Antibodies against ilt2 and uses thereof

Abstract

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

Description

[0001] This invention application is a divisional application of the invention patent application filed on August 12, 2020, with application number 202080070248.9 (international application number PCT / IL2020 / 050889) and titled "Antibody against ILT2 and its use therein".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 034,569, filed June 4, 2020, and U.S. Provisional Patent Application No. 62 / 885,374, filed August 12, 2019, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] This invention pertains to the field of monoclonal antibodies and the regulation of immune responses against cancer. Background Technology

[0005] ILT2, also known as LILRB1, LIR1, and CD85j, is a cell surface protein expressed on immune cells with known functions of suppressing immune responses. This protein contains four IgC domains in its extracellular region 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, sharing approximately 80% homology. Known ligands for ILT2 include MHC-1 and non-classical MHC molecules such as HLA-F, HLA-G, HLA-B27, and UL18 (human CMV). The strongest known interactor for ILT2 in the human genome is HLA-G1.

[0006] HLA-G1 is widely expressed on the surface of various malignant tumors, including breast cancer, cervical cancer, CRC, lung cancer, gastric cancer, pancreatic cancer, thyroid cancer, and ovarian cancer cells, as well as glioblastoma and melanoma cells. Its expression is associated with poorer clinical outcomes. Furthermore, ILT2 expression in the tumor microenvironment is associated with poorer clinical responses to oncolytic immunotherapy, even in the absence of HLA-G1. Utilizing the immune response as a weapon against cancer and for cancer surveillance is a promising approach for cancer prevention and treatment. However, ILT2 presents as a barrier to effective immunotherapy. There is a strong need for therapeutic approaches that can circumvent the ILT2-HLA-G1 axis and address the HLA-G1-independent functions of ILT2. Summary of the Invention

[0007] This invention provides monoclonal antibodies that bind to ILT2 and inhibit ILT2-mediated immunosuppression; and pharmaceutical compositions comprising said monoclonal antibodies. It also provides methods for treating cancer including administering the compositions of this invention, methods for generating the antibodies, binding fragments, and compositions of this invention, and methods for enhancing the efficacy of PD-1 / PD-L1-based therapies.

[0008] According to a first aspect, a monoclonal antibody or antigen-binding fragment is provided, said monoclonal antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0009] a. CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 13 (SGYYWN), CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 14 (YISYDGSNNYNPSLKN), CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 15 (GYSYYYAMDX), CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 16 (RTSQDISNYLN), CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 17 (YTSRLHS), and CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 18 (QQGNTLPT), wherein X is selected from A, C, and S;

[0010] b. CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 1 (DHTIH), CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 2 (YIYPRDGSTKYNEKFKG), CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 3 (TWDYFDY), CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 4 (RASESVDSYGNSFMH), CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 5 (RASNLES), and CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 6 (QQSNEDPYT); or

[0011] c. CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 7 (GYTFTSYGIS), CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 8 (EIYPGSGNSYYNEKFKG), CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 9 (SNDGYPDY), CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 10 (KASDHINNWLA), CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 11 (GATSLET), and CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 12 (QQYWSTPWT).

[0012] According to some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain containing an amino acid sequence selected from the following: SEQ ID NO: 19 (QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARTWDYFDYWGQGTTLTVSS), SEQ ID NO: 21 (QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWVGEIYPGSGNSYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARSNDGYPDYWGQGTTLTVSS), and SEQ ID NO: 23 (DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDXWGQGTSVTVSS), wherein X is selected from A, C and S.

[0013] According to some embodiments, the antibody or antigen-binding fragment of the present invention comprises a light chain containing an amino acid sequence selected from the following: SEQ ID NO: 20 (DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIK), SEQ ID NO: 22 (DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK), SEQ ID NO: 24 (DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTFGQGTKLEIK), and SEQ ID NO: 45 (DIQMTQTTSSLSASLGDRVTISCRTSQDISNYLNWYQQKPDGTVKLLISYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPTFGSGTKLEIK).

[0014] According to some implementation schemes, the antibody or antigen-binding fragment is humanized and the X is selected from A and S.

[0015] According to some embodiments, X is A, SEQ ID NO: 15 is GYSYYYAMDA (SEQ ID NO: 25) and SEQ ID NO: 23 is DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDAWGQGTSVTVSS (SEQ ID NO: 28).

[0016] According to another aspect, there is provided a monoclonal antibody or antigen-binding fragment that binds to an epitope of human leukocyte immunoglobulin-like receptor subfamily B member 1 (ILT2), said epitope being selected from the human ILT2 sequence: VKKGQFPIPSITWEH (SEQ ID NO: 41), LELVVTGAYIKPTLS (SEQ ID NO: 42), VILQCDSQVAFDGFS (SEQ ID NO: 43), and WYRCYAYDSNSPYEW (SEQ ID NO: 44).

[0017] According to some implementations, the epitope is a three-dimensional epitope comprising SEQ ID NO: 41, 42, 43 and 44.

[0018] According to some embodiments, a monoclonal antibody or antigen-binding fragment is provided that binds to ILT2 and inhibits the direct interaction between ILT2 and β-2 microglobulin (B2M).

[0019] According to some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and HLA or MHC-I proteins by inhibiting the direct interaction between ILT2 and B2M.

[0020] According to some implementation schemes, the HLA is HLA-G.

[0021] According to another aspect, a monoclonal antibody or antigen-binding fragment is provided that binds to ILT2 and induces at least three of the following in a subject with cancer:

[0022] a. Increased cytotoxicity of natural killer (NK) cells;

[0023] b. Increased T cell cytotoxicity, proliferation, or both;

[0024] c. Increased macrophage phagocytosis, increased production of M1 inflammatory macrophages, decreased production of M2 suppressive macrophages, or a combination thereof; and

[0025] d. Increased dendritic cell homing to cancerous tumors, increased dendritic cell activation, or a combination thereof.

[0026] According to some implementation schemes, the cancer is a cancer that expresses HLA-G or MHC-I.

[0027] According to some embodiments, the antibody or antigen-binding fragment of the present invention is used for at least one of the following: binding to ILT2, inducing / enhancing anti-tumor T cell responses, increasing T cell proliferation, reducing cancer-induced myelosuppressive activity, increasing the cytotoxicity of natural killer cells, increasing macrophage phagocytosis, increasing the generation of M1 inflammatory macrophages, reducing the generation of M2 suppressive macrophages, increasing the number of dendritic cells in the tumor microenvironment, increasing dendritic cell activation, treating cancers expressing HLA-G, and treating cancers expressing MHC-I.

[0028] According to some embodiments, the antibody or antigen-binding fragment of the present invention, in combination with an opsonizer, is used to treat cancers expressing HLA-G or MHC-I.

[0029] According to some embodiments, the antibody or antigen-binding fragment of the present invention is combined with an anti-PD-L1 / PD-1-based therapy for the treatment of cancers expressing HLA-G or MHC-I.

[0030] According to another aspect, a method is provided for treating a subject with cancer expressing HLA-G or MHC-I, the method comprising administering to the subject a pharmaceutical composition comprising an antibody or antigen-binding fragment of the present invention.

[0031] According to some embodiments, the method of the present invention further includes administering a conditioning agent to the subject.

[0032] According to some embodiments, the opsonizer is an EGFR inhibitor, optionally wherein the EGFR inhibitor is cetuximab.

[0033] According to some embodiments, the method of the present invention further includes administering an anti-PD-L1 / PD-1-based immunotherapy to the subject.

[0034] According to another aspect, a method is provided for treating HLA-G or MHC-I-expressing cancers in a subject of need, the method comprising:

[0035] a. Confirm that the expression of ILT2 or soluble HLA-G in the subjects is higher than a predetermined threshold; and

[0036] b. Administer to the subject an agent that inhibits ILT2-based immunosuppression; thereby treating the subject's cancer.

[0037] According to some implementation schemes, the confirmation includes measuring the expression of ILT2 or soluble HLA-G in the subject prior to the administration.

[0038] According to some embodiments, the method of the present invention includes confirming the expression of ILT2, wherein the expression of ILT2 is in the immune cells of the subject.

[0039] According to some implementation schemes, the immune cells are selected from peripheral blood immune cells and tumor-associated immune cells.

[0040] According to some implementation schemes, the immune cells are selected from CD8-positive T cells, macrophages, NK cells, and TEMRA cells.

[0041] According to some implementation schemes, the immune cells are peripheral blood CD8-positive T cells.

[0042] According to some embodiments, the method of the present invention includes confirming the expression of soluble HLA-G.

[0043] According to some embodiments, the method of the present invention further includes administering an anti-PD-L1 / PD-1-based therapy to the subject.

[0044] According to another aspect, a method is provided for treating HLA-G or MHC-I-expressing cancers in a subject of need, the method comprising:

[0045] a. Administering to the subject an agent that inhibits ILT2-based immunosuppression; and

[0046] b. Administer anti-PD-L1 / PD-1-based therapy to the subjects;

[0047] This allows them to treat the cancer in the test subjects.

[0048] According to another aspect, a method is provided to increase the efficacy of anti-PD-L1 / PD-1-based therapies against cancer cells expressing HLA-G, MHC-I, or both, the method comprising contacting the cancer cells with an ILT2 antagonist.

[0049] According to some implementation schemes, the agent that inhibits ILT2-based immunosuppression is an ILT2 antagonist.

[0050] According to some implementation schemes, the ILT2 antagonist is an antibody or antigen-binding fragment that specifically binds to ILT2 and inhibits ILT2-mediated immune cell suppression.

[0051] According to some implementation schemes, the antibody or antigen-binding fragment of the method is an antibody or antigen-binding fragment as described herein.

[0052] According to some implementation schemes, the anti-PD-L1 / PD-1-based immunotherapy is an anti-PD-1 blocking antibody.

[0053] According to some implementation schemes, the cancer is refractory to anti-PD-L1 / PD-1 therapy.

[0054] According to some embodiments, the method of the present invention further includes administering a conditioning agent to the subject.

[0055] According to some embodiments, the opsonizer is an EGFR inhibitor, optionally wherein the EGFR inhibitor is cetuximab.

[0056] According to another aspect, a pharmaceutical composition comprising an agent that binds to ILT2 and inhibits ILT2-mediated immunosuppression is provided, said pharmaceutical composition being combined with an anti-PD-L1 / PD-1 based therapy for treating a subject with cancer.

[0057] According to another aspect, a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention is provided.

[0058] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0059] Obtain an agent that binds to the extracellular domain of ILT2 or a fragment thereof, and test the agent's ability to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and select at least one agent that induces at least two of the following: the increased phagocytosis, the increased activity, the increased production, the decreased production, the recruitment, the increased activation, the decreased activity, and the increased cytotoxicity; or

[0060] Culture host cells containing one or more vectors that encode a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected in the following manner:

[0061] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0062] ii. Test the ability of the agent to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and

[0063] iii. Select at least one agent that increases at least two of the following: increased phagocytosis, increased activity, increased production, decreased production, recruitment, increased activation, decreased activity, and increased cytotoxicity;

[0064] This produces the medicine.

[0065] According to some embodiments, the method of the present invention includes testing the ability of the agent to induce at least three of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased generation of M1 macrophages, decreased generation of M2 macrophages, increased recruitment of dendritic cells to the tumor microenvironment, increased dendritic cell activation, and increased cytotoxicity against natural killer (NK) cells, and selecting at least one agent that induces at least three of the following.

[0066] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0067] Obtaining an agent that binds to the extracellular domain of ILT2 or a fragment thereof, testing the ability of said agent to enhance the efficacy of anti-PD-L1 / PD-1 based therapies against cancer cells, and selecting at least one agent that enhances the efficacy of anti-PD-L1 / PD-1 based therapies; or culturing host cells comprising one or more vectors containing a nucleic acid sequence encoding an agent, wherein said nucleic acid sequence is a nucleic acid sequence of an agent selected in such a manner as:

[0068] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0069] ii. To test the ability of the said agent to enhance the efficacy of anti-PD-L1 / PD-1 based therapies against cancer cells; and

[0070] iii. Select at least one agent that enhances the efficacy of anti-PD-L1 / PD-1-based therapies against cancer cells;

[0071] This produces the medicine.

[0072] According to some embodiments, the increased efficacy includes a synergistic increase in the secretion of pro-inflammatory cytokines, or the increased cytotoxicity includes an increase in the secretion of pro-inflammatory cytokines.

[0073] According to some implementation schemes, the pro-inflammatory cytokines are selected from GM-CSF, TNFα, and IFNγ.

[0074] According to some implementation schemes, the increased efficacy includes an increase in T cell activation, cytotoxicity, or a synergistic increase in both.

[0075] According to some implementation schemes, the increase in T cell activation, cytotoxicity, or both includes increased membrane CD107a expression.

[0076] According to some implementations, the increased efficacy includes converting cancers refractory to anti-PD-L1 / PD-1-based therapies into cancers responsive to anti-PD-L1 / PD-1-based therapies.

[0077] According to some implementation schemes, the cancer cells are cancers that express HLA-G or MHC-I.

[0078] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0079] Obtain an agent that binds to the extracellular domain of ILT2 or a fragment thereof, test the ability of said agent to inhibit the interaction between ILT2 and B2M, and select at least one agent that inhibits the interaction between ILT2 and B2M; or culture host cells containing one or more vectors encoding a nucleic acid sequence of an agent, wherein said nucleic acid sequence is a nucleic acid sequence of an agent selected in the following manner:

[0080] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0081] ii. Test the ability of the agent to inhibit the interaction between ILT2 and B2M; and

[0082] iii. Select at least one agent that inhibits the interaction between ILT2 and B2M;

[0083] This produces the medicine.

[0084] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0085] Obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO: 41, 42, 43 and 44, or culturing host cells containing one or more vectors with a nucleic acid sequence encoding the drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected by obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO: 41, 42, 43 and 44; thereby producing a drug.

[0086] According to another aspect, a nucleic acid molecule encoding an antibody or antigen-binding fragment of the present invention is provided.

[0087] According to some implementation schemes, the nucleic acid molecule is an expression vector.

[0088] Specifically, the present invention includes, but is not limited to, the following:

[0089] 1. A monoclonal antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0090] a. CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 13 (SGYYWN), CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 14 (YISYDGSNNYNPSLKN), CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 15 (GYSYYYAMDX), CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 16 (RTSQDISNYLN), CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 17 (YTSRLHS), and CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 18 (QQGNTLPT), wherein X is selected from A, C, and S;

[0091] b. CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 1 (DHTIH), CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 2 (YIYPRDGSTKYNEKFKG), CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 3 (TWDYFDY), CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 4 (RASESVDSYGNSFMH), CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 5 (RASNLES), and CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 6 (QQSNEDPYT); or

[0092] c. CDR-H1 contains the amino acid sequence shown in SEQ ID NO: 7 (GYTFTSYGIS), CDR-H2 contains the amino acid sequence shown in SEQ ID NO: 8 (EIYPGSGNSYYNEKFKG), CDR-H3 contains the amino acid sequence shown in SEQ ID NO: 9 (SNDGYPDY), CDR-L1 contains the amino acid sequence shown in SEQ ID NO: 10 (KASDHINNWLA), CDR-L2 contains the amino acid sequence shown in SEQ ID NO: 11 (GATSLET), and CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 12 (QQYWSTPWT).

[0093] 2. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain containing an amino acid sequence selected from the following: SEQ ID NO: 19 (QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARTWDYFDYWGQGTTLTVSS), SEQ ID NO: 21 (QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWVGEIYPGSGNSYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARSNDGYPDYWGQGTTLTVSS), and SEQ ID NO: 23 (DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDXWGQGTSVTVSS), wherein X is selected from A, C and S.

[0094] 3. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment comprises a light chain containing an amino acid sequence selected from the following: SEQ ID NO: 20 (DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIK), SEQ ID NO: 22 (DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK), SEQ ID NO: 24 (DIQMTQSPSSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTFGQGTKLEIK) and SEQ ID NO: 45 (DIQMTQTTSSLSASLGDRVTISCRTSQDISNYLNWYQQKPDGTVKLLISYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPTFGSGTKLEIK).

[0095] 4. An antibody or antigen-binding fragment according to any one of items 1 to 3, wherein the antibody or antigen-binding fragment is humanized and the X is selected from A and S.

[0096] 5. The antibody or antigen-binding fragment according to any one of items 4, wherein X is A, SEQ ID NO: 15 is GYSYYYAMDA (SEQ ID NO: 25) and SEQ ID NO: 23 is DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDAWGQGTSVTVSS (SEQ ID NO: 28).

[0097] 6. A monoclonal antibody or antigen-binding fragment that binds to an epitope of human leukocyte immunoglobulin-like receptor subfamily B member 1 (ILT2), said epitope being selected from the following human ILT2 sequences: VKKGQFPIPSITWEH (SEQ ID NO:41), LELVVTGAYIKPTLS (SEQ ID NO:42), VILQCDSQVAFDGFS (SEQ ID NO:43), and WYRCYAYDSNSPYEW (SEQ ID NO:44).

[0098] 7. The antibody or antigen-binding fragment according to claim 6, wherein the epitope is a three-dimensional epitope comprising SEQ ID NO: 41, 42, 43 and 44.

[0099] 8. A monoclonal antibody or antigen-binding fragment that binds to ILT2 and inhibits the direct interaction between ILT2 and β-2 microglobulin (B2M).

[0100] 9. The antibody or antigen-binding fragment according to claim 8, wherein the antibody or antigen-binding fragment inhibits the interaction of ILT2 with HLA protein or MHC-I protein by inhibiting the direct interaction of ILT2 with B2M.

[0101] 10. The antibody or antigen-binding fragment according to item 9, wherein the HLA is HLA-G.

[0102] 11. A monoclonal antibody or antigen-binding fragment, said monoclonal antibody or antigen-binding fragment binding to ILT2 and inducing at least three of the following in a subject with cancer:

[0103] a. Increased cytotoxicity of natural killer (NK) cells;

[0104] b. Increased T cell cytotoxicity, proliferation, or both;

[0105] c. Increased macrophage phagocytosis, increased production of M1 inflammatory macrophages, decreased production of M2 suppressive macrophages, or a combination thereof; and

[0106] d. Increased dendritic cell homing to the tumor of the cancer, increased dendritic cell activation, or a combination thereof.

