extracellular domain of ILT2 or a fragment thereof is obtained; the ability of said drug to inhibit the interaction between ILT2 and B2M is tested; and at least one drug that inhibits the interaction between ILT2 and B2M is selected; thereby producing a drug.

[0238] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0239] Culture host cells containing one or more vectors that encode a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected in the following manner:

[0240] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0241] ii. Test the ability of the agent to inhibit the interaction between ILT2 and B2M; and

[0242] iii. Select at least one agent that inhibits the interaction between ILT2 and B2M;

[0243] This produces the medicine.

[0244] According to another aspect, a method for generating a drug is provided, the method comprising: obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO:41, 42, 43 and 44; thereby generating the drug.

[0245] According to another aspect, a method for producing a drug is provided, the method comprising: culturing a host cell containing one or more vectors having a nucleic acid sequence encoding a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected by obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO: 41, 42, 43 and 44; thereby producing a drug.

[0246] In some embodiments, the method further includes testing the ability of an agent to inhibit ILT2-mediated immunosuppression, and selecting at least one agent that inhibits ILT2-mediated immunosuppression. In some embodiments, the nucleic acid sequence belongs to the agent selected by testing the ability of the agent to inhibit ILT2-mediated immunosuppression and selecting an agent that inhibits ILT2-mediated immunosuppression. In some embodiments, the method includes testing the ability of the agent to induce at least three of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased cytotoxicity against natural killer (NK) cells; and selecting at least one agent that induces said at least three. In some embodiments, the method includes testing the ability of the agent to induce effects in at least three of the following: T cells, NK cells, dendritic cells, and macrophages. In some embodiments, the method includes testing the ability of the agent to induce effects in T cells, NK cells, dendritic cells, and macrophages.

[0247] In some embodiments, the muscle-building effect includes a synergistic increase in anticancer activity. In some embodiments, the anticancer activity is the secretion of pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are selected from GM-CSF, IL-6, and IFNγ. In some embodiments, the pro-inflammatory cytokines are GM-CSF, IL-6, or IFNγ. Each possibility represents a separate embodiment of the invention. In some embodiments, the pro-inflammatory cytokine is GM-CSF. In some embodiments, the increased efficacy includes a synergistic increase in T cell activation. In some embodiments, the increased efficacy includes a synergistic increase in T cell cytotoxicity. In some embodiments, the increased efficacy includes a synergistic increase in both T cell activation and cytotoxicity. In some embodiments, the increase includes increased membrane CD107a expression. In some embodiments, the increase is characterized by increased membrane CD107a expression. In some embodiments, the increase is compared to efficacy without administration or exposure to the agent. In some embodiments, the increased efficacy includes converting cancers refractory to PD-1 / PD-L1-based therapies into cancers responsive to said therapy. In some embodiments, the cancer expresses HLA. In some implementations, cancer cells express MHC-I.

[0248] In some embodiments, increased macrophage inflammatory activity includes increased macrophage phagocytosis of cancer cells. In some embodiments, increased macrophage inflammatory activity includes increased production of M1 macrophages. In some embodiments, increased macrophage inflammatory activity includes decreased production of M2 macrophages. In some embodiments, increased macrophage inflammatory activity includes increased M1 phenotype on macrophages. In some embodiments, increased macrophage inflammatory activity includes decreased M2 phenotype on macrophages.

[0249] In some embodiments, dendritic cell activity includes dendritic cell activation. In some embodiments, dendritic cell activity includes recruitment of dendritic cells to the tumor. In some embodiments, dendritic cell activity is activity against cancer cells. In some embodiments, activity against cancer cells is activity within a tumor mesenchymal exchange (TME). In some embodiments, the tumor is a TME. In some embodiments, dendritic cell activity includes antigen presentation.

[0250] In some embodiments, testing the ability of the agent includes increasing at least one, two, three, four, five, or all of the following: T cell activity against cancer cells, macrophage inflammatory activity, dendritic cell activity, and natural killer (NK) cell cytotoxicity against cancer cells. Each possibility represents a separate embodiment of the invention. In some embodiments, selecting at least one agent includes selecting an agent that increases at least one, two, three, four, five, or all of the following: T cell activity against cancer cells, macrophage inflammatory activity, dendritic cell activity, and natural killer (NK) cell cytotoxicity against cancer cells. In some embodiments, increasing macrophage inflammatory activity is increasing the production of M1 macrophages and / or increasing macrophage phagocytosis of cancer cells. In some embodiments, increasing macrophage inflammatory activity is decreasing the production of M2 macrophages. In some embodiments, testing the ability of the agent includes the ability of the agent to increase macrophage inflammatory activity. In some embodiments, testing the ability of the agent includes the ability of the agent to increase dendritic cell activity. In some embodiments, to the tumor is to the TME. In some implementations, the ability of the drug to be tested includes the drug's ability to increase the cytotoxicity of NK cells against cancer cells.

[0251] In some embodiments, the method further includes testing the ability of the agent to inhibit the interaction between ILT2 and B2M. In some embodiments, the interaction is a direct interaction. In some embodiments, the method further includes testing the ability of the agent to inhibit the contact between ILT2 and B2M. In some embodiments, the interaction is binding. In some embodiments, the contact is binding. In some embodiments, the method further includes testing the ability of the agent to bind to epitopes.

[0252] The following examples are intended to illustrate how to prepare and use the compounds and methods of the present invention, and should in no way be considered limiting. Although the invention will now be described in conjunction with specific embodiments, it will be apparent to those skilled in the art that many modifications and alterations will be apparent. Therefore, it is intended to include all such modifications and alterations that fall within the spirit and broad scope of the appended claims.

[0253] Example

[0254] Generally, the nomenclature used herein and the laboratory procedures used in this invention include molecular, biochemical, microbiological, and recombinant DNA techniques. These techniques are well explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Volumes I-III, edited by Ausubel, RM, (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, MD, (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, NY, (1988); Watson et al., "Recombinant DNA," Scientific American Books, NY; Birren et al. (eds.), "Genome Analysis: A Laboratory Manual Series," Volumes I-4, Cold Spring Harbor. Laboratory Press, New York (1998); as described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III, edited by Cellis, J.E. (1994); “Culture of Animal Cells: A Manual of Basic Technique”, Freshney, Wiley-Liss, New York (1994), Third Edition; “Current Protocols in Immunology”, Volumes I-III, Coligan, J.E.References cited are:

[1994] ,

[2007] , [2008 ...

[0255] Materials and methods

[0256] Antibodies Commercial anti-ILT2 mAbs are: clone #1-GHI / 75 (BioLegend, catalog number 333704), clone #2-HP-F1 (eBioscience, catalog number 16-5129). Other mAbs used include: HLA-G (MEM-G / 9; Abcam, catalog number ab7758; G-0031), ILT4 (42D1, Biolegend, catalog number 338704), ILT6 (Sino Biological, catalog number 13549-MM06), LILRA1 (R&D systems, catalog number MAB30851), pan-HLA (W6 / 22; eBioscience, catalog number 16-9983-85), and His (Proteintech, catalog number 10001-0-AP).

[0257] Flow cytometry - Typically, cells are kept on ice or at 4°C throughout all steps. Before staining, prepare 5 x 10⁻⁶ cells... 5Cells were blocked for 15 min with 50 μg / mL human IgG (Sigma, catalog number I4506) in FACS buffer (PBS containing 0.1% BSA). Antibody was used at the manufacturer's recommended concentration and incubated in the dark for 30 min. Incubation was performed in 100 μL of 96-well U-shaped plates. Cells were washed twice with 200 μL of FACS buffer and transferred to 150 μL of FACS buffer in FACS tubes for analysis. Cells were analyzed using Kaluza software for Gallios flow cytometry on a Gallios flow cytometer (Beckman Coulter).

[0258] Myeloid cell differentiation -Monocytes were selected using EasySep through a negative selection method. TM Human mononuclear cell enrichment kit (STEMCELL, catalog number 19059) was used to isolate cells from fresh blood samples from healthy donors. Different cell populations were tested for the indicated phenotype by FACS analysis of relevant markers and by analysis of characteristic cytokine secretion. For mature cells, 0.8 x 10⁻⁶ cells were used. 6 Monocytes were cultured at a density of 1 / mL in RPMI medium containing growth factors, with the medium changed on days 3 and 6. Inflammatory M1 macrophages were matured for 6 days in the presence of 50 ng / mL GM-CSF (M1 phenotype), followed by maturation for 48 hours in the presence of 20 ng / mL IFN-γ and 50 ng / mL LPS. Inhibitory M2 macrophages were differentiated for 6 days using 50 ng / mL M-CSF, followed by differentiation for 48 hours using 10 ng / mL M-CSF and 20 ng / mL IL-4 and IL-10. Dendritic cells were induced for 6 days with 50 ng / mL GM-CSF and 20 ng / mL IL-4, and further differentiated into mature (100 ng / mL LPS) or tolerant (IL-10 100 U / mL and IFN-α2b 1000 U / mL) dendritic cells.

