Anti-ILT3 Antibodies and Uses Thereof

Abstract

Antibodies that bind to leukocyte immunoglobulin-like receptor subfamily B member 4 (ILT3) are provided. Nucleic acid molecules encoding the antibodies, compositions comprising the antibodies, and methods of using the antibodies are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 334,365, filed April 25, 2022, the contents of which are incorporated by reference herein in their entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (BDB-P-018-PCT SQL.xml; size: 89,038 bytes; creation date: April 3, 2023) are incorporated herein by reference in their entirety.

[0003] The present invention relates to the field of immune checkpoint inhibition. [Background technology]

[0004] During cancer development, cancer cells can evade immune surveillance through various mechanisms. Understanding these evasion mechanisms is important for the development of novel cancer treatments. Cancer immunotherapy, which aims to reactivate the patient's immune system to eliminate cancer cells, is currently a very active area of ​​cancer research and has shown unprecedented results in the clinic. Despite these advances, there is a constant need for novel therapies that, as a sole treatment or in combination with other agents, allow a more substantial increase in patient mortality and quality of life.

[0005] Leukocyte immunoglobulin-like receptor subfamily B member 4 (ILT3, CD85k, LILRB4) is expressed in various myeloid cells, especially those with immunosuppressive activity such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and tolerogenic dendritic cells (DCtol). ILT3 contains two extracellular immunoglobulin-like domains and three intracellular tyrosine-based inhibitory motifs (ITIMs). Expression of ILT3 in a soluble form in myeloid cells has been reported to be associated with tumor immune escape, metastasis, and poor prognosis in various malignancies. ILT3 signaling in myeloid cells leads to reduced expression of costimulatory receptors and differentiation into an immunosuppressive phenotype. In addition to regulating myeloid cell maturation and function, ILT3 signaling induces T cell anergy and differentiation into regulatory T and suppressor T cell phenotypes through unknown ligands.

[0006] Although the ILT3 ligand was undefined for many years, in recent years, evidence is accumulating that ILT3 binds to proteins within the tumor microenvironment (TME) and tumor extracellular matrix (ECM). Proteins such as apolipoprotein E (APOE) and fibronectin (FN1) are present within the TME and can bind to ILT3. When ILT3 binds to its ligand, it induces an immunosuppressive phenotype in myeloid cells, mediating inhibition of T cell activity and creating an immunosuppressive TME that supports tumor growth and proliferation.

[0007] Consistent with this reported data, it is believed that inhibiting ILT3 using blocking antibodies increases the disappearance of tumor cells. This can occur by inhibiting the immunosuppressive effect of tumor-resident myeloid cells, which induces a proinflammatory phenotype of myeloid cells that supports the activation of tumor-infiltrating T cells (TILs), remodeling the TME from an immunosuppressive TME to a proinflammatory TME. Overall, blocking ILT3 increases the effector activity of various immune cells against malignant cells. Anti-ILT3 antibodies have been disclosed in International Patent Application Publication Nos. 2006 / 138739, 2018 / 089300, 2019 / 099597, and 2020 / 056077(A), but there is still a great need for novel, superior anti-ILT3 antibodies that target multiple pathways through which ILT3 can create an immunosuppressive environment and have strong and sustained effects. Summary of the Invention

[0008] The present invention provides antibodies that bind to leukocyte immunoglobulin-like receptor subfamily B member 4 (ILT3). Nucleic acid molecules encoding the antibodies, compositions comprising the antibodies, and methods of using the antibodies are also provided.

[0009] According to a first aspect, there is provided an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), The antibody or antigen-binding fragment thereof is provided, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1 (GYSFX1GF), where X1 is S or T, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:2 (FPSX2GE), where X2 is S or N, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3 (QAFYYFDX3), where X3 is S or Y, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4 (KSSQSLLSSSNQKNYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5 (WASTRES), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6 (QQYYSYPLT).

[0010] According to another embodiment, an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), An antibody or antigen-binding fragment thereof is provided, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 49 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 50 (AITFGGGNTYYPDSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 51 (HGDGNYDFYAMDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 52 (KSSQSLLNSGNQKNYLT), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5 (WASTRES), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 53 (QNDYSYPLT).

[0011] According to another embodiment, an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), An antibody or antigen-binding fragment thereof is provided, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 49 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 56 (TISSDGGNTYYTDSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 57 (HDGRGALDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 58 (RASQDISNYLN), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 59 (YTSRLHS), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 60 (QQGNTLPWT).

[0012] According to another embodiment, an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), An antibody or antigen-binding fragment thereof is provided, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 63 (NSAVH), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 64 (VIWAGGNTNYNSTLMS), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 65 (HETYGDSFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 66 (RSSQSLLDSDGKTYLN), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 67 (LVSKLDS), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 68 (WQGTHFPFT).

[0013] According to another aspect, there is provided an antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment of the invention for binding to ILT3.

[0014] According to another aspect, there is provided a nucleic acid molecule or a plurality of nucleic acid molecules encoding an antibody or antigen-binding fragment of the invention.

[0015] According to another aspect, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment of the invention and a pharma- ceutically acceptable carrier, excipient, or adjuvant.

[0016] According to another aspect, there is provided a method of treating cancer in a subject in need thereof, comprising administering to the subject an antibody, or antigen-binding fragment thereof, of the invention, thereby treating the cancer.

[0017] According to another aspect, there is provided a method of treating cancer in a subject in need thereof, comprising administering a pharmaceutical composition of the present invention to the subject, thereby treating the cancer.

[0018] According to some embodiments, a. SEQ ID NO:1 is SEQ ID NO:15 (GYSFTGF), SEQ ID NO:2 is SEQ ID NO:16 (FPSNGE), and SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY)); b. SEQ ID NO:1 is SEQ ID NO:15 (GYSFTGF), SEQ ID NO:2 is SEQ ID NO:16 (FPSNGE), and SEQ ID NO:3 is SEQ ID NO:18 (QAFYYFDS)); c. SEQ ID NO:1 is SEQ ID NO:15 (GYSFTGF), SEQ ID NO:2 is SEQ ID NO:19 (FPSSGE), and SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY)); d. SEQ ID NO:1 is SEQ ID NO:20 (GYSFSGF), SEQ ID NO:2 is SEQ ID NO:16 (FPSNGE), and SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY)); e. SEQ ID NO:1 is SEQ ID NO:20 (GYSFSGF), SEQ ID NO:2 is SEQ ID NO:19 (FPSSGE), and SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY)); or f. SEQ ID NO:1 is SEQ ID NO:20 (GYSFSGF), SEQ ID NO:2 is SEQ ID NO:19 (FPSSGE), and SEQ ID NO:3 is SEQ ID NO:18 (QAFYYFDS).

[0019] According to some embodiments, the antibody or antigen-binding fragment comprises at least one of the following: a. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 34, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 35; b. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:36, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:37; c. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:38, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39; d. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:40, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:41; e. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:42, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:41; f. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:42, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39; g. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39; h. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:44, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:45; i. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:44, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39; j. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:46, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39; k. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39; and l. A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:48, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39.

[0020] According to some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:54, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:55.

[0021] According to some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:61, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:62.

[0022] According to some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:69, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:70.

[0023] According to some embodiments, the heavy chain comprises an IgG4 constant region.

[0024] According to some embodiments, the IgG4 constant region comprises a sequence having at least 80% sequence identity to SEQ ID NO: 21 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFSPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK)TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK).

[0025] According to some embodiments, the sequence having at least 80% sequence identity to SEQ ID NO:21 comprises the S124P and L131E mutations in SEQ ID NO:21.

[0026] According to some embodiments, the light chain comprises a kappa constant region, and optionally the kappa constant region comprises a sequence having at least 80% sequence identity to SEQ ID NO: 22 (RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC). According to some embodiments, the antibody or antigen-binding fragment comprises at least one of the following; a. a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:8; b. a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10; c. a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:12; d. a heavy chain comprising the amino acid sequence of SEQ ID NO:23 and a light chain comprising the amino acid sequence of SEQ ID NO:24; e. a heavy chain comprising the amino acid sequence of SEQ ID NO:25 and a light chain comprising the amino acid sequence of SEQ ID NO:24; f. a heavy chain comprising the amino acid sequence of SEQ ID NO:25 and a light chain comprising the amino acid sequence of SEQ ID NO:12; g. a heavy chain comprising the amino acid sequence of SEQ ID NO:26 and a light chain comprising the amino acid sequence of SEQ ID NO:12; h. a heavy chain comprising the amino acid sequence of SEQ ID NO:27 and a light chain comprising the amino acid sequence of SEQ ID NO:12; i. a heavy chain comprising the amino acid sequence of SEQ ID NO:27 and a light chain comprising the amino acid sequence of SEQ ID NO:28; j. a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO:12; k. a heavy chain comprising the amino acid sequence of SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:12; and l. A heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0027] According to some embodiments, the antibody or antigen-binding fragment is capable of binding to Leukocyte Immunoglobulin-Like Receptor Subfamily B Member 4 (LILRB4 or ILT3).

[0028] According to some embodiments, the ILT3 comprises the amino acid sequence provided in SEQ ID NO:71.

[0029] According to some embodiments, ILT3 is present on the surface of myeloid cells, dendritic cells, macrophages, or a combination thereof.

[0030] According to some embodiments, the myeloid cells are myeloid-derived suppressor cells (MDSCs), the dendritic cells are tolerogenic dendritic cells, the macrophages are suppressive macrophages, and optionally, the macrophages are tumor-associated macrophages (TAMs), or a combination thereof.

[0031] According to some embodiments, binding to ILT3 inhibits binding of ILT3 to an ILT3 ligand.

[0032] According to some embodiments, the binding inhibits binding of ILT3 to both apolipoprotein E (APOE) and fibronectin (FN1).

[0033] According to some embodiments, APOE, FN1, or both are present within the tumor microenvironment (TME).

[0034] According to some embodiments, binding to ILT3 relieves T cells, monocytes, macrophages, or a combination thereof, from ILT3-mediated inhibition.

[0035] According to some embodiments, the liberation of T cells comprises increased T cell proliferation, increased proinflammatory cytokine secretion by T cells, increased proinflammatory chemokine secretion by T cells, or a combination thereof.

[0036] According to some embodiments, binding to ILT3 increases dendritic cell activation.

[0037] According to some embodiments, increasing dendritic cell activation comprises restoring dendritic cell activation suppressed by FN1, APOE, or both.

[0038] According to some embodiments, increasing dendritic cell activation comprises increasing secretion of proinflammatory cytokines or chemokines by the dendritic cells.

[0039] According to some embodiments, binding to ILT3 increases monocyte and / or macrophage activation.

[0040] According to some embodiments, increasing monocyte and / or macrophage activation comprises increasing secretion of pro-inflammatory cytokines or chemokines by dendritic cells.

[0041] According to some embodiments, the proinflammatory cytokine is selected from interferon gamma (IFNG), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α), and the proinflammatory chemokine is selected from CC motif chemokine ligand 3 (CCL3) and CCL4, or both.

[0042] According to some embodiments, the antigen-binding fragment is selected from the group consisting of an Fv, Fab, F(ab')2, scFV, or scFV2 ​​fragment.

[0043] According to some embodiments, the antibody or antigen-binding fragment is humanized.

[0044] In some embodiments, the antibody is a monoclonal antibody.

[0045] According to some embodiments, the agent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0046] According to some embodiments, the pharmaceutical composition is formulated for administration to a subject.

[0047] According to some embodiments, the pharmaceutical composition is formulated for systemic or intratumoral administration.

[0048] According to some embodiments, the cancer is a solid tumor.

[0049] According to some embodiments, the cancer is selected from breast cancer, renal cancer, head and neck cancer, lung cancer, sarcoma, gastric cancer, colorectal cancer and ovarian cancer.

