Antibodies and their use in relation to ILT2

Abstract

The present disclosure provides monoclonal anti-ILT2 antibodies or antigen-binding fragments thereof, as well as pharmaceutical compositions containing same and methods for producing same. Methods for treating cancer using the antibodies or compositions of the present disclosure are also provided. Methods for selecting patients are also provided.

Description

[Technical Field]

[0001] This invention belongs to the field of monoclonal antibodies and modulates the immune response against cancer.

[0002] Cross-reference of related applications This application claims priority to PCT patent application PCT / IL2020 / 050889 filed on 12 August 2020; U.S. provisional patent application 63 / 145,604 filed on 4 February 2021; and U.S. provisional patent application 63 / 149,371 filed on 15 February 2021. The disclosures of these priority applications are incorporated herein by reference in their entirety.

[0003] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated in its entirety by reference. An electronic copy of the sequence listing, created on August 10, 2021, is named 022548_WO091_SL.txt and is 105,080 bytes in size. [Background technology]

[0004] Immunoglobulin-like transcription 2 (ILT2), also known as leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), LIR1, and CD85j, is a cell surface protein expressed on immune cells and is known to inhibit the immune response. This protein contains four IgC domains in its extracellular domain and four 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 approximately 80% homology. Known ligands for ILT2 include MHC-I as well as non-classical MHC molecules, such as HLA-F, HLA-G, HLA-B27, and UL18 (human CMV). The strongest known interacting agent for ILT2 in humans is HLA-G1. [Overview of the project] Problems to be Solved by the Invention

[0005] HLA-G1 is widely expressed on the surface of various cancer cells including glioblastoma multiforme cells and melanoma cells, in addition to cells of breast cancer, cervical cancer, CRC (colorectal cancer), lung cancer, gastric cancer, pancreatic cancer, thyroid cancer and ovarian cancer. Its expression is associated with poor clinical outcomes. Furthermore, ILT2 expression on immune cells in the tumor microenvironment has been shown to be associated with poor 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 approach for the prevention and treatment of cancer. However, ILT2 presents an obstacle to effective immunotherapy. There is a great need for treatment modalities that can evade the ILT2-HLA-G1 axis, as well as the HLA-G1-dependent functions of ILT2. Means for Solving the Problems

[0006] The present disclosure provides monoclonal antibodies that bind to ILT2 and inhibit ILT2-mediated immunosuppression, as well as pharmaceutical compositions comprising the same. Also provided are methods of treating cancer comprising administering a composition described herein, methods of producing the antibodies, antigen-binding fragments, and compositions described herein, and methods of increasing the efficacy of PD-1 / PD-L1-based therapies.

[0007] According to one aspect, there is provided a monoclonal antibody or antigen-binding fragment that binds to a sequence of human immunoglobulin-like receptor subfamily B member 1 (ILT2) selected from SEQ ID NOs: 41 to 44 and 68 to 70.

[0008] According to some embodiments, the sequence is selected from SEQ ID NOs: 68 to 70.

[0009] According to some embodiments, the sequence is SEQ ID NO: 71 or 72.

[0010] According to some embodiments, the antibody or antigen-binding fragment is bound to SEQ ID NOs. 71 and 72.

[0011] According to some embodiments, the antibody or antigen-binding fragment of the present invention binds to ILT2 and inhibits the direct interaction between ILT2 and beta-2-microglobulin (B2M).

[0012] 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 of ILT2 with B2M.

[0013] According to some embodiments, HLA is HLA-G.

[0014] According to some embodiments, the antibody is an IgG4 antibody and comprises the heavy chain constant region of a human IgG4 antibody containing S228P and L235E mutations (Eu numbering).

[0015] According to one embodiment, a monoclonal anti-ILT2 IgG4 antibody comprising three heavy chain CDRs (CDR-H1~3) and three light chain CDRs (CDR-L1~3) is provided: a. Sequence numbers 13-18, respectively b. Sequence numbers 1-6, respectively, or c. Sequence numbers 7-12, respectively Includes, The antibody provided comprises the heavy chain constant region of a human IgG4 antibody, and the heavy chain constant region contains one or both of the S228P and L235E mutations (Eu numbering).

[0016] According to some embodiments, the antibodies disclosed in the present invention include a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 19, 21, and 23, or an amino acid sequence that is at least 95% identical thereto.

[0017] According to some embodiments, the antibodies of the disclosure of the present invention include a light chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 20, 22, 24, and 45, or an amino acid sequence that is at least 95% identical thereto.

[0018] According to some embodiments, X in SEQ ID NO: 15 is A, and the heavy chain comprises a variable domain sequence selected from SEQ ID NOs: 28 and 56-59, or an amino acid sequence that is at least 95% identical thereto.

[0019] According to some embodiments, the light chain comprises a variable domain sequence selected from SEQ ID NOs. 24 and 60-62, or an amino acid sequence that is at least 95% identical thereto.

[0020] According to some embodiments, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 55, or a sequence that is at least 95% identical thereto.

[0021] According to some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 48 or at least 95% identical thereto, and the light chain comprises an amino acid sequence of SEQ ID NO: 49 or at least 95% identical thereto.

[0022] According to some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 51 or at least 95% identical thereto, and the light chain comprises an amino acid sequence of SEQ ID NO: 49 or at least 95% identical thereto.

[0023] According to some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 52 or at least 95% identical thereto, and the light chain comprises an amino acid sequence of SEQ ID NO: 49 or at least 95% identical thereto.

[0024] According to some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 64 or at least 95% identical thereto, and the light chain comprises an amino acid sequence of SEQ ID NO: 49 or at least 95% identical thereto.

[0025] According to some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 65 or at least 95% identical thereto, and the light chain comprises an amino acid sequence of SEQ ID NO: 66 or at least 95% identical thereto.

[0026] According to some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 67 or at least 95% identical thereto, and the light chain comprises an amino acid sequence of SEQ ID NO: 49 or at least 95% identical thereto.

[0027] According to some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 53 or at least 95% identical thereto, and the light chain comprises an amino acid sequence of SEQ ID NO: 54 or at least 95% identical thereto.

[0028] According to one embodiment, a monoclonal antibody is provided that includes a heavy chain and a light chain, respectively, containing SEQ ID NOs. 48 and 49.

[0029] According to some embodiments, the antibodies or antigen-binding fragments of the disclosure of the present invention are intended for use in at least one of the following: binding to ILT2, inducing / enhancing the antitumor T cell response, increasing T cell proliferation, reducing cancer-induced suppressor myeloactivity, increasing the cytotoxicity of natural killer cells, increasing macrophage phagocytosis, increasing the production of M1 inflammatory macrophages, decreasing the production of M2 suppressor 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.

[0030] According to some embodiments, the antibodies or antigen-binding fragments of the present invention are intended for use in combination with opsonizing agents for treating cancers expressing HLA-G or MHC-I.

[0031] According to certain embodiments, the antibodies or antigen-binding fragments of the present invention are intended for use in combination with anti-PD-L1 / PD-1 based therapies (e.g., immunotherapy) for treating cancers expressing HLA-G or MHC-I. In certain embodiments, the anti-PD-L1 / PD-1 based therapy is pembrolizumab therapy.

[0032] According to one embodiment, a pharmaceutical composition comprising an antibody or antigen-binding fragment of the present invention is provided.

[0033] According to one embodiment, a method is provided for treating cancer expressing HLA-G or MHC-I in a subject requiring such treatment, comprising administering a pharmaceutical composition of the disclosure of the present invention or an antibody or antigen-binding fragment of the disclosure of the present invention to the subject.

[0034] According to one embodiment, a method is provided for increasing the efficacy of an anti-PD-L1 / PD-1 based therapy against cancer cells expressing HLA-G, MHC-I, or both in a subject requiring such treatment, comprising administering a pharmaceutical composition of the disclosure of the present invention or an antibody or antigen-binding fragment of the disclosure of the present invention to a subject receiving an anti-PD-L1 / PD-1 based therapy.

[0035] According to some embodiments, the method further includes administering an opsonizing agent to a target.

[0036] According to some embodiments, the opsonizing agent is an EGFR inhibitor, and optionally, the EGFR inhibitor is cetuximab.

[0037] According to some embodiments, the method further includes administering anti-PD-L1 / PD-1 based therapy (e.g., immunotherapy). In certain embodiments, the anti-PD-L1 / PD-1 based immunotherapy is pembrolizumab therapy.

[0038] According to one embodiment, a method is provided for identifying antibodies that compete with a reference antibody, whose heavy chain and light chain each contain sequence numbers 48 and 49, for binding to ILT2, comprising contacting an antibody library with a polypeptide sequence containing an ILT2 sequence selected from sequence numbers 41-44 and 68-70, and selecting an antibody from the library that binds to the ILT2 sequence, thereby obtaining an antibody that competes with the reference antibody for binding to ILT2.

[0039] According to one embodiment, a method is provided for producing a drug (e.g., a protein or molecule that binds to ILT2), comprising: obtaining a drug that binds to a sequence of human ILT2 selected from SEQ ID NOs: 41-44 and 68-70; or obtaining a host cell containing one or more nucleotide sequences encoding a drug that binds to a sequence of human ILT2 selected from SEQ ID NOs: 41-44 and 68-70; and culturing the host cell under conditions that enable drug expression, thereby producing the drug. According to some embodiments, the drug binds to a sequence selected from SEQ ID NOs: 68-70. According to some embodiments, the drug binds to one or both of SEQ ID NOs: 71 and 72. According to some embodiments, the drug binds to SEQ ID NOs: 71 and 72.

[0040] According to one embodiment, a drug produced by the method disclosed in the present invention is provided.

[0041] According to one embodiment, an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment of the present invention is provided. In some embodiments, the nucleic acid molecule is an expression vector.

[0042] According to one embodiment, a host cell containing an isolated nucleic acid molecule of the present invention is provided.

[0043] Further embodiments and the full scope of the applicability of the disclosure of the present invention will become apparent from the detailed description below. However, it should be understood that the detailed description and specific examples, while illustrating preferred embodiments of the disclosure of the present invention, are provided merely as illustrations, as various changes and modifications within the essence and scope of the disclosure of the present invention are expected to become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0044] [Figure 1] This figure shows histograms illustrating ILT2 expression in lymphocytes. Commercial antibody #1 was used at a final concentration of 5 μg / mL. Binding is depicted as a black histogram, while isotype control staining is shown as a light gray histogram. [Figure 2] This figure shows histograms depicting ILT2 expression in various immune cells. Commercial antibody #1 at a final concentration of 5 μg / mL was used. Binding is depicted as a black histogram, while isotype control staining is shown as a light gray histogram. [Figure 3A] This is a table from the TCGA database listing the indications for cancers in which ILT2 RNA is overexpressed. [Figure 3B] This is a dot plot showing the correlation between MDSC (myelin-derived suppressor cell) enrichment and ILT2 expression in tumors. A bar graph illustrating the correlation between M2 enrichment and ILT2 expression is also displayed. [Figure 3C] This is a scattering plot of the percentage of various immune cells expressing ILT2 in different tumors. [Figure 4A] This is a bar graph showing the percentage of HLA-G positive cases for various cancers as determined by immunohistochemistry (IHC). [Figure 4B] This is a scattering plot of HLA-G IHC scores for various cancers. [Figure 5] This is a scattering plot of soluble HLA-G levels in various cancers. [Figure 6-1]Figure 6A shows the heavy and light chain sequences of three anti-ILT2 antibodies. The CDR, as determined by the KABAT system, is underlined. Figure 6B is a line graph of the binding of humanized 15G8 antibody to ILT2 expressed on the surface of BW cells transfected with human ILT2. The graph shows the over-the-background (FAB) levels of the different tested antibodies compared to the secondary antibody alone. Figures 6C-6F are bar graphs measuring phagocytosis as mean fluorescence intensity (MFI) of phagocytic events in (6C)A375-HLA-G, (6D)COLO-320-WT, (6E)COLO-320-WT, and (6F)COLO-320-HLA-G cancer cells co-cultured with macrophages in the presence of 15G8 antibody. Figures 6G-6H are bar graphs showing the percentage of cytotoxicity by NK cell lines co-cultured with various cancer cell lines expressing (6G)A375-HLA-G and (6H)A253WT in the presence of 15G8 antibody. [Figure 6-2] Continuation of Figure 6-1. [Figure 6-3] Continuation of Figure 6-2. [Figure 6-4] Continuation of Figure 6-3. [Figure 6-5] Continuation of Figure 6-4. [Figure 6-6] Continuation of Figure 6-5. [Figure 6-7] Continuation of Figure 6-6. [Figure 6-8] Continuation of Figure 6-7. [Figure 6-9] Continuation of Figure 6-8. [Figure 6-10] Continuation of Figure 6-9. [Figure 7-1]Figure 7A is a table of antibody binding values ​​to ILT2 and ILT2 family members. Figure 7B is a histogram of antibody binding to ILT2 on the cell surface of BW cells transfected with human ILT2. Figure 7C is a line graph of binding to ILT2 expressed on the surface of BW cells transfected with human ILT2, for chimeric and humanized 19E3 (left panel) and chimeric and humanized 15G8 (right panel). Figure 7D shows immunostaining in gastric cancer samples with 19E3 antibody. Figure 7E is a scattering plot of percentages of various immune cells expressing ILT2 in PBMC samples from healthy controls and cancer patients using 15G8 humanized antibody. [Figure 7-2] Continuation of Figure 7-1. [Figure 7-3] Continuation of Figure 7-2. [Figure 7-4] Continuation of Figure 7-3. [Figure 8-1]Figure 8A is a bar graph showing the percentage of blocking for each ILT2 antibody and positive controls (PC, GHI / 75 antibody). Figure 8B is a histogram of ILT2-biotin binding to HLA-G expressing cells in the presence of antibodies that block ILT2. Binding of ILT2-biotin to cells was determined by flow cytometry analysis using streptavidin-PE. No antibody (gray line), 15G8 (light gray line), isotype control (black line). Figure 8C is a line graph of the blocking activity of the 15G8 humanized antibody as determined by ILT2-biotin binding to HLA-G expressing cells. Figure 8D is a line graph of the blocking activity of chimeric and humanized 19E3 (left panel) and chimeric and humanized 15G8 (right panel) as determined by ILT2-biotin binding to HLA-G expressing cells in the presence of antibodies. Figure 8E is a bar graph of mouse IL-2 secretion from cells expressing the ILT2 signaling reporter construct in the presence or absence of cells expressing HLA-G and antibodies that block ILT2. PC = positive control (GHI / 75 antibody). Figure 8F is a line graph of the blocking activity of the 15G8 humanized antibody as determined by the reporter assay. Figure 8G is a bar graph of mouse IL-2 secretion from cells expressing the ILT2 signaling reporter construct in the presence or absence of antibodies that block ILT2 and positive control antibodies. Figures 8H-8K are bar graphs showing human IL-2 secretion from Jurkat cells expressing (8H)ILT2-deficient or (8I-8K)ILT2, co-cultured with A375 cancer cells expressing only (8I)MHC-I or (8J-8K)MHC-I and exogenous HLA-G, in the presence or absence of both an ILT2-blocking antibody and a positive control (8I-8J) pan-HLA antibody or (8K) HLA-G-specific antibody. Figures 8L-8N are bar graphs showing human IL-2 secretion from ILT2-expressing Jurkat cells, cultured with A375 HLA-G-expressing cancer cells, in the presence or absence of (8L)15G8 antibody, (8M)GHI / 75 antibody, and (8N)HP-F1 antibody.Figures 8O-8P are dot plots of the expression of the activation markers (8O) phosphorylated ZAP70 and (8P) phosphorylated Syk in TIL cells and NK cells incubated with HLA-G positive cancer cells in the presence and absence of 15G8 antibody, respectively. [Figure 8-2] Continuation of Figure 8-1. [Figure 8-3] Continuation of Figure 8-2. [Figure 8-4] Continuation of Figure 8-3. [Figure 8-5] Continuation of Figure 8-4. [Figure 8-6] Continuation of Figure 8-5. [Figure 8-7] Continuation of Figure 8-6. [Figure 8-8] Continuation of Figure 8-7. [Figure 9A] This bar graph measures phagocytosis as a percentage of HLA-G-expressing cancer cells co-cultured with macrophages in the presence of ILT2 antibody compared to a control, as determined by a FACS-based method. [Figure 9B] This is a line graph of real-time phagocytosis of cancer cells by macrophages in the presence of ILT2 antibodies, as determined by the Incucyte® system. [Figure 9C] This bar graph measures phagocytosis as a percentage of control cells expressing various HLA-G and MHC-I, co-cultured with macrophages in the presence of the ILT2 antibody 15G8. [Figure 9D] This bar graph shows phagocytosis by macrophages co-cultured with A253-HLA-G cells in the presence of ILT2 antibody, cetuximab (Erbitux®), hIgG control, or a combination thereof. [Figure 10] Figures 10A-10B are bar graphs showing IFNγ secretion 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. [Figure 11-1]Figure 11A is a bar graph showing the percentage of cytotoxicity by cells from NK cell lines co-cultured with various cancer cell lines expressing (11A)HLA-G and (11B)MHC-I in the presence of ILT2 antibody. Figure 11B is a bar graph showing the percentage of cytotoxicity by cells from NK cell lines co-cultured with various cancer cell lines expressing (11A)HLA-G and (11B)MHC-I in the presence of ILT2 antibody. Figures 11C-11D are bar graphs showing the secretion of (11C)granzyme B and (11D)IFNγ from cells of NK cell lines co-cultured with head and neck (H&N) cancer and melanoma cells, respectively, in the presence of 15G8 ILT2 antibody. Figures 11E-11F are bar graphs showing (11E)IFNγ expression and (11F)CD107A expression in ILT2-positive primary NK cells incubated with target cancer cells in the presence of ILT2 antibody. Figure 11G-11H shows scattered plots of individual expressions illustrating the correlation between ILT2-positive cells and (11G)IFNγ expression and (11H)CD107A expression in response to ILT2 antibody. [Figure 11-2] Continuation of Figure 11-1. [Figure 11-3] Continuation of Figure 11-2. [Figure 11-4] Continuation of Figure 11-3. [Figure 11-5] Continuation of Figure 11-4. [Figure 11-6] Continuation of Figure 11-5. [Figure 12] These are line graphs of HLA-DR and CD80 expression (MFI) as determined by flow cytometry in 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 showing increased expression of the identified markers compared to control IgG is shown for each condition tested. [Figure 13] Figure 13A is a bar graph of phagocytosis by macrophages co-cultured with various primary tumor cells. Figures 13B-13C are bar graphs of dose-dependent phagocytosis by autologous macrophages in the presence of (13B) RCC patients and (13C) H&N patients isolated from H&N patients. [Figure 14-1] Figure 14A is a dot plot of ILT2 and PD-1 expression in tumor cells (left panel) and PBMCs (right panel) from RCC and esophageal cancer patients. Figures 14B-14C are box plots of (14B) PD-1 and (14C) ILT2 RNA expression in CD8 T cell populations in TME from CRC patients. Figures 14D-14E are dot plots of (14D) ILT2 expression in CD8 T cells from peripheral blood of healthy donors and (14E) ILT2 and PD-1 expression in TILs from esophageal cancer. Figure 14F is a scattering plot of increased cell membrane CD107a on PBMCs from 10 healthy donors activated with Staphylococcus enterotoxin B (SEB) in the presence of 15G8, anti-PD-1 antibody or a combination of the two. Figure 14G is a bar graph of increased CD107a expression in exemplary PBMCs from three donors. Figures 14H-14J are bar graphs showing the levels of secretion 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. Figures 14K-14L are bar graphs showing the levels of IFNγ secretion from T cells co-cultured with (14K) dendritic cells or (14L) macrophages in a mixed lymphocyte reaction. [Figure 14-2] Continuation of Figure 14-1. [Figure 14-3] Continuation of Figure 14-2. [Figure 14-4] Continuation of Figure 14-3. [Figure 14-5] Continuation of Figure 14-4. [Figure 14-6] Continuation of Figure 14-5. [Figure 14-7] Continuation of Figure 14-6. [Figure 15-1]Figure 15A is a line graph of tumor volume of HLA-G and MHC-I expressing tumors grown in immunodeficient mice supplemented with human macrophages and anti-ILT2 antibodies. Figure 15B is an illustration of a mouse treatment schedule to prevent lung tumors. Figure 15C is a photograph of the lungs from immunodeficient mice implanted with HLA-G positive cancer cells in the presence or absence of human PBMCs and ILT2 antibodies. Figure 15D is a scattered plot summarizing the data from 15C. Figure 15E is an illustration of an established mouse treatment schedule for treating lung tumors. Figure 15F is a box plot of tumor weights. [Figure 15-2] Continuation of Figure 15-1. [Figure 15-3] Continuation of Figure 15-2. [Figure 15-4] Continuation of Figure 15-3. [Figure 16-1] Figures 16A–16F are box plots of (16A) CD107A expression in total CD8 T cells, (16B) CD107A expression in TEMRA cells, (16C) CD69 expression in NK cells, (16D) CD69 expression in total CD8 T cells, (16E) CD107 expression in TEMRA cells, and (16F) CD69 expression in combined-treated NK cells in mice that received PBMCs from a donor along with low or high levels of ILT2 in those TEMRA or NK cells, respectively. * indicates P<0.005. ** indicates P<0.0005. *** indicates P<0.0001. [Figure 16-2] Continuation of Figure 16-1. [Figure 17-1]Figure 17A illustrates the treatment schedule for humanized NSG mice implanted with head and neck cancer and treated with anti-ILT2 or control antibodies. Figure 17B is a line graph of tumor weight from mice treated with IgG and anti-ILT2. Figures 17C-17F are bar graphs of baseline ILT2 levels of peripheral CD8 T cells in (R) or unresponsive (NR) mice that responded to BND-22 treatment (17C). Post-treatment (17D) CD107A expression, (17E) M1 / M2 ratio and (17F) total CD80-positive dendritic cell count in tumors in four mice treated with anti-ILT2 antibodies. [Figure 17-2] Continuation of Figure 17-1. [Figure 18-1] Figure 18A is a partial sequence of ILT2 showing residues with significant predicted binding. These residues are divided into four categories according to their raw probability belonging to their epitopes. Asterisks indicate the location of selected mutations. Figures 18B-18C are 3D renderings of the ILT2 surface structure, showing (18B) the locations of residues from 18A and (18C) the four major interaction regions on ILT2. Figures 18D-18F are 3D ribbon or surface diagrams of ILT2, showing (18D) the epitope of the 15G8 antibody from WO2020 / 136145 as well as the epitopes of the 3H5, 12D12, and 27H5 antibodies (upper left circle), in addition to the secondary epitope of the 3H5 antibody (upper right circle) (18E-18F), and the interaction of the 15G8 epitope on ILT2 with B2M in the form of a complex with (18E) HLA-A or (18F) HLA-G. [Figure 18-2] Continuation of Figure 18-1. [Figure 18-3] Continuation of Figure 18-2. [Figure 18-4] Continuation of Figure 18-3. [Figure 19-1]Figures 19A-19G show the distribution of the protection ratio of free ILT2 to the ILT2-15G8 complex. (19A) Bar graph of data obtained from trypsin digestion of ILT2 protein. The median of the distribution is 1.5 and the mean is 1.37. Figure 19B is a bar graph of data obtained from trypsin and Asp-N digestion of ILT2 protein. The median of the distribution is 1.58 and the mean is 1.48. Selected protected regions are marked. Figure 19C is a dose-response plot comparison of free ILT2 peptides to the complex. Lines with squares and circles represent the control and the complex, respectively. (19C) Peptides 56-71: K=9.09s-1 for free ILT2, K=2.31s-1 for the complex, showing a 1 / 3.94 decrease as a result of complex formation. Figure 19D shows that for peptides 57-71, the K value of free ILT2 is 6.93 s-1, and the K value of the complex is 1.97 s-1, indicating a 1 / 3.52 reduction as a result of complex formation. Figure 19E shows that for peptides 84-100, the K value of free ILT2 is 1.26 s-1, and the K value of the complex is 0.37 s-1, indicating a 1 / 3.41 reduction as a result of complex formation. Figure 19F shows that for peptides 57-66, the K value of free ILT2 is 4.32 s-1, and the K value of the complex is 0.72 s-1, indicating a 1 / 6 reduction as a result of complex formation. Figure 19G shows that for peptides 91-100, the K value of free ILT2 is 0.7 s-1, and the K value of the complex is 0.14 s-1, indicating a 1 / 5 reduction as a result of complex formation. [Figure 19-2] Continuation of Figure 19-1. [Figure 19-3] Continuation of Figure 19-2. [Figure 19-4] Continuation of Figure 19-3. [Figure 19-5] Continuation of Figure 19-4. [Figure 19-6] Continuation of Figure 19-5. [Figure 20] This is a graph of ILT2-Fc binding to human B2M in the presence of 15G8 or unrelated antibodies in an ELISA assay. [Figure 21-1]Figures 21A-21B are bar graphs showing the percentage increase in phagocytosis of (21A)A375-HLA-G and (21B)SKMEL28-HLA-G cancer cells co-cultured with macrophages in the presence of various anti-ILT2 antibodies, compared to IgG controls. Figures 21C-21D are line graphs of competitive ILT2-binding ELISA using biotinylated 15G8 antibody in the presence of competing non-biotinylated (21C)GHI / 75, HP-F1, and (21D)MAB20172 and 15G8 antibodies. [Figure 21-2] Continuation of Figure 21-1. [Modes for carrying out the invention]