[0107] 12. The antibody or antigen-binding fragment according to item 11, wherein the cancer is a cancer expressing HLA-G or MHC-I.

[0108] 13. An antibody or antigen-binding fragment according to any one of items 1 to 12, which is used for at least one of: binding ILT2, inducing / enhancing anti-tumor T cell responses, increasing T cell proliferation, reducing cancer-induced myelosuppressive activity, increasing the cytotoxicity of natural killer cells, increasing macrophage phagocytosis, increasing the generation of M1 inflammatory macrophages, reducing the generation of M2 suppressive macrophages, increasing the number of dendritic cells in the tumor microenvironment, increasing dendritic cell activation, treating cancers expressing HLA-G, and treating cancers expressing MHC-I.

[0109] 14. An antibody or antigen-binding fragment according to any one of items 1 to 13, wherein the antibody or antigen-binding fragment is combined with an opsonizer for the treatment of cancers expressing HLA-G or MHC-I.

[0110] 15. An antibody or antigen-binding fragment according to any one of items 1 to 14, said antibody or antigen-binding fragment in combination with an anti-PD-L1 / PD-1 based therapy for the treatment of cancers expressing HLA-G or MHC-I.

[0111] 16. A method for treating a subject with cancer expressing HLA-G or MHC-I, the method comprising administering to the subject a pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 15.

[0112] 17. The method according to claim 16, further comprising administering a conditioning agent to the subject.

[0113] 18. The method according to item 14 or 17, wherein the opsonizing agent is an EGFR inhibitor, optionally wherein the EGFR inhibitor is cetuximab.

[0114] 19. The method according to item 17 or 18, the method further comprising administering an anti-PD-L1 / PD-1-based immunotherapy to the subject.

[0115] 20. A method for treating a subject with cancer expressing HLA-G or MHC-I, the method comprising:

[0116] a. Confirm that the expression of ILT2 or soluble HLA-G in the subjects is higher than a predetermined threshold; and

[0117] b. Administer to the subject an agent that inhibits ILT2-based immunosuppression; thereby treating the subject's cancer.

[0118] 21. The method according to item 20, wherein the confirmation includes measuring the expression of ILT2 or soluble HLA-G in the subject prior to the administration.

[0119] 22. The method according to item 20 or 21, the method comprising confirming the expression of ILT2 and wherein the expression of ILT2 is in the immune cells of the subject.

[0120] 23. The method according to item 22, wherein the immune cells are selected from peripheral blood immune cells and tumor-associated immune cells.

[0121] 24. The method according to item 22 or 23, wherein the immune cells are selected from CD8-positive T cells, macrophages, NK cells, and T cells. EMRA cell.

[0122] 25. The method according to any one of items 22 to 24, wherein the immune cells are peripheral blood CD8 positive T cells.

[0123] 26. The method according to any one of items 20 to 25, wherein the method includes confirming the expression of soluble HLA-G.

[0124] 27. The method according to any one of claims 20 to 26, the method further comprising administering an anti-PD-L1 / PD-1-based therapy to the subject.

[0125] 28. A method for treating a subject with cancer expressing HLA-G or MHC-I, the method comprising:

[0126] a. Administering to the subject an agent that inhibits ILT2-based immunosuppression; and

[0127] b. Administer anti-PD-L1 / PD-1-based therapy to the subjects;

[0128] This allows them to treat the cancer in the test subjects.

[0129] 29. A method for increasing the efficacy of anti-PD-L1 / PD-1-based therapies against cancer cells expressing HLA-G, MHC-I, or both, the method comprising contacting the cancer cells with an ILT2 antagonist.

[0130] 30. The method according to any one of claims 20 to 28, wherein the agent inhibiting ILT2-based immunosuppression is an ILT2 antagonist.

[0131] 31. The method according to item 29 or 30, wherein the ILT2 antagonist is an antibody or antigen-binding fragment that specifically binds to ILT2 and inhibits ILT2-mediated immune cell suppression.

[0132] 32. The method according to claim 31, wherein the antibody or antigen-binding fragment is an antibody or antigen-binding fragment according to any one of claims 1 to 15.

[0133] 33. The method according to any one of items 27 to 32, wherein the anti-PD-L1 / PD-1-based immunotherapy is an anti-PD-1 blocking antibody.

[0134] 34. The method according to any one of items 20 to 33, wherein the cancer is refractory to anti-PD-L1 / PD-1 therapy.

[0135] 35. The method according to any one of claims 20 to 34, the method further comprising administering a conditioning agent to the subject.

[0136] 36. The method according to claim 35, wherein the opsonizing agent is an EGFR inhibitor, optionally wherein the EGFR inhibitor is cetuximab.

[0137] 37. A pharmaceutical composition comprising an agent that binds to ILT2 and inhibits ILT2-mediated immune cell suppression, said pharmaceutical composition in combination with an anti-PD-L1 / PD-1 based therapy for treating a subject with cancer.

[0138] 38. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 15.

[0139] 39. A method for producing a pharmaceutical agent, the method comprising:

[0140] Obtain an agent that binds to the extracellular domain of ILT2 or a fragment thereof, and test the agent's ability to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and select at least one agent that induces at least two of the following: the increased phagocytosis, the increased activity, the increased production, the decreased production, the recruitment, the increased activation, the decreased activity, and the increased cytotoxicity; or

[0141] Culture host cells containing one or more vectors that encode a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected in the following manner:

[0142] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0143] ii. Test the ability of the agent to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and

[0144] iii. Select at least one agent that increases at least two of the following: increased phagocytosis, increased activity, increased production, decreased production, recruitment, increased activation, decreased activity, and increased cytotoxicity;

[0145] This produces the medicine.

[0146] 40. The method according to claim 39, the method comprising testing the ability of the agent to induce at least three of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased generation of M1 macrophages, decreased generation of M2 macrophages, increased recruitment of dendritic cells to the tumor microenvironment, increased dendritic cell activation, and increased cytotoxicity against natural killer (NK) cells, and selecting at least one agent that induces at least three of the following.

[0147] 41. A method for producing a pharmaceutical agent, the method comprising:

[0148] Obtaining an agent that binds to the extracellular domain of ILT2 or a fragment thereof, testing the ability of said agent to enhance the efficacy of anti-PD-L1 / PD-1 based therapies against cancer cells, and selecting at least one agent that enhances the efficacy of anti-PD-L1 / PD-1 based therapies; or culturing host cells comprising one or more vectors containing a nucleic acid sequence encoding an agent, wherein said nucleic acid sequence is a nucleic acid sequence of an agent selected in such a manner as:

[0149] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0150] ii. To test the ability of the said agent to enhance the efficacy of anti-PD-L1 / PD-1 based therapies against cancer cells; and

[0151] iii. Select at least one agent that enhances the efficacy of anti-PD-L1 / PD-1-based therapies against cancer cells;

[0152] This produces the medicine.

[0153] 42. The method according to any one of claims 39 to 41, wherein the increased efficacy comprises a synergistic increase in the secretion of pro-inflammatory cytokines, or wherein the increased cytotoxicity comprises an increase in the secretion of pro-inflammatory cytokines.

[0154] 43. The method according to item 42, wherein the pro-inflammatory cytokine is selected from GM-CSF, TNFα, and IFNγ.

[0155] 44. The method according to any one of claims 41 to 43, wherein the increased efficacy includes an increase in T cell activation, cytotoxicity, or a synergistic increase in both.

[0156] 45. The method according to any one of items 39, 40 and 44, wherein the increase in T cell activation, cytotoxicity or both comprises increased membrane CD107a expression.

[0157] 46. ​​The method according to any one of claims 41 to 45, wherein the increased efficacy comprises converting cancer refractory to anti-PD-L1 / PD-1-based therapies into cancer responsive to anti-PD-L1 / PD-1-based therapies.

[0158] 47. The method according to any one of claims 39 to 46, wherein the cancer cells are cancers expressing HLA-G or MHC-I.

[0159] 48. A method for producing a pharmaceutical agent, the method comprising:

[0160] Obtain an agent that binds to the extracellular domain of ILT2 or a fragment thereof, test the ability of said agent to inhibit the interaction between ILT2 and B2M, and select at least one agent that inhibits the interaction between ILT2 and B2M; or culture host cells containing one or more vectors encoding a nucleic acid sequence of an agent, wherein said nucleic acid sequence is a nucleic acid sequence of an agent selected in the following manner:

[0161] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0162] ii. Test the ability of the agent to inhibit the interaction between ILT2 and B2M; and

[0163] iii. Select at least one agent that inhibits the interaction between ILT2 and B2M;

[0164] This produces the medicine.

[0165] 49. A method for producing a pharmaceutical agent, the method comprising:

[0166] Obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO: 41, 42, 43 and 44, or culturing host cells containing one or more vectors with a nucleic acid sequence encoding the drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected by obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO: 41, 42, 43 and 44; thereby producing a drug.

[0167] 50. A nucleic acid molecule that encodes an antibody or antigen-binding fragment according to any one of claims 1 to 15.

[0168] 51. The nucleic acid molecule according to claim 50, wherein the nucleic acid molecule is an expression vector.

[0169] Other embodiments of the invention and its full scope will become clear from the detailed description given below. However, it should be understood that while the detailed description and specific examples indicate preferred embodiments of the invention, they are given in an illustrative manner, as various variations and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. Attached Figure Description

[0170] Figure 1 Histograms depicting ILT2 expression on lymphocytes were plotted. Commercial antibody #1 was used at a final concentration of 5 µg / mL. Bindings were plotted as black histograms, while isotype controls were stained and displayed as light gray histograms.

[0171] Figure 2 Histograms depicting ILT2 expression on various immune cells were plotted. Commercial antibody #1 was used at a final concentration of 5 µg / mL. Bindings were plotted as black histograms, while isotype controls were stained and displayed as light gray histograms.

[0172] Figures 3A-3C ( Figure 3A A table of cancer indications from the TCGA database, in which ILT2 RNA is overexpressed. Figure 3B A dot plot showing the association between MDSC enrichment and ILT2 expression in tumors. A bar plot depicting the association between M2 enrichment and ILT2 expression is also presented. Figure 3C Scatter plot of the percentage of various immune cells expressing ILT2 in different tumors.

[0173] Figures 4A-4B ( Figure 4A A bar graph showing the percentage of various cancers that are HLA-G positive, as determined by immunohistochemistry (IHC). Figure 4BScatter plot of HLA-G IHC scores for various cancers.

[0174] Figure 5 Scatter plot of soluble HLA-G levels in various cancers.

[0175] Figure 6 The heavy and light chain sequences of the three anti-ILT2 antibodies. CDRs identified by the KABAT system are underlined or shown in red.

[0176] Figures 7A-7E ( Figure 7A A table showing antibody binding values ​​with ILT2 and members of the ILT2 family. Figure 7B Histogram of antibody binding to ILT2 on the surface of BW cells transfected with human ILT2. Figure 7C Line graphs showing the binding of chimeric and humanized 19E3 (left panel) and chimeric and humanized 15G8 (right panel) to ILT2 expressed on the surface of BW cells transfected with human ILT2. Figure 7D Immunostaining of gastric cancer samples using 19E3 antibody. Figure 7E A scatter plot showing the percentage of various immune cells expressing ILT2 in PBMC samples from healthy controls and cancer patients using the 15G8 humanized antibody.

[0177] Figures 8A-8P ( Figure 8A Bar graph showing the percentage of blockade for each ILT2 antibody and positive control (PC, GHI / 75 antibody). Figure 8B Histogram showing the binding of ILT2-biotin to HLA-G-expressing cells in the presence of ILT2 blocking antibody. ILT2-biotin binding to cells was determined by flow cytometry using streptavidin-PE. No antibody (grey line), 15G8 (light gray line), allotype control (black line). Figure 8C (e.g., a line graph illustrating the blocking activity of a humanized 15G8 antibody determined by the binding of ILT2-biotin to HLA-G-expressing cells.) Figure 8D Line graphs illustrating the blocking activities of chimeric and humanized 19E3 (left panel) and chimeric and humanized 15G8 (right panel), as determined by the binding of ILT2-biotin to HLA-G-expressing cells in the presence of antibodies. Figure 8E Bar graph showing mouse IL-2 secretion from cells expressing an ILT2 signaling reporter construct in the presence of HLA-G-expressing cells and with or without ILT2 blocking antibodies. PC = positive control (GHI / 75 antibody). Figure 8F (e.g., a line graph showing the blocking activity of the 15G8 humanized antibody as determined by reporter assay.) Figure 8G Bar graph showing mouse IL-2 secretion from cells expressing an ILT2 signaling reporter construct, with or without ILT2 blocking antibodies and positive control antibodies. Figures 8H-8K ) from the presence or absence of ILT2 blocking antibodies and positive controls ( Figures 8I-8J Pan-HLA antibodies or ( Figure 8K In the case of HLA-G specific antibodies, and ( Figure 8I ) Only has MHC-I expression or ( Figures 8J-8K A375 cancer cells expressing both MHC-I and exogenous HLA-G were co-cultured. Figure 8H ) lacking ILT2 or ( Figures 8I-8K Bar graph showing human IL-2 secretion from Jurkat cells expressing ILT2. Figure 8L-Figure 8N ) comes from existence or non-existence ( Figure 8L )15G8 antibody, ( Figure 8M GHI / 75 antibody and ( Figure 8N Bar graph of human IL-2 secretion in Jurakt cells expressing ILT2, cultured together with HLA-G-expressing A375 cancer cells in the presence of HP-F1 antibody. Figures 8O-8P TIL cells and NK cells, incubated with HLA-G positive cancer cells in the presence and absence of 15G8 antibody, respectively, activated the marker ( Figure 8O Phosphorylated ZAP70 and ( Figure 8P Dot plot of phosphorylated Syk expression.

[0178] Figures 9A-9D ( Figure 9A As determined by an FACS-based method, phagocytosis was measured as a bar graph of the percentage of HLA-G-expressing cancer cells co-cultured with macrophages in the presence of ILT2 antibody relative to controls. Figure 9B ) such as through Incucyte ® A line graph showing the real-time phagocytic activity of macrophages against cancer cells in the presence of ILT2 antibody, as determined by the system. Figure 9C Phagocytosis was measured as a bar graph showing the percentage of various HLA-G and MHC-I-expressing cancer cells relative to controls co-cultured with macrophages in the presence of ILT2 antibody 15G8. Figure 9D Bar graph showing the phagocytic activity of macrophages co-cultured with A253-HLA-G cells in the presence of ILT2 antibody, Erbitux, hIgG control, or a combination thereof.

[0179] Figures 10A-10B .From the presence of ILT2 antibody and ( Figure 10AWild-type 721.221 cells or 721.221 cells expressing HLA-G or ( Figure 10B Bar graph showing IFNγ and granzyme B secretion from activated CD8 T cells co-cultured with HLA-G-expressing A375 cells.

[0180] Figure 11A-11H ( Figures 11A-11B ) comes from the expression of ILT2 in the presence of ILT2 antibody ( Figure 11A HLA-G and ( Figure 11B (Bar graph showing the percentage of cytotoxicity in NK cell lines co-cultured with various MHC-I cancer cell lines.) Figures 11C-11D () Figure 11C Granzyme B from NK cell lines co-cultured with H&N cancer cells and melanoma cells in the presence of 15G8 ILT2 antibody, respectively. Figure 11D Bar graph of IFNγ secretion. Figures 11E-11F ILT2-positive primary NK cells co-incubated with target cancer cells in the presence of ILT2 antibody ( Figure 11E IFNγ expression and ( Figure 11F Bar graph of CD107A expression. Figures 11G-11H A scatter plot showing the expression of ILT2 alone, which contrasts ILT2-positive cells with those responding to ILT2 antibodies. Figure 11G IFNγ expression and ( Figure 11H The association of CD107A expression.

[0181] Figure 12 Linear graphs of HLA-DR and CD80 expression (MFI) in macrophages as determined by flow cytometry, where the macrophages differentiated into M0, M1, or M2 macrophages from monocytes isolated from healthy donors in the presence of IgG or anti-ILT2 antibody. The number of patients with increased expression of the specified marker compared to control IgG is shown for each condition tested.

[0182] Figures 13A-13C ( Figure 13A Bar graph showing the phagocytic activity of macrophages co-cultured with various primary tumor cells. Figures 13B-13C In the presence of the humanized antibody of the present invention, autologous macrophages react with ( Figure 13B RCC patients and ( Figure 13C A bar graph showing the dose-dependent phagocytosis of primary tumor cells isolated from H&N patients.

[0183] Figures 14A-14L ( Figure 14A Dot plots of ILT2 and PD-1 expression in tumor cells (left panel) and PBMCs (right panel) from patients with RCC and esophageal cancer. Figures 14B-14C In the TME of CRC patients, the CD8 T cell population ( Figure 14B PD-1 and ( Figure 14C Box plot of ILT2 RNA expression. Figures 14D-14E () Figure 14D ILT2 expression in CD8 T cells from healthy donor peripheral blood and ( Figure 14E A dotted map of ILT2 and PD-1 expression in TILs from esophageal cancer. Figure 14F Scatter plot of membrane CD107a increase on PBMCs from 10 healthy donors activated with staphylococcal enterotoxin B (SEB) in the presence of 15G8, anti-PD-1 antibody, or a combination of both. Figure 14G Bar graph showing the increase in CD107a expression in exemplary PBMCs from three donors. Figures 14H-14J In the presence of anti-PD-1 antibody, humanized anti-ILT2 antibody, or both, inflammatory cytokines from activated PBMCs co-cultured with various primary cancer cells ( Figure 14H )IFNγ, ( Figure 14I TNFα, ( Figure 14J Bar graph of GM-CSF secretion levels. Figures 14K-14L In mixed lymphocyte reactions, cells originating from ( Figure 14K Dendritic cells or ( Figure 14L Bar graph showing the IFNγ secretion level of T cells co-cultured with macrophages.