[0050] According to some embodiments, the cancer is characterized by the presence of tumor-infiltrating immune cells that express ILT3.

[0051] According to some embodiments, the TME of the cancer is characterized by expression of APOE, FN1, or both.

[0052] According to some embodiments, the expression is overexpressed as compared to non-cancerous tissue of the same type as the tumor.

[0053] According to some embodiments, the method further comprises administering an immune checkpoint inhibitor (ICI).

[0054] According to some embodiments, the ICI inhibits the PD-1 / PD-L1 / L2 checkpoint.

[0055] According to some embodiments, the ICI is selected from pembrolizumab, nivolumab, atezolizumab, cemiplimab, dostallimab, durvalumab and avelumab.

[0056] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description set forth below. It should be understood, however, that while the detailed description and specific examples indicate preferred embodiments of the invention, they are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0057] [Figure 1] Figure 1A-C: Histograms of ILT3 surface expression on (1A) monocytes and MDSCs from two healthy donors, (1B) immature dendritic cells (iDCs), mature dendritic cells (mDCs), and tolerogenic dendritic cells (DCtol) from two healthy donors, and (1C) monocytes differentiated into M0, M1, M2 macrophages from two healthy donors. IC-isotype control, Mix-anti-ILT3 antibody. Black lines indicate control cells incubated with isotype control antibody, and grey lines indicate anti-ILT3 PE-conjugated antibody staining of cells. [Diagram 2]Figure 2A-B: (2A) Micrographs of IHC staining for ILT3 (red) and CD68 (blue) of tissue samples. Arrows indicate double positive cells. (2B) Bar graphs quantifying the percentage of the number of cancers that were ILT3 positive out of all cancers sampled for each cancer type. [Diagram 3] Figure 3A-B: (3A) Dot plot tracking relative MDSC enrichment of cancers from the TCGA database divided into three bins of ILT3 expression: low, medium, and high. (3B) Bar graph of M2 fraction enrichment compared to patients with different levels of ILT3 in the same bin of cancer. [Figure 4] 4A-B: (4A) Histograms of exogenous hILT3 expression on the HEK cell surface measured using a commercial antibody (ZM4.1) and an antibody of the invention. Isotype control histograms are shown in black and ILT3 binding is shown in grey. (4B) Histograms of antibody binding to cynomolgus ILT3 (50 μg / ml). Isotype control histograms are shown in grey and ILT3 binding is shown in black. [Diagram 5] Figure 5: Line graph of ELISA detection of ILT3 when competing with naked ZM4.1 antibody. ZM4.1 and antibody AB#2 compete for binding to ILT3, whereas 5E5 and AB#3 bind to ILT3 at an epitope and therefore do not compete with ZM4.1. [Figure 6]Figures 6A-6G: (6A) TNFα secretion from PBMCs, (6B-6C) IFNg secretion from CD8 T cells in the presence of autologous MDSCs, (6D-6F) IFNg secretion from CD4 T cells in the presence of different dendritic cells in the presence or absence of 5E5 antibody, (6G) IFNg secretion from CD4 T cells in the presence of M2 macrophages produced in the presence or absence of 5E5 antibody, and (6C) bar graphs comparing with Merck's m52B8 antibody. (6E-6F) 5E5 antibody in combination with anti-PD-1 antibody is measured with (6E) and without (6F) anti-CD3 stimulation of T cells. Although one donor is shown in Figures 6A-6D, 3-4 donors were tested and all showed comparable results. All donors are shown in 6E-6F with their individual increases shown in the line graphs on the right. *P<0.05, **P<0.01, unpaired Student's t test compared with mIgG1. [Figure 7] Figures 7A-7E: Line graphs of blocking of ILT3 binding to (7A, 7C-7D) fibronectin and (7B, 7E) APOE in the presence of 5E5 antibody of the invention and (7A-7B, 7D) ZM4.1 antibody or (7C, 7E) m52B8 antibody or (7D) other antibodies. [Figure 8] 8A-D: (8A) IL-8 secretion from THP-1 cells after activation with Rituximab, (8B) TNFα secretion from DCs, (8C) IL-8 secretion from DCs, (8D) TNFα secretion from DCs after activation with Erbitux and culture with fibronectin with or without various anti-ILT3 antibodies. h52B8 is a humanized anti-ILT3 antibody from Merck, IO202 is a humanized anti-ILT3 antibody from Immune-OncTherapeutics, and h5A7 is a humanized anti-ILT3 antibody from NGMBio. [Figure 9] Figures 9A-B: Bar graphs of TNFα secretion from (9A) ILT3-expressing and (9B) non-expressing DCs cultured with or without 5E5 antibody in the presence of fibronectin. *P<0.05; unpaired Student's t-test compared to IgG. [Figure 10] Figure 10: Line graph of binding of various h5E5 antibodies to hILT3. EC50 levels were determined using GraphPad software. [Figure 11] Figures 11A-B: (11A) Line graphs of percentage blockade of ILT3 binding to FN1 by various h5E5 antibodies and (11B) a direct comparison of c5E5 and h5E5-133. IC50 levels were determined using GraphPad software. [Figure 12] Figures 12A-D: (12A) IFNg secretion from CD8 T cells in the presence of autologous MDSCs, (12B) TNFa secretion from DCs, and (12C-12D) IL-8 secretion from THP-1 cells in the presence of fibronectin and rituximab treated with (12C) c5E5 or h5E5 antibodies, or (12D) m5E5 or an alternative antibody of the invention. Figure 12A includes insets of 11 donor samples tested, showing levels after isotype control and h5E5 at a concentration of 16 μg / ml. [Figure 13] Figures 13A-B: Bar graphs of TNFα secretion from monocytes differentiated into M1-activated macrophages (MF) in the presence of (13A) gastric cancer cells or (13B) colon cancer cells with and without h5E5 antibody. *P<0.05, unpaired Student's t-test. [Figure 14] Figure 14: Bar graph of TNFα secretion from immature DCs stimulated with IL-10 and LPS to generate DCtol when cultured with or without anti-ILT3 antibody or isotype control. Samples from two donors are shown. [Figure 15] FIGS. 15A-B: (15A) Bar graphs of IFNg secretion from CD8 T cells incubated with autologous MDSCs, and (15B) IFNg secretion from CD4 T cells co-cultured with the indicated dendritic cells treated with anti-PD1 antibody, h5E5, or a combination of the two. [Figure 16]Figures 16A-16D: (16A-16B) Bar graphs showing IFNg production from (16A) sarcoma tumor organoids and (16B) breast cancer tumor organoids activated in the presence of h5E5 or isotype control. (16C-16D) Bar graphs showing CCL3, CCL4, and IL-8 production from (16C) breast cancer tumor organoids and (16D) CRC tumor organoids activated in the presence of various anti-ILT3 antibodies, isotype control, pembrolizumab, or combinations thereof. *P<0.05; **P<0.01; ***P<0.005; unpaired Student's t-test. [Figure 17] FIG. 17: Line graph showing MC38 tumor size over time in syngeneic transgenic hILT3 mice treated with c5E5 or isotype control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] The present invention provides, in some embodiments, antibodies that bind to leukocyte immunoglobulin-like receptor subfamily B member 4 (ILT3). Nucleic acid molecules encoding the antibodies, compositions comprising the antibodies, and methods of using the antibodies are also provided.

[0059] The present invention is based on the generation of several ILT3 blocking antibodies using hybridoma technology. The lead antibody 5E5 demonstrated the required binding profile, selectivity, cross-reactivity, blocking activity, and activity in functional assays. The antibody was humanized using germline humanization. Humanized 5E5 can block the interaction of ILT3 with various ligands (such as APOE and FN1) and inhibit the immunosuppressive effects mediated by ILT3 signaling. 5E5 re-regulates the immunosuppressive TME by blocking and neutralizing ILT3, and induces a pro-inflammatory phenotype in tumor-resident myeloid cells, resulting in enhanced activation of tumor-infiltrating T cells and inhibition of tumor growth.

[0060] In a first aspect, there is provided an antibody or antigen-binding fragment thereof that binds to ILT3.

[0061] Leukocyte immunoglobulin-like receptor subfamily B member 4 is known as LILRB4, ILT3, and CD85K, among many other names. The Entrez gene identifier for human ILT3 is 11006 and the mouse identifier is 14727. The protein sequence for human ILT3 can be found at Uniprot identifier Q8NHJ6 and the mouse protein sequence can be found at identifier Q61450. The human mRNA sequence can be found at RefSeq sequences NM_001081438, NM_001278426, NM_001278427, NM_001278428 and NM_001278429. The mouse mRNA sequence can be found at RefSeq sequences NM_001291892, NM_001291893 and NM_008147. The human protein sequence can be found in RefSeq sequences NP_001265355, NP_001265356, NP_001265357, NP_001265358 and NP_001265359. The mouse protein sequence can be found in RefSeq sequences NP001278821, NP_00127882 and NP_032173. In some embodiments the amino acid sequence of ILT3 comprises MIPTFTALLCLGLSLGPRTHMQAGPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDKEESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSPVGWSQPSDPLELVMTGAYSKPTLSALPSPLVTSGKSVTLLCQSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPVTSVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRPSPTRSVSTAAGPEDQPLMPTGSVPHSGLRRHWE (SEQ ID NO:71). In some embodiments the amino acid sequence of ILT3 consists of SEQ ID NO:71. In some embodiments SEQ ID NO:71 is human ILT3.

[0062] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that contain at least one binding domain formed from the folding of polypeptide chains with a three-dimensional binding space with an internal surface shape and charge distribution complementary to the antigenic determinant characteristics of the antigen. Antibodies typically have a tetrameric form containing two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies can be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and antibodies that can be displayed as soluble or binding forms, as well as fragments including epitope-binding fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies can be derived from any species. The term antibody also includes binding fragments such as, but not limited to, Fv, Fab, Fab', F(ab')2 single chain antibodies (svFC), dimeric variable regions (diabodies) and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including, but not limited to, an Fc region or a fragment thereof. Those skilled in the art will further appreciate that other fusion products may be produced, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.

[0063] The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0064] In some embodiments, the antibody is a murine antibody. In some embodiments, the antibody may be a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a full IgG. In some embodiments, the antibody is an IgG2. In some embodiments, the antibody is an IgG4. In some embodiments, the antibody or antigen binding domain is an antigen binding domain that lacks an Fc domain.

[0065] In some embodiments, the peptide is selected from an antibody, an antigen-binding fragment of an antibody, a Fab fragment, a single-chain antibody, a single-domain antibody, a nanobody, a VHH antibody, and an antibody mimic. The term "antibody mimic" as used herein refers to an organic compound that can specifically bind to a target antigen. In some embodiments, the antibody mimic is not structurally related to an antibody. Examples of antibody mimics include, but are not limited to, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, fynomers, Kunitz main peptides, monobodies, and nanoCLAMPS. In some embodiments, the antibody mimic is a DARPin. All of these agents are well known in the art and are known to be useful in blocking the interaction between a receptor and its ligand. Small molecules and proteins that can bind to mCD28 can block the cleavage site or prevent or impair access of the protease. In some embodiments, the protein is an antibody mimic. The term "DARPin" used herein refers to designed ankyrin repeat protein.DARPin is generally a genetically engineered antibody mimicking protein that is highly specific to its protein target.Therefore, DARPin of CD28 can be an example of an agent.

[0066] The basic unit of naturally occurring antibody structure is a heterotetrameric glycoprotein complex of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H) linked together by both non-covalent association and disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. There are five human antibody classes (IgG, IgA, IgM, IgD, and IgE), and within these classes, various subclasses are recognized based on structural differences such as the number of immunoglobulin units in a single antibody molecule, the disulfide bridge structure of the individual units, and differences in chain length and sequence. The class and subclass of an antibody is its isotype.