[0045] The disclosures of the present invention relate to monoclonal antibodies or antigen-binding fragments and pharmaceutical compositions that bind to ILT2 and inhibit ILT2-mediated immunosuppression. Methods for treating cancer and enhancing PD-1 / PD-L1 immunotherapy are also provided.

[0046] The disclosure of this invention is at least in part based on the remarkable discovery that ILT2 antagonism acts synergistically with PD-1 and PD-L1-based immunotherapies to combat cancer cells. Specifically, it was found that antibodies blocking ILT2, when combined with anti-PD-1 antibodies, increased the secretion of pro-inflammatory cytokines by immune cells. This increase was not merely additive, but greater than the sum of the individual effects of each agent. In fact, a de novo increase was observed for at least one cytokine in which neither agent had any effect on its own. This combined treatment makes it possible to convert cancer from PD-1 / PD-L1 refractory to PD-1 / PD-L1 responsive.

[0047] Even more surprisingly, the level of ILT2 expression in patients' immune cells was found to correlate with the effectiveness of ILT2 blocking therapy. Responders had high ILT2 levels, while non-responders had low ILT2 levels. Specifically, ILT2 levels in circulating CD8-positive T cells were a predictor of treatment outcomes.

[0048] In conclusion, the antibody disclosed in this invention was found to bind to a specific epitope within the ILT2 interdomain between the D1 and D2 domains. This region is known to be the interaction domain between ILT2 and beta-2-microglobulin (B2M). The antibody disclosed in this invention is the first antibody found to directly block this interaction. Furthermore, the antibody disclosed in this invention was found to possess immunostimulatory activity not reported with other anti-ILT2 antibodies. The antibody of this invention was able to modulate immune surveillance of T cells, NK cells, dendritic cells, and macrophages against cancer cells expressing MHC-I (e.g., HLA-G). In particular, the anti-ILT2 antibody, used for the first time as a monotherapy agent, was shown to enhance phagocytosis of cancer cells.

[0049] antibody In a first embodiment, the disclosure of the present invention provides an antibody comprising three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence (DHTIH) described in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence (YIYPRDGSTKYNEKFKG) described in SEQ ID NO: 2, CDR-H3 comprises the amino acid sequence (TWDYFDY) described in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence (RASESVDSYGNSFMH) described in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence (RASNLES) described in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence (QQSNEDPYT) described in SEQ ID NO: 6. In some embodiments, the antibody further comprises the heavy-chain constant region of an IgG4 antibody (e.g., human IgG4).

[0050] In another embodiment, the disclosure of the present invention provides an 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 (GYTFTSYGIS) described in SEQ ID NO: 7, CDR-H2 comprises the amino acid sequence (EIYPGSGNSYYNEKFKG) described in SEQ ID NO: 8, CDR-H3 comprises the amino acid sequence (SNDGYPDY) described in SEQ ID NO: 9, CDR-L1 comprises the amino acid sequence (KASDHINNWLA) described in SEQ ID NO: 10, CDR-L2 comprises the amino acid sequence (GATSLET) described in SEQ ID NO: 11, and CDR-L3 comprises the amino acid sequence (QQYWSTPWT) described in SEQ ID NO: 12. In some embodiments, the antibody further comprises the heavy-chain constant region of an IgG4 antibody (e.g., human IgG4).

[0051] In another embodiment, the disclosure of the present invention provides an 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 (SGYYWN) described in SEQ ID NO: 13, CDR-H2 comprises the amino acid sequence (YISYDGSNNYNPSLKN) described in SEQ ID NO: 14, CDR-H3 comprises the amino acid sequence (GYSYYYAMDX) described in SEQ ID NO: 15, where X is selected from A, C, and S, CDR-L1 comprises the amino acid sequence (RTSQDISNYLN) described in SEQ ID NO: 16, CDR-L2 comprises the amino acid sequence (YTSRLHS) described in SEQ ID NO: 17, and CDR-L3 comprises the amino acid sequence (QQGNTLPT) described in SEQ ID NO: 18. In some embodiments, the antibody further comprises the heavy-chain constant region of an IgG4 antibody (e.g., human IgG4).

[0052] In some embodiments, SEQ ID NO: 15 is GYSYYYAMDA (SEQ ID NO: 25). In some embodiments, SEQ ID NO: 15 is SEQ ID NO: 25, and the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, SEQ ID NO: 15 is GYSYYYAMDS (SEQ ID NO: 26). In some embodiments, SEQ ID NO: 15 is SEQ ID NO: 26, and the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, SEQ ID NO: 15 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.

[0053] In another embodiment, an antibody or antigen-binding fragment is provided that binds to the interdomain of human leukocyte immunoglobulin-like receptor subfamily B member 1 (ILT2) between domains D1 and D2.

[0054] In another embodiment, an antibody or antigen-binding fragment that binds to the sequence of ILT2 is provided, selected from VKKGQFPIPSITWEH (SEQ ID NO: 41), LELVVTGAYIKPTLS (SEQ ID NO: 42), VILQCDSQVAFDGFS (SEQ ID NO: 43), WYRCYAYDSNSPYEW (SEQ ID NO: 44), KGQFPIPSITWEHAGR (SEQ ID NO: 68), GQFPIPSITWEHAGR (SEQ ID NO: 69), and SESSDPLELVVTGAYIK (SEQ ID NO: 70). In some embodiments, the antibody or antigen-binding fragment can bind to one, two, three, four, five, six, or all seven of the sequence.

[0055] In another embodiment, an antibody or antigen-binding fragment is provided that binds to ILT2 and inhibits the interaction between ILT2 and B2M.

[0056] 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 mouse antibody. In some embodiments, the antibody is a humanized antibody. As used herein, a “humanized” antibody refers to an antibody that has a human backbone but is derived from a non-human antibody or has a CDR (Chronic Derived Relative) taken from a non-human antibody. In some embodiments, the CDR may be in a modified state during humanization, but generally it is still derived from the CDR of a non-human parent antibody. In some embodiments, the antigen-binding fragment is a single-chain antibody. In some embodiments, the antigen-binding fragment is a single-domain antibody.

[0057] In some embodiments, the antibody contains an IgG4 constant region. In some embodiments, the antibody is a humanized antibody containing a human IgG4 constant region. In some embodiments, the IgG4 constant region is an engineered IgG4 constant region containing mutations compared to a wild-type IgG4 constant region. In some embodiments, the human IgG4 constant region contains the following amino acid sequence: ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPSCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 50).

[0058] In some embodiments, the human IgG4 constant region comprises SEQ ID NO: 50. In some embodiments, the human IgG4 constant region contains at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 50. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the human IgG4 constant region contains at least 95% identity to SEQ ID NO: 50. In some embodiments, the human IgG4 constant region contains at least 97% identity to SEQ ID NO: 50. In some embodiments, the human IgG4 constant region contains at least 99% identity to SEQ ID NO: 50.

[0059] In some embodiments, the IgG4 constant region contains at least one mutation. In some embodiments, the mutation reduces binding to Fc receptors, such as Fc gamma receptor (FcγR), or reduces Fab-arm exchange of the IgG4 antibody. In some embodiments, the mutation is a mutation of serine 108. In some embodiments, serine is mutated to proline. In some embodiments, serine 108 is serine 108 of SEQ ID NO: 50 (or S228 according to Eu numbering). The mutation of serine 108 to proline is also known as the S228P mutation. In some embodiments, the mutation is a mutation of leucine 115 (or L235 according to Eu numbering). In some embodiments, leucine is mutated to glutamate. In some embodiments, leucine 115 is leucine 115 of SEQ ID NO: 50. The mutation of leucine 115 to glutamate is also known as the L235E mutation. Those skilled in the art will understand that the exact numerical positions of any amino acid in the constant region of the heavy chain are determined by the length of the variable region of the heavy chain. Thus, the position of serine is indicated as 108 in the constant region, and the position of leucine is indicated as 115 (see, for example, SEQ ID NO: 50). Modifications to the constant region (e.g., addition or deletion of bases) can also change the numerical positions, but it will be further understood that any analog or derivative of IgG4 is assumed to contain the listed mutations. In some embodiments, the IgG4 constant region contains multiple mutations. In some embodiments, the IgG4 constant region contains mutations at serine 108 and leucine 115. In some embodiments, the IgG4 constant region contains S108P and L115E mutations (or S228P and L235E mutations according to Eu's numbering).

[0060] In some embodiments, the IgG4 constant region includes the following amino acid sequence, in which case mutations to the wild-type human IgG4 constant region (S228P and L235E;Eu numbering) are shown in the box: [ka]

[0061] In some embodiments, the human IgG4 constant region includes or consists of SEQ ID NO: 55. In some embodiments, the antibody of the disclosure of the present invention includes SEQ ID NO: 55. In some embodiments, the heavy chain constant region of the antibody of the disclosure of the present invention includes SEQ ID NO: 55. In some embodiments, the heavy chain constant region of the antibody of the disclosure of the present invention consists of SEQ ID NO: 55. In some embodiments, the IgG4 constant region is an analog or derivative of SEQ ID NO: 55, which contains further mutations, for example, to improve ease of manufacture or to reduce the immunogenicity of the antibody. In some embodiments, the IgG4 constant region contains at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity of SEQ ID NO: 55. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the IgG4 constant region contains at least 95% identity of SEQ ID NO: 55. In some embodiments, the IgG4 constant region contains at least 97% identity of SEQ ID NO: 55. In some embodiments, the IgG4 constant region contains at least 99% identity with SEQ ID NO: 55.

[0062] In some embodiments, the IgG4 constant region analog further includes at least one other mutation. In some embodiments, at least one other mutation is in SEQ ID NO: 55. In some embodiments, at least one other mutation is not at position 108. In some embodiments, at least one other mutation is not at position 115. In some embodiments, at least one other mutation is not at position 108 or 115. Other mutations in IgG4 are well known in the art, and any of these may be adopted. Other examples of mutations, though not limited to these, include E233P, F234A, L235A, G237A, P239G, F243L, T250Q, T250E, M252Y, S254T, T256E, E258F, D259I, V264A, D265A, F296Y, T307A, T307Q, V308W, V308Y, V308F, Q311V, and K317Q. Examples include A330R, E356K, K370Q, K370E, E380A, R409K, V427T, M428L, M428F, H434K, N434S, N434A, N434H, N434F, H435R, Y436H, K439E, L445P, deletion of G236, deletion of G446, and deletion of K447 (the numbers are shown according to Eu numbering). Here again, the positions represented by the numbers assigned to these mutations are in the general notation (Eu numbering), but depend on the length of the variable region of the heavy chain. Within sequence number 50, these positions correspond to E113, F114, L115, G117, P119, F123, T130, M132, S134, T136, E138, D139, V144, D145, F176, T187, V188, Q191, K197, A210, E236, K250, E260, R289, V307, M308, H313, N314, H315, Y316, K319, L325, G326, and K327. It is also understood that specific combinations of mutations, such as E233P / F234A / L235A / G236del / G237A or S228P / F234A / L235A / G237A / P238S, may be adopted.

[0063] In some embodiments, the antibody or antigen-binding fragment binds to ILT2. In some embodiments, ILT2 is human ILT2. In some embodiments, ILT2 is mammalian ILT2. In some embodiments, ILT2 is primate ILT2 (e.g., cynomolgus monkey 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, HLA is HLA-G. In some embodiments, the ligand is not MHC. In some embodiments, the ligand is MHC. In some embodiments, 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 boundary between the D1 and D2 domains. In some embodiments, the interdomain is the hinge domain between the D1 and D2 domains. In some embodiments, the interdomain does not include the N-terminal domain of D1. In some embodiments, the interdomain is derived from amino acids 54-184 of SEQ ID NO: 31. In some embodiments, amino acids 54-184 of SEQ ID NO: 31 constitute the interdomain. In some embodiments, the interdomain is derived from amino acids 90-184 of SEQ ID NO: 31. In some embodiments, amino acids 90-184 constitute the interdomain. In some embodiments, an antibody or antigen-binding fragment binds to an epitope within the interdomain. 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 interdomain. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the epitope is located 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 is at least partially located in D2 and binds to the epitope. 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 which interacts with the α3 domain of HLA-G.

[0064] 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 the NCBI reference sequence: NP_006660.4. In some embodiments, ILT2 has the following amino acid sequence: MTPILTVLIC LGLSLGPRTH VQAGHLPKPT LWAEPGSVIT QGSPVTLRCQ GGQETQEYRL YREKKTALWI TRIPQELVKK GQFPIPSITW EHAGRYRCYY GSDTAGRSES SDPLELVVTG AYIKPTLSAQ PSPVVNSGGN VILQCDSQVA FDGFSLCKEG EDEHPQCLNS QPHARGSSRA IFSVGPVSPS RRWWYRCYAY DSNSPYEWSL PSDLLELLVL GVSKKPSLSV QPGPIVAPEE TLTLQCGSDA GYNRFVLYKD GERDFLQLAG AQPQAGLSQA NFTLGPVSRS YGGQYRCYGA HNLSSEWSAP SDPLDILIAG QFYDRVSLSV QPGPTVASGE NVTLLCQSQG WMQTFLLTKE GAADDPWRLR STYQSQKYQA EFPMGPVTSA HAGTYRCYGS QSSKPYLLTH PSDPLELVVS GPSGGPSSPT TGPTSTSGPE DQPLTPTGSD PQSGLGRHLG VVIGILVAVI LLLLLLLLLF LILRHRRQGK HWTSTQRKAD FQHPAGAVGP EPTDRGLQWR SSPAADAQEE NLYAAVKHTQ PEDGVEMDTR SPHDEDPQAV TYAEVKHSRP RREMASPPSP LSGEFLDTKD RQAEEDRQMD TEAAASEAPQ DVTYAQLHSL TLRREATEPP PSQEGPSPAV PSIYATLAIH (Sequence ID 31)

[0065] In some embodiments, ILT2 has an amino acid sequence provided in the NCBI reference sequence: NP_001075106.2. In some embodiments, ILT2 has an amino acid sequence provided in the NCBI reference sequence: NP_001075107.2. In some embodiments, ILT2 has an amino acid sequence provided in the NCBI reference sequence: NP_001075108.2. In some embodiments, ILT2 has an amino acid sequence provided in the NCBI reference sequence: NP_001265328.2.