[0184] Figures 15A-15F ( Figure 15A Linear graph of tumor volume of HLA-G and MHC-I-expressing tumors growing in immunocompromised mice supplemented with human macrophages and anti-ILT2 antibodies. Figure 15B A diagram illustrating the treatment schedule for mice used to prevent lung tumors. Figure 15C (Images of the lungs of immunocompromised mice inoculated with HLA-G positive cancer cells, with or without human PBMC and ILT2 antibodies.) Figure 15D (Summary from) Figure 15C A scatter plot of the data. Figure 15E A diagram illustrating the treatment schedule in mice for treating established lung tumors. Figure 15F Box plot of tumor weight.

[0185] Figures 16A-16F ( Figures 16A-16F The following box plots: Figure 16A CD107A expression in total CD8 T cells, Figure 16B )T EMRA CD107A expression in cells, ( Figure 16C CD69 expression in NK cells, Figure 16D CD69 expression in total CD8 T cells was significantly reduced in mice receiving donor PBMCs with low or high ILT2 levels, respectively. EMRA In cells or NK cells, ( Figure 16E )T EMRA CD107 expression in cells and ( Figure 16F CD69 expression in NK cells treated with combination therapy. * indicates P < 0.005. ** indicates P < 0.0005. *** indicates P < 0.0001.

[0186] Figures 17A-17F ( Figure 17A A diagram illustrating the treatment timeline for humanized NSG mice inoculated with H&N cancer and treated with anti-ILT2 or control antibodies. Figure 17B Line graph of tumor weight from mice treated with IgG and anti-ILT2. Figures 17C-17E () Figure 17C Bar graph of baseline ILT2 levels in peripheral CD8 T cells in mice that responded to (R) or did not respond to (NR) BND-22 treatment. Intratumoral treatment in four mice treated with anti-ILT2 antibodies ( Figure 17D CD107A expression, ( Figure 17E M1 / M2 ratio and ( Figure 17F Total number of CD80-positive dendritic cells.

[0187] Figures 18A-18F ( Figure 18A The partial sequence of ILT2 is shown, revealing residues with significant predicted binding. These residues are categorized into four classes based on their original probability of belonging to an epitope, from purple (highest probability) to light cyan (lowest probability, but still significant). An asterisk indicates the location of the selected mutation. Figures 18B-18C 3D rendering of the ILT2 surface structure, its display ( Figure 18B The position of residues from 18A and ( Figure 18C The four main interaction regions on ILT2. Figures 18D-18F ) 3D strip plot or surface plot of ILT2, which shows ( Figure 18D Epitopes of the 15G8 antibody (yellow / pink) and epitopes of the 3H5, 12D12 and 27H5 antibodies from WO2020 / 136145 (red), and secondary epitopes of the 3H5 antibody (dark blue). Figures 18E-18F ) and the 15G8 position (pink) on ILT2 and having ( Figure 18E HLA-A (blue) or ( Figure 18FThe interaction of B2M (light purple) in the HLA-G (blue) complex.

[0188] Figures 19A-19C ( Figures 19A-19B For example, compared with the IgG control, co-culture with macrophages in the presence of various anti-ILT2 antibodies ( Figure 19A A375-HLA-G and ( Figure 19B Bar graph showing the percentage increase in phagocytosis in SKMEL28-HLA-G cancer cells. Figure 19C Line plot of competitive ILT2 binding ELISA using biotinylated 15G8 antibody in the presence of competitive unbiotinylated GHI / 75, HP-F1 and 15G8 antibodies. Detailed Implementation

[0189] This invention relates to monoclonal antibodies or antigen-binding fragments that bind to ILT2 and inhibit ILT2-mediated immunosuppression, and pharmaceutical compositions. Methods for treating cancer and enhancing PD-1 / PD-L1 immunotherapy are also provided.

[0190] This invention is based, at least in part, on the surprising discovery that ILT2 antagonism synergizes with PD-1 and PD-L1-based immunotherapies to combat cancer cells. Specifically, it was found that the combination of ILT2-blocking antibodies and anti-PD-1 antibodies increases the secretion of pro-inflammatory cytokines by immune cells. This increase is not only additive but also exceeds the sum of the effects of each agent alone. In fact, a de novo increase was observed for at least one cytokine, where the individual agents had no effect. This combination therapy allows for the transformation of PD-1 / PD-L1-refractory cancers into reactive ones.

[0191] Surprisingly, it was also found that ILT2 expression levels in patients' immune cells were correlated with the effectiveness of ILT2 blockade therapy. Responders to the therapy had high ILT2 levels, while non-responders had low ILT2 levels. Specifically, circulating CD8-positive T cells predicted treatment outcomes.

[0192] Finally, a unique epitope was discovered within the ILT2 interdomain between the D1 and D2 domains of the antibody of the present invention. This region is known to be the interaction domain between ILT2 and B2M, and the antibody of the present invention is the first known antibody to directly block this interaction. Furthermore, the antibody of the present invention was found to possess immunostimulatory effects not previously reported with other anti-ILT2 antibodies. The antibody is able to modulate immune surveillance of T cells, NK cells, dendritic cells, and macrophages against cancer cells expressing HLA-G and MHC-I. Specifically, for the first time, it was discovered that the anti-ILT2 antibody used as a monotherapy can enhance phagocytosis of cancer cells.

[0193] Antibody

[0194] In a first aspect, an antibody or antigen-binding fragment is provided, the antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 1 (DHTIH), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 2 (YIYPRDGSTKYNEKFKG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 3 (TWDYFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 4 (RASESVDSYGNSFMH), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 5 (RASNLES), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 6 (QQSNEDPYT).

[0195] In another aspect, an antibody or antigen-binding fragment is provided, the antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 7 (GYTFTSYGIS), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 8 (EIYPGSGNSYYNEKFKG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 9 (SNDGYPDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 10 (KASDHINNWLA), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 11 (GATSLET), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 12 (QQYWSTPWT).

[0196] In another aspect, an antibody or antigen-binding fragment is provided, the antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 13 (SGYYWN), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 14 (YISYDGSNNYNPSLKN), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 15 (GYSYYYAMDX), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 16 (RTSQDISNYLN), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 17 (YTSRLHS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 18 (QQGNTLPT), wherein X is selected from A, C, and S.

[0197] In some embodiments, SEQ ID NO: 16 is GYSYYYAMDA (SEQ ID NO: 25). In some embodiments, SEQ ID NO: 16 is SEQ ID NO: 25, and the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, SEQ ID NO: 16 is GYSYYYAMDS (SEQ ID NO: 26). In some embodiments, SEQ ID NO: 16 is SEQ ID NO: 26, and the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, SEQ ID NO: 16 is GYSYYYAMDC (SEQ ID NO: 27). In some embodiments, SEQ ID NO: 16 is SEQ ID NO: 27, and the antibody or antigen-binding fragment is a mouse antibody.

[0198] In another aspect, an antibody or antigen-binding fragment is provided that binds to the interdomain of human leukocyte immunoglobulin-like receptor superfamily B member 1 (ILT2) between domains D1 and D2.

[0199] On the other hand, an antibody or antigen-binding fragment that binds to an ILT2 epitope is provided, the epitope being selected from the following ILT2 sequences: VKKGQFPIPSITWEH (SEQ ID NO: 41), LELVVTGAYIKPTLS (SEQ ID NO: 42), VILQCDSQVAFDGFS (SEQ ID NO: 43), and WYRCYAYDSNSPYEW (SEQ ID NO: 44).

[0200] In another aspect, an antibody or antigen-binding fragment is provided that binds to ILT2 and inhibits the interaction between ILT2 and β-2-microglobulin (B2M).

[0201] On the other hand, an antibody or antigen-binding fragment is provided that binds to ILT2 and induces at least one of the following in a subject with cancer:

[0202] a. Increased cytotoxicity of natural killer (NK) cells;

[0203] b. Increased T cell cytotoxicity, proliferation, or both;

[0204] c. Increased macrophage phagocytosis, increased production of M1 inflammatory macrophages, decreased production of M2 suppressive macrophages, or a combination thereof; and

[0205] d. Increased dendritic cell homing to the tumor of the cancer, increased dendritic cell activation, or a combination thereof.

[0206] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a mouse antibody. In some embodiments, the antibody is a humanized antibody. As used herein, a "humanized" antibody means an antibody having a human backbone but having a CDR derived from or derived from a non-human antibody. In some embodiments, the CDR may be modified during humanization, but generally still be derived from a non-human antibody's CDR. In some embodiments, the antigen-binding fragment is a single-chain antibody. In some embodiments, the antigen-binding fragment is a single-domain antibody.

[0207] In some embodiments, the antibody or antigen-binding fragment binds to leukocyte immunoglobulin-like receptor subfamily B member 1 (ILT2). In some embodiments, ILT2 is human ILT2. In some embodiments, ILT2 is mammalian ILT2. In some embodiments, ILT2 is primate ILT2. In some embodiments, ILT2 is mouse ILT2. In some embodiments, the antibody or antigen-binding fragment binds to the extracellular domain of ILT2. In some embodiments, the antibody or antigen-binding fragment binds to the ligand pocket of ILT2. In some embodiments, the ligand is B2M. In some embodiments, the ligand is not HLA. In some embodiments, the ligand is HLA. In some embodiments, the HLA is HLA-G. In some embodiments, the ligand is not MHC. In some embodiments, the ligand is MHC. In some embodiments, the MHC is MHC class I (MHC-I). In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 interdomain. In some embodiments, the interdomain is the interface between the D1 and D2 domains. In some embodiments, the intercalation domain is a hinge domain between domains D1 and D2. In some embodiments, the intercalation domain does not contain the N-terminal domain of D1. In some embodiments, the intercalation domain is amino acids 54-184 of SEQ ID NO: 31. In some embodiments, amino acids 54-184 of SEQ ID NO: 31 contain the intercalation domain. In some embodiments, the intercalation domain is amino acids 90-184 of SEQ ID NO: 31. In some embodiments, amino acids 90-184 contain the intercalation domain. In some embodiments, an antibody or antigen-binding fragment binds to an epitope within the intercalation domain. In some embodiments, the epitope comprises at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the intercalation domain. Each possibility represents a separate embodiment of the invention. In some embodiments, the epitope is within D2. In some embodiments, an antibody or antigen-binding domain binds to the epitope in D2. In some embodiments, the epitope is at least partially located in D2. In some embodiments, the antibody or antigen-binding domain binds to the epitope at least partially located in D2. In some embodiments, the epitope spans D1 and D2. In some embodiments, the antibody or antigen-binding fragment does not bind to the ILT2 domain that interacts with the α3 domain of HLA-G.

[0208] In some embodiments, ILT2 is mammalian ILT2. In some embodiments, ILT2 is human ILT2. In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_006660.4. In some embodiments, ILT2 has the following amino acid sequence: (SEQ ID NO: 31).

[0209] In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_001075106.2. In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_001075107.2. In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_001075108.2. In some embodiments, ILT2 has the amino acid sequence provided in NCBI reference sequence: NP_001265328.2.

[0210] In some embodiments, the D1 domain of ILT2 comprises or consists of the following amino acid sequence: GHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGA (SEQ ID NO: 46). In some embodiments, the D1 domain of ILT2 comprises or consists of amino acids 24-121 of SEQ ID NO: 31. In some embodiments, the D2 domain of ILT2 comprises or consists of the following amino acid sequence: YIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGV (SEQ ID NO: 47). In some embodiments, the D2 domain of ILT2 comprises or consists of amino acids 122-222 of SEQ ID NO: 31. In some embodiments, the interstitial domain of ILT2 comprises the amino acids Gln41, Lys65, Trp90, Gly120, Ala121, Val122, Ile123, Gln148, Val149, Ala150, Phe151, Asp201, Asn203, and Glu207 of SEQ ID NO: 31. In some embodiments, the epitope comprises the amino acids Gln41, Lys65, Trp90, Gly120, Ala121, Val122, Ile123, Gln148, Val149, Ala150, Phe151, Asp201, Asn203, and Glu207 of SEQ ID NO: 31. In some embodiments, the epitope comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids selected from the amino acids Gln41, Lys65, Trp90, Gly120, Ala121, Val122, Ile123, Gln148, Val149, Ala150, Phe151, Asp201, Asn203, and Glu207 of SEQ ID NO: 31. In some embodiments, the epitope comprises at least 10 amino acids selected from the amino acids Gln41, Lys65, Trp90, Gly120, Ala121, Val122, Ile123, Gln148, Val149, Ala150, Phe151, Asp201, Asn203, and Glu207 of SEQ ID NO: 31. In some implementations, the antibody or antigen-binding fragment binds to the ILT2 epitope provided in SEQ ID NO: 41.In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 epitope provided in SEQ ID NO: 42. In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 epitope provided in SEQ ID NO: 43. In some embodiments, the antibody or antigen-binding fragment binds to the ILT2 epitope provided in SEQ ID NO: 44. In some embodiments, the antibody or antigen-binding fragment binds to a three-dimensional epitope comprising at least two of SEQ ID NO: 41, 42, 43, and 44. In some embodiments, the three-dimensional epitope comprises at least three of SEQ ID NO: 41, 42, 43, and 44. In some embodiments, the three-dimensional epitope comprises SEQ ID NO: 41, 42, 43, and 44.

[0211] In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope comprising an ILT2 residue selected from Q18, G19, K42, L45, S64, I65, T66, W67, E68, G97, A98, Y99, I100, Q125, V126, A127, F128, D178, N180, S181, and E184. In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope comprising an ILT2 residue selected from G97, A98, Y99, I100, Q125, and V126. In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope comprising a plurality of ILT2 residues selected from Q18, G19, K42, L45, S64, I65, T66, W67, E68, G97, A98, Y99, I100, Q125, V126, A127, F128, D178, N180, S181, and E184. In some embodiments, the antibody or antigen-binding fragment binds to an ILT2 epitope comprising a plurality of ILT2 residues selected from G97, A98, Y99, I100, Q125, and V126. In some embodiments, the antibody or antigen-binding fragment binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 residues selected from Q18, G19, K42, L45, S64, I65, T66, W67, E68, G97, A98, Y99, I100, Q125, V126, A127, F128, D178, N180, S181, and E184. Each possibility represents a separate embodiment of the invention. In some embodiments, the antibody or antigen-binding fragment binds to at least 1, 2, 3, 4, 5, or 6 residues selected from G97, A98, Y99, I100, Q125, and V126. Each possibility represents a separate embodiment of the invention. In some implementations, the antibody or antigen-binding fragment binds to G97, A98, Y99, I100, Q125, and V126. It should be understood that the designations used herein refer to SEQ ID NO: 31.

[0212] In some embodiments, the antibody or antigen-binding fragment is an ILT2 antagonist. In some embodiments, the antibody or antigen-binding fragment is not an ILT2 agonist. In some embodiments, the antagonism is part of ILT2-mediated immunosuppression. In some embodiments, the antibody or antigen-binding fragment inhibits ILT2-mediated immunosuppression. In some embodiments, the antibody or antigen-binding fragment inhibits ILT2 signaling.

[0213] In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and B2M. In some embodiments, the interaction is a direct interaction. In some embodiments, the antibody or antigen-binding fragment inhibits the contact between ILT2 and B2M. In some embodiments, the contact is a direct contact. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and HLA, MHC, or both. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and B2M, thereby inhibiting the interaction between ILT2 and HLA, MHC, or both. In some embodiments, the interaction is mediated by B2M. In some embodiments, the antibody indirectly inhibits the interaction with HLA, MHC, or both by inhibiting the interaction with B2M. In some embodiments, the interaction is a B2M-mediated interaction. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and the B2M / HLA complex. In some embodiments, the antibody or antigen-binding fragment inhibits the interaction between ILT2 and the B2M / MHC complex. In some embodiments, the complex comprises a B2M monomer. In some embodiments, the complex comprises an HLA or MHC monomer. In some embodiments, the complex comprises a B2M dimer. In some embodiments, the complex comprises an HLA or MHC dimer.

[0214] In some embodiments, ILT2-mediated immunosuppression is the suppression of immune cells. In some embodiments, the immune cells are selected from T cells, macrophages, dendritic cells, and natural killer (NK) cells. In some embodiments, ILT2-mediated immunosuppression is the suppression of T cells, macrophages, dendritic cells, and NK cells. In some embodiments, ILT2-mediated immunosuppression is the suppression of T cells, macrophages, and NK cells. In some embodiments, the T cells are CD8-positive T cells. In some embodiments, the T cells are T... EMRA Cells (terminally differentiated effector memory cells reexpressing CD45RA). In some implementations, immune cells are selected from CD8-positive T cells, T cells... EMRACells, dendritic cells, macrophages, and natural killer (NK) cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are macrophages. In some embodiments, the macrophages are tumor-associated macrophages (TAMs). In some embodiments, the immune cells are dendritic cells. In some embodiments, the dendritic cells are tolerogenic dendritic cells. In some embodiments, the immune cells are peripheral blood immune cells. In some embodiments, the immune cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the immune cells are intratumoral immune cells. In some embodiments, the immune cells are immune cells in the tumor microenvironment (TME). In some embodiments, ILT2-mediated immunosuppression is inhibition of macrophage phagocytosis. In some embodiments, ILT2-mediated immunosuppression is inhibition of NK cell cytotoxicity. In some embodiments, ILT2-mediated immunosuppression is inhibition of T cell cytotoxicity. In some embodiments, ILT2-mediated immunosuppression is inhibition of T cell proliferation. In some implementations, ILT2-mediated immunosuppression is the inhibition of immune cell proliferation.

[0215] In some embodiments, the antibody or antigen-binding fragment does not bind to members of the leukocyte immunoglobulin-like receptor superfamily B other than ILT2. In some embodiments, the antibody or antigen-binding fragment is specific for ILT2. In some embodiments, the antibody or antigen-binding fragment preferentially binds to ILT2. In some embodiments, the antibody or antigen-binding fragment does not inhibit members of the leukocyte immunoglobulin-like receptor superfamily B other than ILT2.

[0216] As used herein, "increased binding power" means a greater specific binding to a target or antigen than the binding of an isotype control. In some embodiments, increased binding is an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% of the binding power. Each possibility represents a separate embodiment of the invention. In some embodiments, increased binding is the presence of binding as compared to an isotype control that does not bind. Binding of an antibody to a specific domain will be well known to those skilled in the art. Antibody binding can be determined in any manner known to those skilled in the art, including but not limited to: X-ray crystallography, immunoprecipitation, Western blotting, competitive assays, and kinetic repulsion assays. In some embodiments, increased binding power is specific binding.