[0259] Transfection - An HLA-G1 plasmid (encoding the full-length HLA-G transcript) is generated by cloning HLA-G1 cDNA into the PCDNA3.1 vector. Transfection is performed using... Transfection was performed using PolyPlus Transfections. The ILT2 / CD3z plasmid was generated by combining the extracellular portion of the human ILT2 protein with the transmembrane and cytoplasmic residue frames of the mouse CD3 gene. The plasmid nucleus was transfected into the mouse BW5417.3 T cell line using Nucleofector II (Lonza) as described by the manufacturer. Stable transfectants were selected in a medium containing G418.

[0260] NK and cancer cell line co-culture assay - NK cells were incubated together with the indicated cell line at 37°C for 5 hours in the presence of anti-ILT2 antibody and a matched isotype control. Cytotoxicity levels were measured using a fluorescence assay kit for LDH detection (Promega).

[0261] Flow cytometry blocking assay - Recombinant human ILT2 protein fused to the Fc region of human IgG1 at the N-terminus will be conjugated with biotin (Innova bioscience). A total of 5 x 10 5 A375 / HLA-G1 cells were incubated at room temperature for 30 min at 100 μL in the presence of anti-ILT2 clone #1 or an allotype-matched control mAb and biotin-conjugated ILT2-Fc (10 μg / mL). After several washing steps, streptavidin-PE was added to a final concentration of 0.2 μg / mL and incubated on ice for 30 min before FACS analysis.

[0262] BW ILT2 / CD3z chain chimera assay - 3X10 4 One BW / ILT2z sample was mixed with an equivalent number of A375 / WT or A375 / HLA-G1 cells for 24 hours. Functional mAbs at indicated concentrations and matched isotype controls were used. The amount of secreted mouse IL2 was evaluated using a commercial ELISA kit (BioLegend).

[0263] Example 1

[0264] ILT2 and HLA-G were found on cancer cells and cancer-associated immune cells.

[0265] ILT2 is a known immunosuppressive molecule found on the surface of healthy immune cells and many tumor cells. ILT2 has been shown to bind to MHC-1 and HLA class molecules (HLA-G, HLA-F, and HLA-B27) and compete with CD8, thereby inhibiting T cell activation. To further understand the range of cells expressing ILT2, flow cytometry analysis was performed on various immune cell types using a commercial antibody (antibody #1). As reported in the literature, cytotoxic T cells (CTLs) and natural killer (NK) cells derived from melanoma patients showed positive surface expression of ILT2. Figure 1 Monocytes from healthy donor blood were also examined and found to highly express ILT2. Figure 2 (Leftmost image). After monocytes differentiate into different myeloid cell populations (dendritic cells and macrophages), regardless of whether they are immature, inflammatory, or tolerant, they all retain ILT2 expression. Figure 2 (See right side image).

[0266] Bioinformatics analysis of the TCGA database was used to examine ILT2 expression in different cancer indications. Figure 3A Interestingly, an association was observed between ILT2 RNA expression levels and the presence of myeloid-derived suppressor cells (MDSCs) and suppressor M2 tumor-associated macrophages (TAMs) in tumor samples represented in TCGA. Figure 3B Analysis of fresh tumor samples from various solid tumors using flow cytometry confirmed ILT2 expression in innate and adaptive immune cells within the tumor microenvironment (TME). Tumor samples were collected from patients with non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck cancer, esophageal cancer, and colon cancer, and single-cell suspensions were generated through enzymatic digestion. The percentages of ILT2-positive cells among total immune cells, tumor-associated macrophages (TAM), CD4-positive T cells, CD8-positive T cells, and natural killer (NK) cells were presented as follows. Figure 3C Therefore, it is evident that ILT2 is expressed simultaneously on cells with anti-cancer activity (inflammatory cells) and on cells with cancer-promoting and immunosuppressive activities (tolerogenic and MDSCs).

[0267] HLA-G expression is also being studied in various cancers. Tissue microarrays (TMAs) of cancer samples from different indications were stained with commercial polyclonal HLA-G antibodies via immunohistochemistry. The percentage of sample cases for each cancer type is indicated. Figure 4A Additionally, for several indications, expanded TMA is examined. The HLA-G staining score is calculated by multiplying the staining intensity by the percentage of positive cells. High HLA-G staining scores above 100 were detected in high percentages of esophageal, gastric, head and neck, and renal cancers. Figure 4B The percentage of positive cases for each indication is shown in Table 1.

[0268] Table 1:

[0269]

[0270] HLA-G exists in a soluble, secreted form as well as a more common membrane form. To examine the expression level of soluble HLA-G in cancer patients, the presence of HLA-G in plasma samples was detected using a commercial ELISA. Overexpression of HLA-G was found in several cancer indications compared to normal (healthy) controls. Figure 5 Furthermore, in certain cancer types, patient groups with significantly higher levels can be detected.

[0271] Example 2

[0272] ILT2 blocking antibody production

[0273] Monoclonal ILT2 antagonist antibodies were generated using hybridoma technology. Initially, 69 ILT2-specific hybridomas were generated. Three leader antibodies were selected based on their preferred binding, cross-reactivity profile, and functional activity in various assays examined. The selected antibodies were 19E3, 15G8, and 17F2. These antibodies were sequenced using common methods. The sequences of the selected antibodies are shown below. Figure 6 The CDRs were determined using the KABAT system. 15G8 and 19E3 were humanized using common CDR transplantation methods. In short, the essential CDRs and framework residues from the initial hybridoma-derived antibody were identified and transplanted into the variable and constant regions of the germline human antibody. The final humanized antibody was an IgG4 antibody. The final humanized 15G8 also contained a single amino acid change, removing cysteine ​​from CDR-H3 and replacing it with either alanine or serine. This change was performed to improve developability. Binding of the two resulting antibodies was confirmed, and the 15G8 antibody with alanine was selected for further testing. All humanized 15G8 mentioned below refers to the alanine variant.

[0274] The ability of anti-ILT2 antibodies to bind to ILT2 was tested using three different systems. Binding to recombinant ILT2 was tested using ELISA and the Biacore T200 (Table 2). Figure 7A ), and used BW cells transfected with ILT2 to test the binding of ILT2 to the membrane ( Figures 7B-7C Chimeric mice and humanized antibodies showed similar binding (). Figure 7C A commercial mouse anti-human ILT2 antibody (Biolegend; clone GHI / 75) was used as a positive control. All three assay antibodies successfully bound ILT2, whether in solution or on the cell surface. Cross-reactivity with several similar ILT family members (PIRB, ILT6, and LILRA1) was also examined using a binding ELISA. Antibodies against these proteins were used as positive controls. None of the antibodies cross-reacted with PIRB, ILT6, or LILRA1. The antibodies were also effective for immunostaining. Figure 7D Interestingly, when PBMCs were isolated from the blood of cancer patients, it was found that ILT2 was expressed on more T cells and NK cells in cancer patients compared to healthy controls. Figure 7E ).

[0275] Table 2

[0276] Ka (1 / Ms) Kd (1 / s) KD ( / M) 1.37-1.76E+06 2.22-5.22E-03 1.26-3.16E-09

[0277] Example 3

[0278] ILT2 antibody blocks ILT2-HLA-G interaction

[0279] Four different assays were used to test the ability of the generated anti-ILT2 antibody to block the interaction between HLA-G and ILT2. First, a blocking flow cytometry assay was performed. HLA-G-transfected A375 cells were incubated with biotinylated ILT2 in the presence of the antibody of the present invention and a positive control antibody. A commercially available anti-ILT2 antibody, GHI / 75 (BioLegend, catalog number 333704), was used as a positive control. The binding of ILT2-biotin to cells was determined by flow cytometry analysis using streptavidin-PE. Figure 8A The percentage of blockade was determined by normalization against the negative control (ILT2 binding in the presence of control IgG). Representative FACS analyses are presented in [the table / data]. Figure 8B The diagram shows ILT2 binding in the absence of antibody (gray line), in the presence of 15G8 (light gray line), and in the presence of an isotype control (black line). The percentage of blockade was calculated at different antibody concentrations. Figure 8C Chimeric mouse and humanized antibodies showed similar blocking abilities. Figure 8D ).