[0066] In some embodiments, the D1 domain of ILT2 contains or consists of the following amino acid sequence: GHLPKPTLWA EPGSVITQGS PVTLRCQGGQ ETQEYRLYRE KKTALWITRI PQELVKKGQF PIPSITWEHA GRYRCYYGSD TAGRSESSDP LELVVTGA (Sequence No. 46) In some embodiments, the D1 domain of ILT2 contains or consists of amino acids 24-121 of SEQ ID NO: 31. In some embodiments, the D2 domain of ILT2 contains or consists of the following amino acid sequence: YIKPTLSAQP SPVVNSGGNV ILQCDSQVAF DGFSLCKEGE DEHPQCLNSQ PHARGSSRAI FSVGPVSPSR RWWYRCYAYD SNSPYEWSLP SDLLELLVLG V (Sequence ID 47) In some embodiments, the D2 domain of ILT2 contains or consists of amino acids 122-222 of SEQ ID NO: 31. In some embodiments, the interdomain of ILT2 contains 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 contains 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.

[0067] 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 embodiments, the antibody or antigen-binding fragment binds to the ILT2 sequence provided in SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 sequence provided in SEQ ID NO: 42. In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 sequence provided in SEQ ID NO: 43. In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 sequence provided in SEQ ID NO: 44. In some embodiments, the antibody or antigen-binding fragment binds to a three-dimensional epitope containing residues from at least two of SEQ ID NOs: 41, 42, 43, and 44 (e.g., a three-dimensional epitope containing at least two of SEQ ID NOs: 41, 42, 43, and 44). In some embodiments, the three-dimensional epitope contains residues from at least three of SEQ ID NOs: 41, 42, 43, and 44 (e.g., a three-dimensional epitope containing at least three of SEQ ID NOs: 41, 42, 43, and 44). In some embodiments, the three-dimensional epitope contains residues from SEQ ID NOs: 41, 42, 43, and 44 (e.g., a three-dimensional epitope containing SEQ ID NOs: 41, 42, 43, and 44).

[0068] In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 41 (for example, to an ILT2 epitope containing or preceding SEQ ID NO: 41). In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 42 (for example, to an ILT2 epitope containing or preceding SEQ ID NO: 42). In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 43 (for example, to an ILT2 epitope containing or preceding SEQ ID NO: 43). In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 44 (for example, to an ILT2 epitope containing or preceding SEQ ID NO: 44). In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 68 (for example, to an ILT2 epitope containing or preceding SEQ ID NO: 68). In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 69 (for example, to an ILT2 epitope containing or preceding SEQ ID NO: 69). In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 70 (for example, to an ILT2 epitope containing or preceding SEQ ID NO: 70). In some embodiments, the antibody or antigen-binding fragment binds to a three-dimensional epitope containing residues from at least two of SEQ ID NOs: 41-44 and 68-70 (for example, to a three-dimensional epitope containing at least two of SEQ ID NOs: 41-44 and 68-70). In some embodiments, the three-dimensional epitope contains residues from at least three of SEQ ID NOs: 41-44 and 68-70 (for example, the three-dimensional epitope contains at least three of SEQ ID NOs: 41-44 and 68-70). In some embodiments, the three-dimensional epitope contains residues from at least four of SEQ ID NOs: 41-44 and 68-70 (for example, the three-dimensional epitope contains at least four of SEQ ID NOs: 41-44 and 68-70). In some embodiments, the three-dimensional epitope includes residues from SEQ ID NOs: 41, 42, and 68-70 (for example, the three-dimensional epitope includes SEQ ID NOs: 41, 42, and 68-70).In some embodiments, the antibody or antigen-binding fragment binds to a sequence selected from SEQ ID NOs. 68-70 (for example, to an epitope containing any one of SEQ ID NOs. 68-70 or within that range).

[0069] In some embodiments, the antibody or antigen-binding fragment binds to the sequence GQFPIPSITW (SEQ ID NO: 71) (for example, to an ILT2 epitope containing or within the sequence). In some embodiments, the antibody or antigen-binding fragment binds to the sequence ELVVTGAYIK (SEQ ID NO: 72) (for example, to an ILT2 epitope containing or within the sequence). In certain embodiments, the antibody or antigen-binding fragment binds to SEQ ID NOs. 71 and 72 (for example, to an epitope containing or within SEQ ID NOs. 71 and 72). In some embodiments, the antibody or antigen-binding fragment binds to a sequence selected from SEQ ID NOs. 68-72 (for example, to an epitope containing or within the sequence). In some embodiments, the antibody or antigen-binding fragment binds to a sequence selected from SEQ ID NOs. 41-44 and 68-72 (for example, to an epitope containing or within the sequence). In some embodiments, SEQ ID NO: 71 is an epitope within SEQ ID NO: 41. In some embodiments, sequence number 71 is the epitope in sequence number 68. In some embodiments, sequence number 71 is the epitope in sequence number 69. In some embodiments, sequence number 72 is the epitope in sequence number 42. In some embodiments, sequence number 72 is the epitope in sequence number 70.

[0070] In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope containing 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 containing ILT2 residues selected from G97, A98, Y99, I100, Q125, and V126. In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope containing multiple residues of ILT2 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 containing multiple residues of ILT2 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 another embodiment of the disclosure of the present invention. In some embodiments, the antibody or antigen-binding fragment binds to 1, 2, 3, 4, 5, or 6 residues selected from G97, A98, Y99, I100, Q125, and V126. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the antibody or antigen-binding fragment binds to G97, A98, Y99, I100, Q125, and V126. It will be understood that the ILT2 residue numbers used herein refer to SEQ ID NO: 31.

[0071] 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 the antagonism 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.

[0072] In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and B2M. In some embodiments, the interaction is direct. In some embodiments, the antibody or antigen-binding fragment inhibits the contact of ILT2 with B2M. In some embodiments, the contact is direct. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and HLA. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and MHC. In some embodiments, MHC is HLA. In some embodiments, HLA is HLA-G. 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 HLA, MHC, or both by inhibiting the interaction of ILT2 with B2M. 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, an antibody or antigen-binding fragment inhibits the interaction between ILT2 and the B2M / HLA complex. In some embodiments, an 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.

[0073] In some embodiments, ILT2-mediated immunosuppression is the suppression of immune cells. In some embodiments, 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, T cells are CD8-positive T cells. In some embodiments, T cells are T EMRA These are cells (terminally differentiated effector memory cells that reexpress CD45RA). In some embodiments, the immune cells are CD8-positive T cells, T EMRA The immune cells are selected from T cells, 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 intratumor immune cells. In some embodiments, the immune cells are immune cells in the tumor microenvironment (TME). In some embodiments, ILT2-mediated immunosuppression is the suppression of macrophage phagocytosis. In some embodiments, ILT2-mediated immunosuppression is the suppression of NK cell cytotoxicity. In some embodiments, ILT2-mediated immunosuppression is the suppression of T cell cytotoxicity. In some embodiments, ILT2-mediated immunosuppression is the suppression of T cell proliferation. In some embodiments, ILT2-mediated immunosuppression is the suppression of immune cell proliferation.

[0074] In some embodiments, the antibody or antigen-binding fragment does not bind to members of the leukocyte immunoglobulin-like receptor subfamily B other than ILT2. In some embodiments, the antibody or antigen-binding fragment is specific for ILT2. An antibody with a K D d of ≦1 μM, preferably ≦100 nM or ≦10 nM is said to specifically bind to the antigen. 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 subfamily B other than ILT2.

[0075] "Increased binding potency", as used herein, refers to specific binding to a target or antigen that is greater than the binding of an isotype control. In some embodiments, increased binding is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% increase in binding potency. Each possibility represents a separate embodiment of the present disclosure. In some embodiments, increased binding is the presence of binding when compared to an isotype control with no binding. Binding of an antibody to a specific domain will be well known to those skilled in the art. Antibody binding can be assayed by any method known to those skilled in the art, including but not limited to X-ray crystallography, immunoprecipitation, immunoblotting, competition assays, surface plasmon resonance, and kinetic exclusion assays. In some embodiments, increased binding potency is specific binding.

[0076] The antibody or antigen-binding fragment, variant, or derivative disclosed herein has a 10 3 M -1 −8 -1 , 5×10 3 M -1 −8 -1 , 10 4 M -1 −9 -1 or 5×10 4 M -1 −9-1 It can be said that the antibody binds to the target antigen, e.g., ILT2, at an on-rate (k(on)) greater than or equal to that of the antibody. Each possibility represents another embodiment of the disclosure of the present invention. Most antibodies bind at least 10 -9 A typical K from M D In contrast to having , the antibodies or antigen-binding fragments, variants, or derivatives disclosed herein have 10 -6 M or stronger K D It can be said that it binds to the target antigen. In some embodiments, K D This is a measure of affinity. A stronger bond indicates higher affinity, and a lower K is lower. D Those skilled in the art will understand that the binding is done by 10. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -6 M~10 -12 M's K D It binds to the target antigen. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -6 M~10 -11 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -6 M~10 -10 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -6 M~10 -9 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -7 M~10 -12 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -8 M~10 -12It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -9 M~10 -12 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -7 M~10 -11 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -8 M~10 -11 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -9 M~10 -11 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -7 M~10 -10 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -8 M~10 -10 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -9 M~10 -10 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -7 M~10 -9 It binds to the target antigen at KD of M. In some embodiments, the antibody or antigen-binding fragment, variant, or derivative disclosed herein is 10 -8 M~10 -9 It binds to the target antigen at the M KD.

[0077] 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 heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 19. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 19. 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 heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 21. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 21. 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. In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 23. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 23.

[0078] In some embodiments, the antibody or antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO: 20 (DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIK). In some embodiments, the light chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 20. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment includes a light chain containing the amino acid sequence of SEQ ID NO: 22 (DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK). In some embodiments, the light chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 22. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 22. 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 light chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 24. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 24.In some embodiments, the antibody or antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO: 45 (DIQMTQTTSSLSASLGDRVTISCRTSQDISNYLNWYQQKPDGTVKLLISYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPTFGSGTKLEIK). In some embodiments, the light chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 45. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 45.

[0079] 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, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 28. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 28. 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, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 29. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 29. 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 from a mouse.In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 30. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 30.

[0080] In some embodiments, SEQ ID NO: 15 is SEQ ID NO: 25, and the heavy chain includes or consists of SEQ ID NO: 28. In some embodiments, SEQ ID NO: 15 is SEQ ID NO: 25, and the heavy chain includes DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLSSVTAADTATYYCAHGYSYYYAMDAWGQGTTVTVSS (SEQ ID NO: 56). In some embodiments, the heavy chain includes an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 56. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 56. In some embodiments, the heavy chain variable region consists of SEQ ID NO: 56. In some embodiments, the heavy chain variable region consists of a sequence having at least 95% identity with SEQ ID NO: 56. In some embodiments, SEQ ID NO: 15 is SEQ ID NO: 25, and the heavy chain includes DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLSSVTAADTATYYCAHGYSYYYAMDAWGQGTTVTVSS (SEQ ID NO: 57). In some embodiments, the heavy chain includes an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 57. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 57. In some embodiments, the heavy chain variable region consists of SEQ ID NO: 57. In some embodiments, the heavy chain variable region consists of a sequence that is at least 95% identical to sequence number 57.In some embodiments, SEQ ID NO: 15 is SEQ ID NO: 25, and the heavy chain includes QVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYISYDGSNNYNPSLKNRVTISRDTSKNQFSLKLSSVTAADTATYYCAHGYSYYYAMDAWGQGTTVTVSS (SEQ ID NO: 58). In some embodiments, the heavy chain includes an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 58. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 58. In some embodiments, the heavy chain variable region consists of SEQ ID NO: 58. In some embodiments, the heavy chain variable region consists of a sequence that is at least 95% identical to SEQ ID NO: 58. In some embodiments, SEQ ID NO: 15 is SEQ ID NO: 25, and the heavy chain includes QVQLQGSGPGLVKPSETLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYISYDGSNNYNPSLKNRVTISRDTSKNQFSLKLSSVTAADTATYYCAHGYSYYYAMDAWGQGTTVTVSS (SEQ ID NO: 59). In some embodiments, the heavy chain includes an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 59. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 59. In some embodiments, the heavy chain variable region consists of SEQ ID NO: 59. In some embodiments, the heavy chain variable region consists of a sequence that is at least 95% identical to SEQ ID NO: 59. In some embodiments, the heavy chain includes sequences selected from sequence numbers 28 and 56-59. In some embodiments, the heavy chain includes sequences selected from sequence numbers 56-59. In some embodiments, the heavy chain variable region consists of sequences selected from sequence numbers 28 and 56-59. In some embodiments, the heavy chain variable region consists of sequences selected from sequence numbers 56-59.

[0081] In some embodiments, the heavy chain is DVQLQGSGPGLVKPSETLSLTTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDAWGQGTS VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 48). In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 48. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 48. In some embodiments, the heavy chain consists of SEQ ID NO: 48. In some embodiments, the heavy chain consists of a sequence that is at least 95% identical to sequence number 48. In some embodiments, the heavy chain is DVQLQGSGPGLVKPSETLSLTTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDSWGQGTS VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 51). In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 51. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 51. In some embodiments, the heavy chain consists of SEQ ID NO: 51. In some embodiments, the heavy chain consists of a sequence that is at least 95% identical to sequence number 51. In some embodiments, the heavy chain is DVQLQGSGPGLVKPSETLSLTTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDCWGQGTS VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 52). In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 52. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 52. In some embodiments, the heavy chain consists of SEQ ID NO: 52. In some embodiments, the heavy chain consists of a sequence that is at least 95% identical to sequence number 52. In some embodiments, the heavy chain is DVQLQGSGPGLVKPSETLSLTTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLSSVTAADTATYYCAHGYSYYYAMDAWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 64). In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 64. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 64. In some embodiments, the heavy chain consists of SEQ ID NO: 64. In some embodiments, the heavy chain consists of a sequence that is at least 95% identical to sequence number 64. In some embodiments, the heavy chain is DVQLQGSGPGLVKPSETLSLTTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLSSVTAADTATYYCAHGYSYYYAMDAWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 65). In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 65. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 65. In some embodiments, the heavy chain consists of SEQ ID NO: 65. In some embodiments, the heavy chain consists of a sequence that is at least 95% identical to sequence number 65. In some embodiments, the heavy chain is QVQLQGSGPGLVKPSETLSLTTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYISYDGSNNYNPSLKNRVTISRDTSKNQFSLKLSSVTAADTATYYCAHGYSYYYAMDAWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 67). In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 67. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 67. In some embodiments, the heavy chain consists of SEQ ID NO: 67. In some embodiments, the heavy chain consists of a sequence that is at least 95% identical to sequence number 67. In some embodiments, the heavy chain is QVQLQGSGPGLVKPSETLSLTTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYISYDGSNNYNPSLKNRVTISRDTSKNQFSLKLSSVTAADTATYYCAHGYSYYYAMDAWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 106). In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 106. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 106. In some embodiments, the heavy chain consists of sequence number 106. In some embodiments, the heavy chain consists of a sequence that is at least 95% identical to sequence number 106. In some embodiments, the heavy chain is QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWVGEIYPGSGNSYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARSNDGYPDYWGQGTTLT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 53). In some embodiments, the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 53. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the heavy chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 53. In some embodiments, the heavy chain consists of SEQ ID NO: 53. In some embodiments, the heavy chain consists of a sequence that is at least 95% identical to sequence number 53.

[0082] In some embodiments, the antibody contains a kappa light chain. In some embodiments, the constant region of the light chain is the kappa constant region. In some embodiments, the kappa constant region contains RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 63). In some embodiments, the kappa constant region consists of SEQ ID NO: 63. In some embodiments, the kappa constant region is an analog or derivative of SEQ ID NO: 63. In some embodiments, the kappa constant region contains at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity of SEQ ID NO: 63. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the kappa constant region contains at least 95% identity of SEQ ID NO: 63. In some embodiments, the kappa steady region contains at least 97% identity with SEQ ID NO: 63. In some embodiments, the kappa steady region contains at least 99% identity with SEQ ID NO: 63.

[0083] In some embodiments, the light chain comprises DIQMTQSTSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCQQGNTLPTFGQGTKLEIK (SEQ ID NO: 60). In some embodiments, the light chain comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 60. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 60. In some embodiments, the light chain variable region comprises a sequence that is at least 95% identical to SEQ ID NO: 60. In some embodiments, the light chain comprises DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPTFGQGTKLEIK (SEQ ID NO: 61). In some embodiments, the light chain comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 61. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 61. In some embodiments, the light chain variable region comprises a sequence that is at least 95% identical to SEQ ID NO: 61. In some embodiments, the light chain variable region comprises a sequence that is at least 95% identical to SEQ ID NO: 61. In some embodiments, the light chain comprises DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTFGQGTKLEIK (SEQ ID NO: 24). In some embodiments, the light chain comprises an amino acid sequence that is at least identical to SEQ ID NO: 24, including at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical. Each possibility represents another embodiment of the disclosure of the present invention.In some embodiments, the light chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 24. In some embodiments, the light chain variable region consists of SEQ ID NO: 24. In some embodiments, the light chain variable region consists of a sequence that is at least 95% identical to SEQ ID NO: 24. In some embodiments, the light chain contains DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAPKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPTFGQGTKLEIK (SEQ ID NO: 62). In some embodiments, the light chain contains an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 62. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 62. In some embodiments, the light chain variable region consists of SEQ ID NO: 62. In some embodiments, the light chain variable region consists of a sequence that is at least 95% identical to sequence number 62. In some embodiments, the light chain includes sequences selected from sequence numbers 24 and 60-62. In some embodiments, the light chain includes sequences selected from sequence numbers 60-62. In some embodiments, the light chain variable region consists of sequences selected from sequence numbers 24 and 60-62. In some embodiments, the light chain includes sequences selected from sequence numbers 60-62. In some embodiments, the light chain variable region consists of sequences selected from sequence numbers 60-62.

[0084] In some embodiments, the light chain includes DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 49). In some embodiments, the light chain includes an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 49. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 49. In some embodiments, the light chain consists of SEQ ID NO: 49. In some embodiments, the light chain consists of a sequence that is at least 95% identical to SEQ ID NO: 49. In some embodiments, the light chain includes DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 66). In some embodiments, the light chain includes an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 66. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain contains an amino acid sequence that is at least 95% identical to sequence number 66. In some embodiments, the light chain consists of sequence number 66. In some embodiments, the light chain consists of a sequence that is at least 95% identical to sequence number 66.In some embodiments, the light chain comprises DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 54). In some embodiments, the light chain comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 54. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 54. In some embodiments, the light chain comprises SEQ ID NO: 54. In some embodiments, the light chain consists of a sequence that is at least 95% identical to SEQ ID NO: 54. In some embodiments, the light chain includes DIQMTQSTSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCQQGNTLPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 107). In some embodiments, the light chain includes an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 107. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain contains an amino acid sequence that is at least 95% identical to sequence number 107. In some embodiments, the light chain consists of sequence number 107. In some embodiments, the light chain consists of a sequence that is at least 95% identical to sequence number 107.In some embodiments, the light chain includes DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAPKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 108). In some embodiments, the light chain includes an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 108. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, the light chain includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 108. In some embodiments, the light chain consists of sequence number 108. In some embodiments, the light chain consists of a sequence that is at least 95% identical to sequence number 108.

[0085] In some embodiments, the antibody comprises a heavy chain containing SEQ ID NO: 48 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody comprises a heavy chain consisting of SEQ ID NO: 48 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain containing SEQ ID NO: 48 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain consisting of SEQ ID NO: 48 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody is 15G8-13.

[0086] In some embodiments, the antibody comprises a heavy chain containing SEQ ID NO: 51 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody comprises a heavy chain consisting of SEQ ID NO: 51 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain containing SEQ ID NO: 51 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain consisting of SEQ ID NO: 51 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody is 15G8-13 having serine at position 108.

[0087] Antibodies having any combination of the heavy and light chains described above are anticipated by the disclosure of this invention.

[0088] In some embodiments, the antibody comprises a heavy chain containing SEQ ID NO: 52 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody comprises a heavy chain consisting of SEQ ID NO: 52 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain containing SEQ ID NO: 52 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain consisting of SEQ ID NO: 52 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody is 15G8-13 having cysteine ​​at position 108.