[0217] The antibodies or antigen-binding fragments, variants, or derivatives disclosed herein may be referred to as having a value greater than or equal to 10. 3 M "1 sec "1 5 x 10 3 M "1 sec "1 10 4 M "1 sec "1 Or 5 x 10 4 M "1 sec " The association rate (k(on)) of the target antigen (e.g., ILT2). Each possibility represents a separate embodiment of the invention. Antibodies or antigen-binding fragments, variants, or derivatives disclosed herein may be referred to by a series of 10. -6 M or stronger affinity binds to the target antigen, while the typical affinity of most antibodies is 10. -9 M.

[0218] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 19 (QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARTWDYFDYWGQGTTLTVSS). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 21 (QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWVGEIYPGSGNSYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARSNDGYPDYWGQGTTLTVSS). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 23 (DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDXWGQGTSVTVSS), where X is selected from A, C, and S.

[0219] In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20 (DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIK). In some embodiments, the antibody or antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO: 24 (DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTFGQGTKLEIK). In some embodiments, the antibody or antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO: 45 (DIQMTQTTSSLSASLGDRVTISCRTSQDISNYLNWYQQKPDGTVKLLISYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPTFGSGTKLEIK).

[0220] In some embodiments, SEQ ID NO: 23 is DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDAWGQGTSVTVSS (SEQ ID NO: 28). In some embodiments, SEQ ID NO: 23 is SEQ ID NO: 28, and the antibody or antigen-binding fragment is humanized. In some embodiments, SEQ ID NO: 23 is DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDSWGQGTSVTVSS (SEQ ID NO: 29). In some embodiments, SEQ ID NO: 23 is SEQ ID NO: 29, and the antibody or antigen-binding fragment is humanized. In some embodiments, SEQ ID NO: 23 is DVQLQGSGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTSEDTATYYCAHGYSYYYAMDCWGQGTSVTVSS (SEQ ID NO: 30). In some embodiments, SEQ ID NO: 23 is SEQ ID NO: 30, and the antibody or antigen-binding fragment is mouse-derived.

[0221] In some embodiments, the antibody or antigen-binding fragment of the present invention is used to treat or improve cancer in a subject of need. In some embodiments, the cancer is HLA-G positive cancer. In some embodiments, the cancer is MHC-I positive cancer. In some embodiments, the cancer expresses HLA-G. In some embodiments, the cancer expresses MHC-I. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to transform the tumor microenvironment from immunosuppressive to immunostimulatory. In some embodiments, the transformation of the tumor microenvironment includes one or more of the following: inducing / enhancing anti-tumor T cell responses, increasing T cell proliferation, reducing cancer-induced myelosuppressive activity, increasing dendritic cell (DC) activation, increasing dendritic cell homing to the tumor, increasing macrophage phagocytosis, increasing M1 macrophage production, decreasing M2 macrophage production, and increasing NK cell activity. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase T cell responses against cancer cells. In some embodiments, the T cell response includes increased secretion of pro-inflammatory cytokines. In some embodiments, the T cell response includes increased cytotoxicity. In some embodiments, the T cell response includes increased T cell proliferation. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase macrophage phagocytosis of cancer cells. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase dendritic cell homing to tumors or cancer. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase macrophage phagocytosis. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase macrophage phagocytosis of cancer. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the production of M1 macrophages. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce the production of M2 macrophages. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase NK cell cytotoxicity against cancer cells. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce cancer-induced myelosuppressive activity. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce the activity of tolerant dendritic cells (DCs). In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the activity or number of M1 monocytes. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce the activity or number of M2 monocytes. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the production of M1 macrophages. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to reduce the production of M2 macrophages. In some embodiments, the M1 monocytes / macrophages are inflammatory macrophages / monocytes. In some embodiments, the M2 monocytes / macrophages are suppressive macrophages / monocytes.In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the number of dendritic cells (DCs) in a tumor. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase the recruitment of DCs to the tumor. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase DC recruitment to the tumor. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase DC activation. In some embodiments, increasing DC activation includes reducing the activity of toxic dendritic cells. In some embodiments, the antibody or antigen-binding fragment of the present invention is used to increase antigen presentation. In some embodiments, recruitment to the tumor is recruitment to the tumor microenvironment (TME).

[0222] In some embodiments, the antibody or antigen-binding fragment induces at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 anticancer effects in a subject. Each possibility represents a separate embodiment of the invention. In some embodiments, the antibody or antigen-binding fragment induces at least 2 effects in a subject. In some embodiments, the antibody or antigen-binding fragment induces at least 3 effects in a subject. In some embodiments, the antibody or antigen-binding fragment induces at least 4 effects in a subject. In some embodiments, the effects are selected from: increased NK cell cytotoxicity, increased T cell cytotoxicity, increased T cell proliferation, increased macrophage phagocytosis, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell homing to cancerous tumors, and increased dendritic cell activation. In some embodiments, the effect is selected from: a) increased NK cell cytotoxicity; b) increased T cell cytotoxicity, proliferation, or both; c) increased macrophage phagocytosis, increased M1 macrophage production, decreased M2 macrophage production, or combinations thereof; and d) increased dendritic cell homing to cancerous tumors, increased dendritic cell activation, or combinations thereof. In some embodiments, cytotoxicity is cancer-specific cytotoxicity. In some embodiments, phagocytosis is phagocytosis by cancer cells or cancer cells. In some embodiments, the antibody or antigen-binding fragment induces anticancer activity on T cells, NK cells, dendritic cells, and macrophages in a subject. In some embodiments, the antibody or antigen-binding fragment induces anticancer activity on at least three of the T cells, NK cells, dendritic cells, and macrophages in a subject. In some embodiments, the antibody or antigen-binding fragment induces the effect as a single therapy. In some embodiments, the antibody or antigen-binding fragment induces the effect without combination.

[0223] In some embodiments, the increased cytotoxicity includes increased secretion of pro-inflammatory cytokines. Pro-inflammatory cytokines are well known in the art and include, but are not limited to: IL-1, IL-1β, IL-6, TNFα, IFNγ, MCP-1, IL-12, IL-18, IL-2, IL-15, IL-17, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the pro-inflammatory cytokine is selected from IL-6, interferon-γ (IFNγ), and GM-CSF. In some embodiments, the pro-inflammatory cytokine is GM-CSF.

[0224] "Anti-ILT2 antibody," "antibody that recognizes ILT2," or "antibody against ILT2" is an antibody that binds to ILT2 with sufficient affinity and specificity. In some embodiments, the anti-ILT2 antibody has ILT2 as the antigen it binds to.

[0225] An "antigen" is a molecule or part of a molecule that can trigger antibody formation and be bound by the antibody. An antigen may have one or more epitopes. The specificity mentioned above is intended to indicate that an antigen will react with its corresponding antibody in a highly selective manner, rather than with a variety of other antibodies that may be induced by other antigens.

[0226] The term "antigenic determinant" or "epitope" according to the present invention refers to a region in an antigen molecule that specifically reacts with a particular antibody. Using methods known in the art, peptide sequences derived from epitopes can be used alone or in combination with a vector moiety to immunize animals and generate additional polyclonal or monoclonal antibodies. The IMGT Information System (www.imgt.cines.fr / ) (IMGT® / V-Quest) can also be used to analyze immunoglobulin variable domains to identify variable regions (including CDRs). See, for example, Brochet, X. et al., Nucl. Acids Res. J6:W503-508 (2008).

[0227] Kabat et al. also defined a numbering system for variable domain sequences, applicable to any antibody. Those skilled in the art can explicitly assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described in Kabat et al., U.S. Department of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0228] In some embodiments, the antibody or antigen-binding fragment is used in combination with another agent. In some embodiments, the combination with another agent is for treating cancers expressing HLA-G and / or MHC-I. In some embodiments, the agent is an opsonizer. In some embodiments, the agent is an anti-PD-1 and / or anti-PD-L1 agent. In some embodiments, the antibody or antigen-binding fragment is used in combination with an anti-PD-1 / PD-L1 based therapy.

[0229] As used herein, an opsonizer is any agent that can bind to and opsonize target cells (e.g., cancer cells, cells with intracellular pathogens, etc.). For example, any antibody believed to bind to target cells is an opsonizer, wherein said antibody has an Fc region. In some embodiments, the opsonizer is an antibody that induces antibody-dependent phagocytosis (ADCP). Examples of opsonizers include, but are not limited to, anti-CD47 antibodies, anti-CD20 antibodies, anti-HER2 antibodies, anti-EGFR antibodies, anti-CD52 antibodies, and anti-CD30 antibodies. In some embodiments, the opsonizer is selected from rituximab, trastuzumab, pertuzumab, Herceptin, cetuximab, panitumumab, and Erbitux. In some embodiments, the opsonizer is an anti-EGFR antibody. In some embodiments, the opsonizer is Erbitux.

[0230] As used herein, “anti-PD-1 / PD-L1 therapy” and “PD-1 / PD-L1 therapy” are synonymous and used interchangeably, and refer to a treatment regimen that includes blocking the PD-1 and PD-L1 signaling axes. In some embodiments, the cancer is PD-L1 positive cancer. In some embodiments, PD-1 / PD-L1 therapy is PD-1 / PD-L1 immunotherapy. In some embodiments, PD-1 / PD-L1 therapy is PD-1 / PD-L1 blockade. In some embodiments, PD-1 / PD-L1 therapy is an agent that blocks PD-1-based immunosuppression. In some embodiments, PD-1 / PD-L1 therapy comprises an anti-PD-1 blocking antibody. In some embodiments, PD-1 / PD-L1 therapy comprises an anti-PD-L1 blocking antibody. In some embodiments, PD-1 / PD-L1 therapy enhances immune surveillance. In some embodiments, PD-1 / PD-L1 therapy is an anticancer therapy. In some embodiments, PD-1 / PD-L1 therapy enhances tumor immune surveillance. The term "antibody" (also known as "immunoglobulin") is used in the broadest sense and specifically covers monoclonal antibodies and antibody fragments, provided they exhibit the desired biological activity. In some embodiments, the invention also covers the use of chimeric or humanized antibodies.

[0231] The basic building block of naturally occurring antibody structures is a heterotetrameric protein complex of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains linked together by non-covalent association and disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Five classes of human antibodies exist (IgG, IgA, IgM, IgD, and IgE), and within these classes, multiple subclasses are identified based on structural differences such as the number of immunoglobulin units in a single antibody molecule, the disulfide bridge structure of individual units, and differences in chain length and sequence. Antibody classes and subclasses are their isotypes.

[0232] The sequences of the amino-terminal regions of the heavy and light chains are more diverse than those of the carboxyl-terminal regions, and are therefore called variable domains. This part of the antibody structure confers antigen-binding specificity to the antibody. The heavy chain variable (VH) domain and the light chain variable (VL) domain together form a single antigen-binding site; thus, the basic immunoglobulin unit has two antigen-binding sites. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0233] The carboxyl-terminal portions of the heavy and light chains form constant domains, namely CH1, CH2, CH3, and CL. Although the diversity within these domains is significantly low, differences exist between animal species, and furthermore, several different antibody isotypes exist within the same organism, each with different functions.

[0234] The term "frame region" or "FR" refers to amino acid residues in the variable domains of an antibody that are distinct from the amino acid residues in the hypervariable region as defined herein. The term "hypervariable region," as used herein, refers to amino acid residues in the variable domains of an antibody responsible for antigen binding. The hypervariable region contains amino acid residues from the "complementarity-determining region" or "CDR." The CDR is primarily responsible for binding to the antigen epitope. The ranges of FR and CDR have been precisely defined (see, Kabat et al.). In some embodiments, the CDR is determined using the KABAT system. In some embodiments, the CDR is determined using the Clothia system. In some embodiments, the Clothia system is an enhanced Clothia system (Martin system).

[0235] Monoclonal antibodies as used herein explicitly include “chimeric” antibodies, wherein a portion of the heavy and / or light chains is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of one or more chains is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984)). Additionally, complementarity-determining region (CDR) transplantation can be performed to alter certain properties of the antibody molecule, including affinity or specificity. Non-limiting examples of CDR transplantation are disclosed in US Patent 5,225,539.

[0236] Chimeric antibodies are molecules whose different parts are derived from different animal species, such as those having a variable region derived from mouse mAbs and a constant region from human immunoglobulins. Antibodies having a variable region framework residue that is essentially derived from human antibodies (called receptor antibodies) and a complementarity-determining region that is essentially derived from mouse antibodies (called donor antibodies) are also called humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in applications and to increase yield in production, for example, in cases where mouse mAbs have higher hybridoma yields but higher human immunogenicity, thus using human / mouse chimeric mAbs. Chimeric antibodies and methods of their production are known in the art (e.g., PCT patent applications WO 86 / 01533, WO 97 / 02671, WO 90 / 07861, WO 92 / 22653 and U.S. patents 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539). As used herein, the term "humanized antibody" refers to an antibody comprising a framework region derived from a human antibody and one or more core-drug receptors (CDRs) derived from a non-human (typically mouse or rat) immunoglobulin. The portion of the humanized immunoglobulin (potentially other than the CDR) is substantially identical to the corresponding portion of the native human immunoglobulin sequence. However, in some cases, such as specific amino acid residues in the framework region, modifications can be made to optimize the performance of the humanized antibody. Importantly, the humanized antibody is expected to bind to the same antigen as the donor antibody providing the CDR. Further details can be found, for example, in U.S. Patent No. 5,225,539 assigned to the Medical Research Council, UK. The terms "frame region derived from recipient human immunoglobulin" and "frame region derived from recipient human immunoglobulin" and similar grammatical expressions are used interchangeably herein to refer to a framework region or a portion thereof having the same amino acid sequence as the recipient human immunoglobulin.

[0237] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies constituting said group are identical and / or bind to the same epitopes, except for possible variants that may occur during the production of the monoclonal antibody, which are typically present in small amounts. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as meaning that the antibody used according to the methods provided herein can be prepared by the hybridoma method first described in Kohler et al., Nature 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described in, for example, the following literature: Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0238] The mAbs of this invention can belong to any immunoglobulin class, including IgG, IgM, IgE, or IgA. Hybridomas that produce mAbs can be cultured in vitro or in vivo. High-titer mAbs can be obtained in vivo by intraperitoneal injection of cells from a single hybridoma into primitively sensitized Balb / c mice to produce ascites containing high concentrations of the desired mAb. Isotype IgM or IgG mAbs can be purified from such ascites using column chromatography methods well known to those skilled in the art, or from culture supernatants.

[0239] The terms “antibody fragment” or “antigen-binding fragment” are used synonymously and include, preferably, a portion of the antigen-binding region of a complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; tandem biantibodies (taDb); linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); monoarmed antibodies; single variable domain antibodies; microantibodies; single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, di-scFv, or tandem (di, tri)-scFv); and bispecific T-cell binders (BiTE).

[0240] Papain digestion of the antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site; and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still able to cross-link the antigen.

[0241] The “Fv” is the smallest antibody fragment containing both a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. The three surfaces of the VH-VL dimer are in this configuration. In summary, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three hypervariable regions specific to the antigen) has the ability to recognize and bind to the antigen, but its affinity is lower than that of the entire binding site.

[0242] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name for Fab' in this paper where one or more cysteine ​​residues in the constant domain have at least one free thiol group. The F(ab')2 antibody fragment was initially generated as a Fab' fragment pair with a hinge cysteine ​​residue between the Fab' fragments. Other chemical conjugates of antibody fragments are also known.

[0243] The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be designated as one of two distinct types (called κ and λ) based on the amino acid sequence of its constant domain.

[0244] Antibodies can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, e, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well known.

[0245] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, wherein these domains are contained within a single polypeptide chain. In some embodiments, the Fv polypeptide also contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0246] The term "biantibody" refers to a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the linker is forced to pair with a complementary domain of the other chain, resulting in two antigen-binding sites. Biantibodies are described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0247] The term "multispecific antibody" is used in the broadest sense and explicitly covers antibodies that exhibit multi-epitope specificity. Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VHVL unit has multi-epitope specificity; antibodies having two or more VL and VH domains, wherein each VHVL unit binds to a different epitope; antibodies having two or more single variable domains, wherein each single variable domain binds to a different epitope; full-length antibodies; antibody fragments such as Fab, Fv, dsFv, scFv, biantibodies, bispecific biantibodies, triantibodies, trifunctional antibodies, and antibody fragments that are covalently or non-covalently linked. "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on one or more of the same or different targets.

[0248] The monoclonal antibodies of the present invention can be prepared using methods well known in the art. Examples include a variety of techniques, such as those in the following literature: Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96, MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0249] In addition to conventional methods for antibody production in vivo, phage display technology can be used to generate antibodies in vitro. This generation of recombinant antibodies is significantly faster than conventional antibody production and can generate recombinant antibodies against a very large number of antigens. Furthermore, many antigens have been shown to be non-immunogenic or highly toxic when using conventional methods, making them unsuitable for antibody generation in animals. Additionally, affinity maturation (i.e., increasing affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, a large number of different antibodies against a specific antigen can be generated in a single selection process. To generate recombinant monoclonal antibodies, large libraries of antibodies with different antigen recognition sites can be generated using a variety of methods, all based on display libraries. This library can be prepared in several ways: a synthetic library can be generated by cloning the synthetic CDR3 region from a heavy chain germline gene library, from which recombinant antibody fragments with various specificities can be selected. Human lymphocyte libraries can be used as starting materials for constructing antibody libraries. An initial library of human IgM antibodies can be constructed, resulting in a highly diverse human library. This method has been successfully and widely used to select a large number of antibodies against different antigens. Protocols for phage library construction and recombinant antibody selection are provided in well-known reference texts: Current Protocols in Immunology, Colligan et al. (eds.), John Wiley & Sons, Inc. (1992–2000), Chapter 17, Section 17.1.

[0250] Nonhuman antibodies can be humanized using any method known in the art. In one approach, a nonhuman complementarity-determining region (CDR) is inserted into the frame sequence of a human antibody or a shared antibody. Other variations can then be introduced into the antibody frame to modulate affinity or immunogenicity.