[0067] The amino-terminal regions of the heavy and light chains are called variable domains because they are more diverse in sequence than the carboxy-terminal regions. This part of the antibody structure confers the antigen-binding specificity of the antibody. The heavy chain variable (VH) domain and the light chain variable (VL) domain together form one antigen-binding site, so that a basic immunoglobulin unit has two antigen-binding sites. Certain amino acid residues are believed to 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).

[0068] The carboxy-terminal portions of the heavy and light chains form the constant domains, CH1, CH2, CH3, and CL. Although there is much less diversity in these domains, they vary between different animal species, and even within the same individual there are several different antibody isotypes, each with different functions.

[0069] The term "framework region" or "FR" refers to amino acid residues in the variable domain of an antibody other than the hypervariable region amino acid residues as defined herein. The term "hypervariable region" as used herein refers to amino acid residues in the variable domain of an antibody that are involved in antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". The CDRs are primarily involved in binding to an epitope of an antigen. The extent of FRs and CDRs has been precisely defined (see Kabat et al.).

[0070] Immunoglobulin variable domains can also be analyzed using the IMGT information system (www: / / imgt.cines.fr / ) (IMGT® / V-Quest) to identify variable region segments that contain the CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. J6:W503-508 (2008).

[0071] Chothia et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. A person skilled in the art can unambiguously assign this "Chothia numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Chothia numbering" refers to the numbering system described by Chothia et al., Journal of Molecular Biology, "Canonical Structures for the Hypervariable regions of immunoglobulins" (1987) and Chothia et al., Nature, "Conformations of Immunoglobulin Hypervariable Regions" (1989).

[0072] The term "humanized antibody" as used herein refers to an antibody from a non-human species whose protein sequence has been modified to increase similarity to human antibodies. Humanized antibodies can be produced by producing recombinant DNA that encodes the CDRs of a non-human antibody surrounded by sequences similar to human antibodies. In some embodiments, a humanized antibody is a chimeric antibody. In some embodiments, humanizing involves inserting the CDRs of the present invention into a scaffold or framework of a human antibody. Humanized antibodies are well known in the art, and any method of producing a humanized antibody that retains the CDRs of the present invention can be used.

[0073] In some embodiments, the antibody is a monoclonal antibody. As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody (such variants are usually present in minor amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as being produced by any particular preparation method. The monoclonal antibodies used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0074] The mAbs of the present invention may be of any immunoglobulin class, such as IgG, IgM, IgD, IgE, or IgA. Hybridomas producing mAbs may be grown in vitro or in vivo. High titer mAbs can be obtained by in vivo production, in which cells from individual hybridomas are injected intraperitoneally into pristine-primed Balb / c mice to produce ascites fluid containing high concentrations of the desired mAb. mAbs of isotype IgM or IgG may be purified from such ascites fluid or culture supernatants using column chromatography methods well known to those skilled in the art.

[0075] "Antibody fragments" include a portion of an intact antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments; diabodies; tandem diabodies (taDbs); linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al, Protein Eng. 8(10):1057-1062(1995)); one-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di, tri)-scFv); and bispecific T cell engagers (BiTEs).

[0076] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0077] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight non-covalent association. Three surfaces of the VH-VL dimer are present in this configuration. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0078] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that they have the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0079] The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0080] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called a, delta, e, gamma, and micro, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0081] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises 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 scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0082] The term "diabody" refers to a small antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in 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 domains are forced to pair with complementary domains on another chain, creating two antigen binding sites. The production of diabodies is known in the art and is described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0083] The term "multispecific antibody" is used in the broadest sense and particularly includes within its scope antibodies with polyepitopic 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) in which the VHVL unit has polyepitopic specificity, antibodies with two or more VL and VH domains in which each VHVL unit binds a different epitope, antibodies with two or more single variable domains in which each single variable domain binds a different epitope, full-length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, antibody fragments linked by covalent or non-covalent bonds. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0084] The monoclonal antibodies of the present invention can be prepared using methods well known in the art, including various techniques such as those described in Kohler, G. and Milstein, C, Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4:72 (1983); Cole et al, pg. 77-96, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0085] Besides the traditional methods of generating antibodies in vivo, antibodies can be generated in vitro using phage display technology. Such recombinant antibody production is much faster and can be generated against a huge number of antigens compared to traditional antibody production. Moreover, many antigens have been found to be non-immunogenic or extremely toxic when using traditional methods and therefore cannot be used to generate antibodies in animals. Moreover, affinity maturation of recombinant antibodies (i.e., increasing affinity and specificity) is very simple and relatively rapid. Finally, a large number of different antibodies against a particular antigen can be generated in a single selection step. To generate recombinant monoclonal antibodies, various methods, all based on display libraries, can be used to generate a large pool of antibodies with different antigen recognition sites. Such libraries can be created in several ways: synthetic repertoires can be generated by cloning synthetic CDR3 regions into a pool of heavy chain germline genes, thereby generating a large antibody repertoire, from which recombinant antibody fragments with different specificities can be selected. As starting material for constructing the antibody library, a human lymphocyte pool can be used. It is possible to construct a naive repertoire of human IgM antibodies, thus creating a highly diverse human library. This method has been widely used and successfully to select a large number of antibodies against different antigens. Protocols for the construction of bacteriophage libraries and the selection of recombinant antibodies are described in the well-known reference Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

[0086] Non-human antibodies may be humanized by any method known in the art. In one method, non-human complementarity determining regions (CDRs) are inserted into a human antibody or consensus antibody framework sequence. Further modifications can then be introduced into the antibody framework to adjust affinity or immunogenicity.

[0087] In some embodiments, antibodies and portions thereof include antibodies, antibody fragments, Fab and F(ab')2, single domain antigen-binding recombinant fragments, and natural nanobodies. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fv, Fab, F(ab')2, scFV, or scFV2 ​​fragments.

[0088] In some embodiments, the invention provides nucleic acid sequences encoding an antibody or antigen-binding portion of the invention.

[0089] For example, a polynucleotide may encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain contains not only a heavy chain variable region (VH) but also a heavy chain constant region (CH), which typically contains three constant domains: CH1, CH2, and CH3; as well as a "hinge" region. In some circumstances, the presence of a constant region is desirable.

[0090] Other polypeptides that may be encoded by the polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fv, scFv, Fab', and CHI, with the CK or CL domains truncated. Minibodies are smaller than conventional antibodies and therefore should achieve better tissue penetration for clinical / diagnostic applications, but are bivalent and therefore should retain higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context dictates otherwise, the term "antibody" as used herein includes not only whole antibody molecules but also antigen-binding antibody fragments of the types described above. Each framework region present in the encoded polypeptide can contain at least one amino acid substitution with respect to the corresponding human acceptor framework. Thus, for example, the framework regions can contain, in total, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions with respect to the acceptor framework region. Given the properties of the individual amino acids that make up the disclosed protein products, the skilled artisan will recognize some reasonable substitutions. Amino acid substitutions, or "conservative substitutions," may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0091] Suitably, the polynucleotides described herein may be isolated and / or purified. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0092] As used herein, "non-naturally occurring" substances, compositions, entities, and / or any combination of substances, compositions, entities, or any grammatical variations thereof, is a qualified term that expressly excludes, but merely excludes, forms of substances, compositions, entities, and / or any combination of substances, compositions, entities that are well understood by those of skill in the art as being "naturally occurring" or that have been or may be determined or interpreted as being "naturally occurring" at any time by a judicial, administrative, or judicial body.

[0093] In some embodiments, the antibody or antigen-binding portion thereof comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (GYSFX1GF), where X1 is S or T, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2 (FPSX2GE), where X2 is S or N, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3 (QAFYYFDX3), where X3 is S or Y, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4 (KSSQSLLSSSNQKNYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5 (WASTRES), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6 (QQYYSYPLT). In some embodiments, the CDRs are numbered according to the Chothia numbering system, and the CDRs comprise SEQ ID NOs: 1-6.

[0094] In some embodiments, the antibody or antigen-binding portion thereof comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), where CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 13 (GFYID), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 14 (YIFPSX2GETSYNQKFKG), where X2 is S or N, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3 (QAFYYFDX3), where X3 is S or Y, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4 (KSSQSLLSSSNQKNYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5 (WASTRES), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6 (QQYYSYPLT). In some embodiments, the CDRs are numbered according to the Kabat numbering system, and the CDRs comprise SEQ ID NOs: 13-14 and 3-6. It will be appreciated by those skilled in the art that the Chothia and Kabat systems are compatible and that CDRs given with respect to either system can be converted to CDRs for the other system.

[0095] In some embodiments, SEQ ID NO:1 is SEQ ID NO:15 (GYSFTGF). In some embodiments, SEQ ID NO:1 is SEQ ID NO:20 (GYSFSGF). In some embodiments, SEQ ID NO:2 is SEQ ID NO:16 (FPSNGE). In some embodiments, SEQ ID NO:2 is SEQ ID NO:19 (FPSSGE). In some embodiments, SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY). In some embodiments, SEQ ID NO:3 is SEQ ID NO:18 (QAFYYFDS). In some embodiments, SEQ ID NO:1 is SEQ ID NO:15, SEQ ID NO:2 is SEQ ID NO:16, and SEQ ID NO:3 is SEQ ID NO:17. In some embodiments, SEQ ID NO:3 is SEQ ID NO:18 (QAFYYFDS). In some embodiments, SEQ ID NO:14 is SEQ ID NO:32 (YIFPSNGETSYNQKFKG). In some embodiments, SEQ ID NO:14 is SEQ ID NO:33 (YIFPSSGETSYNQKFKG).

[0096] In some embodiments, SEQ ID NO:1 is SEQ ID NO:15, SEQ ID NO:2 is SEQ ID NO:16, and SEQ ID NO:3 is SEQ ID NO:18. In some embodiments, SEQ ID NO:1 is SEQ ID NO:15, SEQ ID NO:2 is SEQ ID NO:19, and SEQ ID NO:3 is SEQ ID NO:17. In some embodiments, SEQ ID NO:1 is SEQ ID NO:15, SEQ ID NO:2 is SEQ ID NO:19, and SEQ ID NO:3 is SEQ ID NO:18. In some embodiments, SEQ ID NO:1 is SEQ ID NO:20, SEQ ID NO:2 is SEQ ID NO:16, and SEQ ID NO:3 is SEQ ID NO:17. In some embodiments, SEQ ID NO:1 is SEQ ID NO:20, SEQ ID NO:2 is SEQ ID NO:16, and SEQ ID NO:3 is SEQ ID NO:18. In some embodiments, SEQ ID NO:1 is SEQ ID NO:20, SEQ ID NO:2 is SEQ ID NO:19, and SEQ ID NO:3 is SEQ ID NO:17. In some embodiments, the antibody or antigen-binding fragment is 5E5. In some embodiments, SEQ ID NO:1 is SEQ ID NO:20, SEQ ID NO:2 is SEQ ID NO:19, and SEQ ID NO:3 is SEQ ID NO:18. An antibody or antigen binding domain that comprises SEQ ID NO: 16 according to the Chothia system is understood to comprise SEQ ID NO: 32 according to the Kabat system. Similarly, an antibody or antigen binding domain that comprises SEQ ID NO: 19 according to the Chothia system comprises SEQ ID NO: 33 according to the Kabat system.

[0097] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain. In some embodiments, the heavy chain is an IgG heavy chain. In some embodiments, the IgG is an IgG4. In some embodiments, the heavy chain comprises an IgG4 constant region. In some embodiments, the IgG4 constant region is TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 21). Each possibility represents a separate embodiment of the invention. In some embodiments, the IgG4 constant region comprises a sequence having at least 80% identity to SEQ ID NO: 21. In some embodiments, the IgG4 constant region comprises a sequence having at least 90% identity to SEQ ID NO: 21. In some embodiments, the IgG4 constant region does not induce ADCC or CDC. In some embodiments, the IgG4 constant region comprises a serine 124 to proline mutation (S124P). In some embodiments, the numbering is with respect to SEQ ID NO: 21. In some embodiments, S124P is S241P and the numbering is with respect to the complete heavy chain. In some embodiments, the IgG4 constant region comprises a leucine 131 to glutamic acid mutation (L131E). In some embodiments, the numbering is with respect to SEQ ID NO: 21. In some embodiments, L131E is L248E and the numbering is with respect to the complete heavy chain.It will be understood that depending on the deletions or insertions into the complete heavy chain, the numbers provided for these two mutations may be shifted slightly.