[0089] In some embodiments, the antibody comprises a heavy chain containing SEQ ID NO: 64 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody comprises a heavy chain consisting of SEQ ID NO: 64 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain containing SEQ ID NO: 64 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain consisting of SEQ ID NO: 64 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody is 15G8-23.

[0090] In some embodiments, the antibody comprises a heavy chain containing SEQ ID NO: 65 and a light chain containing SEQ ID NO: 66. In some embodiments, the antibody comprises a heavy chain consisting of SEQ ID NO: 65 and a light chain consisting of SEQ ID NO: 66. In some embodiments, the antibody consists of a heavy chain containing SEQ ID NO: 65 and a light chain containing SEQ ID NO: 66. In some embodiments, the antibody consists of a heavy chain consisting of SEQ ID NO: 65 and a light chain consisting of SEQ ID NO: 66. In some embodiments, the antibody is 15G8-32.

[0091] In some embodiments, the antibody comprises a heavy chain containing SEQ ID NO: 67 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody comprises a heavy chain consisting of SEQ ID NO: 67 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain containing SEQ ID NO: 67 and a light chain containing SEQ ID NO: 49. In some embodiments, the antibody consists of a heavy chain consisting of SEQ ID NO: 67 and a light chain consisting of SEQ ID NO: 49. In some embodiments, the antibody is 15G8-53.

[0092] In some embodiments, the antibody comprises a heavy chain containing SEQ ID NO: 53 and a light chain containing SEQ ID NO: 54. In some embodiments, the antibody comprises a heavy chain consisting of SEQ ID NO: 53 and a light chain consisting of SEQ ID NO: 54. In some embodiments, the antibody consists of a heavy chain containing SEQ ID NO: 53 and a light chain containing SEQ ID NO: 54. In some embodiments, the antibody consists of a heavy chain consisting of SEQ ID NO: 53 and a light chain consisting of SEQ ID NO: 54. In some embodiments, the antibody is 19E3.

[0093] In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in treating or mitigating cancer in subjects requiring it. 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 is cancer that expresses HLA-G. In some embodiments, the cancer is cancer that expresses MHC-I. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a tumor. In some embodiments, the cancer is selected from hepatobiliary cancer, cervical cancer, genitourinary cancer (e.g., urothelial carcinoma), testicular cancer, prostate cancer, thyroid cancer, ovarian cancer, nervous system cancer, eye cancer, lung cancer, soft tissue cancer, bone cancer, pancreatic cancer, bladder cancer, skin cancer, intestinal cancer, liver cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, breast cancer (e.g., triple-negative breast cancer), kidney cancer (e.g., renal cancer), head and neck cancer, leukemia, and lymphoma. In some embodiments, cancer is selected from breast cancer, hepatobiliary cancer, cervical cancer, colorectal cancer, esophageal cancer, stomach cancer, head and neck cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), kidney cancer, skin cancer (e.g., melanoma or squamous cell carcinoma), genitourinary cancer, and pancreatic cancer. In some embodiments, cancer is selected from breast cancer, hepatobiliary cancer (e.g., hepatocellular carcinoma, gallbladder cancer, cholangiocarcinoma, etc.), cervical cancer, colorectal cancer (e.g., KRAS wild-type colorectal cancer), esophageal cancer, stomach cancer, head and neck cancer, liver cancer, lung cancer, kidney cancer, skin cancer, genitourinary cancer, pancreatic cancer, and leukemia.

[0094] In some embodiments, the antibodies or antigen-binding fragments of the disclosure of the present invention are for use in shifting the tumor microenvironment from immunosuppressive to immunopromoting. In some embodiments, shifting the tumor microenvironment includes one or more of the following: inducing / enhancing the antitumor T cell response, increasing T cell proliferation, reducing cancer-induced suppressor myeloactivity, increasing dendritic cell (DC) activation, increasing dendritic cell homing to tumors, increasing macrophage phagocytosis, increasing M1 macrophage production, decreasing M2 macrophage production, and increasing NK cell activity. In some embodiments, the antibodies or antigen-binding fragments of the disclosure of the present invention are for use in increasing the T cell response against cancer cells. In some embodiments, the T cell response includes increased pro-inflammatory cytokine secretion. 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 antibodies or antigen-binding fragments of the disclosure of the present invention are for use in increasing macrophage phagocytosis of cancer cells. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in increasing the homing of dendritic cells to tumors or cancers. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in increasing macrophage phagocytosis. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in increasing cancer macrophage phagocytosis. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in increasing M1 macrophage production. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in decreasing M2 macrophage production.In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in increasing the cytotoxicity of NK cells against cancer cells. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in reducing the myeloactivity of suppressors induced by cancer. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for use in reducing the activity of tolerogenic dendritic cells (DCs). In some embodiments, the antibodies or antigen-binding fragments of the present invention are for increasing the activity or number of M1 monocytes. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for decreasing the activity or number of M2 monocytes. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for increasing the production of M1 macrophages. In some embodiments, the antibodies or antigen-binding fragments of the present invention are for decreasing the production of M2 macrophages. In some embodiments, M1 monocytes / macrophages are inflammatory macrophages / monocytes. In some embodiments, M2 monocytes / macrophages are suppressor macrophages / monocytes. In some embodiments, the antibody or antigen-binding fragment of the present invention is for increasing the number of DCs in a tumor. In some embodiments, the antibody or antigen-binding fragment of the present invention is for increasing the recruitment of DCs to a tumor. In some embodiments, the antibody or antigen-binding fragment of the present invention is for increasing the recruitment of DCs to a tumor. In some embodiments, "to the tumor" refers to "to the tumor microenvironment (TME)". In some embodiments, the antibody or antigen-binding fragment of the present invention is for increasing DC activation. In some embodiments, increasing DC activation includes decreasing tolerogenic dendritic cell activity. In some embodiments, the antibody or antigen-binding fragment of the present invention is for increasing antigen presentation.

[0095] 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 another embodiment of the disclosure of the present 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 action is selected from a) increased cytotoxicity of NK cells; b) increased cytotoxicity, proliferation, or both of T cells; c) increased phagocytosis of macrophages, increased production of M1 macrophages, decreased production of M2 macrophages, or a combination thereof; and d) increased homing of dendritic cells to cancer tumors, increased activation of dendritic cells, or a combination thereof. In some embodiments, cytotoxicity is cytotoxicity against cancer. In some embodiments, phagocytosis is phagocytosis of cancer or cancer cells. In some embodiments, the antibody or antigen-binding fragment induces an anticancer effect in T cells, NK cells, dendritic cells, and macrophages in a subject. In some embodiments, the antibody or antigen-binding fragment induces an anticancer effect in at least three of T cells, NK cells, dendritic cells, and macrophages in a subject. In some embodiments, the antibody or antigen-binding fragment induces an action as a monotherapy. In some embodiments, the antibody or antigen-binding fragment induces an action that does not involve combination therapy.

[0096] In some embodiments, increased cytotoxicity includes increased secretion of pro-inflammatory cytokines. Pro-inflammatory cytokines are well known in the art, and examples include, but are not limited to, IL-1, IL-1B, 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-gamma (IFNγ), and GM-CSF. In some embodiments, the pro-inflammatory cytokine is GM-CSF.

[0097] An "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 to which it binds.

[0098] An "antigen" is a molecule or part of a molecule that can induce antibody formation and accept antibody binding. An antigen may have one or more epitopes. The specific reactions described above indicate that the antigen is expected to react in a highly selective manner with its corresponding antibody and not with a large number of other antibodies that may have been induced by other antigens.

[0099] The terms “antigenic determinant” or “epitope” as disclosed in this invention refer to a region of an antigen molecule that specifically reacts with a particular antibody. Epitope-derived peptide sequences can be used alone or in combination with carrier components to immunize animals and produce additional polyclonal or monoclonal antibodies by applying methods known in the art. Variable domains of immunoglobulins can also be analyzed using the IMGT Information System (www.imgt.cines.fr / ) (IMGT® / V-Quest) to identify variable region segments including CDRs. See, for example, Brochet et al., Nucl Acids Res. (2008) J6:W503~508.

[0100] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. Those skilled in the art can clearly assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data other than the sequence itself. When used herein, “Kabat numbering” refers to the numbering system described by Kabat et al., U.S. Department of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0101] In some embodiments, the antibody or antigen-binding fragment is intended for use in combination with another agent. In some embodiments, the use in combination with another agent is for treating cancers expressing HLA-G and / or MHC-I. In some embodiments, the agent is an opsonizing agent. 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 intended for use in combination with anti-PD-1 / PD-L1-based therapy.

[0102] As used herein, "opsonizing agent" is any agent capable of binding to and opsonizing target cells (e.g., cancer cells, cells containing intracellular pathogens, etc.). For example, any antibody capable of binding to target cells and possessing an Fc region is considered an opsonizing agent. In some embodiments, the opsonizing agent is an antibody that induces antibody-dependent cell phagocytosis (ADCP). Examples of opsonizing agents 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 opsonizing agent is selected from rituximab (Rituxan®), trastuzumab (Herceptin®), trastuzumab (Perjeta®), cetuximab (Erbitux®), and panitumumab (Vectibix®). In some embodiments, the opsonizing agent is an anti-EGFR antibody. In some embodiments, the opsonizing agent is cetuximab.

[0103] "Anti-PD-1 / PD-L1 therapy" and "PD-1 / PD-L1 therapy," as used herein, are synonymous and used synonymously to refer to a treatment regimen that involves blocking the PD-1 and PD-L1 signaling axes. In some embodiments, 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 a drug that blocks PD-1-based immunosuppression. In some embodiments, PD-1 / PD-L1 therapy includes an antibody that blocks anti-PD-1 (e.g., selected from nivolumab (Optivo®), pembrolizumab (Keytruda®), and cemiplimab (Lybtayo®)). In some embodiments, PD-1 / PD-L1 therapy includes an antibody that blocks anti-PD-L1 (e.g., selected from atezolizumab (Tecentriq®), avelumab (Bavencio®), and durvalumab (Imfinzi®)). In some embodiments, PD-1 / PD-L1 therapy enhances immune surveillance. In some embodiments, PD-1 / PD-L1 therapy is an anti-cancer therapy. In some embodiments, PD-1 / PD-L1 therapy enhances tumor immune surveillance. Unless otherwise specified, the term “antibody” (also referred to as “immunoglobulin”) encompasses monoclonal antibodies and antibody fragments (also referred to herein as antibody moieties) insofar as they represent the desired biological activity. In certain embodiments, the use of chimeric antibodies or humanized antibodies is also encompassed in the disclosure of the present invention. In the context of “antibody or its antigen-binding fragment,” the term “antibody” refers to a complete antibody having two heavy chains and two light chains.

[0104] The basic unit of naturally occurring antibody structures is a heterotetrameric glycoprotein complex of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains linked together by both non-covalent association and disulfide bonds. Furthermore, each heavy and light chain also possesses regularly spaced interchain disulfide crosslinks. There are five human antibody classes (IgG, IgA, IgM, IgD, and IgE), and within these classes, various subclasses (e.g., IgG1, IgG2, IgG3, and IgG4) are recognized based on structural differences, such as the number of immunoglobulin units in a single antibody molecule, the disulfide crosslink structure of individual units, and differences in chain length and sequence. Antibody classes and subclasses are their isotypes.

[0105] The amino-terminal regions of the heavy and light chains are called variable domains because their sequences are more diverse than those of the carboxy-terminal region. This part of the antibody structure confers the antigen-binding specificity of the antibody. Since the heavy chain variable (VH) domain and the light chain variable (VL) domain together form a single antigen-binding site, the basal immunoglobulin unit has two antigen-binding sites. Certain amino acid residues are thought to form the boundary between the light and heavy chain variable domains (Chothia et al., J Mol Biol. (1985) 186, 651-63; Novotny and Haber, Proc Natl Acad Sci USA (1985) 82: 4592-6).

[0106] The carboxyl-terminal portions of the heavy and light chains form constant domains, namely CH1, CH2, CH3, and CL. While the diversity in these domains is even lower, there are differences between animal species, and furthermore, within the same individual, there are numerous different isotypes of antibodies, each with a different function.

[0107] The term “framework region” or “FR” refers to amino acid residues in the variable domain of an antibody other than the highly variable region amino acid residues as defined herein. The term “highly variable region,” as used herein, refers to amino acid residues in the variable domain of an antibody that are involved in antigen binding. The highly variable region includes amino acid residues from the “complementarity-determining region” or “CDR.” The CDR is primarily involved in the binding of the antigen to the epitope. The degree of FR and CDR is 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 Chothia system. In some embodiments, the Chothia system is an improved version of the Chothia system (Martin system).

[0108] Monoclonal antibodies as used herein specifically include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody from a different species or belonging to a different antibody class or subclass, in addition to fragments of such antibodies insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc Natl Acad Sci USA (1984) 57:6851-5). In addition, complementarity-determining region (CDR) grafting may be performed to modify certain properties of the antibody molecule, such as affinity or specificity. Non-limiting examples of CDR grafting are disclosed in U.S. Patent No. 5,225,539.

[0109] Chimeric antibodies are molecules in which different parts of the molecule are derived from different animal species, for example, having a variable region derived from a mouse mAb and a constant region of human immunoglobulin. Antibodies that substantially have variable region framework residues derived from human antibodies (called acceptor antibodies) and antibodies that substantially have complementarity-determining regions derived from mouse antibodies (called donor antibodies) are also called humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yield in production. For example, if a mouse mAb has a higher yield from hybridomas but is more immunogenic in humans, such a human / mouse chimeric mAb is used. Chimeric antibodies and methods for their production are publicly known in the art (e.g., PCT patent applications WO86 / 01533, WO97 / 02671, WO90 / 07861, WO92 / 22653, and U.S. Patents 5,693,762, 5,693,761, 5,585,089, 5,530,101, and 5,225,539). The term “humanized antibody,” as used herein, refers to an antibody comprising a framework region derived from a human antibody and one or more CDRs derived from a non-human (usually mouse or rat) immunoglobulin. The humanized immunoglobulin portion is substantially identical to the corresponding portion of the natural human immunoglobulin sequence, except in some cases the CDRs. However, in some cases, specific amino acid residues, such as specific amino acid residues in the framework region, can be modified to optimize the performance of the humanized antibody. Importantly, humanized antibodies are expected to bind to the same antigen as the donor antibody providing the CDR. For further details, see, for example, U.S. Patent No. 5,225,539, assigned to the British Medical Research Council. The terms “acceptor human immunoglobulin-derived framework region” and “acceptor human immunoglobulin-derived framework region,” as well as similar grammatical expressions, are used synonymously herein and refer to a framework region or portion thereof having the same amino acid sequence as acceptor human immunoglobulin.

[0110] The term “monoclonal antibody” or “mAb,” as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for variants that may occur during the production of the monoclonal antibody, which are generally present only in small amounts. In contrast to the preparation of polyclonal antibodies, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is against a single determinant on an antigen. Monoclonal antibodies are advantageous in that, in addition to their specificity, they are not contaminated by other immunoglobulins. The modifier “monoclonal” indicates the characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies. Monoclonal antibodies that may be used according to the methods provided herein may be prepared by the hybridoma method first described by Kohler et al., Nature (1975) 256:495, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature (1991) 352:624-8 and Marks et al., J Mol Biol. (1991) 222:581-97.

[0111] The mAbs disclosed in this invention may belong to any immunoglobulin class, including IgG, IgM, IgE, or IgA. Hybridomas producing mAbs can be cultured in vitro or in vivo. High titer mAbs can be obtained by in vivo production, in which case cells from individual hybridomas are injected intraperitoneally into newly immunostimulated Balb / c mice to generate ascites containing high concentrations of the desired mAb. From such ascites, or from the culture supernatant, isotype IgM or IgG mAbs can be purified using column chromatography methods well known to those skilled in the art.

[0112] The terms “antibody fragment,” “antigen-binding fragment,” and “antigen-binding site” are used synonymously and include a portion of an intact antibody, preferably the portion containing its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, scFvs, diabodies, tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. (1995) 8(10):1057~62); one-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments (e.g., Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem(di, tri)-scFv, etc.), and bispecific T cell engagers (BiTE).

[0113] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, while the remainder is an "Fc" fragment, its name reflecting its ability to easily crystallize. Pepsin treatment produces an F(ab')2 fragment with two antigen-binding sites, yet still capable of cross-linking the antigen.

[0114] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain tightly associated by non-covalent bonds. Collectively, six highly variable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific highly variable regions) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0115] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is, as used herein, the name of Fab' having at least one free thiol group in the cysteine ​​residue of the constant domain. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments having a cysteine ​​hinge between them. Other chemical couplings of antibody fragments are also known.

[0116] The "light chains" of antibodies (immunoglobulins) derived from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.

[0117] Antibodies can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the heavy chain corresponding to different classes of antibodies are called a, delta, e, gamma, and micro, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0118] A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of the antibody, which are present on a single polypeptide chain. In some embodiments, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which can form the scFv into a structure desirable for antigen binding. For a general overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0119] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, which contains a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to pair the two domains on the same chain, the domains are paired with a complementary domain on another chain, creating two antigen-binding sites. Diabody is described in Natl.Acad.Sci.USA, 90:6444~6448 (1993).

[0120] The term "multispecific antibody" is used in its broadest sense and specifically encompasses antibodies that possess polyepitope specificity. Such multispecific antibodies include, but are not limited to, antibodies containing heavy chain variable domains (VH) and light chain variable domains (VL) in which the VH / VL unit possesses polyepitope specificity; antibodies having two or more VL and VH domains in which each VH / VL unit binds to a different epitope; antibodies having two or more single variable domains in which each single variable domain binds to a different epitope; full-length antibodies; antibody fragments, such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies; and antibody fragments linked by covalent or non-covalent bonds. "Polyepitope specificity" refers to the ability to specifically bind to two or more different epitopes in the same or different targets.

[0121] The disclosures of this invention also provide multispecific antibodies (e.g., bispecific antibodies) having the binding specificity of the anti-ILT2 antibody described herein (e.g., including antigen-binding sites, e.g., six CDRs or VH and VL). In some embodiments, the multispecific antibody also has the binding specificity of ILT2 or a different protein, e.g., a cancer antigen, or another distinct antibody whose activity affects a disease state such as cancer. Multispecific antibodies and their preparations are known in the art. In some embodiments, the multispecific antibody described herein is used in place of the anti-ILT2 antibody described herein in the therapeutic methods, kits, or products described herein.

[0122] The monoclonal antibodies disclosed in this invention can be prepared using methods well known in the art. Examples include various techniques, such as those described in Kohler, G. and Milstein, C., Nature 256:495-497 (1975); Kozbor et al., Immunology Today (1983) 4:72; Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96; and Alan R. Liss, Inc. (1985).

[0123] In addition to conventional methods for generating antibodies in vivo, antibodies can also be produced in vitro using phage display technology. Such recombinant antibody production is significantly faster than conventional antibody production, and they can be produced against a vast number of antigens. Furthermore, when using conventional methods, many antigens are found to be non-immunogenic or highly toxic and therefore cannot be used to produce antibodies in animals. Moreover, affinity maturation of recombinant antibodies (i.e., increasing affinity and specificity) is extremely simple and relatively rapid. Ultimately, a vast number of different antibodies against specific antigens can be produced in a single selection procedure. To produce recombinant monoclonal antibodies, a large pool of antibodies with different antigen recognition sites can be generated using various display library-based methods. Such libraries can be constructed in numerous ways: one method involves cloning synthetic CDR3 regions in a pool of heavy chain germline genes, thus generating a large antibody repertoire from which recombinant antibody fragments with varying specificities can be selected. Another method uses a human lymphocyte pool as a starting material for constructing antibody libraries. By constructing a naive repertoire of human IgM antibodies, it is possible to create a human library with high diversity. This method has been widely used to successfully select a large number of antibodies against different antigens. Protocols for bacteriophage library construction and recombinant antibody selection are provided in the well-known reference book *Current Protocols in Immunology*, Colligan et al. (eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

[0124] Non-human antibodies can be humanized by any method known in the industry. In one method, a non-human complementarity-determining region (CDR) is inserted into the framework sequence of a human antibody or consensus antibody. Further modifications can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0125] In some embodiments, the antibodies described herein are neutralizing antibodies. "Neutralization" is defined as the reduction of protein function by the antibodies disclosed herein, as discussed herein. In one embodiment, "neutralization" is, as discussed herein, the reduction of protein function on the surface of immune cells, preferably immature and mature. Bone marrow lineage This involves the binding of antibodies to derived cells, T cells, and NK cells, thereby blocking the transmission of inhibitory signals within these cells and conferring a less repressive phenotype and function.