[0251] In some embodiments, the antibodies described herein are neutralizing antibodies. As discussed herein, “neutralization” is defined as the reduction of protein function by the antibodies of the present invention. In one embodiment, as discussed herein, “neutralization” is the binding of an antibody to the surface of immune cells, preferably immature and mature myeloid-derived cells, T cells, and NK cells, thereby blocking the propagation of inhibitory signals within these cells and conferring a less inhibitory phenotype and function.

[0252] In some embodiments, the present invention provides nucleic acid sequences encoding the antibodies of the present invention. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 75% identity with the DNA sequence described herein. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 80% identity with the DNA sequence described herein. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 85% identity with the DNA sequence described herein. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 90% identity with the DNA sequence described herein. In one embodiment, the antibody as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 95% identity with the DNA sequence described herein.

[0253] According to another aspect, a nucleic acid sequence encoding the antibody or antigen-binding fragment of the present invention is provided.

[0254] According to another aspect, a nucleic acid molecule encoding an antibody or antigen-binding fragment of the present invention is provided.

[0255] In some embodiments, the nucleic acid sequence encoding the heavy chain of the antibody or antigen-binding fragment of the present invention is selected from CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGGCGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTATGGTATAAGCTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGGTTGGAGAGATTTATCCTGGAAGTGGTAATTCTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCGTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGAATGATGGTTACCCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 32), GATGTACAGCTTCAGGGGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGATGGTAGCAATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTTCTGAGGACACAGCCACATATTACTGTGCCCATGGTTACTCATATTACTATGCTATGGACTGCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:33), GATGTCCAGCTGCAAGGCTCTGGCCCTGGACTGGTTAAGCCTTCCGAGACACTGTCCCTGACCTGCTCTGTGACCGGCTACTCTATCACCTCCGGCTACTACTGGAACTGGATCAGACAGTTCCCCGGCAAGAAACTGGAATGGATGGGCTACATCTCCTACGACGGCTCCAACAACTACAACCCCAGCCTGAAGAACCGGATCACCATCTCTCGGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAACTCCGTGACCGCTGCCGATACCGCTACCTACTACTGTGCTCACGGCTACTCCTACTACTACGCCATGGATGCTTGGGGCCAGGGCACATCTGTGACAGTGTCCTCT (SEQ ID NO: 34) and CAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGAGCTTCAGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCACTGACCATACTATTCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTAGAGATGGTAGTACTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAAGAACCTGGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 35).

[0256] In some embodiments, the nucleic acid sequence encoding the light chain of the antibody or antigen-binding fragment of the present invention is selected from GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 36), GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGACAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTCCTACACATCAAGATTGCACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO:37), GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGACCTCTCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAACCCGGCAAGGCCGTGAAGCTGCTGATCTCCTACACCTCCAGA CTGCACTCTGGCGTGCCCTCCAGATTTTCTGGCTCTGGATCTGGCACCGACTACACCCTGACCATCAGTTCTCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCAGCAGGGCAACACCCTGCCTACCTTTGGCCAGGGCACCAAGCTGGAAATCAAG (SEQ ID NO: 38) and GACATCCAGATGACACAATCTTCATCCTACTTGTCTGTATCTCTAGGAGGCAGAGTCACCATTACTTGCAAGGCAAGTGACCACATTAATAATTGGTTAGCCTGGTATCAGCAGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAACCAGTT TGGAAACTGGGGTTCCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 39).

[0257] In some embodiments, the antibody or antigen-binding fragment is mouse-derived, and the sequence encoding the heavy chain is selected from SEQ ID NO: 32, 33, and 35. In some embodiments, the antibody or antigen-binding fragment is mouse-derived, and the sequence encoding the light chain is selected from SEQ ID NO: 36, 37, and 39. In some embodiments, the antibody or antigen-binding fragment is humanized, and the sequence encoding the heavy chain is SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment is humanized, and the sequence encoding the light chain is SEQ ID NO: 38.

[0258] As used interchangeably in this article, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA.

[0259] Polynucleotides encoding polypeptides can be obtained from any source, including but not limited to cDNA libraries prepared from tissues believed to possess polypeptide mRNA and express said polypeptide mRNA at detectable levels. Therefore, polynucleotides encoding polypeptides can be readily obtained from cDNA libraries prepared from human tissues. Genes encoding polypeptides can also be obtained from genomic libraries or through known synthetic procedures (e.g., automated nucleic acid synthesis).

[0260] For example, polynucleotides can encode the entire immunoglobulin molecular chain, such as the light chain or the heavy chain. A complete heavy chain includes not only the variable region (VH) but also the constant region (CH), which typically contains three constant domains: CH1, CH2, and CH3; and a "hinge" region. In some cases, the presence of a constant region is desirable.

[0261] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments such as single-domain antibodies (“dAb”), Fv, scFv, Fab’, and CHI, with the CK or CL domains cleaved. Because microantibodies are smaller than conventional antibodies, they can achieve better tissue penetration in clinical / diagnostic applications, and in the case of being bivalent, they can retain a higher binding affinity than monovalent antibody fragments (such as dAb). Therefore, unless the context requires otherwise, the term “antibody” as used herein encompasses not only complete antibody molecules but also antigen-binding antibody fragments of the types discussed above. Each frame region present in the encoded polypeptide may contain at least one amino acid substitution relative to the corresponding human receptor frame. Thus, for example, a frame region may contain a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions relative to the receptor frame region. Given the characteristics of the individual amino acids constituting the disclosed protein product, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions can be made, for example, based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved; this is known as "conservative substitution."

[0262] Suitablely, the polynucleotides described herein can be isolated and / or purified. In some embodiments, the polynucleotides are isolated polynucleotides.

[0263] As used herein, the term “non-naturally existing” is a conditional term that expressly excludes (but only excludes) the following forms of said substance, composition, entity and / or any combination of substance, composition or entity that are known to those skilled in the art as “naturally existing” or that are determined or interpreted as “naturally existing” by (or may at any time by) a judge or administrative or judicial authority.

[0264] Treatment and diagnostic methods

[0265] According to another aspect, a method is provided for treating a subject with cancer expressing HLA, MHC-I, or both, the method comprising administering an antibody or antigen-binding fragment of the present invention to the subject.

[0266] According to another aspect, a method for treating cancer in a subject in need is provided, the method comprising confirming that ILT2 expression in the subject is above a predetermined threshold, and administering to the subject an agent that inhibits ILT2-based immunosuppression, thereby treating the subject's cancer.

[0267] According to another aspect, a method for treating cancer in a subject in need is provided, the method comprising: administering to the subject an agent that inhibits ILT2-mediated immunosuppression; and administering to the subject a PD-1 / PD-L1-based therapy; thereby treating the subject's cancer.

[0268] According to another aspect, a method is provided to increase the efficacy of PD-1 / PD-L1-based therapies against cancer cells, the method comprising contacting the cancer cells with an agent that inhibits ILT2-mediated immunosuppression.

[0269] According to another aspect, an agent that binds to ILT2 and inhibits ILT2-mediated immune cell suppression is provided, which is used in combination with anti-PD-L1 / PD-1 based therapies for the treatment of subjects with cancer.

[0270] As used herein, the terms "treatment" or "treating" a disease, disorder, or condition encompass the reduction of at least one symptom, decrease in severity, or inhibition of progression. Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. For effective treatment, the useful compositions described herein need only reduce the severity of the disease, disorder, or condition, decrease the severity of associated symptoms, or provide an improvement in the quality of life for the patient or subject.

[0271] As used herein, the term "treatment" refers to a clinical intervention that attempts to alter the course of disease in the individual being treated, and can be performed for prevention or during a clinicopathological process. The desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, reducing the pathological consequences of disease, slowing the rate of disease progression, improving disease status, and mitigating or improving prognosis. The term "treatment" can also encompass in vitro procedures that affect cells or tissues in culture.

[0272] In some embodiments, the antibody or antigen-binding fragment is administered as a single therapy. In some embodiments, the antibody or antigen-binding fragment is administered in combination with PD-1 / PD-L1 therapy. In some embodiments, the antibody or antigen-binding fragment is administered in combination with an opsonizer. In some embodiments, the opsonizer is not an anti-CD47 agent. In some embodiments, the anti-CD47 agent is an anti-CD47 antibody. In some embodiments, the antibody or antigen-binding fragment is not administered in combination with an anti-CD47 agent or therapy. In some embodiments, the antibody or antigen-binding fragment is not combined with an anti-CD47 agent or therapy.

[0273] In some embodiments, the treatment includes increasing immune surveillance. In some embodiments, the treatment includes increasing the immune response. In some embodiments, the treatment includes reducing tumor burden. In some embodiments, the treatment includes reducing cancer metastasis. In some embodiments, the treatment includes increasing cytotoxicity against the cancer. In some embodiments, the treatment includes increasing the inflammatory response against the cancer. In some embodiments, the treatment includes increasing phagocytosis by the cancer.

[0274] As used herein, the term "subject" refers to an individual or patient who is a vertebrate, such as a mammal, and particularly includes a human. In some embodiments, the subject is a human. In some embodiments, the subject is a mammal. In some embodiments, the subject has cancer.

[0275] In some embodiments, the cancer is an HLA-expressing cancer. In some embodiments, the HLA is HLA-G. In some embodiments, the cancer is an MHC-I-expressing cancer. In some embodiments, the cancer is a PD-1-expressing cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is refractory to PD-1 and / or PD-L1-based therapies. In some embodiments, the cancer has never responded to PD-1 and / or PD-L1-based therapies. In some embodiments, the cancer has responded to PD-1 and / or PD-L1-based therapies but has become refractory. In some embodiments, the method of the present invention converts refractory cancer into reactive cancer.

[0276] In some embodiments, the method includes confirming that the cancer expresses HLA, MHC-I, or both. In some embodiments, the method includes confirming that the cancer expresses HLA. In some embodiments, the method includes confirming that the cancer expresses MHC-I. In some embodiments, the method includes confirming that the cancer expresses both HLA and MHC-I. In some embodiments, confirmation includes measuring expression in the cancer. In some embodiments, confirmation includes measuring expression on the surface of the cancer. In some embodiments, in the cancer and / or on the cancer is in and / or on cancer cells. In some embodiments, confirmation includes measuring HLA-G secreted by the cancer. In some embodiments, confirmation includes measuring soluble HLA-G. In some embodiments, soluble HLA-G is in body fluids. In some embodiments, the body fluid is blood.

[0277] In some embodiments, the method includes confirming ILT2 expression in the subject. In some embodiments, the method includes confirming that ILT2 expression in the subject is above a predetermined threshold. In some embodiments, confirmation includes measuring ILT2 expression in the subject. In some embodiments, confirmation is performed before administration. In some embodiments, measurement is performed before administration. In some embodiments, ILT2 expression is in immune cells. In some embodiments, ILT2 expression is in the subject's immune cells. In some embodiments, the immune cells are peripheral blood immune cells. In some embodiments, the immune cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the immune cells are intratumoral immune cells. In some embodiments, the immune cells are immune cells in the tumor microenvironment (TME). In some embodiments, the immune cells are selected from CD8-positive T cells, macrophages, NK cells, and T cells. EMRA Cells. In some embodiments, the immune cells are CD8-positive T cells. In some embodiments, the immune cells are peripheral blood CD8-positive T cells.

[0278] In some embodiments, administering the antibody or antigen-binding fragment of the present invention comprises administering a pharmaceutical composition containing the antibody or antigen-binding fragment of the present invention. In some embodiments, a therapeutically effective amount of the antibody or antigen-binding fragment is administered. In some embodiments, the pharmaceutical composition further comprises a carrier, excipient, or adjuvant. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0279] As used herein, the terms “carrier,” “excipient,” or “adjuvant” refer to any component in a pharmaceutical composition that is not an active agent. As used herein, the term “pharmaceuticalally acceptable carrier” refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered astragalus gum; malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline, Ringer's solution; ethanol and phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can be used as carriers herein include sugars, starches, cellulose and their derivatives, powdered astragalus gum, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifiers, and other non-toxic, pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants (such as sodium dodecyl sulfate), as well as colorants, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions considered herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those skilled in the art, such as those described in the following literature: Merck Index, 13th edition, edited by Budavari et al., Merck & Co., Inc., Ravi, NJ (2001); CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th edition (2004); and “Inactive Ingredient Guide,” US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, all contents of which are hereby incorporated by reference in their entirety.Examples of pharmaceutically acceptable excipients, carriers, and diluents that can be used in the compositions of the present invention include distilled water, physiological saline, Ringer's solution, dextran solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th ed., Gilman et al., Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990); and Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, PA (2005), each of which is incorporated herein by reference in its entirety. The compositions described in this invention may also be contained in artificially generated structures such as liposomes, ISCOMS, slow-release particles, and other mediators that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. Liposomes used in conjunction with the peptides described in this invention are formed from standard vesicle-forming lipids, which typically comprise neutral and negatively charged phospholipids and sterols (such as cholesterol). The choice of lipids is generally determined by considerations such as liposome size and stability in the blood. Various methods can be used to prepare liposomes, as reviewed, for example, in *Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York*, and also see U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0280] The carrier may contain, by weight, a total of about 0.1% to about 99.99999% of the pharmaceutical composition presented herein.

[0281] The term "therapeutic effective dose" refers to the amount of a drug that is effective in treating a disease or disorder in a mammal. It also refers to the amount that, at the required dose and time period, effectively achieves the desired therapeutic or preventative outcome. The exact dosage form and regimen will be determined by the physician based on the patient's condition.

[0282] In some embodiments, the method further includes administering an opsonizing agent to the subject. In some embodiments, the method further includes contacting cells with the opsonizing agent. In some embodiments, the opsonizing agent is an epidermal growth factor receptor (EGFR) inhibitor. In some embodiments, the EGFR inhibitor is cetuximab. In some embodiments, the opsonizing agent is not an anti-CD47 agent. In some embodiments, the method further includes administering a PD-1 / PD-L1-based therapy to the subject. In some embodiments, the method further includes contacting cells with a PD-1 / PD-L1-based therapy. In some embodiments, the method further includes growing cells in the presence of a PD-1 / PD-L1-based therapy. In some embodiments, the PD-1 / PD-L1-based therapy is a PD-1 or PD-L1 blocking antibody. In some embodiments, the method does not include administering an anti-CD47 agent or therapy. In some embodiments, the method does not administer an anti-CD47 agent or therapy. In some embodiments, the method further includes administering an anti-CD47 agent or therapy.

[0283] In some embodiments, an agent that inhibits ILT2-based immunosuppression binds to ILT2. In some embodiments, the agent binds to the extracellular domain of ILT2. In some embodiments, the agent is an ILT2 antagonist. In some embodiments, the agent is an ILT2 blocking antibody. In some embodiments, the agent inhibits the interaction between ILT2 and B2M. In some embodiments, the agent is the antibody of the present invention.

[0284] In some embodiments, the agent inhibiting ILT2-based immunosuppression is administered before, after, or simultaneously with the opsonizer. In some embodiments, the agent inhibiting ILT2-based immunosuppression and the opsonizer are administered in a single composition. In some embodiments, the agent inhibiting ILT2-based immunosuppression and the opsonizer are administered in separate compositions.

[0285] In some embodiments, the agent inhibiting ILT2-based immunosuppression is administered before, after, or simultaneously with PD-1 / PD-L1 therapy. In some embodiments, the agent inhibiting ILT2-based immunosuppression and PD-1 / PD-L1 therapy are administered in a single composition. In some embodiments, the agent inhibiting ILT2-based immunosuppression and PD-1 / PD-L1 therapy are administered in separate compositions. In some embodiments, at least one of the agents or therapies is suitable for co-administration.

[0286] As used herein, the term "suitable for co-administration" means that the antibody is present in a form that allows it to be safely and readily administered to a subject. In some non-limiting embodiments, co-administration may be performed orally, by injection, or by inhalation. In some embodiments, the antibody will be contained within a pharmaceutical composition that can be safely and readily administered to a subject. In some embodiments, the pharmaceutical composition comprises an antibody and a pharmaceutically acceptable carrier or excipient.

[0287] In some embodiments, the HLA is HLA-G. In some embodiments, the HLA is atypical HLA. In some embodiments, the HLA is canonical HLA. In some embodiments, mRNA expression is confirmed. In some embodiments, protein expression is confirmed. In some embodiments, surface expression of the protein is confirmed. Methods for measuring expression are well known in the art and include PCR, Q-PCR, RNA blotting, immunoblotting, in situ hybridization, immunostaining, and FACS. In some embodiments, the methods include FACS analysis of cancer to confirm surface expression.

[0288] Preparations

[0289] The present invention also contemplates pharmaceutical formulations for human medical use comprising at least one antibody recognizing ILT2 as an active agent, said pharmaceutical formulations for the manufacture of therapeutic compositions for the treatment, diagnosis or prevention of conditions described herein in various ways.

[0290] In such pharmaceutical preparations, the active agent is preferably used in conjunction with one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredient. The one or more carriers must be pharmaceutically acceptable in the sense of compatibility with other ingredients in the preparation and without undue harm to the recipient. The active agent is provided in an amount sufficient to effectively achieve the desired pharmacological action as described above and in an amount suitable for achieving the desired daily dose.

[0291] Typically, the molecules of the present invention, containing the antigen-binding portion of an antibody, are suspended in a sterile saline solution for therapeutic use. Pharmaceutical compositions can alternatively be formulated to control the release of the active ingredient (the molecule containing the antigen-binding portion of an antibody) or to prolong its presence in the patient system. A variety of suitable drug delivery systems are known, including, for example, implantable drug delivery systems, hydrogels, hydroxymethyl cellulose, microcapsules, liposomes, microemulsions, microspheres, etc. Controlled-release formulations can be prepared by using polymer complexes or adsorption of molecules according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of stearic acid dimer and polyanhydride copolymers of sebacic acid. The release rate of the molecules according to the present invention (i.e., antibodies or antibody fragments) from such matrices depends on the molecular weight of the molecules, the amount of the molecules within the matrices, and the size of the dispersed particles.

[0292] The pharmaceutical compositions of the present invention can be administered by any suitable means, such as oral, topical, intranasal, subcutaneous, intramuscular, intravenous, intra-articular, intra-articular, intralesional, or parenteral administration. Generally, intravenous (iv), intra-articular, topical, or parenteral administration is preferred.