[0280] The ability of ILT2 antibodies to functionally block the interaction between HLA-G and ILT2 was also examined in a BW ILT2 / mouse Z-chain chimeric reporter assay. BW cells (BW-ILT2) were transfected with human ILT2 fused to the ζ chain of mouse T cells. Cells were then co-incubated with A375-HLA-G cells in the presence of a selected ILT2 antibody. Following functional ILT2-HLA-G interaction, BW cells secrete the reporter cytokine, mouse IL-2. Blocking the interaction reduced the secretion of this reporter cytokine. Mouse IL-2 secretion was determined by ELISA after 24 hours of incubation. Results are expressed as the mean of mIL-2 levels ± SE from triplicate wells for each treatment. Figure 8E A commercial mouse anti-human ILT2 antibody (Biolegend; clone GHI / 75) was used as a positive control (PC) for both assays. The percentage of blockade was calculated at different antibody concentrations. Figure 8F Using the same BWILT2 / mouse Z-chain chimeric reporter assay, the possibility that the novel antibody might possess ILT2 activation activity on its own was ruled out. Cells were incubated with the ILT2 antibody in the absence of cancer cells, and mouse IL-2 secretion was measured again. Figure 8G The novel ILT2 antibody was found to have no agonistic effect, but other antibodies (1G7) generated using the same hybridoma method could bind to ILT2 and induce its activity.

[0281] Functional blockade was also examined in human Jurkat cells (T cells). Jurkat cells were incubated with or without exogenous HLA-G and single-chain anti-CD3 (OKT3) expressing A375 cancer cells. The secretion of pro-inflammatory human IL-2 was measured. High levels of IL-2 were secreted when Jurkat cells were co-cultured with cancer cells, using unmodified Jurkat cells (ILT2-negative cells). Figure 8H Unsurprisingly, the addition of the 15G8 antibody had no effect on IL-2 secretion, as the ILT2 to be blocked was absent. Therefore, Jurkat cells were transfected to express human ILT2. First, ILT2-positive Jurkat cells were cultured with and without exogenous OKT3-expressing A375 cancer cells. These cancer cells were naturally MHC-I positive. MHC-I from cancer cells strongly inhibits IL-2 secretion (…). Figure 8I In this scenario, the addition of 15G8 antibody blocked ILT2 / MHC-I interaction in a dose-dependent manner and increased IL-2 secretion. A pan-HLA antibody was used as a positive control, and at equivalent concentrations, 15G8 antibody was comparable to the pan-HLA antibody. Figure 8I To enhance the inhibitory effect, A375 cells were also transfected with HLA-G, making them MHC-I and HLA-G positive. These cells produced an even stronger inhibitory effect on ILT2-positive cells, thereby reducing IL-2 secretion to the level of IL-2 secretion in cultured Jurkat cells alone. Figure 8J A dose-dependent effect was again observed with the administration of the 15G8 antibody, and at equal doses, the 15G8 antibody and the pan-HLA antibody were again equally effective. Figure 8J It is noteworthy that when only HLA-G specific antibodies were used instead of pan-HLA, the effect was significantly reduced and comparable to that of 15G8 antibody used at a concentration of 1 / 100. Figure 8K ).

[0282] The Jurkat system was also used to compare the 15G8 antibody with two commercially available antibodies: GHI / 75 and HP-F1. Jurkat cells expressing human ILT2 were co-cultured with A375 cells expressing HLA-G / OKT3 in the presence and absence of different concentrations of 15G8, GHI / 75, and HP-F1. As already observed, 15G8 caused a statistically significant, dose-dependent increase in IL-2 secretion. Figure 8L Compared to the culture medium alone, GHI / 75 had no effect on IL2 secretion, but resulted in a slight increase compared to the IgG control. Figure 8M HP-F1 produced a small but significant increase, which reached a plateau and did not increase with further dosing. Figure 8NHP-F1 performed worse even at 20 μg / ml compared to 15G8, which was only 4 μg / ml.

[0283] Finally, activation was measured directly in TILs and NK cells. TILs were incubated with A375-HLA-G-OKT3 cells for 5 minutes, followed by detection of the T cell activation marker, phosphorylated ZAP70. NK cells were incubated with A253-HLA-G cells for 2 minutes, followed by detection of the NK cell activation marker, phosphorylated Syk. Activation was observed in both cell types when co-cultured with cancer cells, but this activation was enhanced in the presence of ILT2 antibody. Figures 8O-8P These results confirm that ILT2 antibodies can effectively block ILT2-HLA-G interaction, thereby leading to enhanced T cell and NK cell activation.

[0284] Example 4

[0285] ILT2 antibody enhances the phagocytic activity of HLA-G and MHC-I positive tumor cells.

[0286] The ability of generated anti-ILT2 antibodies to enhance the phagocytic activity of tumor cells was tested using two different systems. Monocytes were isolated from the blood of healthy donors and incubated for 6–7 days in the presence of M-CSF to generate macrophages. First, flow cytometry-based assays were performed. Different cancer cell lines stained with PKH67-FITC were co-incubated with macrophages stained with eFluor 670-APC in the presence of the indicated antibody. The level of phagocytosis was determined by the percentage of macrophages double-stained, indicating the phagocytosis of target cells. The level of phagocytosis was presented as a percentage relative to a control (culture medium only). Figure 9A As shown, different ILT2 blocking antibodies can enhance the phagocytic activity of macrophages on HLA-G-positive A375 cells. Additionally, using real-time... An analytical system was used to examine the ability of macrophages to enhance the phagocytic activity of tumor cells. Target cell lines were analyzed using pHrodo. TM Red cell marker dyes were applied, washed, and added to macrophages along with repeated treatments. pHrodo TM The fluorescence of red cell marker dyes increases in acidic environments (such as the inherent acidity in phagosomes), making it possible to quantify phagocytic events by measuring fluorescence. The instrument samples the assay plate every 30 minutes for fluorescence red signal intensity and phase mapping. Phagocytic events are reflected as the accumulation of red fluorescence signal, and the phagocytic rate is reflected from the kinetics of red fluorescence signal accumulation. Using this real-time system, the ability of humanized anti-ILT2 antibody to enhance the phagocytosis of HLA-G positive A375 cells was confirmed. Figure 9B Additionally, using The system confirmed that the generated ILT2-blocking antibody could enhance the phagocytic activity of both HLA-G-positive and various MHC-I-positive (WT) cancer cell lines. Figure 9C ).

[0287] Use the above Real-time systemic examination was conducted to examine the effect of combining the generated ILT2 antibody with the antibody Erbitux, which is induced by antibody-dependent phagocytosis (ADCP), on the phagocytosis of cancer cells. Compared to the activity of each antibody alone, the combination of the ILT2 blocking antibody and Erbitux significantly increased the phagocytosis of cancer cell lines overexpressing HLA-G. Figure 9D In fact, the combination of Erbitux and the 15G8 humanized antibody has a synergistic effect, with the combined treatment increasing phagocytosis more than the additive effect alone.

[0288] Example 5

[0289] The selected ILT2 antibody can restore T cell activity suppressed by HLA-G.

[0290] To examine the ability of the generated anti-ILT2 antibody to restore T cell activity suppressed by HLA-G, human CD8 T cells were co-incubated with wild-type 721.221 cells (221WT) or 721.221 cells overexpressing soluble HLA-G5 (221-HLA-G). IFNγ secretion levels of T cells were measured using a standard ELISA after 5 days. Results are shown as a fold increase over the effect of 221-HLA-G alone and are expressed as the mean of four independent experiments. Figure 10A The results presented confirm that several ILT2 antibodies can restore HLA-G-suppressed T cell activity. This was also tested by co-incubation with A375-HLA-G-OKT3 cells. After 72 hours, the secretion of human granzyme B was also measured, and it was found to increase in a dose-dependent manner in the presence of the 15G8 antibody. Figure 10B ).

[0291] Example 6

[0292] The selected ILT2 antibody can enhance NK cell cytotoxicity against HLA-G and MHC-I positive tumor cells.