[0126] In some embodiments, the antibody or antigen-binding fragment of the present invention is the agent of the present invention.

[0127] In some embodiments, the disclosure of the present invention provides nucleic acid sequences encoding antibodies of the disclosure of the present invention. In one embodiment, the antibody described herein is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 75% identity to the nucleotide sequence described herein. In one embodiment, the antibody described herein is encoded by a nucleic acid molecule comprising a nucleic acid sequence having at least 80% identity to the nucleic acid sequence described herein. In one embodiment, the antibody described herein is encoded by a nucleic acid molecule comprising a nucleic acid sequence having at least 85% identity to the nucleic acid sequence described herein. In one embodiment, the antibody described herein is encoded by a nucleic acid comprising a nucleic acid sequence having at least 90% identity to the nucleic acid sequence described herein. In one embodiment, the antibody described herein is encoded by a nucleic acid comprising a nucleic acid sequence having at least 95% identity to the nucleic acid sequence described herein.

[0128] In another aspect, nucleic acid sequences encoding an antibody or antigen-binding fragment of the present invention are provided.

[0129] In another embodiment, nucleic acid molecules encoding an antibody or antigen-binding fragment of the present invention are provided.

[0130] In some embodiments, the nucleic acid sequence encoding the variable region of the heavy chain of the antibody or antigen-binding fragment of the disclosure of the present invention includes a nucleic acid sequence selected from the following: caggttcagctgcagcagtctggagctgagctggcgaggcctggggcttcagtgaagctgtcctgcaaggcttctggctacaccttcacaagctatggtataagctgggtgaagcagagaactggacagggccttgagtgggttggagagatttatcctggaagtggtaattcttactacaatgagaagttcaagggcaaggccacactgactgcagacaaatcctccagcacagcgtacatggagctccgcagcctgacatctgaggactctgcggtctatttctgtgcaagatcgaatgatggttaccctgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 32), gatgtacagcttcaggggtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggttattactggaactggatccggcagtttccaggaaacaaactggaatggatgggctacataagctacgatggtagcaataactacaacccatctctcaaaaatcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgaattctgtgacttctgaggacacagccacatattactgtgcccatggttactcatattactatgctatggactgctggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO: 33),gatgtccagctgcaaggctctggccctggactggttaagccttccgagacactgtccctgacctgctctgtgaccggctactctatcacctccggctactactggaactggatcagacagttccccggcaagaaactggaatggatgggctacatctcctacgacggctccaacaactaca accccagcctgaagaaccggatcaccatctctcgggacacctccaagaaccagttctccctgaagctgaactccgtgaccgctgccgataccgctacctactactgtgctcacggctactcctactactacgccatggatgcttggggccagggcacatctgtgacagtgtcctct(Sequence number 34) and caggttcagctgcaacagtctgacgctgagttggtgaaacctggagcttcagtgaagatatcctgcaaggtttctggctacaccttcactgaccatactattcactggatgaagcagaggcctgaacagggcctggaatggattggatatatttatcctagagatggtagtacta agtacaatgagaagttcaagggcaaggccacattgactgcagacaaatcctccagcacagcctacatgcagctcaacagcctgacatctgaggactctgcagtctatttctgtgcaagaacctgggactactttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 35),

[0131] In some embodiments, the nucleic acid sequence encoding the variable region of the light chain of the antibody or antigen-binding fragment of the disclosure of the present invention includes a nucleic acid sequence selected from the following: gacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatatcctgcagagccagtgaaagtgttgatagttatggcaatagttttatgcactggtaccagcagaaaccaggacagccacccaaactcctcatctatcgtgcatc caacctagaatctgggatccctgccaggttcagtggcagtgggtctaggagacttcaccctcaccattaatcctgtggaggctgatgatgttgcaacctattactgtcagcaaagtaatgaggatccgtacacgttcggaggggggaccaagctgggaaataaaaa( Sequence number 36), gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcaggacaagtcaggacattagcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctcctacacatcaagattgcactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccacattagcaacctggagcaagaagatattgccacttactttgccaacagggtaatacgcttcccacgttcggctcggggacaaagttggaaataaa (sequence number 37),gacatccagatgacccagtctccatcctctctgtctgcctctgtgggcgacagagtgaccatcacctgtcggacctctcaggacatctccaactacctgaactggtatcagcagaaacccggcaaggccgtgaagctgctgatctctcctacacctccagactgca ctctggcgtgccctccagattttctggctctggatctggcaccgactacaccctgaccatcagttctctgcagcctgaggacttcgccacctactactgtcagcagggcaacaccctgcctaccttggccagggcaccaagctggaaatcaag (SEQ ID NO: 38) and gacatccagatgacacaatcttcatcctacttgtctgtatctctaggaggcagagtcaccattacttgcaaggcaagtgaccacattaataattggttagcctggtatcagcagaaaccaggaaatgctcctaggctcttaatatctggtgcaaccagtttggaa actggggttccttcaagattcagtggcagtggatctggaaaggattacactctcagcattaccagtcttcagactgaagatgttgctacttattactgtcaacagtattggagtactccgtggacgttcggtggaggcaccaagctggaaatcaaa (SEQ ID NO: 39),

[0132] In some embodiments, the nucleic acid sequence encoding the heavy chain of the antibody or antigen-binding fragment of the present invention is

[0133] In some embodiments, the nucleic acid sequence encoding the light chain of the antibody or antigen-binding fragment of the present invention is (SEQ ID NO: 111) is included or consists of the above. In some embodiments, the nucleic acid sequence encoding the light chain of the antibody or antigen-binding fragment of the disclosure of the present invention further includes a sequence encoding a signal peptide. In some embodiments, the sequence encoding the light chain of the antibody or antigen-binding fragment of the disclosure of the present invention further includes a stop codon.

[0134] In some embodiments, the antibody or antigen-binding fragment is from a mouse, and the sequence encoding the heavy chain is selected from SEQ ID NOs: 32, 33, and 35. In some embodiments, the antibody or antigen-binding fragment is from a mouse, and the sequence encoding the light chain is selected from SEQ ID NOs: 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.

[0135] When used synonymously in this specification, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA.

[0136] Polynucleotides encoding polypeptides can be obtained from a variety of sources, including, but are not limited to, cDNA libraries prepared from tissues that are thought to possess polypeptide mRNA and express it at detectable levels. Therefore, polynucleotides encoding polypeptides can be successfully obtained from cDNA libraries prepared from human tissues. The genes encoding polypeptides may also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0137] For example, a polynucleotide may encode an entire immunoglobulin molecular chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (VH) but also a heavy chain constant region (CH), which is typically expected to contain three constant domains: CH1, CH2, and CH3; as well as a "hinge" region. In some situations, the presence of a constant region is desirable.

[0138] Other polypeptides encoded by polynucleotides include antigen-binding antibody fragments, e.g., single-domain antibodies ("dAb"), Fv, scFv, Fab', from which the CHI and CK or CL domains have been excised. Because minibodies are smaller than conventional antibodies, they can achieve better tissue penetration in clinical / diagnostic use; however, being bivalent, they may retain higher binding affinity than monovalent antibody fragments, e.g., dAb. Therefore, unless otherwise indicated in the context, the term "antibody," as used herein, encompasses not only the entire antibody molecule but also the types of antigen-binding antibody fragments discussed above. Each framework region present in the encoded polypeptide may contain at least one amino acid substitution compared to the corresponding human acceptor framework. Thus, for example, a framework region may contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to the acceptor framework region. Given the characteristics of the individual amino acids constituting the disclosed protein product, some reasonable substitutions are expected to be recognizable by experts. Amino acid substitutions, or "conservative substitutions," can be made, for example, based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved.

[0139] Preferably, the polynucleotides described herein can be isolated and / or purified. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0140] The term “not naturally occurring”—substance, composition, entity, and / or any combination of substance, composition, or entity, or any grammatical variation thereof—expressly excludes, but only excludes, any form of substance, composition, entity, and / or any combination of substance, composition, or entity that is well understood by those of ordinary art in the art as “naturally occurring,” or any term that includes conditions that, at any time, will be determined or interpreted, or may be determined, as “naturally occurring” by an examiner or administrative or judicial body.

[0141] Methods for treatment and diagnosis In another embodiment, a method is provided for treating cancer expressing HLA, MHC-I, or both in a subject requiring such treatment, comprising administering an antibody or antigen-binding fragment of the present invention to the subject.

[0142] In another embodiment, a method is provided for treating cancer in a subject requiring such treatment, comprising confirming that ILT2 expression in the subject exceeds a predetermined threshold, and administering an ILT2-based immunosuppressant agent to the subject, thereby treating the cancer in the subject.

[0143] In another embodiment, a method is provided for treating cancer in a subject requiring such treatment, 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 cancer in the subject.

[0144] In another embodiment, a method is provided for increasing the efficacy of a PD-1 / PD-L1-based therapy against cancer cells, comprising contacting the cancer cells with a drug that inhibits ILT2-mediated immunosuppression.

[0145] In another embodiment, a drug is provided that binds to ILT2 and inhibits ILT2-mediated immunosuppression, for use in combination with anti-PD-L1 / PD-1 based therapies for treating subjects with cancer.

[0146] The terms “treatment” or “treatment” of a disease, disorder, or condition, as used herein, encompass alleviation of at least one symptom of those, reduction in their severity, or inhibition of their progression (e.g., cancer metastasis). Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. To be an effective treatment, a useful composition herein is sufficient if it reduces the severity of the disease, disorder, or condition, reduces the severity of symptoms associated with them, or improves the quality of life of the patient or subject.

[0147] As used herein, the term “treatment” refers to a clinical intervention in an attempt to alter the course of a disease in the treated individual, and may be performed either preventively or during the course of a clinically pathological condition. Desired effects of treatment include preventing the onset or recurrence of the disease, alleviating symptoms, reducing the pathological outcomes of the disease, slowing the rate of disease progression, mitigating the condition, achieving remission, or improving the prognosis. The term “treatment” may also encompass ex vivo procedures affecting cells or tissues in culture.

[0148] In some embodiments, the antibody or antigen-binding fragment is administered as monotherapy. In some embodiments, the antibody or antigen-binding fragment is administered in conjunction with PD-1 / PD-L1 therapy. In some embodiments, the antibody or antigen-binding fragment is administered in conjunction with an opsonizing agent. In some embodiments, the opsonizing agent 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 conjunction 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.

[0149] 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 the tumor burden. In some embodiments, the treatment includes reducing cancer metastasis. In some embodiments, the treatment includes increasing cytotoxicity against cancer. In some embodiments, the treatment includes increasing the inflammatory response against cancer. In some embodiments, the treatment includes increasing cancer phagocytosis.

[0150] The term "subject," as used herein, refers to an individual or patient that is a vertebrate, such as a mammal, and in particular a human. In some embodiments, the subject is a human. In some embodiments, the subject is a mammal (e.g., a mouse, rat, dog, rabbit, or non-human primate). In some embodiments, the subject has cancer.

[0151] In some embodiments, the cancer is an HLA-expressing cancer. In some embodiments, the HLA is HLA-G, for example, HLA-G1 and other isoforms of HLA-G. In some embodiments, the cancer is an MHC-I-expressing cancer. In some embodiments, the cancer is a PD-L1-expressing cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is refractory to PD-1 and / or PD-L1-based therapy. In some embodiments, the cancer has never responded to PD-1 and / or PD-L1-based therapy. In some embodiments, the cancer responded to PD-1 and / or PD-L1-based therapy but became refractory. In some embodiments, the method of the disclosure of the present invention converts a refractory cancer into a responsive cancer.

[0152] In some embodiments, the cancer is unresectable, metastatic, refractory to or not a candidate for standard approved therapies, or any combination thereof.

[0153] In some embodiments, the method includes confirming that cancer expresses HLA, MHC-I, or both. In some embodiments, the method includes confirming that cancer expresses HLA. In some embodiments, the method includes confirming that cancer expresses MHC-I. In some embodiments, the method includes confirming that cancer expresses MHC-I (i.e., class I HLA in humans). In some embodiments, confirmation includes measuring expression in cancer. In some embodiments, confirmation includes measuring expression on the surface of cancer. In some embodiments, "in cancer" and / or "on cancer" means "in cancer cells" and / or "on cancer cells." In some embodiments, confirmation includes measuring HLA-G secreted by cancer. In some embodiments, confirmation includes measuring soluble HLA-G. In some embodiments, soluble HLA-G is in body fluids. In some embodiments, body fluid is blood.

[0154] 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 exceeds 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 expression in immune cells. In some embodiments, ILT2 expression is expression in immune cells of the subject. In some embodiments, immune cells are peripheral blood immune cells. In some embodiments, immune cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, immune cells are intratumor immune cells. In some embodiments, immune cells are immune cells in the tumor microenvironment (TME). In some embodiments, immune cells are CD8-positive T cells, macrophages, NK cells, and T EMRAThe immune cells are selected from the 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.

[0155] In some embodiments, administering the antibody or antigen-binding fragment of the disclosure of the present invention includes administering a pharmaceutical composition comprising the antibody or antigen-binding fragment of the disclosure 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, an excipient, or an adjuvant. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0156] The terms “carrier,” “excipient,” or “adjuvant,” as used herein, refer to any non-active component of a pharmaceutical composition. The term “pharmaceutically acceptable carrier,” as used herein, refers to a non-toxic, inert solid, semi-solid liquid extender, diluent, encapsulating material, any type of compounding aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that may serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solution; and other non-toxic, suitable substances used in pharmaceutical formulations. Some non-limiting examples of substances that may serve as carriers in this specification include sugars, stearic acid, magnesium stearate, calcium sulfate, polyols, pyrogen-free water, isotonic saline, phosphate buffer solution, and other non-toxic, pharmaceutically acceptable substances used in other pharmaceutical formulations. Wetting agents and lubricants, such as sodium lauryl sulfate, as well as excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions anticipated herein.

[0157] The carrier may, in total, constitute about 0.1% to about 99.99999% based on the weight of the pharmaceutical composition presented herein.

[0158] The term "therapeutic dose" refers to the amount of drug that is effective in treating a disease or disorder in a mammal. The term "therapeutic dose" also refers to the amount and duration of medication that is effective in achieving the desired therapeutic or prophylactic outcome. The exact dosage form and regimen will be determined by the physician according to the patient's condition.

[0159] In some embodiments, the method further includes administering an opsonizing agent to a target. 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 a target. In some embodiments, the method further includes contacting cells with a PD-1 / PD-L1 based therapeutic agent. In some embodiments, the method further includes growing cells in the presence of a PD-1 / PD-L1 based therapeutic agent. In some embodiments, the PD-1 / PD-L1 based therapeutic agent is an antibody that blocks PD-1 or PD-L1 (e.g., pembrolizumab). In some embodiments, the method does not include the administration of an anti-CD47 agent or therapy. In some embodiments, the method lacks the administration of an anti-CD47 agent or therapy. In some embodiments, the method further includes administering an anti-CD47 agent or therapy.

[0160] In some embodiments, a drug that inhibits ILT2-based immunosuppression binds to ILT2. In some embodiments, the drug binds to the extracellular domain of ILT2. In some embodiments, the drug is an ILT2 antagonist. In some embodiments, the drug is an antibody that blocks ILT2. In some embodiments, the drug inhibits the interaction between ILT2 and B2M. In some embodiments, the drug is an antibody of the disclosure of the present invention.

[0161] In some embodiments, the ILT2-based immunosuppressant is administered before, after, or concurrently with the opsonizing agent. In some embodiments, the ILT2-based immunosuppressant and the opsonizing agent are administered in a single composition. In some embodiments, the ILT2-based immunosuppressant and the opsonizing agent are administered in separate compositions.

[0162] In some embodiments, the ILT2-based immunosuppressant is administered before, after, or concurrently with the PD-1 / PD-L1 therapy. In some embodiments, the ILT2-based immunosuppressant and the PD-1 / PD-L1 therapy are administered in a single composition. In some embodiments, the ILT2-based immunosuppressant and the PD-1 / PD-L1 therapy are administered in separate compositions. In some embodiments, at least one of the agents or therapies is adapted for co-administration.

[0163] The term “adapted for co-administration,” as used herein, means that antibodies are present in a form that allows them to be safely and readily administered to a subject. Co-administration may, in some non-limiting embodiments, be carried out by injection, i.e., by intratumoral injection, intravenous injection or infusion, or by subcutaneous injection, or by other known methods such as oral administration or inhalation. In some embodiments, the antibodies are expected to be contained within a pharmaceutical composition that can be safely and readily administered to a subject, for example. In some embodiments, the pharmaceutical composition comprises antibodies and a pharmaceutically acceptable carrier or excipient.

[0164] In some embodiments, the HLA is HLA-G. In some embodiments, the HLA is non-canonical 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, protein surface expression is confirmed. Methods for measuring expression are well known in the art, and examples include PCR, Q-PCR, Northern blotting, immunoblotting, in situ hybridization, immunostaining, and FACS. In some embodiments, the method includes FACS analysis of cancer to confirm surface expression.

[0165] It is understood that the antibodies or antigen-binding fragments and pharmaceutical compositions of the present invention may be used in the treatment methods described herein, for use in the treatments described herein, and / or for use in the manufacture of pharmaceuticals for the treatments described herein. The present invention also provides kits and products comprising the antibodies or antigen-binding fragments or pharmaceutical compositions described herein.

[0166] compound The disclosure of the present invention also anticipates pharmaceutical formulations for human medical use, such pharmaceutical formulations comprising at least one antibody that recognizes ILT2 as an active agent for the manufacture of therapeutic compositions for the treatment, diagnosis, or prevention of conditions as variously described herein.

[0167] In such pharmaceuticals and pharmaceutical formulations, the active agent is preferably used together with one or more pharmaceutically acceptable carriers and optionally any other therapeutic components. The carriers must be pharmaceutically acceptable in the sense that they are compatible with the other components of the formulation and are not excessively harmful to the recipient. The active agent is provided in an amount effective to achieve the desired pharmacological effect as described above, and in a quantity appropriate to achieve the desired daily dose.

[0168] Typically, the molecules of the disclosure of the present invention, including the antigen-binding site of an antibody, are expected to be suspended in sterile saline for therapeutic use. Alternatively, the pharmaceutical composition may be formulated to control the release of the active ingredient (the molecule containing the antigen-binding site of an antibody) or to extend its presence in the patient's system. Numerous suitable drug delivery systems are known, examples of which include, for example, implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, and microspheres. Controlled-release formulations can be prepared through the use of polymers for complexing or adsorption of the molecules of the disclosure of the present invention. Examples of biocompatible polymers include poly(ethylene-co-vinyl acetate) matrices, as well as matrices of polyanhydride copolymers of stearate dimers and sebaric acid. The release rate of the molecules of the disclosure of the present invention, i.e., antibodies or antibody fragments, from such matrices depends on the molecular weight of the molecules, the amount of molecules in the matrix, and the size of the dispersed particles.

[0169] The pharmaceutical composition of this invention can be administered by any suitable means, for example, orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intra-articularly, intrafocally, or parenterally. Intravenous (IV) or intra-articular administration is generally expected to be preferred.

[0170] It will be apparent to those skilled in the art that the therapeutically effective dose of the molecule disclosed in this invention depends, in particular, on the administration schedule, the unit dose of the molecule administered (regardless of whether the molecule is administered in combination with other therapeutic agents), the patient's immune and health status, the therapeutic activity of the molecule administered, and the judgment of the treating physician.