[0098] In some embodiments, the antibody or antigen-binding fragment comprises a light chain. In some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda light chain. In some embodiments, the antibody or antigen-binding fragment comprises a kappa constant region. In some embodiments, the kappa constant region comprises a sequence having at least 70, 75, 80, 85, 90, 95, 93, 95, 97, 99 or 100% identity to RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:22). Each possibility represents a separate embodiment of the invention. In some embodiments, the kappa constant region comprises a sequence having at least 80% identity to SEQ ID NO:22. In some embodiments, the kappa constant region comprises a sequence having at least 90% identity to SEQ ID NO:22.

[0099] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIDWVKQSPGKSLEWIGYIFPSNGETSYNQKFKGKATLTVDKSSSTVNMQLNSLTSEDSAVYYCARQAFYYFDYWGQGTTLTVSS (SEQ ID NO: 34). In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of the amino acid sequence of DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLSSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 35). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region that comprises or consists of SEQ ID NO: 34 and a light chain comprising a variable region that comprises or consists of SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment is a murine antibody. In some embodiments, the antibody or antigen-binding fragment is 5E5.

[0100] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIDWVKQSPGKSLEWIGYIFPSNGETSYNQKFKGKATLTVDKSSSTVNMQLNSLTSEDSAVYYCARQAFYYFDYWGQGTTLTVSS (SEQ ID NO: 36). In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of the amino acid sequence of DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLSSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 37). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region that comprises or consists of SEQ ID NO: 36 and a light chain comprising a variable region that comprises or consists of SEQ ID NO: 37. In some embodiments, the antibody or antigen-binding fragment is a chimeric antibody. In some embodiments, the antibody or antigen-binding fragment is 5E5.

[0101] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSS (SEQ ID NO: 38). In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFAGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIK (SEQ ID NO: 39). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region that comprises or consists of SEQ ID NO: 38 and a light chain comprising a variable region that comprises or consists of SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, the antibody or antigen-binding fragment is 5E5.

[0102] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSS (SEQ ID NO: 40). In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIK (SEQ ID NO: 41). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain that comprises a variable region that comprises or consists of SEQ ID NO: 40 and a light chain that comprises a variable region that comprises or consists of SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0103] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSS (SEQ ID NO: 42). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 42 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0104] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain that comprises a variable region that comprises or consists of SEQ ID NO: 42 and a light chain that comprises a variable region that comprises or consists of SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0105] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDSWGQGTTVTVSS (SEQ ID NO: 43). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 43 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0106] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSS (SEQ ID NO: 44). In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIK (SEQ ID NO: 45). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region that comprises or consists of SEQ ID NO: 44 and a light chain comprising a variable region that comprises or consists of SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region that comprises or consists of SEQ ID NO: 44 and a light chain comprising a variable region that comprises or consists of SEQ ID NO: 45. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0107] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSS (SEQ ID NO: 46). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 46 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0108] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSS (SEQ ID NO: 47). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 47 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0109] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDSWGQGTTVTVSS (SEQ ID NO: 48). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 48 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0110] In some embodiments, the antibody or antigen-binding fragment is EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIDWVKQSPGKSLEWIGYIFPSNGETSYNQKFKGKATLTVDKSSSTVNMQLNSLTSEDSAVYYCARQAFYYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCIC The heavy chain constant region comprises or consists of the amino acid sequence of TVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFIYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 7). In some embodiments, the antibody or antigen-binding fragment comprises a light chain that comprises or consists of the amino acid sequence of DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLSSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 8). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain that comprises or consists of SEQ ID NO: 7 and a light chain that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment is a murine antibody.

[0111] In some embodiments, the antibody or antigen-binding fragment is EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIDWVKQSPGKSLEWIGYIFPSNGETSYNQKFKGKATLTVDKSSSTVNMQLNSLTSEDSAVYYCARQAFYYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC The heavy chain comprises or consists of the amino acid sequence of PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9). In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising or consisting of the amino acid sequence of DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLSSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising or consisting of SEQ ID NO: 9 and a light chain comprising or consisting of SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment is a chimeric antibody.

[0112] In some embodiments, the antibody or antigen-binding fragment is QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC The heavy chain comprises or consists of the amino acid sequence of PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 11). In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising or consisting of the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFAGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising or consisting of SEQ ID NO: 11 and a light chain comprising or consisting of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0113] In some embodiments, the antibody or antigen-binding fragment is EVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC The heavy chain comprises or consists of the amino acid sequence of PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 23). In some embodiments, the antibody or antigen-binding fragment comprises a light chain that comprises or consists of the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain that comprises or consists of SEQ ID NO: 23 and a light chain that comprises or consists of SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0114] In some embodiments, the antibody or antigen-binding fragment is QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 25). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising or consisting of SEQ ID NO: 25 and a light chain comprising or consisting of SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0115] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain that comprises a variable region that comprises or consists of SEQ ID NO: 25 and a light chain that comprises a variable region that comprises or consists of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0116] In some embodiments, the antibody or antigen-binding fragment is QVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDSWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 26 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0117] In some embodiments, the antibody or antigen-binding fragment is EVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC The heavy chain comprises or consists of the amino acid sequence of PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 27). In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising or consisting of the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:28). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO:27, and a light chain comprising a variable region comprising or consisting of SEQ ID NO:12. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region that comprises or consists of SEQ ID NO:27 and a light chain comprising a variable region that comprises or consists of SEQ ID NO:28.In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0118] In some embodiments, the antibody or antigen-binding fragment is EVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 29 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0119] In some embodiments, the antibody or antigen-binding fragment is EVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC The antibody or antigen-binding fragment comprises a heavy chain comprising or consisting of the amino acid sequence of PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 30). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 30 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0120] In some embodiments, the antibody or antigen-binding fragment is QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDSWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 31 and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), where CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 49 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 50 (AITFGGGNTYYPDSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 51 (HGDGNYDFYAMDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 52 (KSSQSLLNSGNQKNYLT), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5 (WASTRES), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 53 (QNDYSYPLT). In some embodiments, the antibody comprising SEQ ID NOs: 49-53, and 5 CDRs is 3F6.

[0122] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPAKRLEWVAAITFGGGNTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCARHGDGNYDFYAMDYWGQGTSVTVSS (SEQ ID NO: 54). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of SEQ ID NO: 54. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSRTDFTLTISRVQAEDLAVYYCQNDYSYPLTFGAGTKLELK (SEQ ID NO: 55). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region consisting of SEQ ID NO: 55. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region that comprises or consists of SEQ ID NO:54 and a light chain variable region that comprises or consists of SEQ ID NO:55. In some embodiments, the antibody 3F6 comprises a heavy chain variable region that comprises or consists of SEQ ID NO:54 and a light chain variable region that comprises or consists of SEQ ID NO:55.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), where CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 49 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 56 (TISSDGGNTYYTDSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 57 (HDGRGALDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 58 (RASQDISNYLN), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 59 (YTSRLHS), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 60 (QQGNTLPWT). In some embodiments, the antibody comprising the CDRs of SEQ ID NOs: 56-60, and 49 is 7A5.

[0124] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPAKRLEWVATISSDGGNTYYTDSVKGRFTISRDNARNTLDLQMSSLRSEDTAMYYCARHDGRGALDYWGQGTSVTVSS (SEQ ID NO: 61). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of SEQ ID NO: 61. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 62). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region consisting of SEQ ID NO: 62. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region that comprises or consists of SEQ ID NO:61 and a light chain variable region that comprises or consists of SEQ ID NO:62. In some embodiments, the antibody 7A5 comprises a heavy chain variable region that comprises or consists of SEQ ID NO:61 and a light chain variable region that comprises or consists of SEQ ID NO:62.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), where CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 63 (NSAVH), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 64 (VIWAGGNTNYNSTLMS), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 65 (HETYGDSFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 66 (RSSQSLLDSDGKTYLN), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 67 (LVSKLDS), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 68 (WQGTHFPFT). In some embodiments, the antibody comprising the CDRs of SEQ ID NOs: 63-68 is 10G12.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence QVQLKESGPVLVAPSQSLSITCTVSGFSLTNSAVHWVRQPPGKGLEWLGVIWAGGNTNYNSTLMSRLTINKDNSKSQVFLRMNSLQTDDTAIYYCAKHETYGDSFDYWGQGTTLTVSS (SEQ ID NO: 69). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of SEQ ID NO: 69. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence DVVMTQTPLTLSVTIGQPASISCRSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK (SEQ ID NO: 70). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region consisting of SEQ ID NO: 70. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region that comprises or consists of SEQ ID NO:69 and a light chain variable region that comprises or consists of SEQ ID NO:70. In some embodiments, the antibody 10G12 comprises a heavy chain variable region that comprises or consists of SEQ ID NO:69 and a light chain variable region that comprises or consists of SEQ ID NO:70.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof is capable of binding to ILT3. In some embodiments, the antibody or antigen-binding fragment thereof is an anti-ILT3 antibody. In some embodiments, the target antigen of the antibody is ILT3. In some embodiments, the ILT3 is membrane ILT3 (mILT3). In some embodiments, the ILT3 is soluble ILT3 (sILT3). An "anti-ILT3 antibody", "antibody that recognizes ILT3", or "antibody against ILT3" is an antibody that binds to ILT3 with sufficient affinity and specificity. In some embodiments, the antibody has increased binding to ILT3. In some embodiments, the antibody has increased binding to ILT3 compared to a commercially available ILT3 antibody. In some embodiments, the commercially available ILT3 antibody is selected from ZM4.1, m52B8, and IO202. In some embodiments, the antibody or antigen-binding fragment thereof does not significantly bind to any other target. In some embodiments, any other target is any other ILT protein. In some embodiments, the ILT3 is a mammalian ILT3. In some embodiments, the ILT3 is human ILT3. In some embodiments, the mammal is a monkey. In some embodiments, the monkey is selected from a rhesus monkey and a cynomolgus monkey. In some embodiments, the monkey is a cynomolgus monkey. In some embodiments, the mammal is a human. In some embodiments, the mammal is not a mouse. In some embodiments, the antibody does not bind to mouse ILT3. In some embodiments, the antibody binds to human and monkey ILT3.

[0128] In some embodiments, the membrane ILT3 is on the cell surface. In some embodiments, the membrane is a plasma membrane. In some embodiments, the membrane is a membrane of an immune cell. In some embodiments, the immune cell is selected from a myeloid cell, a dendritic cell, and a macrophage. In some embodiments, the immune cell is a myeloid cell. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the myeloid cell is a myeloid-derived suppressor cell (MDSC). In some embodiments, the dendritic cell is a tolerogenic dendritic cell. In some embodiments, the macrophage is a suppressive macrophage. In some embodiments, the macrophage is an M2 macrophage. In some embodiments, the M2 macrophage is a suppressive macrophage. In some embodiments, the immune cell is a tumor-associated immune cell. In some embodiments, the immune cell is a tumor-infiltrating immune cell. In some embodiments, the macrophage is a tumor-associated macrophage (TAM).