[0293] The ability of the generated anti-ILT2 antibody to enhance NK cell effector activity was tested in the system by co-incubating NK cells with various target cancer cell lines. Cells were co-incubated at an effector-to-target ratio of 7.5:1 for 5 hours, after which cytotoxicity levels were detected using a fluorescence assay kit for LDH detection. The percentage of specific cytotoxicity was calculated as follows:

[0294]

[0295] like Figure 11A As shown, the ILT2 antibody of the present invention can significantly enhance the cytotoxicity of NK cells against both HLA-G positive cells and various MHC-I positive cancer cell lines in a dose-dependent manner. Figure 11B Granulase B was also measured. Figure 11C ) and interferon-γ ( Figure 11D The secretion of IFNγ, ILT2, CD56, and CD107A was observed to increase in a dose-dependent manner. Primary NK cells were co-cultured with target HLA-G+ melanoma cells, and the expression of IFNγ, ILT2, CD56, and CD107A was analyzed by FACS. ILT2-positive and CD56-positive NK cell populations were specifically analyzed, and a dose-dependent increase in IFNγ expression and membrane CD107A expression was observed. Figures 11E-11F When plotted separately for each experiment, the association between the percentage of ILT2-positive cells and increased IFNγ and CD107A expression was clearly evident. Figures 11G-11H ).

[0296] Example 7

[0297] ILT2 antibodies increase the production of inflammatory macrophages.

[0298] The effect of blocking ILT2 on macrophage maturation was examined in vitro. Monocytes isolated from healthy donors were differentiated for 5 days in the presence of M-CSF (50 mg / mL) to generate mature macrophages (M0) in the presence of humanized ILT2-blocking antibodies or control IgG. Macrophages were then further differentiated in the presence of LPS (50 ng / mL) to generate M1 macrophages, or differentiated with IL-4 (25 ng / mL) to generate M2 macrophages. Figure 12 As shown, the presence of ILT2-blocking antibodies during macrophage maturation increased HLA-DR (a marker of M1 inflammatory macrophages) expression on macrophages from most tested donors, regardless of whether they differentiated into M0, M1, or M2 macrophages. Additionally, macrophages differentiated into M1 macrophages also exhibited increased CD80 levels in most tested donors. In summary, these results confirm that the selected ILT2 antagonist antibody can induce macrophages displaying higher HLA-DR and CD80 levels, indicating macrophages with a higher inflammatory M1 phenotype.

[0299] Example 8

[0300] ILT2 blocking antibodies enhance the activity of immune cells against tumor cells from patients.

[0301] The activity of generated anti-ILT2 antibodies was examined in an ex vivo system using tumor samples from cancer patients (RCC and H&N). To test the antibody's ability to increase phagocytosis of tumor cells from patients, macrophages generated from monocytes were incubated together with tumor cells isolated from the tumor samples. The use of [the antibody] was detailed above. The real-time analysis system examines the level of phagocytosis. For example, in... Figure 13A As shown, the ILT2 antibody can enhance the phagocytic activity of tumor cells from patients with different cancer indications. Furthermore, this effect is dose-dependent and persists even when autologous macrophages are used, and is also effective for RCC ( Figure 13B Both ) and squamous cell carcinoma (13C) from H&N were observed. Additionally, the effect of ILT2 antibody in enhancing PBMC activity was examined. Single-cell suspensions of tumor samples from patients were incubated together with PBMCs isolated from the same patients in the presence of IL-2 (activated PBMCs). Figure 14G As shown, in the presence of ILT2 antibody, PBMC secretion of the pro-inflammatory TNF-α cytokine is increased in the presence of tumor cells. In summary, these results confirm the ability of blocking ILT2 antibody to increase the activity of immune cells against tumor cells from various cancer indications.

[0302] Example 9

[0303] ILT2 blocking antibodies can be combined with PD-1 / PD-L1 therapy

[0304] For the most part, ILT2 and PD-1 are expressed on various immune cells, including both peripheral blood cells and tumor microenvironment-resident immune cells. Figure 14A Analysis of ILT2 and PD-1 expression in intratumoral CD8-positive T cells from CRC patients revealed that both central memory T cells (Tcm) and consumed T cells (Tex) expressed high levels of PD-1. Figure 14B However, it expresses low levels of ILT2 ( ), Figure 14C CD45RA reexpressing T cells (T cells) EMRA This showed a completely opposite pattern, expressing high levels of ILT2 and low levels of PD-1. This dichotomy is not unique to cancer; a large proportion (83%) of ILT2 levels were found in blood from healthy donors. EMRA The cells were ILT2 positive, while only a small percentage (17%) of the total CD8-positive T cells were positive.Figure 14D However, in the TME, ILT2 expression was enhanced in T cells. A single-cell suspension was generated by enzymatic digestion of tumors isolated from esophageal cancer patients. FACS analysis showed that the majority of CD8-positive tumor-infiltrating lymphocytes (TILs) were T cells. EMRA Cells (50%), and these T cells EMRA 100% of the cells were ILT2 positive, but almost all were PD-1 negative (95%). Figure 14E ).

[0305] The effects of the anti-ILT2 antibody combined with anti-PD-1 of the present invention were tested in SEB-activated (10 ng / ml) PBMCs from 10 healthy donors. Membrane CD107a expression was used as a marker of increased cytotoxicity. Overall, the 15G8 antibody produced a small, on average, increase in surface CD107a, while the anti-PD-1 produced a slightly larger response, which was donor-dependent. Figure 14F The combination of the two antibodies, on average, produces increased CD107a levels; however, these changes are variable depending on the specific donor sample. Figure 14G Three exemplary samples are presented. The first donor showed an additive effect when anti-PD-1 was combined with 15G8, with total CD107a levels approximately equal to the sum of the effects of each antibody alone. The second donor responded more strongly to anti-PD-1 than to anti-ILT2, but unexpectedly, the combination of the two antibodies had a more than additive effect. Anti-PD-1 produced a 19% increase in expression, and anti-ILT2 produced a 3.7% increase, but the combination treatment resulted in a 33.2% increase. This synergistic effect was even more pronounced in the cells of donor #3. In donor #3, 15G8 was more effective than anti-PD-1 (13.1% increase vs. 9.3% increase), and the combination therapy was significantly more effective (41%), producing almost twice the effect predicted from the additive combination alone.

[0306] Next, the combined treatment of patient tumor cells with PD-1 blocking antibody and generated ILT2 antibody was evaluated. Various patient cancer cells were incubated with autologous PBMCs in the presence of anti-PD1 antibody, the antibody of the present invention, and combinations thereof. IgG was used as a control, and the secretion of pro-inflammatory molecules was measured as a readout. Enhanced secretion of pro-inflammatory cytokines was observed in the combined treatment. Figures 14H-14J Compared to the IgG control, treatment of colon adenocarcinoma cells from first-patients with the humanized antibody 15G8 did not enhance IFNγ secretion at all, but rather showed a robust increase in the secretion of anti-PD-1-producing cytokines. Figure 14HHowever, unexpectedly, the combination of anti-PD-1 and ILT2 antibodies increased secretion by more than 50%. A second patient showed a similar trend, where either ILT2 or anti-PD-1 induced a small increase, and a synergistic increase was observed when both antibodies were used in combination. Figure 14I As compared to the control, neither antibody alone altered GM-CSF expression; however, surprisingly, the combination of the two antibodies produced a robust increase of nearly 100% in control GM-CSF levels. Figure 14J ).

[0307] Next, the combination therapy was evaluated using a mixed lymphocyte response. Dendritic cells and CD8-positive T cells were isolated from various healthy donors, and macrophages were generated from monocytes isolated from H&N cancer patients. Cells were combined with the indicated treatment (20 μg / mg each) at a 5:1 effector cell to target ratio. IFNγ secretion by T cells was enhanced in the presence of either anti-ILT2 or anti-PD-1 antibodies, and this effect was increased when both antibodies were used in combination. Figures 14K-14L ). Such as with dendritic cell cultures ( Figure 14K Compared to macrophage cultures, a stronger cumulative effect was observed. Figure 14L These results clearly demonstrate that anti-ILT2 and anti-PD-1 therapies have a synergistic and de novo effect in enhancing the inflammatory response of immune cells.

[0308] Example 10

[0309] ILT2 blocking antibodies reduce tumor burden in vivo.

[0310] The efficacy of anti-ILT2 antibodies was examined in a xenograft model. Immunocompromised SCID-NOD or NSG mice were inoculated with cancer cell lines (A375-HLA-G, A375-WT, COLO-320-HLA-G), and human macrophages derived from healthy donor blood were injected into the mice in the presence of ILT2 antibodies. Figure 15A As shown, administration of the generated ILT2 antibody in this model resulted in significant tumor suppression, most likely mediated by the activity of human macrophages in this system. Furthermore, antitumor efficacy was observed in both HLA-G and MHC-I positive tumor cells.