[0171] The appropriate dosage of the molecule (antibody or fragment thereof) disclosed in this invention varies depending on the route of administration, the type of molecule (polypeptide, polynucleotide, organic molecule, etc.), age, weight, sex, or the patient's condition, and should ultimately be determined by a physician. However, in the case of oral administration, the daily dosage may generally be about 0.01 mg to about 500 mg per kg of body weight, preferably about 0.01 mg to about 50 mg, and more preferably about 0.1 mg to about 10 mg. In the case of parenteral administration, the daily dosage may generally be about 0.001 mg to about 100 mg per kg of body weight, preferably about 0.001 mg to about 10 mg, and more preferably about 0.01 mg to about 1 mg. The daily dosage can be administered, for example, in a regimen typically consisting of 1 to 4 individual doses per day. Another preferred method of administration is intra-articular administration of about 0.01 mg to about 100 mg per kg of body weight. Various considerations regarding reaching an effective dose are found, for example, in Goodman and Gilman: The Pharmacological Bases of Therapeutics, 8th edition, Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pa., 1990.

[0172] Preferred dose regimens for combination chemotherapy are publicly known in the industry, for example, as described by Saltz et al., Proc ASCO (1999) 18:233a and Douillard et al., Lancet (2000) 355:1041-7.

[0173] The molecules disclosed in this invention as active ingredients are, as is well known, pharmaceutically acceptable and are dissolved, dispersed, or mixed with excipients compatible with the active ingredient. Suitable excipients include, for example, water, physiological saline, phosphate-buffered saline (PBS), dextrose, glycerol, and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, the composition may optionally contain small amounts of auxiliary agents, such as wetting agents or emulsifiers, or pH buffers.

[0174] Production method In another embodiment, a method is provided for producing a drug, comprising: obtaining a drug that binds to the extracellular domain of ILT2 or a fragment thereof; testing the ability of said drug to inhibit the interaction between ILT2 and B2M; and selecting at least one drug that inhibits the interaction between ILT2 and B2M; and thereby producing the drug. The drug may be, for example, a molecule or a protein, as used herein.

[0175] In another embodiment, a method for producing a drug, comprising culturing host cells containing one or more vectors comprising a nucleic acid sequence encoding a drug, thereby producing a drug, wherein the nucleic acid sequence is: i. Obtaining a drug that binds to the ILT2 extracellular domain or a fragment thereof; ii. To test the ability of the drug to inhibit the interaction between ILT2 and B2M; and iii. Select at least one drug that inhibits the interaction between ILT2 and B2M. A method is provided in which the nucleic acid sequence of the drug selected by is used.

[0176] In another embodiment, a method for producing a drug is provided, comprising obtaining a drug that binds to a sequence of human ILT2 selected from sequence numbers 41-44 and 68-70; and thereby producing a drug.

[0177] In another embodiment, a method is provided for identifying antibodies that compete with a reference antibody, whose heavy and light chains are respectively contained in SEQ ID NOs. 48 and 49, for binding to ILT2, comprising contacting a library of antibodies with a polypeptide sequence containing an ILT2 sequence selected from SEQ ID NOs. 41-44 and 68-70, and selecting antibodies from the library that bind to the ILT2 sequence, thereby obtaining antibodies that compete with the reference antibody for binding to ILT2.

[0178] In another embodiment, a method is provided for producing a drug, comprising culturing host cells comprising one or more vectors comprising a nucleic acid sequence encoding a drug, thereby producing a drug, wherein the nucleic acid sequence is a selected drug sequence, obtained by obtaining a drug that binds to a human ILT2 sequence selected from SEQ ID NOs. 41-44 and 68-70.

[0179] In some embodiments, the method includes obtaining a drug that binds to sequences selected from SEQ ID NOs. 41-44 and 68-70. In some embodiments, the nucleic acid sequence is the nucleic acid sequence of the drug selected by obtaining a drug that binds to sequences selected from SEQ ID NOs. 41-44 and 68-70. In some embodiments, the method includes obtaining a drug that binds to sequences selected from SEQ ID NOs. 68-70. In some embodiments, the nucleic acid sequence is the nucleic acid sequence of the drug selected by obtaining a drug that binds to sequences selected from SEQ ID NOs. 68-70. In some embodiments, the method includes obtaining a drug that binds to sequences selected from SEQ ID NOs. 71 and 72. In some embodiments, the nucleic acid sequence is the nucleic acid sequence of the drug selected by obtaining a drug that binds to sequences selected from SEQ ID NOs. 71 and 72. In some embodiments, the method includes obtaining a drug that binds to sequences of SEQ ID NOs. 71 and 72. In some embodiments, the nucleic acid sequence is the nucleic acid sequence of the drug selected by obtaining a drug that binds to sequences of SEQ ID NOs. 71 and 72. In some embodiments, the reference antibody is 15G8. In some embodiments, the reference antibody is 15G8-13.

[0180] In some embodiments, the method further includes testing the ability of a drug to inhibit ILT2-mediated immunosuppression and selecting at least one drug that inhibits ILT2-mediated immunosuppression. In some embodiments, the nucleic acid sequence is the nucleic acid sequence of the drug selected by testing the ability of a drug to inhibit ILT2-mediated immunosuppression and selecting a drug that inhibits ILT2-mediated immunosuppression. In some embodiments, the method includes testing the ability of the drug to induce at least three of the following: increased phagocytosis of cancer cells by macrophages, increased T cell activity against cancer cells, increased M1 macrophage production, increased M2 macrophage production, increased recruitment of dendritic cells into the tumor microenvironment, increased dendritic cell activation, and increased cytotoxicity of natural killer (NK) cells, and selecting at least one drug that induces at least three of these. In some embodiments, the method includes testing the ability of a drug 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 a drug to induce effects in T cells, NK cells, dendritic cells, and macrophages.

[0181] In some embodiments, the increase in efficacy 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 another embodiment of the disclosure of the present invention. In some embodiments, the pro-inflammatory cytokine is GM-CSF. In some embodiments, the increase in efficacy includes a synergistic increase in T cell activation. In some embodiments, the increase in efficacy includes a synergistic increase in T cell cytotoxicity. In some embodiments, the increase in efficacy includes a synergistic increase in both T cell activation and cytotoxicity. In some embodiments, the increase includes an increase in cell membrane CD107a expression. In some embodiments, the increase is characterized by an increase in cell membrane CD107a expression. In some embodiments, the increase is an increase compared to the efficacy when the drug is not administered or contacted. In some embodiments, increasing efficacy involves converting cancers refractory to PD-1 / PD-L1-based therapy into cancers responsive to that therapy. In some embodiments, the cancer expresses HLA. In some embodiments, the cancer expresses MHC-I.

[0182] In some embodiments, increased macrophage inflammatory activity includes increased phagocytosis of cancer cells by macrophages. In some embodiments, increased macrophage inflammatory activity includes increased M1 macrophage production. In some embodiments, increased macrophage inflammatory activity includes decreased M2 macrophage production. In some embodiments, increased macrophage inflammatory activity includes increased M1 phenotype in macrophages. In some embodiments, increased macrophage inflammatory activity includes decreased M2 phenotype in macrophages.

[0183] In some embodiments, dendritic cell activity includes activation of dendritic cells. In some embodiments, dendritic cell activity includes recruitment of dendritic cells to a tumor. In some embodiments, dendritic cell activity is activity against cancer cells. In some embodiments, activity against cancer cells is activity in the TME. In some embodiments, the tumor is the TME. In some embodiments, the tumor includes the TME. In some embodiments, the tumor includes the tumor and its TME. In some embodiments, dendritic cell activity includes antigen presentation.

[0184] In some embodiments, testing the ability of a drug includes the ability of a drug to increase 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 the cytotoxicity of natural killer (NK) cells against cancer cells. Each possibility represents another embodiment of the disclosure of the present invention. In some embodiments, selecting at least one drug includes selecting a drug 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 the cytotoxicity of natural killer (NK) cells against cancer cells. In some embodiments, increasing macrophage inflammatory activity means increasing the production of M1 macrophages and / or increasing the phagocytosis of cancer cells by macrophages. In some embodiments, increasing macrophage inflammatory activity means decreasing the production of M2 macrophages. In some embodiments, testing the ability of a drug includes the ability of a drug to increase macrophage inflammatory activity. In some embodiments, testing the ability of a drug includes the ability of a drug to increase dendritic cell activity. In some embodiments, to tumor means to TME. In some embodiments, testing the ability of a drug includes the ability of a drug to increase the cytotoxicity of NK cells against cancer cells.

[0185] In some embodiments, the method further includes testing the ability of a drug 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 a drug to inhibit contact between ILT2 and B2M. In some embodiments, the interaction is binding. In some embodiments, contact is binding. In some embodiments, the method further includes testing the ability of a drug to bind to an epitope.

[0186] The following examples are intended to illustrate the preparation and use of the compounds and methods of this invention and should not be construed as limiting. While the disclosure of the present invention is described here with its specific embodiments, it will be apparent that many modifications and variations will be obvious to those skilled in the art. Therefore, it is intended to encompass all such modifications and variations that fall within the essence and broad scope of the appended claims. [Examples]

[0187] Generally, the scientific names used herein and the experimental procedures utilized in the disclosure of this invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are described in detail in the literature. 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, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds.), "Genome Analysis: A Laboratory Manual Series," Volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998); Methods 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 J.E. Cellis (1994); "Culture of Animal Cells - A Manual of Basic Technique," by Freshney and Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology," Volumes I-III, edited by J.E. Coligan.See also (1994); Stites et al. (eds.), "Basic and Clinical Immunology" (8th edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996); "Monoclonal Antibodies: Methods and Protocols," Vincent Ossipow, Nicolas Fischer, Humana Press (2014); and "Monoclonal Antibodies: Methods and Protocols," Maher Albitar, Springer Science & Business Media (2007). All of these are incorporated by reference. Other general references are provided throughout this document.

[0188] material and method antibody -Commercially available anti-ILT2 mAbs are clone number 1-GHI / 75 (BioLegend, catalog number 333704) and clone number 2-HP-F1 (eBioscience, catalog number 16-5129). Additional mAbs used: 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).

[0189] Flow cytometry - Generally, cells were kept on ice or at 4°C throughout the entire process. Before staining, 5 × 10 5Each cell was blocked for 15 minutes with 50 μg / mL human IgG (Sigma, catalog number I4506) in FACS buffer (PBS containing 0.1% BSA). The antibody was used at the concentration recommended by the manufacturer and incubated in the dark for 30 minutes. 100 μL of the antibody was incubated in a 96-well U-bottom plate, the cells were washed twice with 200 μL of FACS buffer, and transferred to a FACS tube containing 150 μL of FACS buffer for analysis. Cells were analyzed using a Gallios flow cytometer (Beckman Coulter) with Kaluza for Gallios flow cytometry acquisition software.

[0190] Myelocyte differentiation Monocytes were isolated from fresh blood samples from healthy donors using the EasySep® Human Monocyte Enrichment Kit (STEMCELL, catalog number 19059) by a negative selection method. Different cell populations were tested for the indicated phenotype by FACS analysis of relevant markers and analysis of characteristic cytokine secretion. For maturation, 0.8 × 10⁶ cells were placed in RPMI medium containing growth factors. 6 Monocytes were cultured at a density of cells / mL, and the medium was 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 48 hours in the presence of 20 ng / mL IFN-gamma and 50 ng / mL LPS. Suppressive M2 macrophages were differentiated using 50 ng / mL M-CSF for 6 days, followed by 10 ng / mL M-CSF and 20 ng / mL IL-4 and IL-10 for 48 hours. 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 tolerogenic (IL-10 100 U / mL and IFN-α2b (1000 U / mL)) dendritic cells.

[0191] Transfection-HLA-G1 plasmids (encoding the full-length HLA-G transcript) were generated by cloning HLA-G1 cDNA into the PCDNA3.1 vector. Transfection was performed using jetPEI® transfection reagent (PolyPlus Transfections). ILT2 / CD3z plasmids were generated by combining the extracellular portion of human ILT2 protein with transmembrane and intracytoplasmic residues of the mouse CD3 gene within a frame. The plasmids were nucleofected into the mouse BW5417.3T cell line using Nucleofector II (Lonza) as described by the manufacturer. Stable transformants were selected in a medium containing G418.

[0192] NK and cancer cell line co-culture assay -NK cells were incubated with the indicated cell line at 37°C for 5 hours in the presence of an anti-ILT2 antibody and a matching isotype control. Cytotoxic levels were measured using a fluorescence-based LDH detection kit (Promega).

[0193] Flow cytometry blocking assay - Recombinant human ILT2 protein, fused with the Fc portion of human IgG1 at its N-terminus, was conjugated with biotin (Innova bioscience). Total 5 × 10 5 A375 / HLA-G1 cells were incubated at room temperature for 30 minutes in 100 μL volume in the presence of anti-ILT2 clone number 1 or isotype-matched control mAb and ILT2-Fc (10 μg / mL) conjugated with biotin. After several washing steps, streptavidin-PE was added to a final concentration of 0.2 μg / mL, incubated on ice for 30 minutes, and then FACS analysis was performed.

[0194] BW ILT2 / CD3z-chain chimeric assay -3 × 10 4Each BW / ILT2z cell was mixed with an equal number of A375 / WT or A375 / HLA-G1 cells for 24 hours. Functional mAbs and matching isotype controls were used at the indicated concentrations. The amount of secreted mouse IL2 was assessed by a commercially available ELISA kit (BioLegend).

[0195] Phagocytosis assay Monocytes were isolated from pia mater samples obtained from healthy blood bank donors using a human monocyte enrichment kit. To generate macrophages, monocytes were grown for 6-7 days in RPMI medium supplemented with 10% human serum and M-CSF (50 ng / ml). Mature macrophages were removed and re-implanted into 96-well plates (15K cells / well) and incubated overnight at 37°C with 5% CO2. Target primary cancer cells or cell lines from various indications were labeled with pHrodo Red Cell Labeling Dye, washed, and added to macrophages (75K cells / well to achieve a 1:5 effector:target ratio). Assay plates were assayed using an IncuCyte S3 instrument.

[0196] The fluorescence of IncuCyte pHrodo Red Cell Labeling Dye increases in acidic environments such as those present in phagosomes, enabling the quantification of phagocytic events by fluorescence measurement. Assay plates (4 images / well, ×10 magnification) were sampled every 30 minutes using an IncuCyte instrument for red fluorescence signal intensity and phase image. Phagocytic events were reflected as an accumulation of red fluorescence signals, and the phagocytic rate was reflected by the kinetics of the accumulation of red fluorescence signals. [Examples]

[0197] ILT2 and HLA-G are found on cancer cells and cancer-associated immune cells. ILT2 is a well-known immunosuppressive molecule found on the surface of many tumor cells in addition to healthy immune cells. ILT2 has been shown to bind to MHC-1 as well as HLA class molecules (HLA-G, HLA-F, and HLA-B27), compete with CD8, and thereby inhibit T cell activation. To further understand the breadth of cells expressing ILT2, flow cytometry analysis was performed on various immune cells using a commercially available antibody (antibody number 1). As reported in the literature, cytotoxic T cells (CTLs) from melanoma patients, as well as natural killer (NK) cells, were positive for ILT2 surface expression (Figure 1). Monocytes from the blood of healthy donors were also examined and found to be highly expressive of ILT2 (Figure 2, leftmost panel). ILT2 expression was retained when monocytes differentiated into different myeloid cell populations (dendritic cells and macrophages), regardless of whether they were immature, inflammatory, or tolerogenic (Figure 2, right panel).

[0198] ILT2 expression in different cancer indications was examined by genetic information science analysis of the TCGA database (Figure 3A). Interestingly, a correlation 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 presented in TCGA (Figure 3B). Analysis of fresh tumor samples from different solid tumors by flow cytometry demonstrated ILT2 expression by innate and adaptive immune cells in the tumor microenvironment (TME). Tumor samples were collected from patients with non-small cell lung cancer (NSCLC), renal cancer (RCC), head and neck cancer, esophageal cancer, and colon cancer, and single-cell suspensions were generated by enzymatic digestion. Figure 3C shows the percentage of ILT2-positive cells for total immune cells, tumor-associated macrophages (TAMs), CD4-positive T cells, CD8-positive T cells, and natural killer cells (NK). Therefore, it is clear that ILT2 is expressed on both cells with anti-cancer activity (inflammatory cells) and cells with cancer-promoting and immunosuppressive activity (tolerogenic and MDSCs).

[0199] HLA-G expression was also investigated in a variety of cancers. Tissue microarrays (TMA) of cancer samples from different indications were stained by immunohistochemistry with a commercially available polyclonal HLA-G antibody. The percentage of positive cases for each cancer type is shown (Figure 4A). In addition, extended TMAs were examined for multiple individual indications. HLA-G staining scores were calculated based on the multiplication of staining intensity and the percentage of positive cells. High HLA-G staining scores exceeding 100 were detected in a high percentage of esophageal, gastric, head and neck, and renal cancers (Figure 4B). Table 1 shows the percentage of positive cases for each indication.

[0200]

Table 1

[0201] In addition to a soluble secreted form, HLA-G also has the more common cell membrane form. To examine the expression level of soluble HLA-G in cancer patients, plasma samples were tested for the presence of HLA-G using a commercially available ELISA. HLA-G was found to be overexpressed in multiple cancer indications compared to normal (healthy) controls (Figure 5). Furthermore, a population of patients with significantly higher levels could be detected in certain specific cancer types.

Examples

[0202] Generation of Antibodies that Block ILT2 Using hybridoma technology, monoclonal ILT2 antagonist antibodies were generated. Sixty-nine ILT2-specific hybridomas were initially generated. Three lead antibodies were selected based on their favorable binding, cross-reactivity profiles and functional activity in various assays tested. The selected antibodies were 19E3, 15G8 and 17F2. These antibodies were sequenced using conventional methods. Figure 6A shows the sequences of the variable regions of the selected antibodies. CDRs were determined by the KABAT system. 15G8 and 19E3 were humanized using a conventional CDR grafting approach. Briefly, essential CDR and framework residues from the original hybridoma-derived antibody were identified and grafted into the variable and constant regions of a germline human antibody. The final humanized antibody is an IgG4 antibody.

[0203] The IgG4 heavy chain constant region used for grafting contained two point mutations known to reduce binding to FcγR. These mutations are conventionally known as S228P and L235E, and their exact positions depend on the length of the variable region of the heavy chain. For the 15G8 antibody, serine at position 227 was mutated to proline, and leucine at position 234 was mutated to glutamic acid. For the 19E3 antibody, serine at position 225 was mutated to proline, and leucine at position 232 was mutated to glutamic acid. The final humanized 15G8 also contained a single amino acid change that removed a cysteine in CDR-H3 and replaced it with alanine or serine. This change was made to improve developability. The binding of both resulting antibodies was confirmed, and the 15G8 antibody with alanine was selected for further testing. All subsequent references to humanized 15G8 refer to the alanine variant.

[0204] During grafting of 15G8CDR, five heavy chains and four kappa light chains were generated. These chains were named VH1-5 and Vk1-4. Table 2 provides the sequences of these chains. By combining each heavy chain with each light chain, it was possible to obtain 20 different antibodies. All 20 potential antibodies were transiently expressed in HEK EBNA cells, and the supernatant was tested for binding to recombinant ILT2 peptide using Biacore T200. Chimeric 15G8 antibodies were used as a control. Table 3 summarizes the binding results.

[0205] [Table 2]

[0206] [Table 3]

[0207] Seven of the 20 combinations were selected from further research. Antibodies containing light chain Vk1 were not selected because they consistently produced the worst yields regardless of the heavy chain. The combinations of VH3, VH4, and VH5 were selected because Vk4 consistently produced the highest yields. Combinations of Vk4 with VH1 and VH2 also resulted in high expression, but the KD of these antibodies was found to be worse than that of the control antibody, and even worse with all antibodies containing VH1 and VH2. VH1 / Vk3, VH2 / Vk3, and VH3 / Vk2 were all selected because they had the lowest relative KD values. VH5 / Vk3 was also selected due to the combination of high expression and a low relative KD value.