[0293] It will be clear to those skilled in the art that the therapeutically effective amount of the molecule according to the invention will depend in particular on the administration schedule, the unit dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, the patient's immune status and health, the therapeutic activity of the molecule administered, and the judgment of the treating physician.

[0294] Although the appropriate dosage of the molecules (antibodies or fragments thereof) of the present invention varies depending on the route of administration, molecular type (peptide, polynucleotide, organic molecule, etc.), and the patient's age, weight, sex, or condition, and should ultimately be determined by a physician, in the case of oral administration, the daily dose can generally be between about 0.01 mg and about 500 mg per kg of body weight, preferably about 0.01 mg and about 50 mg, more preferably about 0.1 mg and about 10 mg. In the case of parenteral administration, the daily dose can generally be between about 0.001 mg and about 100 mg per kg of body weight, preferably about 0.001 mg and about 10 mg, more preferably about 0.01 mg and about 1 mg. The daily dose can be administered, for example, in a regimen typically administered 1-4 times daily alone. Other preferred methods of administration include intra-articular administration of about 0.01 mg and about 100 mg per kg of body weight. Various considerations for achieving effective doses are described in, for example, the following literature: Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th edition, Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pennsylvania, 1990.

[0295] Suitable dosing regimens for combination chemotherapy are known in the art and described, for example, in Saltz et al., Proc ASCO 1999, 18, 233a and Douillard et al., Lancet 2000, 355, 1041-7.

[0296] The molecules of the present invention, which are the active ingredients, are dissolved, dispersed, or mixed in a well-known pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients are, for example, water, saline, phosphate-buffered saline (PBS), dextran, glycerol, ethanol, and combinations thereof. Other suitable carriers are well known to those skilled in the art. Additionally, the composition may contain small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffers, if desired.

[0297] Generation method

[0298] According to another aspect, a method for producing a drug is provided, the method comprising: obtaining a drug that binds to the extracellular domain of ILT2 or a fragment thereof; testing the ability of the drug to increase at least one of: macrophage inflammatory activity, T cell activity against cancer cells, dendritic cell activity, and natural killer (NK) cell cytotoxicity against cancer cells; and selecting at least one drug that increases at least one of macrophage activity, T cell activity, dendritic cell activity, and cytotoxicity; thereby producing the drug.

[0299] According to another aspect, a method for producing a drug is provided, the method comprising: culturing a host cell containing one or more vectors comprising a nucleic acid sequence encoding the drug, wherein the nucleic acid sequence is a nucleic acid sequence of the drug selected in such a manner as:

[0300] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0301] ii. Test the ability of the agent to increase at least one of the following: macrophage inflammatory activity, T cell activity against cancer cells, dendritic cell activity, and NK cell cytotoxicity against cancer cells; and

[0302] iii. Select at least one agent that increases at least one of phagocytosis, activity, and cytotoxicity;

[0303] This produces the medicine.

[0304] According to another aspect, a method for producing a drug is provided, the method comprising: obtaining a drug that binds to the extracellular domain of ILT2 or a fragment thereof; testing the ability of the drug to increase the efficacy of anti-PD-L1 / PD-1-based therapy against cancer cells; and selecting at least one drug that increases the efficacy of anti-PD-L1 / PD-1-based therapy; thereby producing the drug.

[0305] According to another aspect, a method for producing a drug is provided, the method comprising: culturing a host cell containing one or more vectors comprising a nucleic acid sequence encoding the drug, wherein the nucleic acid sequence is a nucleic acid sequence of the drug selected in such a manner as:

[0306] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0307] ii. To test the ability of the said agent to enhance the efficacy of anti-PD-L1 / PD-1 based therapies against cancer cells; and

[0308] iii. Select at least one agent that enhances the efficacy of anti-PD-L1 / PD-1-based therapies against cancer cells;

[0309] This produces the medicine.

[0310] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0311] An agent is obtained that binds to the extracellular domain of ILT2 or a fragment thereof, and the agent is tested for its ability to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and at least one agent is selected to induce at least two of the following: said increased phagocytosis, said increased activity, said increased production, said decreased production, said recruitment, said increased activation, said decreased activity, and said increased cytotoxicity; thereby producing the agent.

[0312] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0313] Culture host cells containing one or more vectors that encode a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected in the following manner:

[0314] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0315] ii. Test the ability of the agent to induce at least two of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased natural killer (NK) cell cytotoxicity against cancer cells; and

[0316] iii. Select at least one agent that increases at least two of the following: increased phagocytosis, increased activity, increased production, decreased production, recruitment, increased activation, decreased activity, and increased cytotoxicity;

[0317] This produces the medicine.

[0318] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0319] A drug that binds to the extracellular domain of ILT2 or a fragment thereof is obtained; the ability of said drug to inhibit the interaction between ILT2 and B2M is tested; and at least one drug that inhibits the interaction between ILT2 and B2M is selected; thereby producing a drug.

[0320] According to another aspect, a method for producing a pharmaceutical agent is provided, the method comprising:

[0321] Culture host cells containing one or more vectors that encode a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected in the following manner:

[0322] i. Obtain agents that bind to the extracellular domain of ILT2 or fragments thereof;

[0323] ii. Test the ability of the agent to inhibit the interaction between ILT2 and B2M; and

[0324] iii. Select at least one agent that inhibits the interaction between ILT2 and B2M;

[0325] This produces the medicine.

[0326] According to another aspect, a method for generating a drug is provided, the method comprising: obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO:41, 42, 43 and 44; thereby generating the drug.

[0327] According to another aspect, a method for producing a drug is provided, the method comprising: culturing a host cell containing one or more vectors having a nucleic acid sequence encoding a drug, wherein the nucleic acid sequence is a nucleic acid sequence of a drug selected by obtaining a drug that binds to an ILT2 epitope selected from human ILT2 sequences of SEQ ID NO: 41, 42, 43 and 44; thereby producing a drug.

[0328] In some embodiments, the method further includes testing the ability of an agent to inhibit ILT2-mediated immunosuppression, and selecting at least one agent that inhibits ILT2-mediated immunosuppression. In some embodiments, the nucleic acid sequence belongs to the agent selected by testing the ability of the agent to inhibit ILT2-mediated immunosuppression and selecting an agent that inhibits ILT2-mediated immunosuppression. In some embodiments, the method includes testing the ability of the agent to induce at least three of the following: increased macrophage phagocytosis of cancer cells, increased T cell activity against cancer cells, increased M1 macrophage production, decreased M2 macrophage production, increased dendritic cell recruitment to the tumor microenvironment, increased dendritic cell activation, and increased cytotoxicity against natural killer (NK) cells; and selecting at least one agent that induces said at least three. In some embodiments, the method includes testing the ability of the agent to induce effects in at least three of the following: T cells, NK cells, dendritic cells, and macrophages. In some embodiments, the method includes testing the ability of the agent to induce effects in T cells, NK cells, dendritic cells, and macrophages.

[0329] In some embodiments, the muscle-building effect includes a synergistic increase in anticancer activity. In some embodiments, the anticancer activity is the secretion of pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are selected from GM-CSF, IL-6, and IFNγ. In some embodiments, the pro-inflammatory cytokines are GM-CSF, IL-6, or IFNγ. Each possibility represents a separate embodiment of the invention. In some embodiments, the pro-inflammatory cytokine is GM-CSF. In some embodiments, the increased efficacy includes a synergistic increase in T cell activation. In some embodiments, the increased efficacy includes a synergistic increase in T cell cytotoxicity. In some embodiments, the increased efficacy includes a synergistic increase in both T cell activation and cytotoxicity. In some embodiments, the increase includes increased membrane CD107a expression. In some embodiments, the increase is characterized by increased membrane CD107a expression. In some embodiments, the increase is compared to efficacy without administration or exposure to the agent. In some embodiments, the increased efficacy includes converting cancers refractory to PD-1 / PD-L1-based therapies into cancers responsive to said therapy. In some embodiments, the cancer expresses HLA. In some implementations, cancer cells express MHC-I.

[0330] In some embodiments, increased macrophage inflammatory activity includes increased macrophage phagocytosis of cancer cells. In some embodiments, increased macrophage inflammatory activity includes increased production of M1 macrophages. In some embodiments, increased macrophage inflammatory activity includes decreased production of M2 macrophages. In some embodiments, increased macrophage inflammatory activity includes increased M1 phenotype on macrophages. In some embodiments, increased macrophage inflammatory activity includes decreased M2 phenotype on macrophages.

[0331] In some embodiments, dendritic cell activity includes dendritic cell activation. In some embodiments, dendritic cell activity includes recruitment of dendritic cells to the tumor. In some embodiments, dendritic cell activity is activity against cancer cells. In some embodiments, activity against cancer cells is activity within a tumor mesenchymal exchange (TME). In some embodiments, the tumor is a TME. In some embodiments, dendritic cell activity includes antigen presentation.

[0332] In some embodiments, testing the ability of the agent includes the agent increasing at least one, two, three, four, five, or all of the following: T cell activity against cancer cells, macrophage inflammatory activity, dendritic cell activity, and natural killer (NK) cell cytotoxicity against cancer cells. Each possibility represents a separate embodiment of the invention. In some embodiments, selecting at least one agent includes selecting an agent that increases at least one, two, three, four, five, or all of the following: T cell activity against cancer cells, macrophage inflammatory activity, dendritic cell activity, and natural killer (NK) cell cytotoxicity against cancer cells. In some embodiments, increasing macrophage inflammatory activity is increasing the production of M1 macrophages and / or increasing macrophage phagocytosis of cancer cells. In some embodiments, increasing macrophage inflammatory activity is decreasing the production of M2 macrophages. In some embodiments, testing the ability of the agent includes the agent's ability to increase macrophage inflammatory activity. In some embodiments, testing the ability of the agent includes the agent's ability to increase dendritic cell activity. In some embodiments, to the tumor is to the TME. In some implementations, the ability of the drug to be tested includes the drug's ability to increase the cytotoxicity of NK cells against cancer cells.

[0333] In some embodiments, the method further includes testing the ability of the agent to inhibit the interaction between ILT2 and B2M. In some embodiments, the interaction is a direct interaction. In some embodiments, the method further includes testing the ability of the agent to inhibit the contact between ILT2 and B2M. In some embodiments, the interaction is binding. In some embodiments, the contact is binding. In some embodiments, the method further includes testing the ability of the agent to bind to epitopes.

[0334] The following examples are intended to illustrate how to prepare and use the compounds and methods of the present invention, and should in no way be considered limiting. Although the invention will now be described in conjunction with specific embodiments, it will be apparent to those skilled in the art that many modifications and alterations will be apparent. Therefore, it is intended to include all such modifications and alterations that fall within the spirit and broad scope of the appended claims.

[0335] Example

[0336] Generally, the nomenclature used herein and the laboratory procedures used in this invention include molecular, biochemical, microbiological, and recombinant DNA techniques. These techniques are well explained in the literature. See, for example, “Molecular Cloning: A Laboratory Manual”, Sambrook et al., (1989); “Current Protocols in Molecular Biology”, Volumes I-III, Ausubel, RM, ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, MD (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, NY (1988); Watson et al., “Recombinant DNA”, Scientific American Books, NY; Birren et al. (eds.), “Genome Analysis: A Laboratory Manual Series”, Volumes I-4, Cold SpringHarbor Laboratory Press. New York (1998); as described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III, Cellis, JE, ed. (1994); “Culture of Animal Cells - A Manual of Basic Technique”, Freshney, Wiley-Liss, New York (1994), Third Edition; “Current Protocols in Immunology”, Volumes I-III, Coligan JEReferences cited are incorporated by way of citation. Stites et al. (eds.), “Basic and Clinical Immunology” (8th ed.), 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); “Monoclonal Antibodies: Methods and Protocols”, Maher Albitar, Springer Science & Business Media (2007). Other general references are provided throughout this document.

[0337] Materials and methods

[0338] Antibody - Commercial anti-ILT2 mAbs are: clone #1 - GHI / 75 (BioLegend, catalog number 333704), clone #2 - HP-F1 (eBioscience, catalog number 16-5129). Other mAbs used are: 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).

[0339] Flow cytometry - Typically, cells are kept on ice or at 4ºC throughout all steps. Before staining, prepare 5 x 10⁻⁶ cells. 5Cells were blocked for 15 min with 50 µg / mL human IgG (Sigma, catalog number I4506) in FACS buffer (PBS containing 0.1% BSA). Antibody was used at the manufacturer's recommended concentration and incubated in the dark for 30 min. Incubation was performed in 100 μL of 96-well U-shaped plates. Cells were washed twice with 200 μL of FACS buffer and transferred to 150 μL of FACS buffer in FACS tubes for analysis. Cells were analyzed on a Gallios flow cytometer (Beckman coulter) using Kaluza software for Gallios flow cytometry acquisition.

[0340] Myeloid cell differentiation - Monocytes were isolated from fresh blood samples from healthy donors using a negative selection method with the EasySep™ Human Monocyte Enrichment Kit (STEMCELL, catalog number 19059). Different cell populations were tested for the indicated phenotype by FACS analysis of relevant markers and by analysis of characteristic cytokine secretion. For mature cells, 0.8 x 10⁻⁶ cells were used. 6 Monocytes were cultured at a density of 1 / mL in RPMI medium containing growth factors, with the medium changed on days 3 and 6. Inflammatory M1 macrophages were matured for 6 days in the presence of 50 ng / mL GM-CSF (M1 phenotype), followed by maturation for 48 hours in the presence of 20 ng / mL IFN-γ and 50 ng / mL LPS. Suppressive M2 macrophages were differentiated for 6 days using 50 ng / mL M-CSF, followed by differentiation for 48 hours using 10 ng / mL M-CSF and 20 ng / mL IL-4 and IL-10. Dendritic cells were induced for 6 days with 50 ng / mL GM-CSF and 20 ng / mL IL-4, and further differentiated into mature (100 ng / mL LPS) or tolerant (IL-10 100 U / mL and IFN-α2b 1000 U / mL) dendritic cells.

[0341] transfection - HLA-G1 plasmids (encoding full-length HLA-G transcripts) were generated by cloning HLA-G1 cDNA into the PCDNA3.1 vector. Transfection was performed using jetPEI. ® Transfection was performed using PolyPlus Transfections. The ILT2 / CD3z plasmid was generated by combining the extracellular portion of the human ILT2 protein with the transmembrane and cytoplasmic residue frames of the mouse CD3 gene. The plasmid nucleus was transfected into the mouse BW5417.3 T cell line using Nucleofector II (Lonza) as described by the manufacturer. Stable transfectants were selected in a medium containing G418.

[0342] Co-culture assay of NK and cancer cell lines - NK cells were incubated together with the indicated cell line at 37ºC for 5 hours in the presence of anti-ILT2 antibody and a matched isotype control. Cytotoxicity levels were measured using a fluorescence assay kit for LDH detection (Promega).

[0343] Flow cytometry blocking assay - Recombinant human ILT2 protein fused to the Fc region of human IgG1 at the N-terminus will be conjugated with biotin (Innova bioscience). A total of 5 x 10 5 A375 / HLA-G1 cells were incubated at room temperature for 30 min at a volume of 100 µL in the presence of anti-ILT2 clone #1 or an allotype-matched control mAb and biotin-conjugated ILT2-Fc (10 μg / mL). After several washing steps, streptavidin-PE was added to a final concentration of 0.2 μg / mL and incubated on ice for 30 min before FACS analysis.

[0344] BW ILT2 / CD3z chain chimerism determination - 3X10 4 One BW / ILT2z sample was mixed with an equivalent number of A375 / WT or A375 / HLA-G1 cells for 24 hours. Functional mAbs at indicated concentrations and matched isotype controls were used. The amount of secreted mouse IL2 was evaluated using a commercial ELISA kit (BioLegend).

[0345] Example 1

[0346] ILT2 and HLA-G were found on cancer cells and cancer-associated immune cells.

[0347] ILT2 is a known immunosuppressive molecule found on the surface of healthy immune cells and many tumor cells. ILT2 has been shown to bind to MHC-1 and HLA class molecules (HLA-G, HLA-F, and HLA-B27), and compete with CD8, thereby inhibiting T cell activation. To further understand the range of cells expressing ILT2, flow cytometry analysis was performed on various immune cell types using a commercial antibody (antibody #1). As reported in the literature, cytotoxic T cells (CTLs) and natural killer (NK) cells derived from melanoma patients showed positive surface expression of ILT2. Figure 1 Monocytes from healthy donor blood were also examined and found to highly express ILT2 (…). Figure 2 (Leftmost image). After monocytes differentiate into different myeloid cell populations (dendritic cells and macrophages), regardless of whether they are immature, inflammatory, or tolerant, they all retain ILT2 expression. Figure 2(See right-hand image).

[0348] Bioinformatics analysis of the TCGA database was used to examine ILT2 expression in different cancer indications. Figure 3A Interestingly, an association was observed between ILT2 RNA expression levels and the presence of myeloid-derived suppressor cells (MDSCs) and suppressor M2 tumor-associated macrophages (TAMs) in tumor samples represented in TCGA. Figure 3B Analysis of fresh tumor samples from various solid tumors using flow cytometry confirmed ILT2 expression in innate and adaptive immune cells within the tumor microenvironment (TME). Tumor samples were collected from patients with non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck cancer, esophageal cancer, and colon cancer, and single-cell suspensions were generated through enzymatic digestion. The percentages of ILT2-positive cells among total immune cells, tumor-associated macrophages (TAM), CD4-positive T cells, CD8-positive T cells, and natural killer (NK) cells were presented. Figure 3C Therefore, it is evident that ILT2 is expressed simultaneously on cells with anti-cancer activity (inflammatory cells) and on cells with cancer-promoting and immunosuppressive activities (tolerogenic and MDSCs).

[0349] HLA-G expression is also being studied in various cancers. Tissue microarrays (TMAs) of cancer samples from different indications were stained with commercial polyclonal HLA-G antibodies via immunohistochemistry. The percentage of sample cases for each cancer type is indicated. Figure 4A Additionally, for several indications, expanded TMA is examined. The HLA-G staining score is calculated by multiplying the staining intensity by the percentage of positive cells. High HLA-G staining scores above 100 were detected in high percentages of esophageal, gastric, head and neck, and renal cancers. Figure 4B The percentage of positive cases for each indication is shown in Table 1.