[0311] The efficacy of anti-ILT2 antibodies was also examined in an in vivo model of lung lesion melanoma xenograft. Melanoma cells (MEL526-HLA-G) were inoculated into immunocompromised SCID-NOD mice. Human PBMCs isolated from healthy donor blood were injected into the mice in the presence of the selected ILT2 antibody, starting one day post-inoculation and repeated on days 2, 10, and 18. Figure 15BILT2 antibody was administered on days 1, 4, 8, 11, 15, 18, 22, and 25. Figure 15C As shown, administration of the generated ILT2 antibody resulted in a significant reduction in tumor cell metastasis, indicated by the formation of black lesions in the lungs of mice. Mice treated with the ILT2 antibody exhibited far fewer such lesions in their lungs compared to mice treated with control IgG. This effect was also confirmed by a decrease in lung weight in these mice. Figure 15D This is most likely mediated by human lymphocytes administered to mice, in combination with the inhibitory effect of the administered antibody on ILT2. Therefore, anti-ILT2 antibodies are effective in preventing metastasis and tumor formation.

[0312] Next, the efficacy of the novel antibody in treating established tumors was tested in the same in vivo mouse model. SCID-NOD mice were transfected with MEL526-HLA-G cells (as described above) via intravenous administration. After 15 days, human PBMCs isolated from healthy donors were administered to the relevant mouse groups, and this administration was repeated on days 25, 35, and 51 (see [link to relevant documentation]). Figure 15E Antibodies (ILT2 antibody, anti-PD-1 antibody, or a combination of both) were administered on days 14, 17, 20, 24, 27, 30, 34, 37, and 50 (see [link to relevant documentation]). Figure 15E Mice were sacrificed on day 53, and their lungs were weighed. Tumor weight was calculated by subtracting the lung weight of unexperimented mice from the lung weight of the test mice. Anti-PD-1 antibodies reduced tumor weight, but not significantly, while ILT2 antibodies and combination therapy had a significant effect. Figure 15F ).

[0313] Testing tumor-derived CD8 T cells, T EMRA CD107A and CD69 expression in cellular and NK cells. In total CD8 T cells, anti-PD-1 antibody induced a non-significant increase in CD107A expression, while ILT2 antibody, rather than combination therapy, induced a significant change. Figure 16A ). In T EMRA In cells, both ILT2 antibody and combination therapy induced a significant increase ( Figure 16B In NK cells, both anti-PD1 and anti-ILT2 antibodies significantly increased the percentage of CD69-positive cells, but surprisingly, combination therapy had a significantly enhanced effect, with the total percentage of CD69-positive cells being greater than the combination of either therapy alone. Figure 16C Surprisingly, when examining CD69 expression in CD8 T cells, neither anti-PD1 nor anti-ILT2 increased expression; however, combination therapy induced a very significant increase in CD69 expression. Figure 16DFurthermore, the effects of the ILT2 antibody were determined to be correlated with ILT2 expression. Significant differences in activation markers were observed when the experiment was broken down into mice receiving PBMCs with low or high ILT2 expression. EMRA In cells, compared with low ILT2 expression PBMCs, high ILT2 expression PBMCs included more than double CD107A expression ( Figure 16E Similarly, when examining NK cells, in combination therapy, high ILT2-expressing PBMCs induced nearly 90% of cells to express CD69; while low ILT2-expressing PBMCs induced less than 40% of NK cells to express CD69. Figure 16F Therefore, the expression level of ILT2 in PBMCs is essential for the most potent effect of the antibody.

[0314] Example 11

[0315] In vivo humanized H&N model

[0316] In the second in vivo model, humanized mice (mice inoculated with human CD34+ cells) were inoculated with A253-HLA-G cells. When the tumor reached 80 cubic millimeters in size, the mice were treated with either control IgG or ILT2 antibody (15G8, both at 10 mg / kg). The treatment was repeated twice a week. Figure 17A Until day 43, tumor size was determined by measuring the tumor with calipers at different time points. The ILT2 antibody completely inhibited tumor growth in 2 out of 4 mice (mice #23 and #28), where the tumor was eradicated by day 43. Figure 17B To determine whether the different responses to treatment were due to varying levels of ILT2 expression in mouse immune cells, ILT2 expression in CD8 T cells from peripheral blood was measured at baseline. In fact, both mice with a complete response exhibited high ILT2 expression in their T cells, while the other two mice showed significantly lower expression levels. Figure 17C Furthermore, examination of the TME after treatment revealed three other pharmacodynamic markers that distinguished responders from non-responders, namely CD107A expression in T cells (…). Figure 17D M1 / M2 macrophage ratio Figure 17E ) and total CD80-positive dendritic cells ( Figure 17F These results indicate that anti-ILT2 is generated and transformed in the bone marrow and lymphatic compartments of the tumor microenvironment, and may also increase the ability of dendritic cells to present antigens and recruit more T cells to the tumor.

[0317] Example 12

[0318] Epitope localization of 15G8 humanized antibody

[0319] The 15G8 antibody was sent for epitope localization to determine its binding site on ILT2. Localization was performed by MAbSilico. The structure of the ILT2 used was modeled using the following structures: 6AEE (four Ig-like domains, some loops missing), 1VDG (undisclosed, domains 1 and 2), 1G0X (domains 1 and 2), and 4LL9 (domains 3 and 4). The structures of 6AEE and 1G0X were obtained from Wang, Q., et al., (2019). “Structures of the four Ig-like domain LILRB2 and the four-domain LILRB1 and HLA-G1 complex.” Cell. Mol. Immunol., and the structure of 4LL9 was obtained from Chapman, TL, et al., (2000). “Crystal structure and ligand binding properties of the D1D2 region of the inhibitory receptor LIR-1 (ILT2)”. Immunity, 13(5), 727-736. Region D1 is defined as residues 24-121 of ILT2. Region D2 is defined as residues 122-222 of ILT2. Region D3 is defined as residues 223-321 of ILT2. Region D4 is defined as residues 322-409 of ILT2. A 3D model of the antibody is constructed using Modeller.

[0320] Based on the top 30 docking poses, the residues are scored according to the probability that they belong to an epitope. Residues that may belong to an epitope are shown in... Figure 18A On the sequence and Figure 18B The target structure is defined by these residues. Four main interaction regions are defined on the target. Figure 18C All four of these interacting regions were found in the intergenic domain of ILT2, which is the hinge region between D1 and D2. Validation mutations were selected in these regions and are summarized in Table 3. These mutations were generated in full-length ILT2 or truncated D1+D2 protein, and binding to the 15G8 antibody was tested. Loss or reduced binding to the mutants indicates that the region is the true epitope of the 15G8 antibody.

[0321] Table 3: Test Mutations

[0322] Name Mutation 15G8 Region 1 K56A Q58A S63A E67A 15G8 Region 2 D177A N179A E183A 15G8 Region 3 Y98A I99A K100A T102A 15G8 Region 4 Q124A V125A

[0323] However, the binding epitopes of most ILT2 antibodies are unknown, but international patent publication WO2020 / 136145 does disclose epitope information for several antibodies. Two universal binding regions were found, one in the D1 region and one in the D4 region. Specifically, three antibodies named 3H5, 12D12, and 27H5 are characterized by loss of binding to mutants with substitutions at E34, R36, Y76, A82, and R84 in D1. One of those antibodies, 3H5, shows reduced binding to a mutant with substitutions at G29, Q30, T32, Q33, and D80 in D1. These residues are located only in the D1 region and are all outside (within) the four regions defined as the binding epitope of the 15G8 antibody (note that in...). Figure 18A In this sequence, the sequence begins after one amino acid, such that E34 in, for example, WO2020 / 136145, is... Figure 18A The middle one is E33). Therefore, antibody 15G8 binds to a three-dimensional epitope that is different from the three-dimensional epitope bound by the antibody disclosed in WO2020 / 136145 ( Figure 18D ).