[0208] These seven antibodies were purified by protein A chromatography, and their concentrations were calculated. Numerous assays were performed to characterize the functional performance of the selected antibodies.

[0209] First, seven 15G8 antibodies were tested for binding to cell membrane ILT2. BW cells were transfected with human ILT2, and 5 × 10⁶ antibodies were used. 5 Each BW-ILT2 cell was incubated with 10 μg / ml of 10 μg of either one of seven antibodies or a control IgG in staining buffer (0.05% BSA in PBS) for 30 minutes, then washed in staining buffer and incubated with a secondary PE-conjugated donkey anti-mouse antibody. The cells were then washed twice with staining buffer and analyzed using a Cytoflex flow cytometer (Beckman Coulter), with data analyzed using CytExpert software (Ver2.3). As can be seen in Figure 6B, the overall binding kinetics were similar among the seven antibodies, but when the EC50 values ​​were calculated (Table 4), four of the antibodies were superior to the other three. These four antibodies were further tested for their functionality.

[0210] [Table 4]

[0211] The ability of humanized 15G8 antibodies to enhance tumor cell phagocytosis was tested in several cancer types. Real-time monitoring of tumor cell lines by macrophages was performed (see Materials and Methods). As can be seen in Figures 6C–6D, the different humanized ILT2-blocking antibodies tested were able to enhance phagocytosis of both HLA-G-positive tumor cells (Figure 6C) and MHC-I-only-positive tumor cells (Figure 6D). The variants 15G8-13, 15G8-23, and 15G8-34 showed somewhat higher efficacy than the other variants in A375-HLA-G phagocytosis (Figure 6C). Therefore, 15G8-13 and 15G8-23 were further evaluated in additional phagocytosis experiments. As presented in Figures 6E–6F, both antibodies were able to enhance phagocytosis of the tested cell lines, but 15G8-13 showed slightly superior activity, particularly at lower concentrations.

[0212] Next, the ability of humanized 15G8 variants to enhance NK cell effector activity was tested in a system in which NK cells were incubated with target cancer cell lines, and cytotoxicity was subsequently evaluated by measuring LDH levels. As can be seen in Figures 6G-6H, the different variants were all able to significantly enhance the cytotoxicity of NK cells in a dose-dependent manner against both HLA-G positive cells (Figure 6G) and MHC-I-only cells (Figure 6H). Once again, variant 15G8-13 demonstrated somewhat superior activity, this time enhancing the cytotoxicity of NK cells. Due to its slight but consistent superiority, 15G8-13 was selected for all subsequent analyses. Hereafter, 15G8-13 will simply be referred to as the 15G8 humanized antibody. [Examples]

[0213] Comparison of antibodies that block ILT2 The ability of three different anti-ILT2 antibodies to bind to ILT2 was tested using three different systems. Binding to recombinant ILT2 was tested using ELISA (Figure 7A), and binding to cell membrane ILT2 was tested using ILT2-transfected BW cells (Figures 7B-7C). Chimeric and humanized antibodies showed similar binding (Figures 7C-7D). A commercially available mouse anti-human ILT2 antibody (Biolegend; clone GHI / 75) was used as a positive control. The three tested antibodies bound well to ILT2, whether in solution (Figure 7A) or on the cell surface (Figure 7B). Cross-reactivity to several similar ILT family members—PIRB, ILT6, and LILRA1—was similarly examined using binding ELISA (Figure 7A). Antibodies against these proteins were used as positive controls. No antibodies cross-reacted with PIRB, ILT6, or LILRA1. The antibodies were also effective for immunohistochemical staining (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 than in healthy controls (Figure 7E). [Examples]

[0214] ILT2 antibodies block ILT2-HLA-G interactions. The ability of the generated anti-ILT2 antibody to block the interaction between HLA-G and ILT2 was tested using four different assays. First, a blocking flow cytometry assay was performed. HLA-G transfected A375 cells were incubated with biotinylated ILT2 in the presence of the antibody disclosed in this invention and a positive control antibody. A commercially available anti-ILT2 antibody, GHI / 75 (BioLegend, catalog no. 333704), was used as a positive control. ILT2-biotin binding to cells was determined by flow cytometry analysis using streptavidin-PE (Figure 8A). The percentage of blocking was determined by normalizing against a negative control (ILT2 binding in the presence of control IgG). Figure 8B shows representative FACS analyses showing 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 blocking percentage was calculated at various antibody concentrations (Figure 8C). Chimeric mice and humanized antibodies showed similar blocking ability (Figure 8D).

[0215] 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 were transfected with human ILT2 fused to the mouse T cell zeta chain (BW-ILT2). The cells were then incubated with A375-HLA-G cells in the presence of a selected ILT2 antibody. During functional ILT2-HLA-G interaction, BW cells secrete the reporter cytokine, mouse IL-2. Blocking the interaction is expected to reduce the secretion of the reporter cytokine. After 24 hours of incubation, mouse IL-2 secretion was determined by ELISA. Results represent mean ± SE levels of mIL-2 from 3-well sets per treatment (Figure 8E). A commercially available mouse anti-human ILT2 antibody (Biolegend; clone GHI / 75) was used as a positive control (PC) for both assays. The blocking percentage was calculated at various antibody concentrations (Figure 8F). Using this same BW ILT2 / mouse Z-chain chimeric reporter assay, the possibility that the novel antibody itself possessed ILT2 activating activity was ruled out. Cells were incubated with the ILT2 antibody without cancer cells, and mouse IL-2 secretion was measured again (Figure 8G). The novel ILT2 antibody was found not to have agonist activity, but another antibody (1G7) produced by the same hybridoma process could bind to ILT2 and induce its activity.

[0216] Functional blocking was also examined using human Jurkat cells (T cells). Jurkat cells were incubated with or without A375 cancer cells exogenously expressing HLA-G and single-chain anti-CD3 (OKT3). Secretion of pro-inflammatory human IL-2 was measured. When unmodified Jurkat cells (ILT2-negative cells) were used, they secreted high levels of IL-2 when co-cultured with cancer cells (Figure 8H). Unsurprisingly, the addition of 15G8 antibody had no effect on IL-2 secretion because there was no ILT2 to block. Therefore, Jurkat cells were transfected to express human ILT2. First, ILT2-positive Jurkat cells were cultured with and without A375 cancer cells exogenously expressing OKT3. These cancer cells are naturally MHC-I positive. MHC-I from cancer cells strongly inhibited IL-2 secretion (Figure 8I). In this case, the addition of the 15G8 antibody blocked the ILT2 / MHC-I interaction and dose-dependently increased IL-2 secretion. Using a pan-HLA antibody as a positive control, the 15G8 antibody was equivalent to the pan-HLA antibody at equal concentrations (Figure 8I). To enhance the inhibitory effect, A375 cells were also transfected with HLA-G to make them MHC-I and HLA-G positive. These cells exhibited an even stronger inhibitory effect than ILT2-positive cells, reducing IL-2 secretion to the level of Jurkat cells cultured alone (Figure 8J). When the 15G8 antibody was administered, a dose-dependent effect was again observed, and again, the 15G8 antibody and the pan-HLA antibody were equally effective at equal doses (Figure 8J). In particular, when only the HLA-G specific antibody was used instead of the pan-HLA antibody, the effect was significantly reduced and was equivalent to that of the 15G8 antibody used at 1 / 100th of the concentration (Figure 8K).

[0217] Using this Jurkat line, the 15G8 antibody was compared with two commercially available antibodies: GHI / 75 and HP-F1. Jurkat cells expressing human ILT2 were cultured with A375 cells expressing HLA-G / OKT3 in the presence and absence of various concentrations of 15G8, GHI / 75 and HP-F1. As previously observed, 15G8 caused a statistically significant dose-dependent increase in IL-2 secretion (Figure 8L). GHI / 75 had no effect on IL-2 secretion when compared to medium alone, but resulted in a slight increase compared to the IgG control (Figure 8M). HP-F1 caused a small but significant increase, which reached a plateau and did not increase with increasing dose (Figure 8N). Even 20 μg / ml of HP-F1 was inferior compared to only 4 μg / ml of 15G8.

[0218] 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. When co-cultured with cancer cells, activation was observed in both cell types; however, this activation was enhanced in the presence of the ILT2 antibody (Figures 8O-8P). These results demonstrate that the ILT2 antibody can efficiently block the ILT2-HLA-G interaction, resulting in enhanced T cell and NK cell activation.

Examples

[0219] The ILT2 antibody enhances phagocytosis of HLA-G and MHC-I-positive tumor cells The ability of the generated anti-ILT2 antibody to enhance phagocytosis of tumor cells was tested using two different systems. Monocytes were isolated from the blood of a healthy donor and incubated in the presence of M-CSF for 6-7 days to generate macrophages. First, a flow cytometry-based assay was employed. Different cancer cell lines stained with PKH67-FITC were incubated with macrophages stained with eFluor670-APC in the presence of the indicated antibody. Phagocytosis levels were determined by the percentage of double-stained macrophages, indicating target cell uptake. Phagocytosis levels are presented as a percentage from the control (culture medium only). As demonstrated in Figure 9A, antibodies blocking different ILT2s were able to enhance phagocytosis of HLA-G positive A375 cells by macrophages. In addition, the ability of macrophages to enhance phagocytosis of tumor cells was examined using the real-time IncuCyte® analysis system. Target cell lines were labeled with pHrodo® Red Cell Labeling Dye, washed, and added to macrophages, along with various replication treatments. Since the fluorescence of IncuCyte® pHrodo® Red Cell Labeling Dye increased in acidic environments such as those present in phagosomes, fluorescence measurement allowed for the quantification of phagocytic events. Assay plates were sampled every 30 minutes using an IncuCyte® instrument for red fluorescence signal intensity and phase image. Phagocytic events were reflected as an accumulation of red fluorescence signals, and the phagocytic rate was reflected by the kinetics of the red fluorescence signal accumulation. Using this real-time system, the ability of a humanized anti-ILT2 antibody to enhance phagocytosis of HLA-G positive A375 cells was confirmed (Figure 9B). In addition, using the IncuCyte® system, it was demonstrated that the generated blocking ILT2 antibody could enhance phagocytosis of both HLA-G positive and various MHC-I positive (WT) cancer cell lines (Figure 9C).

[0220] The effect of a combination of the generated ILT2 antibody and Erbitux, an antibody that induces antibody-dependent cell phagocytosis (ADCP), on cancer cell phagocytosis was examined using the IncuCyte® real-time system described above. The combination of the ILT2-blocking antibody and Erbitux significantly increased the phagocytosis of HLA-G overexpressing cancer cell lines compared to the activity of each antibody alone (Figure 9D). In fact, the combination of Erbitux and the 15G8 humanized antibody showed a synergistic effect, and the increase in phagocytosis with the combination treatment was greater than with the additive alone. [Examples]

[0221] The selected ILT2 antibody can restore T cell activity that is inhibited by HLA-G. To examine the ability of the generated anti-ILT2 antibodies to restore T cell activity inhibited by HLA-G, human CD8 T cells were co-incubated with either wild-type 721.221 cells (221WT) or 721.221 cells overexpressing soluble HLA-G5 (221-HLA-G). After 5 days, IFNγ secretion levels from T cells were measured using standard ELISA. Results are shown as a percentage of the effect exceeding that of 221-HLA-G alone, representing the average of four independent experiments. The results presented in Figure 10A demonstrate that several ILT2 antibodies can restore T cell activity inhibited by HLA-G. This was also tested using incubation with A375-HLA-G-OKT3 cells. After 72 hours, human granzyme B secretion was also measured and found to increase in a dose-dependent manner in the presence of 15G8 antibody (Figure 10B). [Examples]

[0222] The selected ILT2 antibody can enhance NK cell cytotoxicity against HLA-G and MHC-I positive tumor cells. The ability of the generated anti-ILT2 antibody to enhance NK cell effector activity was tested in a system in which NK cells were incubated with various target cancer cell lines. Cells were co-incubated for 5 hours in an effector-to-target ratio of 7.5:1, and cytotoxicity levels were subsequently detected using a fluorescence-based LDH detection kit. The percentage of specific cytotoxicity was calculated as follows:

number

[0223] As demonstrated in Figure 11A, the ILT2 antibody of the present invention significantly enhanced the cytotoxicity of NK cells against both HLA-G-positive cells and various MHC-I-positive cancer cell lines (Figure 11B) in a dose-dependent manner. Granzyme B (Figure 11C) and interferon-gamma (Figure 11D) secretion were also measured and found to increase in a dose-dependent manner. Primary NK cells were co-cultured with target HLA-G+ melanoma cells, and subsequently analyzed by FACS for the expression of IFNγ, ILT2, CD56, and CD107A. Specific analysis of ILT2-positive, CD56-positive NK cell populations revealed dose-dependent increases in IFNγ expression and cell membrane CD107A expression (Figures 11E-11F). When each experiment was plotted separately, a clear correlation was shown between %ILT2-positive cells and increased IFNγ and CD107A expression (Figures 11G-11H). [Examples]

[0224] ILT2 antibodies increase the production of inflammatory macrophages. The effect of blocking ILT2 during 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 blocking ILT2 antibody or control IgG. Macrophages were further differentiated in the presence of LPS (50 ng / mL) to generate M1 macrophages, or M2 macrophages were generated using IL-4 (25 ng / mL). As demonstrated in Figure 12, the presence of an antibody that blocks ILT2 during the macrophage maturation process increased the expression of HLA-DR (a marker for M1 inflammatory macrophages) in most macrophages from the tested donors, regardless of whether they differentiated into M0, M1, or M2 macrophages. In addition, macrophages differentiated into M1 macrophages also resulted in increased CD80 levels in most of the tested donors. In summary, these results demonstrate that selected ILT2 antagonist antibodies can induce macrophages that present higher levels of HLA-DR and CD80, representing macrophages with a more inflammatory M1 phenotype. [Examples]

[0225] Antibodies that block ILT2 enhance the activity of immune cells against tumor cells from patients. The activity of the generated anti-ILT2 antibody was tested 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 with tumor cells isolated from tumor samples. Phagocytosis levels were examined using the IncuCyte® real-time analysis system as detailed above. As demonstrated in Figure 13A, the ILT2 antibody was able to enhance phagocytosis of tumor cells from patients with different cancer indications. Furthermore, the effect was dose-dependent and was present even with autologous macrophages, observed in both RCC (Figure 13B) and squamous cell carcinoma from H&N (13C). In addition, the effect of the ILT2 antibody on enhancing PBMC activity was tested. Single-cell suspensions of tumor samples from patients were incubated with PBMCs isolated from the same patients in the presence of IL-2 (activated PBMCs). As demonstrated in Figure 14G, PBMC secretion of pro-inflammatory TNF-α cytokines was elevated in the presence of ILT2 antibodies in the presence of tumor cells. Taken together, these results demonstrate the ability of blocking ILT2 antibodies to increase the activity of immune cells against tumor cells from various cancer indications. [Examples]

[0226] Antibodies that block ILT2 can be combined with PD-1 / PD-L1 therapy agents. ILT2 and PD-1 are expressed on different immune cells, including mostly peripheral blood cells and immune cells endogenous to the tumor microenvironment (Figure 14A). Analysis of ILT2 and PD-1 expression in intratumoral CD8-positive T cells from CRC patients found that both T central memory cells (Tcm) and exhausted T cells (Tex) expressed high levels of PD-1 (Figure 14B), but also low levels of ILT2 (Figure 14C). T cells reexpressing CD45RA (T EMRA ) showed opposite patterns, expressing high levels of ILT2 and low levels of PD-1. This dichotomy in results is not a cancer-specific phenomenon, and T from healthy donor blood is also observed. EMRAA large percentage of cells (83%) were found to be ILT2-positive, while only a very small percentage (17%) of total CD8-positive T cells were positive (Figure 14D). Nevertheless, ILT2 expression was enhanced in T cells in the TME. Single-cell suspensions were generated by enzymatic digestion of tumors isolated from esophageal cancer patients. FACS analysis revealed that the majority of CD8-positive tumor-infiltrating lymphocytes (TILs) were T EMRA Being a cell (50%), and these T EMRA The study showed that 100% of the cells were ILT2-positive, but almost completely PD-1-negative (95%) (Figure 14E).

[0227] The effects of the anti-ILT2 antibody and anti-PD-1 combination disclosed in this invention were tested in SEB-activated (10 ng / ml) PBMCs from 10 healthy donors. Cell membrane CD107a expression was used as a marker for increased cytotoxicity. Overall, the 15G8 antibody resulted in a small increase in surface CD107a on average, while anti-PD-1 resulted in a somewhat larger response, which was donor-dependent (Figure 14F). The combination of the two antibodies resulted in increased CD107a levels on average; however, these changes were variable based on the specific donor sample. Figure 14G presents three exemplary samples. 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 had a stronger response to anti-PD-1 than to anti-ILT2, but surprisingly, the combination of the two antibodies was higher than the additive effect. Anti-PD-1 resulted in a 19% increase in expression, anti-ILT2 resulted in a 3.7% increase, but the combination treatment resulted in a 33.2% increase. This synergistic effect was even more pronounced in donor #3 cells. In donor #3, 15G8 was more effective than anti-PD-1 (13.1% increase compared to 9.3% increase), and the combination therapy was even more effective (41%), nearly twice the effect predicted by a simple combination of additives.

[0228] Next, we assessed the combined treatment of patients' tumor cells with antibodies blocking PD-1 and the generated ILT2 antibody. Cancer cells from various patients were incubated with autologous PBMCs in the presence of anti-PD1 antibody, antibodies of the disclosure of this 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 treatments (Figures 14H-14J). Treatment of colon adenocarcinoma cells from the first patient with humanized antibody 15G8 did not enhance IFNγ secretion at all compared to the IgG control, while anti-PD-1 resulted in a robust increase in cytokine secretion (Figure 14H). Surprisingly, however, the combination of anti-PD-1 and ILT2 antibody increased secretion by more than 50%. The second patient showed a similar trend to the small increase induced by ILT2 antibody or anti-PD-1, and the enhanced synergistic increase present when the two antibodies were used in combination (Figure 14I). GM-CSF expression was not altered by either antibody alone compared to the control, but surprisingly, the combination of the two antibodies resulted in a robust increase of nearly 100% of the control GM-CSF levels (Figure 14J).

[0229] Next, we evaluated combination therapies using a mixed lymphocyte reaction. Dendritic cells and CD8-positive T cells were isolated from different healthy donors, and macrophages were generated from monocytes isolated from head and neck cancer patients. The cells were combined in a 5:1 effector cell-to-target ratio using the indicated treatments (20 ug / mg each). In the presence of either anti-ILT2 antibody or anti-PD-1 antibody, IFNγ secretion by T cells was enhanced, and this effect was increased with the combined use of both antibodies (Figures 14K-14L). A greater cumulative effect was observed in macrophage cultures compared to dendritic cell cultures (Figure 14K) (Figure 14L). These results clearly indicate that anti-ILT2 and anti-PD-1 therapy has a synergistic de novo effect in enhancing the inflammatory response of immune cells. [Examples]

[0230] Antibodies that block ILT2 reduce tumor burden in vivo. The efficacy of anti-ILT2 antibodies was tested in a xenograft in vivo model. Immunodeficient SCID-NOD or NSG mice were implanted with cancer cell lines (A375-HLA-G, A375-WT, COLO-320-HLA-G), and human macrophages generated from healthy donor blood were injected into the mice in the presence of ILT2 antibodies. As demonstrated in Figure 15A, administration of the generated ILT2 antibodies resulted in significant tumor inhibition in this model, which in this system was most likely mediated by the activity of human macrophages. In addition, antitumor efficacy was observed in MHC-I positive tumor cells as well as HLA-G positive cells.