[0350] Table 1:

[0351]

[0352] HLA-G exists in a soluble, secreted form as well as a more common membrane form. To examine the expression levels of soluble HLA-G in cancer patients, the presence of HLA-G in plasma samples was detected using a commercial ELISA. Overexpression of HLA-G was found in several cancer indications compared to normal (healthy) controls. Figure 5 Furthermore, in certain cancer types, patient groups with significantly higher levels can be detected.

[0353] Example 2

[0354] ILT2 blocking antibody production

[0355] Monoclonal ILT2 antagonist antibodies were generated using hybridoma technology. Initially, 69 ILT2-specific hybridomas were generated. Three leader antibodies were selected based on their preferred binding, cross-reactivity profile, and functional activity in various assays examined. The selected antibodies were 19E3, 15G8, and 17F2. These antibodies were sequenced using common methods. The sequences of the selected antibodies are shown below. Figure 6 The CDRs were determined using the KABAT system. 15G8 and 19E3 were humanized using common CDR transplantation methods. In short, the essential CDRs and framework residues from the initial hybridoma-derived antibody were identified and transplanted into the variable and constant regions of the germline human antibody. The final humanized antibody was an IgG4 antibody. The final humanized 15G8 also contained a single amino acid change, removing cysteine ​​from CDR-H3 and replacing it with either alanine or serine. This change was performed to improve developability. Binding of the two resulting antibodies was confirmed, and the 15G8 antibody with alanine was selected for further testing. All humanized 15G8 mentioned below refers to the alanine variant.

[0356] The ability of anti-ILT2 antibodies to bind to ILT2 was tested using three different systems. Binding to recombinant ILT2 was tested using ELISA and the Biacore T200 (Table 2). Figure 7A ), and used BW cells transfected with ILT2 to test binding to membrane ILT2 ( ), Figures 7B-7C Chimeric mice and humanized antibodies showed similar binding ( ). Figure 7D A commercial mouse anti-human ILT2 antibody (Biolegend; clone GHI / 75) was used as a positive control. All three assay antibodies successfully bound ILT2, whether in solution or on the cell surface. Cross-reactivity with several similar ILT family members (PIRB, ILT6, and LILRA1) was also examined using a binding ELISA. Antibodies against these proteins were used as positive controls. None of the antibodies cross-reacted with PIRB, ILT6, or LILRA1. The antibodies were also effective for immunostaining. Figure 7E Interestingly, when PBMCs were isolated from the blood of cancer patients, it was found that ILT2 was expressed on more T cells and NK cells in cancer patients compared to healthy controls. Figure 7E ).

[0357] Table 2

[0358]

[0359] Example 3

[0360] ILT2 antibody blocks ILT2-HLA-G interaction

[0361] Four different assays were used to test the ability of the generated anti-ILT2 antibody to block the interaction between HLA-G and ILT2. First, a blocking flow cytometry assay was performed. HLA-G-transfected A375 cells were incubated with biotinylated ILT2 in the presence of the antibody of the present invention and a positive control antibody. A commercially available anti-ILT2 antibody, GHI / 75 (BioLegend, catalog number 333704), was used as a positive control. The binding of ILT2-biotin to cells was determined by flow cytometry analysis using streptavidin-PE. Figure 8A The percentage of blockade was determined by normalization against the negative control (ILT2 binding in the presence of control IgG). Representative FACS analyses are presented in [the table / data]. Figure 8B The diagram shows ILT2 binding in the absence of antibody (gray line), in the presence of 15G8 (light gray line), and in the presence of an isotype control (black line). The percentage of blockade was calculated at different antibody concentrations. Figure 8C Chimeric mouse and humanized antibodies showed similar blocking abilities. Figure 8D ).

[0362] The ability of ILT2 antibodies to functionally block the interaction between HLA-G and ILT2 was also examined in a BW ILT2 / mouse Z-chain chimeric reporter assay. BW cells (BW-ILT2) were transfected with human ILT2 fused to the ζ chain of mouse T cells. Cells were then co-incubated with A375-HLA-G cells in the presence of a selected ILT2 antibody. Following functional ILT2-HLA-G interaction, BW cells secrete the reporter cytokine, mouse IL-2. Blocking the interaction reduced the secretion of this reporter cytokine. Mouse IL-2 secretion was determined by ELISA after 24 hours of incubation. Results are expressed as the mean of mIL-2 levels ± SE from triplicate wells for each treatment. Figure 8E A commercial mouse anti-human ILT2 antibody (Biolegend; clone GHI / 75) was used as a positive control (PC) for both assays. The percentage of blockade was calculated at different antibody concentrations. Figure 8F Using the same BW ILT2 / mouse Z-chain chimeric reporter assay, the possibility that the novel antibody might possess ILT2 activation activity on its own was ruled out. Cells were incubated with the ILT2 antibody in the absence of cancer cells, and mouse IL-2 secretion was measured again. Figure 8G The novel ILT2 antibody was found to have no agonistic effect, but other antibodies (1G7) generated using the same hybridoma method could bind to ILT2 and induce its activity.

[0363] Functional blockade was also examined in human Jurkat cells (T cells). Jurkat cells were incubated with or without exogenous HLA-G and single-chain anti-CD3 (OKT3) expressing A375 cancer cells. Secretion of pro-inflammatory human IL-2 was measured. High levels of IL-2 were secreted when Jurkat cells were co-cultured with cancer cells, using unmodified Jurkat cells (ILT2-negative cells). Figure 8H Unsurprisingly, the addition of the 15G8 antibody had no effect on IL-2 secretion, as the ILT2 to be blocked was absent. Therefore, Jurkat cells were transfected to express human ILT2. First, ILT2-positive Jurkat cells were cultured with and without exogenous OKT3-expressing A375 cancer cells. These cancer cells were naturally MHC-I positive. MHC-I from cancer cells strongly inhibits IL-2 secretion (…). Figure 8I In this scenario, the addition of 15G8 antibody blocked ILT2 / MHC-I interaction in a dose-dependent manner and increased IL-2 secretion. A pan-HLA antibody was used as a positive control, and at equivalent concentrations, 15G8 antibody was comparable to the pan-HLA antibody. Figure 8I To enhance the inhibitory effect, A375 cells were also transfected with HLA-G, making them MHC-I and HLA-G positive. These cells produced an even stronger inhibitory effect on ILT2-positive cells, thereby reducing IL-2 secretion to the level of IL-2 secretion in cultured Jurkat cells alone. Figure 8J A dose-dependent effect was again observed with the administration of the 15G8 antibody, and at equal doses, the 15G8 antibody and the pan-HLA antibody were again equally effective. Figure 8J It is noteworthy that when only HLA-G specific antibodies were used instead of pan-HLA, the effect was significantly reduced and comparable to that of 15G8 antibody used at a concentration of 1 / 100. Figure 8K ).

[0364] The Jurkat system was also used to compare the 15G8 antibody with two commercially available antibodies: GHI / 75 and HP-F1. Jurkat cells expressing human ILT2 were co-cultured with A375 cells expressing HLA-G / OKT3 in the presence and absence of different concentrations of 15G8, GHI / 75, and HP-F1. As already observed, 15G8 caused a statistically significant, dose-dependent increase in IL-2 secretion (…). Figure 8L Compared to the culture medium alone, GHI / 75 had no effect on IL2 secretion, but resulted in a slight increase compared to the IgG control. Figure 8M HP-F1 produced a small but significant increase, which reached a plateau and did not increase with further dosing. Figure 8NHP-F1 performed worse even at 20 μg / ml compared to 15G8, which had a concentration of only 4 μg / ml.

[0365] Finally, activation was measured directly in TILs and NK cells. TILs were incubated with A375-HLA-G-OKT3 cells for 5 minutes, followed by detection of the T cell activation marker, phosphorylated ZAP70. NK cells were incubated with A253-HLA-G cells for 2 minutes, followed by detection of the NK cell activation marker, phosphorylated Syk. Activation was observed in both cell types when co-cultured with cancer cells, but this activation was enhanced in the presence of ILT2 antibody. Figures 8O-8P These results confirm that ILT2 antibodies can effectively block ILT2-HLA-G interaction, thereby leading to enhanced T cell and NK cell activation.

[0366] Example 4

[0367] ILT2 antibody enhances the phagocytic activity of HLA-G and MHC-I positive tumor cells.

[0368] The ability of generated anti-ILT2 antibodies to enhance the phagocytic activity of tumor cells was tested using two different systems. Monocytes were isolated from the blood of healthy donors and incubated for 6–7 days in the presence of M-CSF to generate macrophages. First, flow cytometry-based assays were performed. Different cancer cell lines stained with PKH67-FITC were co-incubated with macrophages stained with eFluor 670-APC in the presence of the indicated antibody. The level of phagocytosis was determined by the percentage of macrophages double-stained, indicating the phagocytosis of target cells. The level of phagocytosis was presented as a percentage relative to a control (culture medium only). Figure 9A As shown, different ILT2 blocking antibodies can enhance the phagocytic activity of macrophages on HLA-G-positive A375 cells. Additionally, using real-time IncuCyte... ® An analytical system was used to examine the ability of macrophages to enhance the phagocytic activity of tumor cells. Target cell lines were analyzed using pHrodo. ™ Red cell marker dyes were used to label the cells, which were then washed and added to macrophages along with repeated treatments. ® pHrodo ™ The fluorescence of red cell marker dyes increases in acidic environments (such as the inherent acidity in phagosomes), enabling the quantification of phagocytic events by measuring fluorescence. (IncuCyte) ®The instrument samples the assay plate every 30 minutes for fluorescence red signal intensity and phase mapping. Phagocytic events are reflected as the accumulation of red fluorescence signal, and the phagocytic rate is reflected from the kinetics of red fluorescence signal accumulation. Using this real-time system, the ability of humanized anti-ILT2 antibody to enhance phagocytosis in HLA-G positive A375 cells was confirmed. Figure 9B Additionally, using IncuCyte... ® The system confirmed that the generated ILT2-blocking antibody could enhance the phagocytic activity of both HLA-G-positive and various MHC-I-positive (WT) cancer cell lines. Figure 9C ).

[0369] Using the above IncuCyte ® Real-time systemic examination was conducted to examine the effect of combining the generated ILT2 antibody with the antibody Erbitux, which is induced by antibody-dependent phagocytosis (ADCP), on the phagocytosis of cancer cells. Compared to the activity of each antibody alone, the combination of the ILT2 blocking antibody and Erbitux significantly increased the phagocytosis of cancer cell lines overexpressing HLA-G. Figure 9D In fact, the combination of Erbitux and the 15G8 humanized antibody has a synergistic effect, with the combined treatment increasing phagocytosis more than the additive effect alone.

[0370] Example 5

[0371] The selected ILT2 antibody can restore T cell activity suppressed by HLA-G.

[0372] To examine the ability of the generated anti-ILT2 antibody to restore T cell activity suppressed by HLA-G, human CD8 T cells were co-incubated with wild-type 721.221 cells (221 WT) or 721.221 cells overexpressing soluble HLA-G5 (221-HLA-G). IFNγ secretion levels of T cells were measured using a standard ELISA after 5 days. Results are shown as a fold increase over the effect of 221-HLA-G alone and are expressed as the mean of four independent experiments. Figure 10A The results presented confirm that several ILT2 antibodies can restore HLA-G-suppressed T cell activity. This was also tested by co-incubation with A375-HLA-G-OKT3 cells. After 72 hours, the secretion of human granzyme B was also measured, and it was found to increase in a dose-dependent manner in the presence of the 15G8 antibody. Figure 10B ).

[0373] Example 6

[0374] The selected ILT2 antibody can enhance NK cell cytotoxicity against HLA-G and MHC-I positive tumor cells.

[0375] The ability of the generated anti-ILT2 antibody to enhance NK cell effector activity was tested in the system by co-incubating NK cells with various target cancer cell lines. Cells were co-incubated for 5 hours at an effector-to-target ratio of 7.5:1, followed by detection of cytotoxicity levels using a fluorescence assay kit for LDH detection. The percentage of specific cytotoxicity was calculated as follows:

[0376]

[0377] like Figure 11A As shown, the ILT2 antibody of the present invention can significantly enhance the cytotoxicity of NK cells against both HLA-G positive cells and various MHC-I positive cancer cell lines in a dose-dependent manner. Figure 11B Granulase B was also measured. Figure 11C ) and interferon-γ ( Figure 11D The secretion of IFNγ, ILT2, CD56, and CD107A was observed to increase in a dose-dependent manner. Primary NK cells were co-cultured with target HLA-G+ melanoma cells, and the expression of IFNγ, ILT2, CD56, and CD107A was analyzed by FACS. ILT2-positive and CD56-positive NK cell populations were specifically analyzed, and a dose-dependent increase in IFNγ expression and membrane CD107A expression was observed. Figures 11E-11F When plotted separately for each experiment, the association between the percentage of ILT2-positive cells and increased IFNγ and CD107A expression was clearly evident. Figures 11G-11H ).

[0378] Example 7

[0379] ILT2 antibodies increase the production of inflammatory macrophages.

[0380] The effect of ILT2 blockade on macrophage maturation was examined in vitro. Monocytes isolated from healthy donors were differentiated for 5 days in the presence of M-CSF (50 mg / mL) to generate mature macrophages (M0) in the presence of humanized ILT2-blocking antibodies or control IgG. Macrophages were then further differentiated in the presence of LPS (50 ng / mL) to generate M1 macrophages, or differentiated with IL-4 (25 ng / mL) to generate M2 macrophages. Figure 12 As shown, the presence of ILT2-blocking antibodies during macrophage maturation increased HLA-DR (a marker of M1 inflammatory macrophages) expression on macrophages from most tested donors, regardless of whether they differentiated into M0, M1, or M2 macrophages. Additionally, macrophages differentiated into M1 macrophages also exhibited increased CD80 levels in most tested donors. In summary, these results confirm that the selected ILT2 antagonist antibody can induce macrophages displaying higher HLA-DR and CD80 levels, indicating macrophages with a higher inflammatory M1 phenotype.

[0381] Example 8

[0382] ILT2 blocking antibodies enhance the activity of immune cells against tumor cells from patients.

[0383] The activity of generated anti-ILT2 antibodies was examined in an ex vivo system using tumor samples from cancer patients (RCC and H&N). To test the antibody's ability to increase phagocytosis of tumor cells from patients, macrophages generated from monocytes were incubated together with tumor cells isolated from the tumor samples. IncuCyte was used as detailed above. ® The real-time analysis system examines the level of phagocytosis. For example, in... Figure 13A As shown, the ILT2 antibody can enhance the phagocytic activity of tumor cells from patients with different cancer indications. Furthermore, this effect is dose-dependent and persists even when autologous macrophages are used, and is also effective for RCC (…). Figure 13B ) and squamous cell carcinoma from H&N ( Figure 13C Both were observed. Additionally, the effect of ILT2 antibody in enhancing PBMC activity was examined. Single-cell suspensions of tumor samples from patients were incubated together with PBMCs isolated from the same patients in the presence of IL-2 (activated PBMCs). Figure 14G As shown, in the presence of ILT2 antibody, PBMC secretion of the pro-inflammatory TNF-α cytokine is increased in the presence of tumor cells. In summary, these results confirm the ability of blocking ILT2 antibody to increase the activity of immune cells against tumor cells from various cancer indications.

[0384] Example 9

[0385] ILT2 blocking antibodies can be combined with PD-1 / PD-L1 therapy

[0386] For the most part, ILT2 and PD-1 are expressed on different immune cells, including both peripheral blood cells and tumor microenvironment-resident immune cells. Figure 14A Analysis of ILT2 and PD-1 expression in intratumoral CD8-positive T cells from CRC patients revealed that both central T memory cells (Tcm) and consumed T cells (Tex) expressed high levels of PD-1. Figure 14B However, it expresses low levels of ILT2 ( Figure 14C CD45RA reexpressing T cells (T cells) EMRA The study showed a completely opposite pattern, expressing high levels of ILT2 and low levels of PD-1. This dichotomy is not unique to cancer; a large proportion (83%) of the ILT2 levels were found in blood from healthy donors. EMRAThe cells were ILT2 positive, while only a small percentage (17%) of the total CD8-positive T cells were positive. Figure 14D However, in the TME, ILT2 expression was enhanced in T cells. A single-cell suspension was generated by enzymatic digestion of tumors isolated from esophageal cancer patients. FACS analysis showed that the majority of CD8-positive tumor-infiltrating lymphocytes (TILs) were T cells. EMRA Cells (50%), and these T cells EMRA The cells were 100% ILT2 positive, but almost entirely PD-1 negative (95%). Figure 14E ).

[0387] The effects of the anti-ILT2 antibody combined with anti-PD-1 of the present invention were tested in SEB-activated (10 ng / ml) PBMCs from 10 healthy donors. Membrane CD107a expression was used as a marker of increased cytotoxicity. Overall, the 15G8 antibody produced a small, on average, increase in surface CD107a, while the anti-PD-1 produced a slightly larger response, which was donor-dependent. Figure 14F The combination of the two antibodies, on average, produces increased CD107a levels; however, these changes are variable depending on the specific donor sample. Figure 14G Three exemplary samples are presented. The first donor showed an additive effect when anti-PD-1 was combined with 15G8, with total CD107a levels approximately equal to the sum of the effects of each antibody alone. The second donor responded more strongly to anti-PD-1 than to anti-ILT2, but unexpectedly, the combination of the two antibodies had a more than additive effect. Anti-PD-1 produced a 19% increase in expression, and anti-ILT2 produced a 3.7% increase, but the combination treatment resulted in a 33.2% increase. This synergistic effect was even more pronounced in donor #3 cells. In donor #3, 15G8 was more effective than anti-PD-1 (13.1% increase vs. 9.3% increase), and the combination therapy was significantly more effective (41%), producing almost twice the effect predicted from the additive combination alone.