[0129] As used herein, the terms "increased binding affinity" and "higher binding affinity" are interchangeable. In some embodiments, an antibody or antigen-binding portion thereof of the invention has a higher binding affinity for sCD28 compared to mCD28. In one embodiment, higher affinity as used herein is 10%. In one embodiment, higher affinity as used herein is 30%. In one embodiment, higher affinity as used herein is 50%. In one embodiment, higher affinity as used herein is 75%. In one embodiment, higher affinity as used herein is 100%. In one embodiment, higher affinity as used herein is 150%. In one embodiment, higher affinity as used herein is 250%. In one embodiment, higher affinity as used herein is 500%. In one embodiment, higher affinity as used herein is 1,000%. In one embodiment, higher affinity as used herein is 1.5-fold. In one embodiment, higher affinity as used herein is 2-fold. In one embodiment, higher affinity as used herein is 5-fold. In one embodiment, higher affinity as used herein is 10-fold. In one embodiment, higher affinity as used herein is 50-fold. In one embodiment, higher affinity as used herein is 100-fold. In one embodiment, higher affinity as used herein is 500-fold. In one embodiment, higher affinity as used herein is 1,000-fold.

[0130] An "anti-ILT3 antibody", "antibody that recognizes ILT3", or "antibody against ILT3" is an antibody that binds to ILT3 with sufficient affinity and specificity. In some embodiments, the anti-ILT3 antibody has ILT3 as the antigen to which it binds.

[0131] An "antigen" is a molecule or a portion of a molecule that can cause the formation of an antibody and be bound by an antibody. An antigen can have one or more epitopes. The above specific reaction is meant to indicate that the antigen reacts very selectively with its corresponding antibody, rather than with the many other antibodies that may be caused by other antigens.

[0132] The term "antigenic determinant" or "epitope" according to the present invention refers to a region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from the epitope can be used alone or in combination with a carrier moiety to immunize animals to produce additional polyclonal or monoclonal antibodies, applying methods known in the art. Immunoglobulin variable domains can also be analyzed using the IMGT information system (www: / / imgt.cines.fr / ) (IMGT® / V-Quest) to identify variable region segments that contain CDRs. See, for example, Brochet, X. et al, Nucl. Acids Res. J6:W503-508 (2008).

[0133] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth in Kabat et al, USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0134] In some embodiments, the agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is a Fab fragment. In some embodiments, the antibody is a single domain antibody. In some embodiments, the antibody lacks an Fc domain. In some embodiments, the agent is an antigen-binding domain that lacks an Fc domain. In some embodiments, the agent is a single domain antibody. In some embodiments, the agent is a camelid, shark, or nanobody. In some embodiments, the antibody or fragment is fused to another protein or protein fragment. In some embodiments, the second protein or fragment enhances half-life, particularly in serum. In some embodiments, the half-life extending protein is human serum albumin. In some embodiments, the agent is modified by a chemical that produces a modification that enhances half-life. In some embodiments, the modification is PEGylation and the chemical is polyethylene glycol. One of ordinary skill in the art will appreciate that any half-life extending protein or chemical or modification known in the art may be used.

[0135] In some embodiments, binding of the antibody or antigen binding domain to a cell does not kill the cell. In some embodiments, binding of the antibody or antigen binding domain to a cell does not result in cell death. In some embodiments, the antibody or antigen binding domain does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the antibody or antigen binding domain does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the antibody or antigen binding domain does not induce ADCC and / or CDC. In some embodiments, the antibody or antigen binding domain comprises an IgG2 or IgG4 domain. In some embodiments, the antibody or antigen binding domain comprises an IgG2 domain. In some embodiments, the antibody or antigen binding domain comprises an IgG4 domain. In some embodiments, the antibody or antigen binding domain comprises an IgG1 or IgG3 mutated to reduce cell death mediated by antibody binding. In some embodiments, the mutation mutates the Fc receptor binding domain. In some embodiments, the Fc domain of the antibody is engineered or mutated to reduce CDC, ADCC, or both. Fc engineering is well known in the art and may use any mutation or amino acid change known to reduce antibody-mediated cell killing.

[0136] In some embodiments, the antibody or antigen binding domain does not comprise IgG1 and / or IgG3. In some embodiments, the antibody or antigen binding domain does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the antibody or antigen binding domain does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the antibody or antigen binding domain comprises IgG1 or IgG3 that includes a mutation that reduces ADCC, CDC, or both induced by antibody binding. In some embodiments, the mutation reduces ADCC, CDC, or both to zero. ADCC and CDC are well characterized, and antibody sequences that allow induction of these cytotoxic pathways are well known. Mutations, such as, by way of non-limiting example, IgG1 or IgG3 to IgG2 or IgG4, are well known. Any such mutations may be used in the backbone of the antibody of the present invention.

[0137] In some embodiments, binding to ILT3 inhibits binding of ILT3 to a ligand. In some embodiments, binding to ILT3 inhibits binding of a ligand to ILT3. In some embodiments, the ligand is an ILT3 ligand. In some embodiments, the ligand is apolipoprotein E (APOE). In some embodiments, the ligand is fibronectin (FN1). In some embodiments, the ligand is APOE and FN1. In some embodiments, the ligand inhibits binding of ILT3 to both APOE and FN1. In some embodiments, APOE is present in the tumor microenvironment (TME). In some embodiments, FN1 is present in the TME. In some embodiments, APOE is present in the extracellular matrix. In some embodiments, FN1 is present in the extracellular matrix. In some embodiments, the extracellular matrix is ​​a tumor extracellular matrix.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof is an immune checkpoint inhibitor (ICI). In some embodiments, the antibody or antigen-binding fragment is an ILT3 blocking antibody. In some embodiments, binding of the antibody or antigen-binding fragment results in blockade of ILT3. In some embodiments, binding to ILT3 relieves an immune cell from ILT3-mediated inhibition. In some embodiments, the inhibition is immunosuppression. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8 T cell. In some embodiments, the T cell is a CD4 T cell. In some embodiments, binding of ILT3 relieves a T cell from ILT3-mediated inhibition. In some embodiments, binding of ILT3 relieves a monocyte from ILT3-mediated inhibition. In some embodiments, binding of ILT3 relieves a macrophage from ILT3-mediated inhibition. In some embodiments, binding of ILT3 relieves a dendritic cell from ILT3-mediated inhibition. In some embodiments, the immune cell is a macrophage. In some embodiments, the macrophage is an M0 macrophage. In some embodiments, the M0 macrophage is a non-activated macrophage. In some embodiments, the M0 macrophage is a naive macrophage. In some embodiments, the macrophage is an M1 macrophage. In some embodiments, the M1 macrophage is an inflammatory macrophage. In some embodiments, the M1 macrophage is a pro-inflammatory macrophage. In some embodiments, the macrophage is an M2 macrophage. In some embodiments, the M2 macrophage is an anti-inflammatory macrophage. In some embodiments, the M2 macrophage is a suppressive macrophage. In some embodiments, the M2 macrophage is a tolerogenic macrophage. In some embodiments, the monocyte is a macrophage. In some embodiments, the macrophage is an immature macrophage. In some embodiments, the macrophage is an inflammatory macrophage.In some embodiments, the macrophage is a tolerogenic macrophage.

[0139] In some embodiments, the release is a decrease. In some embodiments, the decrease is at least a 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99, or 100% decrease. Each possibility represents a separate embodiment of the present invention. In some embodiments, the release comprises immune cell activation. In some embodiments, the activation is an increase in activation. In some embodiments, the increase is at least a 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500% increase. Each possibility represents a separate embodiment of the present invention. In some embodiments, dendritic cell activation is increased. In some embodiments, T cell activation is increased. In some embodiments, monocyte activation is increased. In some embodiments, macrophage activation is increased. In some embodiments, increasing T cell activation comprises restoring T cell activation suppressed by MDSC. In some embodiments, increasing T cell activation comprises restoring T cell activation suppressed by tolerogenic DC (DCtol). In some embodiments, activation is Fc receptor mediated activation. In some embodiments, increasing DC activation comprises restoring DC activation suppressed by fibronectin or APOE. In some embodiments, increasing monocyte activation comprises restoring monocyte activation suppressed by fibronectin or APOE.

[0140] In some embodiments, the liberation comprises increased proliferation. In some embodiments, the activation comprises increased proliferation. In some embodiments, the liberation comprises increased proinflammatory cytokine secretion. In some embodiments, the liberation comprises increased proinflammatory chemokine secretion. In some embodiments, the activation comprises increased proinflammatory cytokine secretion. In some embodiments, the activation comprises increased proinflammatory chemokine secretion. In some embodiments, the proinflammatory cytokine is selected from TNFα, IFNg, and IL-8. In some embodiments, the proinflammatory cytokine is TNFα. In some embodiments, the proinflammatory cytokine is IFNg. In some embodiments, the proinflammatory cytokine is IL-8. In some embodiments, the proinflammatory chemokine is selected from CCL3 and CCL4. In some embodiments, the proinflammatory chemokine is CCL3. In some embodiments, the proinflammatory chemokine is CCL4.

[0141] Another aspect provides an antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment of the invention for binding to ILT3.

[0142] In some embodiments, competing for binding comprises binding to the same epitope. Methods for determining the epitope to which an antibody binds are well known in the art, and any such method can be used to determine the epitope to which an antibody of the present invention binds. In some embodiments, the epitope is present within the ligand binding domain. In some embodiments, the epitope is sufficiently close to the ligand binding domain such that binding of the antibody or antigen binding domain occludes, blocks, or alters the ligand binding domain. In some embodiments, the epitope has low homology to the corresponding mouse sequence of ILT3.

[0143] Another aspect provides a nucleic acid molecule encoding an antibody or antigen-binding fragment of the invention.

[0144] In some embodiments, the nucleic acid molecule is a plurality of nucleic acid molecules. In some embodiments, the nucleic acid molecule comprises an open reading frame encoding an antibody or antigen-binding fragment of the invention. In some embodiments, the open reading frame is a plurality of open reading frames. In some embodiments, the nucleic acid molecule encodes a heavy chain. In some embodiments, the nucleic acid molecule encodes a light chain. In some embodiments, the same nucleic acid molecule encodes the heavy and light chains. In some embodiments, different nucleic acid molecules encode the heavy and light chains.

[0145] In some embodiments, the nucleic acid molecule is a plasmid. In some embodiments, the vector is an expression vector. In some embodiments, the vector is configured for expression in a target cell. In some embodiments, the nucleic acid molecule comprises an open reading frame.

[0146] For example, a polynucleotide may encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain contains not only a heavy chain variable region (VH) but also a heavy chain constant region (CH), which typically contains three constant domains: CH1, CH2, and CH3; as well as a "hinge" region. In some circumstances, the presence of a constant region is desirable.

[0147] Other polypeptides that may be encoded by the polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fv, scFv, Fab', and CHI, with the CK or CL domains truncated. Minibodies are smaller than conventional antibodies and therefore should achieve better tissue penetration for clinical / diagnostic applications, but are bivalent and therefore should retain higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context dictates otherwise, the term "antibody" as used herein includes not only whole antibody molecules but also antigen-binding antibody fragments of the types described above. Each framework region present in the encoded polypeptide can contain at least one amino acid substitution with respect to the corresponding human acceptor framework. Thus, for example, the framework regions can contain, in total, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions with respect to the acceptor framework region. Given the properties of the individual amino acids that make up the disclosed protein products, the skilled artisan will recognize some reasonable substitutions. Amino acid substitutions, or "conservative substitutions," may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0148] Suitably, the polynucleotides described herein may be isolated and / or purified. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0149] As used herein, "non-naturally occurring" substances, compositions, entities, and / or any combination of substances, compositions, entities, or any grammatical variations thereof, is a qualified term that expressly excludes, but merely excludes, forms of substances, compositions, entities, and / or any combination of substances, compositions, entities that are well understood to be "naturally occurring" by those of skill in the art, or that have been determined or construed, or may be determined or construed, at any time, to be "naturally occurring" by a judicial, administrative, or judicial body.