[0231] The efficacy of anti-ILT2 antibodies was also tested in an in vivo model in melanoma xenografts, which are lung lesions. Melanoma cells (MEL526-HLA-G) were implanted into immunodeficient SCID-NOD mice. Human PBMCs were isolated from the blood of healthy donors and injected into the mice in the presence of selected ILT2 antibodies, starting on day 1 after implantation and repeated on days 2, 10, and 18 (Figure 15B). ILT2 antibodies were administered on days 1, 4, 8, 11, 15, 18, 22, and 25. As demonstrated in Figure 15C, administration of the generated ILT2 antibodies resulted in a significant reduction in tumor cell metastasis. This is represented by the formation of black lesions in the lungs of the mice. The lungs of mice treated with ILT2 antibodies had far fewer such lesions compared to mice treated with control IgG. This effect was also demonstrated by a reduction in lung weight in these mice (Figure 15D), which was most likely mediated by human lymphocytes administered to the mice, in combination with inhibition of ILT2 by the administered antibody. Therefore, the anti-ILT2 antibody was effective in preventing metastasis and tumorigenesis.

[0232] Next, the efficacy of the novel antibody in treating already formed tumors was tested in the same in vivo mouse model. As previously described, MEL526-HLA-G cells were implanted into SCID-NOD mice via IV administration. After 15 days, human PBMCs isolated from healthy donors were administered to the relevant group of mice, and this administration was repeated on days 25, 35, and 51 (see Figure 15E). Antibodies (ILT2 antibody, anti-PD-1 antibody, or a combination of these two) were administered on days 14, 17, 20, 24, 27, 30, 34, 37, and 50 (see Figure 15E). On day 53, the mice were lethal and their lungs were weighed. Tumor weight was calculated by subtracting the lung weight of naive mice from the lung weight of the test mice. Anti-PD-1 antibodies reduced tumor weight, albeit not significantly, while ILT2 antibodies and combination therapy had a significant effect (Figure 15F).

[0233] CD8 T cells, T EMRA Tumors derived from cells and NK cells were tested for CD107A and CD69 expression. Overall, CD8 T cells and anti-PD-1 antibodies induced a non-significant increase in CD107A expression, while ILT2 antibodies induced a significant change, although not as a combination therapy (Figure 16A). 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, the combination therapy had a greatly enhanced effect, with a higher total percentage of CD69-positive cells than either therapy alone (Figure 16C). Surprisingly, when CD69 expression was examined in CD8 T cells, neither anti-PD1 nor anti-ILT2 increased expression; however, the combination treatment induced a highly significant increase in CD69 expression (Figure 16D). Furthermore, it was determined that the effect of the ILT2 antibody correlated with ILT2 expression. When the experiment was divided into mice that received PBMCs with low or high ILT2 expression, significant differences in activation markers were observed. EMRAIn cells, high-ILT2-expressing PBMCs contained twice as much CD107A expression compared to low-ILT2-expressing PBMCs (Figure 16E). Similarly, when NK cells were examined, high-ILT2-expressing PBMCs induced CD69 expression in nearly 90% of cells when treated with the combination therapy, while low-ILT2-expressing PBMCs induced CD69 expression in less than 40% of NK cells (Figure 16F). Therefore, the level of ILT2 expression in PBMCs is essential for the most potent action of the antibody. [Examples]

[0234] Humanized H&N Model in Vivo In a second in vivo model, humanized mice (human CD34+ grafted mice) were implanted with A253-HLA-G cells. When the tumors reached a size of 80 cubic millimeters, the mice were treated with either control IgG or ILT2 antibody (15G8, both at 10 mg / kg). Treatment was repeated twice a week until day 43 (Figure 17A), and tumor size was determined by measuring the tumors by calipers at various time points. In two of the four mice (mice #23 and 28), the ILT2 antibody sufficiently slowed tumor growth, and the tumors were eradicated by day 43 (Figure 17B). To determine whether the different responses to treatment were due to different levels of ILT2 expression in the immune cells of the mice, CD8 T cells from peripheral blood were assayed for ILT2 expression at baseline. Indeed, both mice that achieved a complete response possessed T cells with high ILT2 expression, while the other two mice had significantly lower expression levels (Figure 17C). Furthermore, by examining the TME after treatment, we demonstrated three other pharmacodynamic response markers that distinguish responders from non-responders: CD107A expression in T cells (Figure 17D), M1 / M2 macrophage ratio (Figure 17E), and total CD80-positive dendritic cells (Figure 17F). These results suggest that anti-ILT2 can also lead to shifts in the bone marrow and lymphocyte compartments of the tumor microenvironment, and increase the ability of dendritic cells to present antigens and recruit more T cells to the tumor. [Examples]

[0235] Epitope mapping of 15G8 humanized antibodies The 15G8 antibody was sent for epitope mapping to determine its binding location on ILT2. Mapping was performed by MAbSilico. The structure of the ILT2 used was modeled using the following structures: 6AEE (four Ig-like domains, some loop deletions), 1VDG (unpublished, domains 1 and 2), 1G0X (domains 1 and 2), and 4LL9 (domains 3 and 4). The 6AEE and 1G0X structures were taken from Wang et al., Cell Mol Immunol. (2020) 17(9):966-75, and the 4LL9 structure was taken from Chapman et al., Immunity (2000) 13(5):727-36. Region D1 was defined as residues 24-121 of ILT2. Region D2 was defined as residues 122-222 of ILT2. Region D3 was defined as residues 223-321 of ILT2. Region D4 was defined as residues 322-409 of ILT2. A 3D model of the antibody was constructed using MODELLER.

[0236] Based on the top 30 ranked docking poses, target residues were scored for their likelihood of belonging to an epitope. Residues potentially belonging to an epitope are shown on the sequence in Figure 18A and on the target structure in Figure 18B. From these residues, four major interaction regions are defined on the target (Figure 18C). All four of these interaction regions are found in the interdomain section of ILT2, which is the hinge section between D1 and D2.

[0237] While the binding epitopes of most ILT2 antibodies are not publicly known, International Patent Publication WO2020 / 136145 discloses epitope information for various antibodies. Two common binding regions were found, one within the D1 region and the other within the D4 region. In particular, three antibodies, named 3H5, 12D12, and 27H5, were characterized by loss of binding to mutants with substitutions at E34, R36, Y76, A82, and R84 in D1. One of these antibodies, 3H5, showed reduced binding to mutants with substitutions at G29, Q30, T32, Q33, and D80 in D1. These residues are exclusively located within the D1 region and are all outside the four regions (all within the interdomain) defined as the binding epitopes of the 15G8 antibody (it should be noted that the sequences start one amino acid later, for example, E34 in WO2020 / 136145 is E33 in 18A in Figure 18A). Therefore, antibody 15G8 binds to a different three-dimensional epitope than that of the antibody in publication WO2020 / 136145 (Figure 18D).

[0238] Next, specific epitopes conjugated to the 15G8 antibody were empirically tested. Experimental mapping was performed by Neoproteomics Inc. using hydroxyl radical footprinting (HRF) and mass spectrometry techniques (see Materials and Methods). The overall sequence coverage of ILT2 proteins obtained from both trypsin digestion and dual trypsin and Asp-N digestion was approximately 90.7%. For ILT2 proteins digested by trypsin, a total of 23 peptides were detected by LC-MS and MS / MS analysis. Of these 23 peptides, 20 were observed to be labeled (Table 5; the highest normalized protection ratio (NR) value is shown in bold. Peptide locations and corresponding sequences are shown in columns 1 and 2). For ILT2 proteins digested by trypsin and Asp-N, a total of 15 peptides were detected by LC-MS and MS / MS analysis. Of these 15 peptides, 14 were observed to be labeled (Table 6; the highest normalized protection ratio (NR) value is shown in bold. Bold residues are confirmed to be modified).

[0239] [Table 5-1] [Table 5-2]

[0240] [Table 6]

[0241] The HRF process introduced oxidative modifications to the stable side chains, resulting in specific mass shifts identified from tandem mass spectrometry data. Selected ion chromatograms (SICs) were extracted and incorporated into the unoxidized and all oxidized forms (with specific m / z) of the peptide ions. These peak region values ​​were used to characterize the reaction kinetics in the form of dose-response (DR) plots, measuring the loss of the unmodified peptide in response to hydroxyl radical exposure. Regions protected from the solvent in the complex represent reduced oxidation reactions compared to the same regions in the free protein. Differences in oxidation rates (referred to as rate constants, K) indicate possible locations of binding boundaries.

[0242] K values ​​were calculated for each peptide and specific residue using MS data from a single replication experiment. Tables 5 and 6 show the overall fitting results for all detected peptides, including errors. The third and fourth columns represent the K values ​​of free ILT2 and its complex, respectively. Error bars representing fitting errors are shown next to each K value. The fifth column shows the ratio, R (=KFree / KComplex). The sixth column shows the normalized ratio (NR) calculated as R / ((mean + median) / 2). An R value less than 1 for a given peptide suggests that the corresponding region becomes more solvent-accessible due to a structural change introduced during complex formation. An R value close to 1 indicates that the solvent accessibility of the region remains unchanged, while R>1 suggests that the corresponding region represents protection from the solvent in response to complex formation. However, most R values ​​for the peptides (5th column in Table 5) fell between 0.49 and 3.94, with a mean of 1.37 and a median of 1.5 (values ​​of 3.41, 3.52, and 3.94 were excluded from statistical analysis). Most R values ​​for the peptides (5th column in Table 6) fell between 0.78 and 6, with a mean of 1.48 and a median of 1.58 (values ​​of 5 and 6 were excluded from statistical analysis). Furthermore, Figures 19A and 19B show the distribution of R values ​​for all trypsin peptides and trypsin / Asp-N peptides within ILT2, respectively, which also indicate that the majority of peptides within ILT2 represent changes in modification when forming complexes greater than one. Ratios were normalized to a value of 1 using the mean of the mean and median, with strategies similar to those used in metabolomics (mean scaling, division by central tendency) to correct for abiotic variability between samples. The ratios (NRs) were normalized to the R values ​​obtained from trypsin and trypsin / Asp-N experiments using normalization factors of 1.44 and 1.53, respectively. In these studies, the NR for 2+ was considered to represent significant protection in oxidation during complex formation.

[0243] Overall, Table 5 shows three peptides from ILT2 covering the ILT2 regions 56-71, 57-71, and 84-100, exhibiting the highest protection in the ILT2-15G8 complex at 2.74, 2.44, and 2.35, respectively, compared to ILT2 alone. From Table 6, two peptides covering amino acids 57-66 and 91-100 were found, exhibiting the highest protection at 3.92 and 3.27, respectively. These regions are part of the binding boundary with the 15G8 mAb.

[0244] Figures 19C–19G show individual DR plots for five significant peptides. These illustrate comparative DR plots of the five most protected peptides as a result of complex formation. DR plots for the free ILT2 form and the ILT2-15G8 complex are shown in blue and red, respectively. Solid red and blue lines indicate the best fit to the theoretical linear equation. Tables 5 and 6, as well as Figures 19C–G, show the greatest reduction / protection in solvent accessibility for five peptides covering ILT2 residues 56–71 (SEQ ID NO: 3), 57–71 (SEQ ID NO: 4), 84–100 (SEQ ID NO: 5), 57–66 (SEQ ID NO: 6), and 91–100 (SEQ ID NO: 7). The overall protection levels of peptides 57–66 (NR=3.92), 91–100 (NR=3.27), 56–71 (NR=2.74), 57–71 (NR=2.44), and 84–100 (NR=2.35) demonstrate that these regions are part of the binding boundary with 15G8.

[0245] Interestingly, the region defined as the 15G8 epitope is the interdomain between D1 and D2, and this region has been identified as the primary interaction region of ILT2 that binds to beta-2-microglobulin (B2M) when it is in the form of a complex with HLA (see Kuroki et al., J Immu No. (2019) 203(12): 3386~94) (Figures 18E~18F). In fact, residues G97, A98, Y99, I100, Q125 and V126 were specifically identified by Kuroki et al. (Supplement to Figure S2 in Kuroki) as interacting with B2M. Kuroki's residues 97~100 correspond to residues 96~99 in Tables 5 and 6, which are among the two peptides that interact with 15G8 and are therefore residues of the 15G8 epitope. This strongly suggests that 15G8 inhibits ILT2 binding to HLA in a B2M-dependent manner, effectively blocking direct ILT2 binding to B2M. In contrast, others have reported that antibodies (3H5, 12D12, and 27H5) 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 highly significant, given that Kuroki et al. found that the primary interaction site for ILT2 is the B2M site, and binding to the α3 domain is additional and flexible. This may explain the unique ability of 15G8 to perform T cell, NK cell, and macrophage / dendritic cell functions by blocking the primary interaction site of ILT2 while not blocking secondary sites.

[0246] To test whether 15G8 could block the interaction between ILT2 and B2M, a B2M blocking ELISA was performed. A 96-well nickel plate was coated overnight with recombinant human B2M-His tag (3 μg / ml; Sino Biologics, 11976-H08H). Biotinylated ILT2-Fc (20 μg / ml; Sino Biologics, 1614-H02H; Innova Biosciences, 370-0010) was added at 37°C for 2 hours, thereby binding ILT2 to B2M in the presence or absence of titrated BND-22 or an unrelated antibody (anti-human CD28, Biolegend, 302934) (Ab range of 80-0.1 μg / ml, 3x). ILT2 binding to B2M was detected by HRP-conjugated streptavidin (R&D systems, DY998). Absorbance was measured using an ELISA reader (Biotek Synergy-H1 plate reader).

[0247] As shown in Figure 20, 15G8 blocked the ILT2-B2M interaction, particularly at higher antibody concentrations. In contrast, unrelated antibodies were inactive in this system and did not block this interaction. By directly demonstrating that 15G8 can disrupt the binding between ILT2 and B2M, these results confirm previous observations that the 15G8 epitope contains residues known to be specifically related to the ILT2-B2M interaction.

[0248] The only ILT2 antibody identified as having some effect on phagocytosis is GHI / 75, which was shown to enhance anti-CD47 blockade-mediated phagocytosis of cancer cells, but was not shown to have an effect on its own (see Barkal et al., Nat Immunol. (2018) 19(1):76~84). The combined effects of GHI / 75 and anti-CD47 were found to be non-B2M dependent, as B2M deletion did not have an effect on increased phagocytosis. Therefore, the effect of 15G8 alone on phagocytosis (Figures 13A~13C) may be B2M-dependent, which would explain the unique performance of this antibody. In this regard, the superiority of the 15G8 antibody was directly tested. Exogenous HLA-G expressing A375 or SKMEL28 cancer cells were co-cultured with macrophages in the presence of IgG control, 15G8, or GHI / 75. HP-F1 antibody was also tested in A375 cells. Cancer cell lines stained with PKH67-FITC were incubated with macrophages and stained with eFluor670-APC in the presence of the indicated antibody. Phagocytosis levels were determined by the percentage of double-stained macrophages indicating target cell uptake. The percentage increase in phagocytosis compared to IgG control was calculated. In both cell types, 15G8 increased phagocytosis compared to control (Figures 21A-21B). As expected, neither GHI / 75 nor HP-F1 had any effect on phagocytosis. This confirms that 15G8 is the first anti-ILT2 antibody capable of enhancing phagocytosis as monotherapy.

[0249] This is causing epitope issues with GHI / 75 and other commercially available antibodies. Although the epitopes of these antibodies are not publicly available, we performed competitive ELISA assays to determine whether 15G8 and commercially available antibodies GHI / 75, HP-F1, and MAB20172 (R&D systems, clone 292319) could simultaneously bind to ILT2. In the ILT2-binding ELISA, biotinylated 15G8 antibody was used at a constant concentration (1 μg / mL). GHI / 75, HP-F1, or MAB20172 were added at increasing concentrations, and competition was evaluated. Regardless of the amount of these three antibodies added, none of them competed with 15G8 for binding to ILT2 (Figures 21C-21D). In contrast, when naked (unbiotinylated) 15G8 was added, binding decreased in a dose-dependent manner, as expected. This indicates that GHI / 75, HP-F1, and MAB20172 bind to different epitopes than 15G8. Therefore, 15G8 is the first anti-ILT2 antibody identified to date that binds to this epitope, specifically blocking interaction with B2M and enabling simultaneous activation / recruitment of T cells, NK cells, and macrophages / dendritic cells against cancer. [Examples]

[0250] Screening method for identifying antibodies capable of inhibiting the interaction between ILT2 and B2M B2M is a protein that constitutes some of the other ILT2 ligands in addition to HLA-G. As shown in Example 13, 15G8 has the unique property of binding to ILT2 in a specific region involved in the interaction between ILT2 and B2M. This binding allows 15G8 to inhibit or block the binding of ILT2 to its ligand (e.g., HLA-G), resulting in an immunoactivating effect. As described in Example 13 above, the specific binding region was characterized as being located within the boundary of the D1 and D2 domains of ILT2, and further experimental methods were used to determine that it contains residues in SEQ ID NOs. 68, 69, 70, 71, and 72. The 15G8 binding region was also confirmed using an in silico method (Figure 18A).

[0251] It is expected that other antibodies will be produced that are characterized by binding to similar epitopes and have the ability to specifically block the interaction between ILT2 and B2M. Such antibodies can be induced by methods including immunization of animals with fully recombinant ILT2, or only the D1 and D2 domains including the D1-D2 boundary region, or linear peptides similar to (e.g., containing, including, or identical to) SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and / or SEQ ID NO: 72. In all cases, the induced antibodies will be further screened for their ability to block ILT2-B2M binding. The induced antibodies can be screened for specific binding to the B2M binding region in ILT2 using experimental techniques such as hydroxyl radical footprinting (HRF) and mass spectrometry techniques as described above ("Materials and Methods"), or other similar known techniques for epitope binding determination such as hydrogen-deuterium exchange (HDX) further combined with mass spectrometry.

[0252] Similarly, a naive antibody library can be screened for antibodies that bind to either recombinant ILT2 or ILT2 expressed on cells. Screening can be performed, for example, for binding to ILT2 fragments consisting of D1 and D2 domains, including the D1-D2 boundary region. Screening can also be performed for binding to linear peptides similar to (e.g., containing, including, or identical to) SEQ ID NOs. 68, 69, 70, 71, and / or SEQ ID NOs. 72, or to polypeptides consisting of these two peptides and amino acids present between them in the ILT2 sequence. Antibodies representing binding to these sequences may be further screened using different experimental epitope mapping techniques, as described above.

[0253] In some cases, screening may include evaluating candidate antibodies for their ability to specifically block ILT2-B2M interactions using assays to detect whether ILT2 blocks binding to B2M or a different protein containing a B2M moiety, whether recombinant or expressed in cells. In some cases, screening may include evaluating candidate antibodies for their ability to specifically compete with 15G8 antibodies that have been shown to block ILT2 binding to B2M.

[0254] The above-mentioned descriptions of specific embodiments are expected to fully illustrate the overall nature of the disclosure of the present invention so that others may readily modify and / or adapt such specific embodiments for various applications without extraneous experimentation and without departing from the general concept, by applying current knowledge. Therefore, such applications and modifications should and are intended to be understood in the sense and scope of equivalents of the disclosed embodiments. It will be understood that the expressions or terminology used herein are for illustrative purposes only and not for limiting purposes. Means, materials, and processes for performing various disclosed functions may take various alternative forms without departing from the disclosure of the present invention.

[0255] Throughout this specification and its embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” shall be understood to mean including the integer or group of integers stated, but not excluding any other integer or group of integers. “Approximately,” when referring to a measurable value, such as a quantity or a period of time, means to include a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, where the variation is appropriate for performing the disclosed method. Furthermore, unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms.

[0256] [Table 7]

Claims

1. A method for identifying an antibody that competes with a reference antibody containing heavy chains and light chains of sequence numbers 48 and 49, respectively, for binding to ILT2, Contacting an antibody library with a polypeptide sequence containing an ILT2 sequence selected from SEQ ID NOs. 68-70, and From the library, select an antibody that binds to the ILT2 sequence, thereby obtaining an antibody that competes with the reference antibody for binding to ILT2. Methods that include...

2. The method according to claim 1, wherein the antibody that binds to the ILT2 sequence binds to one or both of sequence numbers 71 and 72.

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