[0388] Next, the combined treatment of patient tumor cells with PD-1 blocking antibody and generated ILT2 antibody was evaluated. Various patient cancer cells were incubated with autologous PBMCs in the presence of anti-PD1 antibody, the antibody of the present invention, and combinations thereof. IgG was used as a control, and the secretion of pro-inflammatory molecules was measured as a readout. Enhanced secretion of pro-inflammatory cytokines was observed in the combined treatment (…). Figures 14H-14J Compared to the IgG control, treatment of colon adenocarcinoma cells from the first patient with the humanized antibody 15G8 did not enhance IFNγ secretion at all, but rather showed a robust increase in the secretion of anti-PD-1 cytokines. Figure 14HHowever, unexpectedly, the combination of anti-PD-1 and ILT2 antibodies increased secretion by more than 50%. A second patient showed a similar trend, where either ILT2 antibody or anti-PD-1 induced a small increase, and there was an enhanced synergistic increase when both antibodies were used in combination. Figure 14I As compared to the control, neither antibody alone altered GM-CSF expression; however, surprisingly, the combination of the two antibodies produced a robust increase of nearly 100% in control GM-CSF levels. Figure 14J ).

[0389] Next, the combination therapy was evaluated using mixed lymphocyte responses. Dendritic cells and CD8-positive T cells were isolated from various healthy donors, and macrophages were generated from monocytes isolated from H&N cancer patients. Cells were combined with the indicated treatment (20 ug / mg each) at an effector cell to target ratio of 5:1. IFNγ secretion by T cells was enhanced in the presence of either anti-ILT2 or anti-PD-1 antibodies, and this effect was increased when both antibodies were used in combination. Figures 14K-14L ). Such as with dendritic cell cultures ( Figure 14K Compared to macrophage cultures, a stronger cumulative effect was observed. Figure 14L These results clearly demonstrate that anti-ILT2 and anti-PD-1 therapies have a synergistic and de novo effect in enhancing the inflammatory response of immune cells.

[0390] Example 10

[0391] ILT2 blocking antibodies reduce tumor burden in vivo.

[0392] The efficacy of anti-ILT2 antibodies was examined in xenograft models. Immunocompromised SCID-NOD or NSG mice were inoculated with cancer cell lines (A375-HLA-G, A375-WT, COLO-320-HLA-G), and human macrophages derived from healthy donor blood were injected into the mice in the presence of ILT2 antibodies. Figure 15A As shown, administration of the generated ILT2 antibody in this model resulted in significant tumor suppression, most likely mediated by the activity of human macrophages in this system. Furthermore, antitumor efficacy was observed in both HLA-G and MHC-I positive tumor cells.

[0393] The efficacy of anti-ILT2 antibodies was also examined in an in vivo model of lung lesion melanoma xenograft. Melanoma cells (MEL526-HLA-G) were inoculated into immunocompromised SCID-NOD mice. Human PBMCs isolated from healthy donor blood were injected into mice in the presence of the selected ILT2 antibody, starting one day post-inoculation and repeated on days 2, 10, and 18. Figure 15BILT2 antibody was administered on days 1, 4, 8, 11, 15, 18, 22, and 25. Figure 15C As shown, administration of the generated ILT2 antibody resulted in a significant reduction in tumor cell metastasis, indicated by the formation of black lesions in the lungs of mice. Mice treated with the ILT2 antibody exhibited far fewer such lesions in their lungs compared to mice treated with control IgG. This effect was also confirmed by a decrease in lung weight in these mice. Figure 15D This is most likely mediated by human lymphocytes administered to mice, in combination with the inhibitory effect of the administered antibody on ILT2. Therefore, anti-ILT2 antibodies are effective in preventing metastasis and tumor formation.

[0394] Next, the efficacy of the novel antibody in treating established tumors was tested in the same in vivo mouse model. SCID-NOD mice were transfected with MEL526-HLA-G cells (as described above) via intravenous administration. After 15 days, human PBMCs isolated from healthy donors were administered to the relevant mouse groups, and this administration was repeated on days 25, 35, and 51 (see [link to relevant documentation]). Figure 15E Antibodies (ILT2 antibody, anti-PD-1 antibody, or a combination of both) were administered on days 14, 17, 20, 24, 27, 30, 34, 37, and 50 (see [link to relevant documentation]). Figure 15E Mice were sacrificed on day 53, and their lungs were weighed. Tumor weight was calculated by subtracting the lung weight of unexperimented mice from the lung weight of the test mice. Anti-PD-1 antibodies reduced tumor weight, but not significantly, while ILT2 antibodies and combination therapy had a significant effect. Figure 15F ).

[0395] Testing tumor-derived CD8 T cells, T EMRA CD107A and CD69 expression in cellular and NK cells. In total CD8 T cells, anti-PD-1 antibody induced a non-significant increase in CD107A expression, while ILT2 antibody, rather than combination therapy, induced a significant change. Figure 16A ). In T EMRA In cells, both ILT2 antibody and combination therapy induced a significant increase ( Figure 16B In NK cells, both anti-PD1 and anti-ILT2 antibodies significantly increased the percentage of CD69-positive cells, but surprisingly, combination therapy had a significantly enhanced effect, with the total percentage of CD69-positive cells being greater than the combination of either therapy alone. Figure 16C Surprisingly, when examining CD69 expression in CD8 T cells, neither anti-PD1 nor anti-ILT2 increased expression; however, combination therapy induced a very significant increase in CD69 expression. Figure 16DFurthermore, the effects of the ILT2 antibody were determined to be correlated with ILT2 expression. Significant differences in activation markers were observed when the experiment was broken down into mice receiving PBMCs with low or high ILT2 expression. EMRA In cells, compared with low ILT2 expression PBMCs, high ILT2 expression PBMCs included more than double CD107A expression ( Figure 16E Similarly, when examining NK cells, in combination therapy, high ILT2-expressing PBMCs induced nearly 90% of cells to express CD69; while low ILT2-expressing PBMCs induced less than 40% of NK cells to express CD69. Figure 16F Therefore, the expression level of ILT2 in PBMCs is essential for the most potent effect of the antibody.

[0396] Example 11

[0397] In vivo humanized H&N model

[0398] In the second in vivo model, humanized mice (mice inoculated with human CD34+ cells) were inoculated with A253-HLA-G cells. When the tumor reached 80 cubic millimeters in size, the mice were treated with either control IgG or ILT2 antibody (15G8, both at 10 mg / kg). The treatment was repeated twice a week. Figure 17A Until day 43, tumor size was determined by measuring the tumor with calipers at different time points. The ILT2 antibody completely stopped tumor growth in 2 out of 4 mice (mice #23 and #28), where the tumor was eradicated by day 43. Figure 17B To determine whether the different responses to treatment were due to varying levels of ILT2 expression in mouse immune cells, ILT2 expression in CD8 T cells from peripheral blood was measured at baseline. In fact, both mice with a complete response exhibited high ILT2 expression in their T cells, while the other two mice showed significantly lower expression levels. Figure 17C Furthermore, examination of the TME after treatment revealed three other pharmacodynamic markers that distinguished responders from non-responders, namely CD107A expression in T cells (…). Figure 17D M1 / M2 macrophage ratio ( Figure 17E ) and total CD80-positive dendritic cells ( Figure 17F These results indicate that anti-ILT2 is generated and transformed in the bone marrow and lymphatic compartments of the tumor microenvironment, and may also increase the ability of dendritic cells to present antigens and recruit more T cells to the tumor.

[0399] Example 12

[0400] Epitope localization of 15G8 humanized antibody

[0401] The 15G8 antibody was sent for epitope localization to determine its binding site on ILT2. Localization was performed by MAbSilico. The structure of the ILT2 used was modeled using the following structures: 6AEE (four Ig-like domains, some loops missing), 1VDG (undisclosed, domains 1 and 2), 1G0X (domains 1 and 2), and 4LL9 (domains 3 and 4). The structures of 6AEE and 1G0X were obtained from Wang, Q., et al., (2019). “Structures of the four Ig-like domain LILRB2 and the four-domain LILRB1 and HLA-G1 complex.” Cell. Mol. Immunol., and the structure of 4LL9 was obtained from Chapman, TL, et al., (2000). “Crystal structure and ligand binding properties of the D1D2 region of the inhibitory receptor LIR-1 (ILT2)”. Immunity, 13(5), 727-736. Region D1 is defined as residues 24-121 of ILT2. Region D2 is defined as residues 122-222 of ILT2. Region D3 is defined as residues 223-321 of ILT2. Region D4 is defined as residues 322-409 of ILT2. A 3D model of the antibody is constructed using Modeller.

[0402] Based on the top 30 docking poses, the residues are scored according to the probability that they belong to an epitope. Residues that may belong to an epitope are shown in... Figure 18A On the sequence and Figure 18B The target structure is defined based on these residues. Four main interaction regions are defined on the target. Figure 18C All four of these interacting regions were found in the intercalation region of ILT2, which is the hinge portion between D1 and D2. Validation mutations were selected in these regions and are summarized in Table 3. These mutations were generated in full-length ILT2 or truncated D1+D2 protein, and binding to the 15G8 antibody was tested. Loss or reduced binding to the mutants indicates that the region is the true epitope of the 15G8 antibody.

[0403] Table 3: Test Mutations

[0404]

[0405] However, the binding epitopes of most ILT2 antibodies are unknown, but international patent publication WO2020 / 136145 does disclose epitope information for several antibodies. Two universal binding regions were found, one in the D1 region and one in the D4 region. Specifically, three antibodies named 3H5, 12D12, and 27H5 are characterized by loss of binding with mutants that have substitutions at E34, R36, Y76, A82, and R84 in D1. One of those antibodies, 3H5, shows reduced binding with a mutant that has substitutions at G29, Q30, T32, Q33, and D80 in D1. These residues are located only in the D1 region and are all outside (within) the four regions defined as the binding epitope of the 15G8 antibody (note that in...). Figure 18A In this sequence, the sequence begins after one amino acid, such that E34 in, for example, WO2020 / 136145, is... Figure 18A The middle one is E33). Therefore, antibody 15G8 binds to a three-dimensional epitope that is different from the three-dimensional epitope bound by the antibody disclosed in WO2020 / 136145 ( Figure 18D ).

[0406] Interestingly, the region defined as the 15G8 epitope (which is the interstitial region between D1 and D2) has been identified as the major interacting region of ILT2, which binds to β-2-microglobulin (B2M) upon complexing with HLA (see Kuroki et al., “Structural and functional basis for LILRB immune checkpoint receptor recognition of HLA-G isoforms”, J. Immuno., 2019, Dec. 15;203(12):3386-3394). Figures 18E-18FIn fact, residues G97, A98, Y99, I100, Q125, and V126 were explicitly identified by Kuroki et al. (Supplementary Figure S2 in Kuroki) as interacting with B2M. These residues fall within interaction regions 3 and 4 of 15G8 and are all considered very high-probability or highly probable epitope residues. This strongly suggests that 15G8 inhibits ILT2 binding to HLA in a B2M-dependent manner and effectively blocks direct binding of ILT2 to B2M. In contrast, the 3H5, 12D12, and 27H5 antibodies bind to the N-terminal D1 region of ILT2, which interacts with the α3 domain of HLA-G (see Supplementary Figure S2 in Kuroki). This is significant because Kuroki et al. found that the primary interacting site for ILT2 is the B2M site, and that binding to the α3 domain is additional and flexible. This could explain the unique ability of 15G8 to enable T cell, NK cell, and macrophage / dendritic cell functions: it blocks the major interacting site of ILT2 but not the secondary site.

[0407] The only ILT2 antibody identified as having any effect on phagocytosis was GHI / 75, which was shown to enhance anti-CD47 blockade-mediated cancer cell phagocytosis, but not to have any effect on itself (see Barkal et al., "Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy", Nat. Immunol. Jan;19(1):76-84). The combined effects of GHI / 75 and anti-CD47 were found to be B2M-dependent, as the absence of B2M had no effect on the increased phagocytosis. Therefore, the effect of 15G8 alone on phagocytosis ( Figures 13A-13C This can be B2M dependent, which could explain the unique ability of this antibody. The superiority of the antibody of the present invention in this respect was directly tested. A375 or SKMEL28 cancer cells expressing exogenous HLA-G were co-cultured with macrophages in the presence of an IgG control, the antibody of the present invention, or GHI / 75. The HP-F1 antibody was also tested in A375 cells. Cancer cell lines stained with PKH67-FITC were co-incubated with macrophages stained with eFluor 670-APC in the presence of the indicated antibody. The level of phagocytosis was determined by the percentage of double-stained macrophages, the double staining indicating phagocytosis of target cells. The percentage increase in phagocytosis compared to the IgG control was calculated. In both cell types, all three antibodies of the present invention increased phagocytosis compared to the control. Figures 19A-19B Furthermore, there are some variability between antibodies and between cell types. As expected, neither GHI / 75 nor HP-F1 had any effect on phagocytosis. Thus, it is confirmed that the antibody of the present invention is the first anti-ILT2 antibody that can be used as a monotherapy to enhance phagocytosis.

[0408] This raises the question of epitopes for GHI / 75 and other commercial antibodies. While the epitopes of these antibodies are not publicly disclosed, a competitive ELISA assay was performed to observe whether 15G8, as well as GHI / 75 and HP-F1, could simultaneously bind to ILT2. Biotinylated 15G8 antibody was used at a constant concentration (1 μg / ml) in an ILT2 binding ELISA. GHI / 75 and HP-F1 were added at incremental concentrations, and competition was evaluated. Regardless of the amount of either antibody added, they did not compete with 15G8 for ILT2 binding. Figures 19A-19C In contrast, upon addition of naked (unbiotinylated) 15G8, binding decreased in a dose-dependent manner as expected. This indicates that GHI / 75 and HP-F1 bind to different epitopes compared to 15G8. This makes 15G8 the first anti-ILT2 antibody previously identified as binding to this epitope, specifically blocking interaction with B2M, and capable of simultaneously activating / recruiting T cells, NK cells, and macrophages / dendritic cells against cancer.

[0409] The foregoing description of specific embodiments will fully reveal the general nature of the invention, enabling others to readily modify and / or adapt such specific embodiments for various applications by applying current knowledge without excessive experimentation and without departing from the general concept. Therefore, such modifications and alterations should and are intended to be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not for limiting purposes. Various alternative forms may be taken for performing the various functions disclosed without departing from the invention.

Claims

1. A method for producing a pharmaceutical agent, the method comprising: Obtain an agent that binds to leukocyte immunoglobulin-like receptor subfamily B member 1 (ILT2) or a fragment thereof, test the ability of said agent to inhibit the interaction between ILT2 and β-2 microglobulin (B2M), and select at least one agent that inhibits the interaction between ILT2 and B2M; or culture host cells containing one or more vectors encoding a nucleic acid sequence of the agent, wherein said nucleic acid sequence is a nucleic acid sequence of the agent selected in the following manner: i. Obtaining a drug that binds to ILT2 or a fragment thereof; ii. Test the ability of the agent to inhibit the interaction between ILT2 and B2M; and iii. Select at least one agent that inhibits the interaction between ILT2 and B2M; This produces the medicine.

2. The method according to claim 1, wherein the ILT2 is human ILT2.

3. The method of claim 2, wherein the human ILT2 comprises or is composed of SEQ ID NO:

31.

4. The method according to any one of claims 1 to 3, wherein testing the ability of the agent to inhibit the interaction between ILT2 and B2M includes testing the ability to inhibit the binding of ILT2 and B2M.

5. The method according to any one of claims 1 to 3, wherein the obtaining is obtaining an agent that binds to the extracellular domain of ILT2.

6. The method according to any one of claims 1 to 3, further comprising testing the ability of the selected at least one agent to inhibit the interaction between ILT2 and human leukocyte antigen (HLA) protein or MHC-I protein, and further selecting at least one agent that inhibits the interaction between ILT2 and HLA protein or MHC-I protein.

7. The method of claim 6, wherein the HLA is HLA-G.

8. The method of claim 6, further comprising at least one agent that selectively inhibits the interaction between ILT2 and the B2M / HLA complex.

9. The method according to any one of claims 1 to 3, wherein obtaining the agent binding to ILT2 or a fragment thereof comprises obtaining an agent binding to at least one of the following residues selected from human ILT2: G97, A98, Y99, I100, Q125 and V126, and wherein the D1 and D2 domains numbered with respect to ILT2 comprise fused D1 and D2 domains of SEQ ID NO: 46 and SEQ ID NO:

47.

10. The method of claim 9, wherein obtaining is obtaining an agent that is in combination with all of G97, A98, Y99, I100, Q125 and V126.

11. The method according to any one of claims 1 to 3, wherein obtaining the agent binding to ILT2 or a fragment thereof comprises obtaining an agent binding to an ILT2 epitope within a sequence selected from human ILT2: SEQ ID NO: 41, 42, 43 and 44.

12. The method of claim 11, further comprising obtaining a pharmaceutical agent that binds to at least two of the three-dimensional epitopes comprising SEQ ID NO: 41, 42, 43 and 44.

13. The method of claim 12, further comprising obtaining a pharmaceutical agent that binds to a three-dimensional epitope comprising SEQ ID NO: 41, 42, 43 and 44.

14. The method according to any one of claims 1 to 3, further comprising testing the ability of the selected at least one agent to increase the phagocytosis of macrophages on cancer cells, and further selecting at least one agent that induces increased phagocytosis, optionally wherein the cancer cells are HLA-G expressing cancer cells.

15. The method of claim 14, wherein the test of the ability of the selected at least one agent to increase phagocytosis is performed without CD47 blockade.

16. The method according to any one of claims 1 to 3, further comprising testing the ability of the selected at least one agent to increase the efficacy of PD-L1 / PD blockade against cancer cells, and selecting at least one agent to increase the efficacy of anti-PD-L1 / PD-1-based immunotherapy.

17. The method of claim 16, wherein the increased efficacy comprises at least one of the following: a. Synergistic increase in the secretion of pro-inflammatory cytokines; b. Synergistic increase in T cell activation; and c. Synergistic increase in T cell cytotoxicity.

18. The method of claim 17, wherein at least one of the following is true: a. The pro-inflammatory cytokines are selected from granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), and interferon γ (IFNγ); b. The increased T cell activation includes increased membrane CD107a expression; and c. The increased T cell cytotoxicity includes increased membrane CD107a expression.

19. The method of claim 18, wherein the increased efficacy comprises converting PD-L1 / PD-1 blockade-refractory cancers into PD-L1 / PD-1 blockade-responsive cancers.

20. The method according to any one of claims 1 to 3, wherein the agent is an antibody or an antigen-binding fragment thereof.

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