[0150] The term "nucleic acid" is well known in the art. As used herein, "nucleic acid" generally refers to a molecule (i.e., a chain) of DNA, RNA, or derivatives or analogs thereof, that includes a nucleic acid base. Nucleic acid bases include, for example, naturally occurring purine or pyrimidine bases found in DNA (e.g., adenine "A", guanine "G", thymine "T" or cytosine "C") or RNA (e.g., A, G, uracil "U" or C).

[0151] The term "nucleic acid molecule" includes, but is not limited to, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNA such as miRNA, siRNA, other small interfering nucleic acids, snoRNAs, snRNAs, tRNA, piRNA, tnRNA, small rRNA, hnRNA, lncRNA, circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, ribozymes, viral RNA or DNA, infectious agent nucleic acids, amplification products, modified nucleic acids, plasmid or organelle nucleic acids, and artificial nucleic acids such as oligonucleotides.

[0152] As used herein, the term "oligonucleotide" refers to a short (e.g., 100 bases or less), chemically synthesized, single-stranded DNA or RNA molecule. In some embodiments, an oligonucleotide is attached to the 5' or 3' end of a nucleic acid molecule, for example, by a ligation reaction.

[0153] The term "expression" as used herein refers to the biosynthesis of a gene product, including transcription and / or translation of the gene product. Thus, expression of a nucleic acid molecule can refer to the transcription of a nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and / or the translation of RNA into a precursor or mature protein (polypeptide).

[0154] The expression of genes in cells is well known to those skilled in the art, and the delivery thereof can be carried out by the method of the present invention or by using the composition of the present invention herein.In some embodiments, the gene is in an expression vector, such as a plasmid or a viral vector.The vector can be a viral vector.The viral vector can be a retroviral vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector or a pox virus vector.The promoter can be active in mammalian cells.The promoter can be a viral promoter.

[0155] In some embodiments, the gene or open reading frame is operably linked to a promoter or other regulatory element. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element(s) in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host when the vector is introduced into a host cell by the method of the present invention). In some embodiments, the regulatory element or promoter is active in the target cell. In some embodiments, the target cell is a packaging cell line.

[0156] The term "promoter" as used herein refers to a group of transcriptional control modules clustered around the initiation site of RNA polymerase, i.e., RNA polymerase II. Promoters are composed of distinct functional modules, each consisting of approximately 7-20 bp of DNA and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0157] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAPII and Pol II). RNAP II is an enzyme found in eukaryotic cells. RNAP II catalyzes the transcription of DNA to synthesize the precursor of mRNA and most snRNAs and microRNAs.

[0158] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0159] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses, such as retroviruses, are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that have been shown to be effective for expression in eukaryotic cells.

[0160] In some embodiments, recombinant viral vectors that provide advantages such as horizontal infection and target specificity are used for in vivo expression. In one embodiment, horizontal infection is inherent in, for example, the life cycle of retroviruses, and is the process by which one infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that large areas, most of which were not originally infected by the original viral particle, become rapidly infected. In one embodiment, viral vectors that cannot be horizontally transmitted are produced. In one embodiment, this feature can be useful when the desired purpose is to introduce a specific gene into a very limited number of target cells.

[0161] Another aspect provides a composition comprising an antibody or antigen-binding fragment of the invention.

[0162] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a therapeutic composition. In some embodiments, the composition is a prophylactic composition. In some embodiments, the composition comprises a therapeutically effective amount of an antibody or antigen-binding domain. In some embodiments, the composition comprises a pharma- ceutically acceptable carrier, excipient, or adjuvant.

[0163] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. The term "therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen will be determined by the physician depending on the patient's condition.

[0164] The term "carrier", "excipient" or "adjuvant" as used herein refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous medium such as saline. Some examples of materials which may function as pharma- ceutically 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 carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository wax; 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 glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffers, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can function as carriers herein include sugar, starch, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers, and other non-toxic compatible materials used in other pharmaceutical preparations.Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives can also be present.Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein.In this regard, suitable pharma- ceutically acceptable carriers, excipients and diluents are well known to those skilled in the art, and are described in, for example, The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck&Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and "Inactive Ingredient Guide" US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are incorporated herein by reference in their entirety.Examples of pharma-ceutically acceptable carriers, excipients and diluents useful in the present composition include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution and DMSO. These additional inactive ingredients, as well as effective formulation and administration procedures, are well known in the art and are described in standard texts, such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. 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 herein may also be included in artificially created structures such as liposomes, ISCOMS, delayed release particles, and other vehicles that extend the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the currently described peptides are formed from standard vesicle-forming lipids, which are neutral and generally contain negatively charged phospholipids and sterols such as cholesterol. The choice of lipid is generally determined by considerations such as liposome size and blood stability. Various methods are available for the preparation of liposomes, as discussed, for example, in Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0165] The carriers may in total constitute from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0166] In some embodiments, the composition is for use in the methods of the invention. In some embodiments, the antibody or antigen binding domain is for use in the methods of the invention. In some embodiments, the composition is for use in enhancing immune function. In some embodiments, the antibody or antigen binding domain is for use in enhancing immune function. In some embodiments, the enhancing immune function is alleviating immunosuppression. In some embodiments, the composition is for use in the treatment of cancer. In some embodiments, the antibody or antigen binding domain is for use in the treatment of cancer.

[0167] In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the composition is formulated for systemic administration. In some embodiments, the composition is formulated for intratumoral administration. As used herein, the terms "administer", "administration" and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice. One aspect of the subject matter provides for intravenous administration of a therapeutically effective amount of the subject matter composition to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular, or intraperitoneal.

[0168] Dosage will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0169] According to another aspect, there is provided a method for increasing immune function in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention or an antibody or antigen-binding fragment of the invention, thereby increasing immune function in the subject.

[0170] In some embodiments, increasing immune function comprises increasing immune surveillance. In some embodiments, increasing immune function comprises increasing an immune response. In some embodiments, increasing immune function comprises decreasing immunosuppression. In some embodiments, the immune function comprises immune-mediated cell killing. In some embodiments, the killing is killing disease cells. In some embodiments, the immune response is against disease cells. In some embodiments, the cells are target cells.

[0171] In some embodiments, the disease is a disease treatable by killing target cells. In some embodiments, the disease is a disease treatable by inducing an immune response against target cells. In some embodiments, the target cells are disease cells. In some embodiments, the disease is characterized by the presence of immune cells that express ILT3.

[0172] In some embodiments, the disease is cancer. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a tumor. In some embodiments, the target cell is a tumor cell. In some embodiments, the cancer is characterized by the presence of immune cells expressing ILT3. In some embodiments, the cancer is characterized by the presence of tumor infiltrating immune cells expressing ILT. In some embodiments, the cancer is characterized by expression of APOE, FN1, or both. In some embodiments, the cancer is a cancer extracellular matrix. In some embodiments, the cancer is a cancer TME. In some embodiments, the expression is overexpression. In some embodiments, the overexpression is compared to non-disease tissue. In some embodiments, the non-disease tissue is a non-cancerous tissue. In some embodiments, the tissue is of the same type as the disease tissue. In some embodiments, the tissue is of the same type of tissue as the tumor. In some embodiments, the same tissue type is derived from the same tissue type.

[0173] As used herein, a "cancer" or "premalignant tumor" is a disease associated with cell proliferation. Non-limiting types of cancer include carcinoma, sarcoma, lymphoma, leukemia, blastoma, and germ cell tumors.

[0174] In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a tumor. In some embodiments, the cancer is selected from hepatobiliary cancer, cervical cancer, genitourinary cancer (e.g., urothelial cancer), testicular cancer, prostate cancer, thyroid cancer, ovarian cancer, nervous system cancer, eye cancer, lung cancer, soft tissue cancer, bone cancer, pancreatic cancer, bladder cancer, skin cancer, intestinal cancer, liver cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, breast cancer (e.g., triple-negative breast cancer), renal cancer (e.g., renal cell carcinoma), skin cancer, head and neck cancer, leukemia, and lymphoma. In some embodiments, the cancer is selected from breast cancer, renal cancer, head and neck cancer, lung cancer, sarcoma, gastric cancer, and ovarian cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is colon cancer.

[0175] In some embodiments, the method further comprises administering an immune checkpoint inhibitor (ICI). In some embodiments, the inhibition is blocking. In some embodiments, the method further comprises administering an immune stimulant. In some embodiments, the agent is a molecule. In some embodiments, the agent is an antibody. In some embodiments, the agent is an ICI. In some embodiments, the immune stimulant has specificity for a target selected from CTLA-4, PD-1, PD-LI, PD-L2, CD40, OX40, CD137, GFTR, ILT2, or LAG3, or ligands of these proteins. In some embodiments, the ICI targets the PD-1 / PD-L1 axis. In some embodiments, the ICI inhibits PD-1, PD-L1, PD-L2, or a combination thereof. In some embodiments, the ICI inhibits the PD-1 / PD-L1 / PD-L2 checkpoint. In some embodiments, the ICI targets PD-1. In some embodiments, the ICI binds to PD-1. In some embodiments, the ICI inhibits PD-1. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 agent is nivolumab. In some embodiments, the anti-PD-L1 agent is atezolizumab. Other examples of anti-PD-1 antibodies include, but are not limited to, cemiplimab, dostarlimab, durvalumab, and avelumab.

[0176] In some embodiments, the antibodies or antigen-binding fragments of the invention are for use in combination with an immunostimulant. In some embodiments, the compositions of the invention are for use in combination with an immunostimulant. In some embodiments, the use is a method of the invention. In some embodiments, the antibodies or antigen-binding fragments of the invention are for use in the manufacture of a medicament for the treatment of a disease. In some embodiments, the compositions of the invention are for use in the manufacture of a medicament for the treatment of a disease.

[0177] As used herein, the term "about" when combined with any value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0178] It should be noted that the singular forms "a," "an," and "the," as used herein and in the appended claims, include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a polynucleotide" includes a plurality of such polynucleotides, a reference to "the polypeptide" includes a reference to one or more polypeptides known to those of skill in the art and equivalents thereof, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a prerequisite for using exclusive language, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of "negative" limitations.

[0179] When a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those of ordinary skill in the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in this specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0180] It is recognized that certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of the embodiments of the invention are specifically embraced by the invention and are disclosed herein as if every combination were individually and expressly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the invention and are disclosed herein as if every such subcombination were individually and expressly disclosed herein.

[0181] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0182] Various embodiments and aspects of the present invention as delineated hereinabove and in the claims section below find experimental support in the following examples.

[0183] Working Example In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are explained in detail 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, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley&Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series" 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); the methods described in U.S. Pat. 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, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual", CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this specification.

[0184] Example 1: Spectrum of ILT3 Expression As a starting point, we investigated the breadth of ILT3 expression in various immune cells, cancer-associated immune cells, and cancer. First, PBMCs from two healthy donors were stained with a commercially available anti-ILT3 antibody conjugated to PE (ZM4.1-PE, Biolegend catalog no. 333008) and analyzed by FACS. Monocytes isolated based on CD14 expression were weakly positive for ILT3 (Figure 1A). Myeloid-derived suppressor cells (MDSCs) were generated from PBMCs by co-incubation with SK-MEL-5 melanoma cells for 7 days in the presence of GM-CSF. Surface expression of ILT3 was found to be upregulated in MDSCs compared to monocytes (Figure 1A).

[0185] Immature dendritic cells (iDCs) were generated from monocytes isolated from two other healthy donors by culturing them for 6 days in the presence of GM-CSF (50 ng / ml) and IL-4 (20 ng / ml). Mature dendritic cells (mDCs) were produced by culturing them for an additional 48 h in the presence of LPS (100 ng / ml), and tolerogenic DCs (DCtol) were generated by culturing iDCs with IL-10 (100 U / ml) and IFNa-2b (1000 U / ml) for 48 h. For both donor cells, DCtol displayed the highest levels of surface ILT3 (Figure 1B).