[0324] Interestingly, the region defined as the 15G8 epitope (which is the interstitial region between D1 and D2) has been identified as the major interacting region of ILT2, which binds to β-2-microglobulin (B2M) upon complexation with HLA (see Kuroki et al., “Structural and functional basis for LILRB immune checkpoint receptor recognition of HLA-G isoforms”, J. Immuno., 2019, Dec. 15; 203(12): 3386-3394). Figures 18E-18FIn fact, residues G97, A98, Y99, I100, Q125, and V126 were explicitly identified by Kuroki et al. (Supplementary Figure S2 in Kuroki) as interacting with B2M. These residues fall within interaction regions 3 and 4 of 15G8 and are all considered very high-probability or highly probable epitope residues. This strongly suggests that 15G8 inhibits ILT2 binding to HLA in a B2M-dependent manner and effectively blocks direct binding of ILT2 to B2M. In contrast, the 3H5, 12D12, and 27H5 antibodies bind to the N-terminal D1 region of ILT2, which interacts with the α3 domain of HLA-G (see Supplementary Figure S2 in Kuroki). This is significant because Kuroki et al. found that the primary interacting site for ILT2 is the B2M site, and that binding to the α3 domain is additional and flexible. This could explain the unique ability of 15G8 to enable T cell, NK cell, and macrophage / dendritic cell functions: it blocks the major interacting site of ILT2 but not the secondary site.

[0325] The only ILT2 antibody identified as having any effect on phagocytosis was GHI / 75, which was shown to enhance anti-CD47 blockade-mediated cancer cell phagocytosis, but not to have any effect on itself (see Barkal et al., "Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy", Nat. Immunol. Jan; 19(1):76-84). The combined effects of GHI / 75 and anti-CD47 were found to be B2M-dependent, as the absence of B2M had no effect on the increased phagocytosis. Therefore, the effect of 15G8 alone on phagocytosis ( Figures 13A-13C This can be B2M dependent, which could explain the unique capabilities of this antibody. The superiority of the antibody of the present invention in this respect was directly tested. A375 or SKMEL28 cancer cells expressing exogenous HLA-G were co-cultured with macrophages in the presence of an IgG control, the antibody of the present invention, or GHI / 75. The HP-F1 antibody was also tested in A375 cells. Cancer cell lines stained with PKH67-FITC were incubated with macrophages stained with eFluor 670-APC in the presence of the indicated antibody. The level of phagocytosis was determined by the percentage of macrophages double-stained, the double staining indicating phagocytosis of target cells. The percentage increase in phagocytosis compared to the IgG control was calculated. In both cell types, all three antibodies of the present invention increased phagocytosis compared to the control. Figures 19A-19BFurthermore, there are some variability between antibodies and between cell types. As expected, neither GHI / 75 nor HP-F1 had any effect on phagocytosis. Thus, it is confirmed that the antibody of the present invention is the first anti-ILT2 antibody that can be used as a monotherapy to enhance phagocytosis.

[0326] This raises the question of the epitopes of GHI / 75 and other commercial antibodies. While the epitopes of these antibodies are not publicly disclosed, a competitive ELISA assay was performed to observe whether 15G8, as well as GHI / 75 and HP-F1, could simultaneously bind to ILT2. Biotinylated 15G8 antibody was used at a constant concentration (1 μg / ml) in an ILT2 binding ELISA. GHI / 75 and HP-F1 were added at incremental concentrations, and competition was assessed. Regardless of the amount of either antibody added, they did not compete with 15G8 for ILT2 binding (Figure 19). In contrast, when naked (unbiotinylated) 15G8 was added, binding decreased in a dose-dependent manner, as expected. This indicates that GHI / 75 and HP-F1 bind to a different epitope compared to 15G8. This makes 15G8 the first anti-ILT2 antibody previously identified as binding to this epitope, specifically blocking interaction with B2M, and capable of simultaneously activating / recruiting T cells, NK cells, and macrophages / dendritic cells against cancer.