[0186] Monocytes were again isolated from healthy donors and differentiated into various types of macrophages. First, isolated monocytes were cultured with M-CSF (50 ng / ml) for 6 days to generate M0 macrophages. Next, M0 macrophages were cultured with IFNg (20 ng / ml) and LPS (100 ng / ml) for 48 h to generate M1 macrophages. M2 macrophages were generated by culturing M0 macrophages with M-CSF (10 ng / ml) and IL4 (20 ng / ml) for 48 h. After differentiation, the expression of CD80, CD206, CD14, and HLA-DR was used to validate the resulting populations. ILT3 levels in these cells were assessed by FACS. All macrophages (M0, M1, and M2) showed increased ILT3 expression compared to control monocytes, with M1 macrophages showing a more than 10-fold increase (Figure 1C).

[0187] Immunohistochemistry was then performed on sections from various tumors to confirm ILT3 expression. Sections were double stained for CD68, a marker for tumor-associated macrophages, and ILT3 (Figure 2A). Samples were considered ILT3 positive if more than 200 ILT3-positive cells were identified. Thirty to 90% of collected breast, renal, head and neck, lung, sarcoma, gastric, and ovarian cancers containing ILT3-positive infiltrating immune cells were ILT3 positive (Figure 2B).

[0188] When comparing the enrichment of MDSCs and M2 (suppressive) macrophages in cancers with the average ILT3 expression in those cancers, we found a strong correlation between patients with high ILT3 levels and enrichment of MDSCs (Figure 3A) and M2 macrophages (Figure 3B). All these results highlight the need for antibodies that can effectively block ILT3 on tumor-associated immune cells and convert an immunosuppressive TME into a TME with active immune effector functions.

[0189] Example 2: Relief of immune cell suppression Anti-ILT3 antibodies were generated by hybridoma technology. Antibodies were screened for specificity to human ILT3 and cross-reactivity to non-human primate (NHP) ILT3. Antibodies found to be specific to ILT3 were screened for functional activity.

[0190] We generated HEK cells exogenously overexpressing recombinant human ILT3 and used the cells to evaluate the binding of the antibodies to membrane ILT3. Hybridoma medium was used as a negative control, and a commercial anti-ILT3 antibody (ZM4.1) was used as a positive control. The lead antibody, named 5E5, was found to produce an ILT3 expression signal that was comparable or lower than the commercial antibody (mean fluorescence of 125 vs. 118 for the commercial antibody) (Figure 4A). The other three antibodies (10G12, 3F6, and 7A5) were also found to produce positive signals, the levels of which were comparable to the control antibodies (10G12 showed slightly less signal; 79 and 118). Since these antibodies bound to recombinant ILT3, they may also bind to soluble ILT3 (sILT3). Interestingly, 5E5 bound strongly to cynomolgus ILT3 (Figure 4B), but did not bind at all to mouse recombinant ILT3 (data not shown). Three other antibodies comparable to ZM4.1 also showed some binding to cynomolgus ILT3, albeit at a significantly lower level than 5E5. Several other antibodies produced did not bind to cynomolgus, nor did the commercial antibody tested (ZM4.1). 5E5 was also evaluated for binding to other ILT proteins by ELISA and was found not to bind to ILT5, ILT11, or ILT8, although some of the other antibodies produced by the hybridoma showed cross-reactivity.

[0191] A competition assay was performed to determine whether the 5E5 antibody binds to the same epitope as the commercial antibody ZM4.1. The ILT3 antibody was biotinylated and used at a fixed concentration in an ILT3 binding ELISA (1 μg / mL). Competing antibodies were added at various dilutions and the OD was measured. As a positive control, a "naked" antibody identical to the biotinylated antibody was added using the same concentration range. 5E5 and ZM4.1 did not compete with each other, indicating that 5E5 binds to a unique epitope (Figure 5). Interestingly, another hybridoma antibody (AB#2) competed with ZM4.1.

[0192] Next, the 5E5 antibody was evaluated for its ability to enhance PBMC activation. PBMCs were isolated from healthy donors and activated with LPS for 6 hours at 37°C in the presence or absence of 5E5 antibody. TNF-α secretion levels were quantified by ELISA as a measure of immune cell activation. Media only and naive PBMCs were used as negative controls, as was mouse IgG1. 5E5 was able to increase TNF-α secretion levels to more than two-fold compared to LPS stimulation without antibody (Figure 6A), indicating that this antibody may indeed enhance immune cell activation. Of note, several other antibodies generated, specific for ILT3, did not enhance activation (data not shown).

[0193] Next, T cell activation was examined. MDSCs were generated from PBMCs of healthy donors as described above. Autologous CD8 T cells were activated with anti-CD3 / CD28 beads. Activated T cells were co-cultured with MDSCs in the presence or absence of 5E5 antibody, and interferon gamma (IFNg) was measured. As can be seen in Figure 6B, activated T cells alone secreted very high levels of IFNg, which were essentially completely abolished by co-culture with MDSCs (medium or mIgG1 control). However, 5E5 antibody almost completely eliminated the inhibitory effect of MDSCs, maintaining IFNg levels at more than 70% of the levels observed with activated T cells alone.

[0194] The 5E5 antibody was then compared to the m52B8 antibody (Merck). 52B8 was shown to restore some IFNg secretion, thereby blocking the effect of MDSCs, but the 5E5 antibody was found to be superior (Figure 6C). The increased level of IFNg compared to the negative control (moderate / mIgG1) was almost two-fold greater for the 5E5 antibody compared to the m52B8 antibody.

[0195] iDCs were generated from healthy donor monocytes and converted into DCtol as described above. Allogeneic CD4 T cells were activated by soluble anti-CD3 antibody and then incubated with DCtol in the presence of 5E5 or mIgG1 control. CD4 T cells were also incubated with iDCs as a positive control. As can be seen in Figure 6D, DCtol reduced IFNg secretion from T cells, but the addition of 5E5 antibody restored the levels to those of the control (iDC). Thus, this antibody is capable of completely abolishing the immunosuppressive effect of DCtol on CD4 T cells. This experiment was repeated with iDCs generated in five different healthy donors and their allogeneic CD4 T cells. Again, an increase in IFNg secretion produced by the 5E5 antibody was observed and found to be statistically significant (Figure 6E). This experiment was similarly performed in combination with anti-PD-1 checkpoint blockade. As expected, administration of anti-PD-1 antibody alone (along with an isotype control antibody) resulted in robust IFNg secretion, and interestingly, the h5E5 antibody produced an additive effect on top of the PD-1 effect, indicating that the two act through different pathways. This additive increase was also statistically significant. Similar results were observed when T cells were not first activated with anti-CD3 antibody, although overall IFNg secretion was much lower (Figure 6F). Unexpectedly, in the absence of anti-CD3 activation, the combination of anti-PD-1 and 5E5 did not result in a mere additive increase, but rather a synergistic increase, with IFNg secretion levels increasing much more than would be expected based on the effect of the 5E5 antibody itself.

[0196] In parallel experiments, M2c macrophages were generated from monocytes isolated from healthy donors. Briefly, monocytes were cultured in complete RPMI medium supplemented with M-CSF (50 ng / ml) for 5–6 days to generate M-0 macrophages. M-0 macrophages were stimulated with IL10 (20 ng / ml) for 48 h to generate M2c macrophages. M2c macrophages were then incubated with allogeneic CD4 T cells for 120 h, and IFNg secretion levels were measured by ELISA. As expected, M2c macrophages largely inhibited IFNg secretion from T cells. However, when the M2c generation protocol was performed in the presence of 5E5, M2c cells were less inhibitory and T cells produced higher levels of IFNg (Figure 6G). Isotype controls were similar to the results obtained when no antibody was added and M1 macrophages were used as a positive control.

[0197] Example 3: Blockade of ILT3 ligands To further characterize the 5E5 antibody, its ability to block the binding of known ILT3 ligands fibronectin (FN1) and apolipoprotein E (APOE) to ILT3 was evaluated. Binding of recombinant ILT3-His to fibronectin or APOE was determined by ELISA using anti-His conjugated to HRP by OD analysis. Percent blocking was determined by normalizing to the control (ILT3 binding without anti-ILT3 antibody). 5E5 was compared to both ZM4.1 and m52B8. ZM4.1 was able to inhibit the binding of both ligands, and 5E5 was found to be comparable with respect to FN1 (Figure 7A) and APOE (Figure 7B). 5E5 produced a higher level of blocking effect at equal concentrations. m52B8 was found to be a very poor blocker of the ILT3-FN1 interaction, producing no blockade except at the highest concentration tested, whereas 5E5 was clearly superior (Figure 7C). Several of the other antibodies produced also blocked FN1 binding, although 10G12 again performed slightly worse (Figure 7D). The two antibodies were nearly identical in terms of blocking APOE, although the m52B8 antibody may have been slightly superior (Figure 7E).

[0198] To functionally test the blockade of ligand binding to ILT3, THP-1 monocytic cells were activated by immobilized rituximab via the Fc receptor signaling pathway. Activation was monitored by measuring the secretion of the proinflammatory cytokine IL-8. Addition of fibronectin to the cultures inhibited IL-8 secretion, decreasing it by about 67% (Figure 8A). Next, various anti-ILT3 antibodies were tested for their ability to restore IL-8 secretion. As expected, m52B8 had no effect on IL-8 secretion, as it was unable to block fibronectin binding to ILT3. The antibody IO202 (Immune-Onc Therapeutics) had a modest effect, increasing IL-8 secretion, but not to the levels observed in the absence of fibronectin. In contrast, both 5E5 and ZM4.1 not only restored IL-8 levels, but actually increased them to levels not observed with rituximab activation alone. Both antibodies nearly doubled IL-8 secretion, and similar results are observed with APOE.

[0199] Similar experiments were performed with DCs. DCs were activated via Fc receptor signaling with immobilized rituximab. Addition of fibronectin slightly reduced the secretion of TNFα (Figure 8B) and IL-8 (Figure 8C), which was restored by 5E5 antibody as well as m52B8 and IO202. Blocking ILT3 elevated the proinflammatory cytokine secretion levels to a greater extent than rituximab alone. Again, the increase was more than two-fold, with 5E5 being very slightly superior to these two antibodies. The same experiment was also performed using immobilized Erbitux to induce Fc receptor signaling-mediated activation. Both c5E5 and h5E5 antibodies (see Example 4 below) restored the TNFα secretion inhibited by fibronectin (Figure 8D). In fact, the antibody of the present invention was slightly superior to the known antibody ZM4.1, and significantly superior to the humanized version of the known m52B8 antibody (h52B8) and the known humanized h5A7 antibody (NGM Biopharmaceuticals Inc., US Patent Application US2021 / 0221887). Similar results are observed with APOE instead of fibronectin.

[0200] To demonstrate that the enhanced activation is ILT3-dependent, ILT3-expressing and ILT3-non-expressing DCs were generated. DCs were incubated with fibronectin in the presence of 5E5 antibody, an IgG control. Fibronectin only reduced TNFα secretion from ILT3-expressing DCs (Figure 9A), but not from non-expressing dendritic cells (Figure 9B). Similar results are observed with APOE. 5E5 antibody had no effect on non-expressing DCs, but partially restored TNFα secretion from ILT3-positive DCs. Thus, inhibition and restoration of proinflammatory cytokine secretion are clearly ILT3-dependent.