[0327] The foregoing description of specific embodiments will fully reveal the general nature of the invention, enabling others to readily modify and / or adapt such specific embodiments for various applications by applying current knowledge without excessive experimentation and without departing from the general concept. Therefore, such modifications and alterations should and are intended to be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not for limiting purposes. Various alternative forms may be taken for performing the various functions disclosed without departing from the invention. sequence list <110> Bion Biopharmaceuticals <120> Antibodies against ILT2 and their applications <130> BDB-P-002-PCT <150> 62 / 885,374 <151> 2019-08-12 <150> 63 / 034,569 <151> 2020-06-04 <160> 47 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> artificial <220> <223> synthesis <400> 1 Asp His Thr Ile His 1 5 <210> 2 <211> 17 <212> PRT <213> artificial <220> <223> synthesis <400> 2 Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 3 <211> 7 <212> PRT <213> artificial <220> <223> synthesis <400> 3 Thr Trp Asp Tyr Phe Asp Tyr 1 5 <210> 4 <211> 15 <212> PRT <213> artificial <220> <223> synthesis <400> 4 Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> artificial <220> <223> synthesis <400> 5 Arg Ala Ser Asn Leu Glu Ser 1 5 <210> 6 <211> 9 <212> PRT <213> artificial <220> <223> synthesis <400> 6 Gln Gln Ser Asn Glu Asp Pro Tyr Thr 1 5 <210> 7 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <400> 7 Gly Tyr Thr Phe Thr Ser Tyr Gly Ile Ser 1 5 10 <210> 8 <211> 17 <212> PRT <213> artificial <220> <223> synthesis <400> 8 Glu Ile Tyr Pro Gly Ser Gly Asn Ser Tyr Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 9 <211> 8 <212> PRT <213> artificial <220> <223> synthesis <400> 9 Ser Asn Asp Gly Tyr Pro Asp Tyr 1 5 <210> 10 <211> 11 <212> PRT <213> artificial <220> <223> synthesis <400> 10 Lys Ala Ser Asp His Ile Asn Asn Trp Leu Ala 1 5 10 <210> 11 <211> 7 <212> PRT <213> artificial <220> <223> synthesis <400> 11 Gly Ala Thr Ser Leu Glu Thr 1 5 <210> 12 <211> 9 <212> PRT <213> artificial <220> <223> synthesis <400> 12 Gln Gln Tyr Trp Ser Thr Pro Trp Thr 1 5 <210> 13 <211> 6 <212> PRT <213> artificial <220> <223> synthesis <400> 13 Ser Gly Tyr Tyr Trp Asn 1 5 <210> 14 <211> 16 <212> PRT <213> artificial <220> <223> synthesis <400> 14 Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn 1 5 10 15 <210> 15 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <220> <221> X <222> (10)..(10) <223> X is selected from A, C, and S. <400> 15 Gly Tyr Ser Tyr Tyr Tyr Ala Met Asp Xaa 1 5 10 <210> 16 <211> 11 <212> PRT <213> artificial <220> <223> synthesis <400> 16 Arg Thr Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 17 <211> 7 <212> PRT <213> artificial <220> <223> synthesis <400> 17 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 18 <211> 8 <212> PRT <213> artificial <220> <223> synthesis <400> 18 Gln Gln Gly Asn Thr Leu Pro Thr 1 5 <210> 19 <211> 116 <212> PRT <213> Artificial <220> <223> Synthesis <400> 19 Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Thr Trp Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 20 <211> 111 <212> PRT <213> Artificial <220> <223> Synthesis <400> 20 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 21 <211> 117 <212> PRT <213> Artificial <220> <223> Synthetic <400> 21 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Asn Ser Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Asn Asp Gly Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 22 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic <400> 22 Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly 1 5 10 15 Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 119 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> X <222> (108)..(108) <223> X is selected from A, C, and S. <400> 23 Asp Val Gln Leu Gln Gly Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala His Gly Tyr Ser Tyr Tyr Tyr Ala Met Asp Xaa Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Ser Val Thr Ser Val 115 <210> 24 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 24 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile 35 40 45 Ser Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 25 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <400> 25 Gly Tyr Ser Tyr Tyr Tyr Ala Met Asp Ala 1 5 10 <210> 26 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <400> 26 Gly Tyr Ser Tyr Tyr Tyr Ala Met Asp Ser 1 5 10 <210> 27 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <400> 27 Gly Tyr Ser Tyr Tyr Tyr Ala Met Asp Cys 1 5 10 <210> 28 <211> 119 <212> PRT <213> artificial <220> <223> synthesis <400> 28 Asp Val Gln Leu Gln Gly Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala His Gly Tyr Ser Tyr Tyr Tyr Ala Met Asp Ala Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 29 <211> 119 <212> PRT <213> Artificial <220> <223> Synthetic <400> 29 Asp Val Gln Leu Gln Gly Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala His Gly Tyr Ser Tyr Tyr Tyr Ala Met Asp Ser Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Ser Val Thr Ser Val 115 <210> 30 <211> 119 <212> PRT <213> artificial <220> <223> synthesis <400> 30 Asp Val Gln Leu Gln Gly Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala His Gly Tyr Ser Tyr Tyr Tyr Ala Met Asp Cys Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 31 <211> 650 <212> PRT <213> Homo sapiens <400> 31 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 Lys Thr Ala Leu Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp 100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125 Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln 130 135 140 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys Glu Gly 145 150 155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185 190 Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240 Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255 Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260 265 270 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser 290 295 300 Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315 320 Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325 330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340 345 350 Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg 355 360 365 Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380 Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390 395 400 Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu 405 410 415 Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly 420 425 430 Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly 450 455 460 Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510 Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525 Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly 530 535 540 Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val 545 550 555 560 Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser 565 570 575 Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln 580 585 590 Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala 595 600 605 Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg 610 615 620 Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val 625 630 635 640<000133​​​​​​​​​​​​​​​​​​caggttcagc tgcagcagtc tggagctgag ctggcgaggc ctggggcttc agtgaagctg 60 caggttcagc tgcagcagtc tggagctgag ctggcgaggc ctggggcttc agtgaagctg 60 tcctgcaagg cttctggcta caccttcaca agctatggta taagctgggt gaagcagaga 120 tcctgcaagg cttctggcta caccttcaca agctatggta taagctgggt gaagcagaga 120 actggacagg gccttgagtg ggttggagag atttatcctg gaagtggtaa ttcttactac 180 actggacagg gccttgagtg ggttggagag atttatcctg gaagtggtaa ttcttactac 180 aatgagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcgtac 240 aatgagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcgtac 240 atggagctcc gcagcctgac atctgaggac tctgcggtct atttctgtgc aagatcgaat 300 atggagctcc gcagcctgac atctgaggac tctgcggtct atttctgtgc aagatcgaat 300 gatggttacc ctgactactg gggccaaggc accactctca cagtctcctc a 351 gatggttacc ctgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 33<210> 33 <211> 357<211> 357 <212> DNA<212> DNA <213> 人工<213> Artificial <220> <220> <223> 合成 <223> Synthetic <400> 33 <400> 33 gatgtacagc ttcaggggtc aggacctggc ctcgtgaaac cttctcagtc tctgtctctc 60 gatgtacagc ttcaggggtc aggacctggc ctcgtgaaac cttctcagtc tctgtctctc 60 acctgctctg tcactggcta ctccatcacc agtggttatt actggaactg gatccggcag 120 acctgctctg tcactggcta ctccatcacc agtggttatt actggaactg gatccggcag 120 tttccaggaa acaaactgga atggatgggc tacataagct acgatggtag caataactac 180 tttccaggaa acaaactgga atggatgggc tacataagct acgatggtag caataactac 180 aacccatctc tcaaaaatcg aatctccatc actcgtgaca catctaagaa ccagtttttc 240 aacccatctc tcaaaaatcg aatctccatc actcgtgaca catctaagaa ccagtttttc 240 ctgaagttga attctgtgac ttctgaggac acagccacat attactgtgc ccatggttac 300 tcatattact atgctatgga ctgctggggt caaggaacct cagtcaccgt ctcctca 357 <210> 34 <211> 357 <212> DNA <213> Artificial <220> <223> Synthetic <400> 34 gatgtccagc tgcaaggctc tggccctgga ctggttaagc cttccgagac actgtccctg 60 acctgctctg tgaccggcta ctctatcacc tccggctact actggaactg gatcagacag 120 ttccccggca agaaactgga atggatgggc tacatctcct acgacggctc caacaactac 180 aaccccagcc tgaagaaccg gatcaccatc tctcgggaca cctccaagaa ccagttctcc 240 ctgaagctga actccgtgac cgctgccgat accgctacct actactgtgc tcacggctac 300 tcctactact acgccatgga tgcttggggc cagggcacat ctgtgacagt gtcctct 357 <210> 35 <211> 348 <212> DNA <213> Artificial <220> <223> Synthetic <400> 35 caggttcagc tgcaacagtc tgacgctgag ttggtgaaac ctggagcttc agtgaagata 60 caggttcagc tgcaacagtc tgacgctgag ttggtgaaac ctggagcttc agtgaagata 60 tcctgcaagg tttctggcta caccttcact gaccatacta ttcactggat gaagcagagg 120 tcctgcaagg tttctggcta caccttcact gaccatacta ttcactggat gaagcagagg 120 cctgaacagg gcctggaatg gattggatat atttatccta gagatggtag tactaagtac 180 cctgaacagg gcctggaatg gattggatat atttatccta gagatggtag tactaagtac 180 aatgagaagt tcaagggcaa ggccacattg actgcagaca aatcctccag cacagcctac 240 aatgagaagt tcaagggcaa ggccacattg actgcagaca aatcctccag cacagcctac 240 atgcagctca acagcctgac atctgaggac tctgcagtct atttctgtgc aagaacctgg 300 atgcagctca acagcctgac atctgaggac tctgcagtct atttctgtgc aagaacctgg 300 gactactttg actactgggg ccaaggcacc actctcacag tctcctca 348 gactactttg actactgggg ccaaggcacc actctcacag tctcctca 348 <210> 36<210> 36 <211> 333<211> 333 <212> DNA<212> DNA <213> 人工<213> Artificial <220> <220> <223> 合成 <223> Synthetic <400> 36 <400> 36 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atatcctgca gagccagtga aagtgttgat agttatggca atagttttat gcactggtac 120 atatcctgca gagccagtga aagtgttgat agttatggca atagttttat gcactggtac 120 cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180 cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180 gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240 gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240 cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaagtaatga ggatccgtac 300 acgttcggag gggggaccaa gctggaaata aaa 333 <210> 37 <211> 318 <212> DNA <213> artificial <220> <223> synthetic <400> 37 gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca ggacaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctcctac acatcaagat tgcactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtaatacgc ttcccacgtt cggctcgggg 300 acaaagttgg aaataaaa 318 <210> 38 <211> 318 <212> DNA <213> artificial <220> <223> synthetic <400> 38 gacatccaga tgacccagtc tccatcctct ctgtctgcct ctgtgggcga cagagtgacc 60 atcacctgtc ggacctctca ggacatctcc aactacctga actggtatca gcagaaaccc 120 ggcaaggccg tgaagctgct gatctcctac acctccagac tgcactctgg cgtgccctcc 180 agattttctg gctctggatc tggcaccgac tacaccctga ccatcagttc tctgcagcct 240 gaggacttcg ccacctacta ctgtcagcag ggcaacaccc tgcctacctt tggccagggc 300 accaagctgg aaatcaag 318 <210> 39 <211> 321 <212> DNA <213> Artificial <220> <223> Synthetic <400> 39 gacatccaga tgacacaatc ttcatcctac ttgtctgtat ctctaggagg cagagtcacc 60 attacttgca aggcaagtga ccacattaat aattggttag cctggtatca gcagaaacca 120 ggaaatgctc ctaggctctt aatatctggt gcaaccagtt tggaaactgg ggttccttca 180 agattcagtg gcagtggatc tggaaaggat tacactctca gcattaccag tcttcagact 240 gaagatgttg ctacttatta ctgtcaacag tattggagta ctccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 40 <211> 393 <212> PRT <213> Homo sapiens <400> 40 His Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile Thr 1 5 10 15 Gln Gly Ser Pro Val Thr Leu Arg Cys Gln Gly Gly Gln Glu Thr Gln 20 25 30 Glu Tyr Arg Leu Tyr Arg Glu Lys Lys Thr Ala Leu Trp Ile Thr Arg 35 40 45 Ile Pro Gln Glu Leu Val Lys Lys Gly Gln Phe Pro Ile Pro Ser Ile 50 55 60 Thr Trp Glu His Ala Gly Arg Tyr Arg Cys Tyr Tyr Gly Ser Asp Thr 65 70 75 80 Ala Gly Arg Ser Glu Ser Ser Asp Pro Leu Glu Leu Val Val Thr Gly 85 90 95 Ala Tyr Ile Lys Pro Thr Leu Ser Ala Gln Pro Ser Pro Val Val Asn 100 105 110 Ser Gly Gly Asn Val Ile Leu Gln Cys Asp Ser Gln Val Ala Phe Asp 115 120 125 Gly Phe Ser Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln Cys Leu 130 135 140 Asn Ser Gln Pro His Ala Arg Gly Ser Ser Arg Ala Ile Phe Ser Val 145 150 155 160 Gly Pro Val Ser Pro Ser Arg Arg Trp Trp Tyr Arg Cys Tyr Ala Tyr 165 170 175 Asp Ser Asn Ser Pro Tyr Glu Trp Ser Leu Pro Ser Asp Leu Leu Glu 180 185 190 Leu Leu Val Leu Gly Val Ser Lys Lys Pro Ser Leu Ser Val Gln Pro 195 200 205 Gly Pro Ile Val Ala Pro Glu Glu Thr Leu Thr Leu Gln Cys Gly Ser 210 215 220 Asp Ala Gly Tyr Asn Arg Phe Val Leu Tyr Lys Asp Gly Glu Arg Asp 225 230 235 240 Phe Leu Gln Leu Ala Gly Ala Gln Pro Gln Ala Gly Leu Ser Gln Ala 245 250 255 Asn Phe Thr Leu Gly Pro Val Ser Arg Ser Tyr Gly Gly Gln Tyr Arg 260 265 270 Cys Tyr Gly Ala His Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser Asp 275 280 285 Pro Leu Asp Ile Leu Ile Ala Gly Gln Phe Tyr Asp Arg Val Ser Leu 290 295 300 Ser Val Gln Pro Gly Pro Thr Val Ala Ser Gly Glu Asn Val Thr Leu 305 310 315 320 Leu Cys Gln Ser Gln Gly Trp Met Gln Thr Phe Leu Leu Thr Lys Glu 325 330 335 Gly Ala Ala Asp Asp Pro Trp Arg Leu Arg Ser Thr Tyr Gln Ser Gln 340 345 350 Lys Tyr Gln Ala Glu Phe Pro Met Gly Pro Val Thr Ser Ala His Ala 355 360 365 Gly Thr Tyr Arg Cys Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu 370 375 380 Thr His Pro Ser Asp Pro Leu Glu Leu 385 390 <210> 41 <211> 15 <212> PRT <213> Homo sapiens <400> 41 Val Lys Lys Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His 1 5 10 15 <210> 42 <211> 15 <212> PRT <213> Homo sapiens <400> 42 Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 1 5 10 15 <210> 43 <211> 15 <212> PRT <213> Homo sapiens <400> 43 Val Ile Leu Gln Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ser 1 5 10 15 <210> 44 <211> 15 <212> PRT <213> Homo sapiens <400> 44 Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 1 5 10 15 <210> 45 <211> 106 <212> PRT <213> Artificial <220> <223> Synthetic <400> 45 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Thr Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Ser Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 6L Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 46 <211> 98 <212> PRT <213> Homo sapiens <400> 46 Gly His Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile 1 5 10 15 Thr Gln Gly Ser Pro Val Thr Leu Arg Cys Gln Gly Gly Gln Glu Thr 20 25 30 Gln Glu Tyr Arg Leu Tyr Arg Glu Lys Lys Thr Ala Leu Trp Ile Thr 35 40 45 Arg Ile Pro Gln Glu Leu Val Lys Lys Gly Gln Phe Pro Ile Pro Ser 50 55 60 Ile Thr Trp Glu His Ala Gly Arg Tyr Arg Cys Tyr Tyr Gly Ser Asp 65 70 75 80 Thr Ala Gly Arg Ser Glu Ser Ser Asp Pro Leu Glu Leu Val Val Thr 85 90 95 Gly Ala <210> 47 <211> 101 <212> PRT <213> Homo sapiens <400> 47 Tyr Ile Lys Pro Thr Leu Ser Ala Gln Pro Ser Pro Val Val Asn Ser 1 5 10 15 Gly Gly Asn Val Ile Leu Gln Cys Asp Ser Gln Val Ala Phe Asp Gly 20 25 30 Phe Ser Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln Cys Leu Asn 35 40 45 Ser Gln Pro His Ala Arg Gly Ser Ser Arg Ala Ile Phe Ser Val Gly 50 55 60 Pro Val Ser Pro Ser Arg Arg Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp 65 70 75 80 Ser Asn Ser Pro Tyr Glu Trp Ser Leu Pro Ser Asp Leu Leu Glu Leu 85 90 95 Leu Val Leu Gly Val 100

Claims

1. A monoclonal anti-immunoglobulin-like transcript 2 (ILT2) antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises heavy chain CDR (HCDR) 1-3 amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, wherein X in SEQ ID NO: 15 is selected from the group consisting of A, C, and S, and light chain CDR (LCDR) 1-3 amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively.

2. The antibody or antigen-binding fragment of claim 1, comprising (a) a heavy chain variable domain (VH) amino acid sequence of SEQ ID NO: 23, wherein X in SEQ ID NO: 23 is selected from the group consisting of A, C, and S; (b) a light chain variable domain (VL) amino acid sequence of SEQ ID NO: 24 or 45; or (c) (a) and (b).

3. The antibody or antigen-binding fragment of claim 1, comprising VH and VL amino acid sequences of (a) SEQ ID NOs: 23 and 24, respectively, wherein X in SEQ ID NO: 23 is selected from the group consisting of A, C, and S; (b) SEQ ID NOs: 23 and 45, respectively, wherein X in SEQ ID NO: 23 is selected from the group consisting of A, C, and S; (c) SEQ ID NOs: 28 and 24, respectively; (d) SEQ ID NOs: 29 and 24, respectively; or (e) SEQ ID NOs: 30 and 45, respectively.

4. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is humanized and the X is A or S.

5. The antibody or antigen-binding fragment of claim 4, wherein the X is A and SEQ ID NO: 15 is identical to SEQ ID NO:

25.

6. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a HCDR1 amino acid sequence of SEQ ID NO: 13, a HCDR2 amino acid sequence of SEQ ID NO: 14, a HCDR3 amino acid sequence of SEQ ID NO: 25, a LCDR1 amino acid sequence of SEQ ID NO: 16, a LCDR2 amino acid sequence of SEQ ID NO: 17, and a LCDR3 amino acid sequence of SEQ ID NO:

18.

7. A monoclonal anti-immunoglobulin-like transcript 2 (ILT2) antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) amino acid sequence of SEQ ID NO: 28, and a light chain variable domain (VL) amino acid sequence of SEQ ID NO:

24.

8. The antibody or antigen-binding fragment of claim 7, wherein the antibody is of the human IgG4 subtype.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-8 and a pharmaceutically acceptable excipient.

10. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1-8.

11. An expression vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the expression vector of claim 11.

13. A method of manufacturing an antibody or antigen-binding fragment thereof, the method comprising: culturing the host cell of claim 12 under conditions that allow expression of the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the cell culture.

14. Use of the antibody or antigen-binding fragment of any one of claims 1-8 in the manufacture of a medicament for treating melanoma, colorectal cancer, renal cell carcinoma, head and neck cancer, or esophageal cancer in a human subject in need thereof.

15. The use of claim 14, wherein the cancer is refractory to anti-PD-Ll or anti-PD-1 based therapy.

16. The use of claim 14, wherein the treatment further comprises administering to the subject a conditioning agent, an anti-PD-Ll or anti-PD-1 based immunotherapy, or both.

17. The use of claim 16, wherein the conditioning agent is an EGFR inhibitor.

18. The use of claim 17, wherein the EGFR inhibitor is cetuximab.

19. The use of claim 16, wherein the anti-PD-Ll or anti-PD-1 based immunotherapy is an anti-PD-Ll blocking antibody.

20. The use of claim 14, wherein the treatment comprises confirming expression of ILT2 and / or soluble HLA-G in a sample from the subject is above a predetermined threshold.

21. The use of claim 20, wherein the treatment comprises confirming expression of ILT2 and / or soluble HLA-G in immune cells of the subject.

22. The use of claim 21, wherein the immune cells are peripheral blood immune cells, or intratumoral immune cells.

23. The use of claim 21, wherein the immune cells are selected from CD8 positive T cells, macrophages, NK cells, and T EMRA cells.

24. The use of claim 21, wherein the immune cells are peripheral blood CD8 positive T cells.

25. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-8 in the manufacture of a medicament for treating HLA-G or MHC-I positive colon cancer in a human subject in need thereof, wherein the treatment further comprises administering to the subject an anti-PD-Ll or anti-PD-1 based immunotherapy.

26. The use of claim 25, wherein the cancer is refractory to anti-PD-Ll or PD-1 based therapy.

27. The use of claim 25, wherein the anti-PD-Ll or anti-PD-1 based immunotherapy is an anti-PD-1 blocking antibody.

28. The use of claim 25, wherein the treatment further comprises administering to the subject a conditioning agent.

29. The use of claim 28, wherein the conditioning agent is an EGFR inhibitor.

30. The use of claim 29, wherein the EGFR inhibitor is cetuximab.

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