[0201] Example 4: Generation and validation of humanized 5E5 antibody To generate the humanized 5E5 antibody, the mouse antibody (heavy chain SEQ ID NO: 7, light chain SEQ ID NO: 8) was sequenced to identify the CDRs (SEQ ID NOs: 15-17 and 4-6 according to the Chothia numbering system or SEQ ID NOs: 13, 32, 17, and 4-6 according to the Kabat numbering system). The variable regions of the heavy and light chains were transferred to human constant regions to produce a chimeric antibody (c5E5, heavy chain SEQ ID NO: 9, light chain SEQ ID NO: 10). Additionally, only the CDRs were removed and inserted into a fully human antibody scaffold and affinity matured (h5E5). Several slightly different human antibodies were generated and their ability to bind human ILT3 was examined (Figure 10). All antibodies were found to also bind to ILT3 in cynomolgus and rhesus monkeys. H5E5-133 (heavy chain SEQ ID NO: 11, light chain SEQ ID NO: 12) was determined to be the best and was used for all future experiments. The various human antibodies are summarized in Table 1.

[0202] [Table 1]

[0203] Various h5E5 antibodies were confirmed to block the interaction between ILT3 and FN1 when c5E5 was used as a positive control (Figure 11A). Notably, h5E5-133 was a superior blocker compared to c5E5 (Figure 11B).

[0204] Next, the function of h5E5 was examined. Activation of CD8 T cells after MDSC suppression was examined as above. c5E5 increased the secretion of IFNg from T cells, but the h5E5 antibody was even better at alleviating MDSC suppression, resulting in a statistically significant increase compared to the IgG control (Figure 12A). DC activation in the presence of rituximab and fibronectin was examined as above. In this assay, h5E5-133 was also slightly better than c5E5, with a stronger effect at lower concentrations (Figure 12B). Similar results were observed for the restoration of THP1 activation (Figure 12C). Several other antibodies were also found to be slightly better than mouse 5E5 (Figure 12D). Similar results are observed with APOE instead of fibronectin.

[0205] To further evaluate the function of h5E5, monocytes were stimulated with M-CSF for 5 days in the presence or absence of gastric tumor cells (GA-04) or primary colon cancer cells and h5E5 antibody to generate M0 macrophages. M0 macrophages were then subjected to a differentiation process into M1 macrophages, still in the presence and absence of cancer cells and h5E5 antibody, and activated by LPS stimulation. M1 macrophages secreted high levels of TNFα, which was abolished when cells were co-cultured with autologous gastric cancer cells (GA-04) (Figure 13A) or colon cells (Figure 13B). This inhibition by cancer cells is mediated by ILT3, since the inhibition could be relieved and TNFα secretion restored by the addition of h5E5. The addition of hIgG4 was used as a negative control. Thus, human antibodies can relieve the cancer-induced immune suppression of M1 macrophages, highlighting the importance of ILT3 in myeloid maturation.

[0206] Monocytes were then cultured in ImmunoCult DC Differentiation Medium for 5 days to generate immature DCs (iDCs). DCtol was generated by culturing iDCs with IL-10 and LPS for 2 days. iDC stimulation was performed in the presence of h5E5 antibody, commercial humanized h52B8 antibody, isotype control, or without any addition (medium only). TNFα secretion levels were measured by ELISA. DCtol produced extremely low levels of TNFα, and the same was true when the isotype control was used. However, stimulation in the presence of h5E5 antibody significantly increased the level of cytokine secretion, which was superior to the secretion level produced in the presence of h52B8 antibody (Figure 14).

[0207] It is known that ILT3 blockade can be combined with other immune checkpoint inhibitors (ICIs). To test this, we used T cell activation in the presence of MDSCs to monitor the effect of combination treatments of h5E5, anti-PD1, and h5E5+PD-1. Indeed, the two blocking agents were found to have a combined effect on T cell activation, producing a stronger effect than either antibody by itself (Figure 15A). Indeed, this combination treatment was able to increase IFNg secretion levels above those observed in control activated CD8 T cells. A similar synergistic effect was observed when examining the secretion of IFNg from CD4 cells co-cultured with DCtol (Figure 15B). Similar results were observed in the co-incubation assay of DCtol and CD4 T cells (Figures 6E-6F).

[0208] Example 5: In vitro and in vivo evaluation of the therapeutic efficacy of humanized 5E5 antibody To test the therapeutic efficacy of the antibody of the present invention, primary tumor samples were processed into tumor organoid particles using tissue processing by TissueGrinder. Tumor organoids were activated with PMA and LPS for 48 hours (sarcoma tumor organoids were added with ionomycin), and control tumor organoids were not stimulated. Then, h5E5 antibody or commercial anti-ILT3 antibody was added, and cytokine secretion was measured by ELISA. In the first experiment, h5E5 was found to enhance the production of IFNg in both sarcoma tumor organoids (Figure 16A) and breast cancer tumor organoids (Figure 16B). When various commercially available antibodies were applied to breast cancer tumor organoids, h5E5 caused a strong dose-dependent increase in all molecules tested, but no increase in the production of proinflammatory chemokines (CCL3, CLL4) or cytokines (IL-8) was observed (Figure 16C). Results were similar in colorectal cancer (CRC) tumor organoids, although the effect of the h5E5 antibody was less pronounced and the h52B8 antibody produced a superior increase in cytokine / chemokine production (Figure 16D).Surprisingly, incubation with the anti-PD-1 antibody pembrolizumab had no significant effect on cytokine / chemokine secretion, but when pembrolizumab was combined with h5E5, the increase was highly significant, indicating a synergistic increase over each agent alone (Figure 16D).

[0209] The therapeutic efficacy of 5E5 antibody was evaluated using hILT3 transgenic mice. Mice were subcutaneously inoculated with MC-38 cancer cells (syngeneic mouse colon cancer cell line). Mice implanted with MC-38 were treated with c5E5 or hIgG4 control (20mg / kg / dose) twice a week starting on day 0 (treatments were performed on days 0, 3, 7, 10, 14). Tumor volume was recorded three times a week. As can be seen in FIG. 17, anti-ILT3 antibodies were highly effective, reducing the tumor growth rate and reducing the overall size of the tumor. Thus, the antibodies of the present invention are effective therapeutic agents for the treatment of cancer. Mice inoculated with tumor cells that have a moderate or poor response to anti-PD-1 therapy are also administered pembrolizumab alone or in combination with h5E5, and a synergistic reduction in tumor size is observed.

[0210] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. An anti-leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4 or ILT3) antibody or its antigen-binding fragment, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), CDR-H1 is sequence number 1 (GYSFX １ Includes the amino acid sequence described in GF, where X １ is S or T, CDR-H2 is sequence number 2 (FPSX ２ Includes the amino acid sequence described in GE), where X ２ is S or N, CDR-H3 is sequence number 3 (QAFYYFDX) ３ ) includes the amino acid sequence described, where X ３ is S or Y, CDR-L1 contains the amino acid sequence described in SEQ ID NO: 4 (KSSQSLLSSSNQKNYLA), CDR-L2 contains the amino acid sequence described in Sequence ID No. 5 (WASTRES), CDR-L3 contains the amino acid sequence described in Sequence ID No. 6 (QQYYSYPLT), An antibody or its antigen-binding fragment.

2. a. Whether sequence number 1 is sequence number 15 (GYSFTGF), sequence number 2 is sequence number 16 (FPSNGE), and sequence number 3 is sequence number 17 (QAFYYFDY); b. Whether sequence number 1 is sequence number 15 (GYSFTGF), sequence number 2 is sequence number 16 (FPSNGE), and sequence number 3 is sequence number 18 (QAFYYFDS); c. Whether sequence number 1 is sequence number 15 (GYSFTGF), sequence number 2 is sequence number 19 (FPSSGE), and sequence number 3 is sequence number 17 (QAFYYFDY); d. Whether sequence number 1 is sequence number 20 (GYSFSGF), sequence number 2 is sequence number 16 (FPSNGE), and sequence number 3 is sequence number 17 (QAFYYFDY); e. Sequence ID 1 is Sequence ID 20 (GYSFSGF), Sequence ID 2 is Sequence ID 19 (FPSSGE), Sequence ID 3 is Sequence ID 17 (QAFYYFDY); or f. Sequence ID 1 is Sequence ID 20 (GYSFSGF), Sequence ID 2 is Sequence ID 19 (FPSSGE), and Sequence ID 3 is Sequence ID 18 (QAFYYFDS); The antibody or antigen-binding fragment according to claim 1.

3. The antibody or antigen-binding fragment according to claim 2, wherein SEQ ID NO: 1 is SEQ ID NO: 20 (GYSFSGF), SEQ ID NO: 2 is SEQ ID NO: 19 (FPSSGE), and SEQ ID NO: 3 is SEQ ID NO: 17 (QAFYYFDY).

4. a. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 34, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 35; b. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 36, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 37; c. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39; d. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 40, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 41; e. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 42, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 41; f. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 42, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39; g. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 43, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39; h. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 44, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 45; i. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 44, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39; j. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 46, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39; k. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 47, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39; and l. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 48, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39; The antibody or antigen-binding fragment according to claim 1 or 2, comprising at least one of the following.

5. The antibody or antigen-binding fragment according to claim 4, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

39.

6. An anti-leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4 or ILT3) antibody or its antigen-binding fragment, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), CDR-H1 contains the amino acid sequence described in SEQ ID NO: 49 (SYAMS), CDR-H2 contains the amino acid sequence described in SEQ ID NO: 50 (AITFGGGNTYYPDSVKG), CDR-H3 contains the amino acid sequence described in SEQ ID NO: 51 (HGDGNYDFYAMDY), CDR-L1 contains the amino acid sequence described in SEQ ID NO: 52 (KSSQSLLNSGNQKNYLT), CDR-L2 contains the amino acid sequence described in SEQ ID NO: 5 (WASTRES), and CDR-L3 contains the amino acid sequence described in SEQ ID NO: 53 (QNDYSYPLT); CDR-H1 contains the amino acid sequence described in SEQ ID NO: 49 (SYAMS), CDR-H2 contains the amino acid sequence described in SEQ ID NO: 56 (TISSDGGGNTYYTDSVKG), CDR-H3 contains the amino acid sequence described in SEQ ID NO: 57 (HDGRGALDY), CDR-L1 contains the amino acid sequence described in SEQ ID NO: 58 (RASQDISNYLN), CDR-L2 contains the amino acid sequence described in SEQ ID NO: 59 (YTSRLHS), and CDR-L3 contains the amino acid sequence described in SEQ ID NO: 60 (QQGNTLPWT); or CDR-H1 contains the amino acid sequence described in SEQ ID NO: 63 (NSAVH), CDR-H2 contains the amino acid sequence described in SEQ ID NO: 64 (VIWAGGNTNYNSTLMS), CDR-H3 contains the amino acid sequence described in SEQ ID NO: 65 (HETYGDSFDY), CDR-L1 contains the amino acid sequence described in SEQ ID NO: 66 (RSSQSLLLDSDGKTYLN), CDR-L2 contains the amino acid sequence described in SEQ ID NO: 67 (LVSKLDS), and CDR-L3 contains the amino acid sequence described in SEQ ID NO: 68 (WQGTHFPFT); An antibody or its antigen-binding fragment.

7. a. comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 54, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 55; b. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 61, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 62; or c. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 69, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 70; The antibody or antigen-binding fragment according to claim 6.

8. a. The heavy chain includes an IgG4 steady region, and optionally the IgG4 steady region is sequence number 21 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPPCPAPEFEGGGPSVFLFSPKPKDTLMISRTPEPEVTCCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSSTY The sequence may include a sequence having at least 80% sequence identity with RVVSLVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDGSDGSFFLYSRLTTVDKSRWQEGNVFSSCSVMHEALHNHYTQKSLSLSLSLGK), and optionally the sequence number 21 may include the S124P and L131E mutations; and b. The light chain includes a kappa constant region, and optionally, the kappa constant region may include a sequence having at least 80% sequence identity with SEQ ID NO: 22 (RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVVTKSFNRGEEC); An antibody or antigen-binding fragment according to any one of claims 1 to 2 and 6 to 7, which is at least one of the following.

9. a. A heavy chain containing the amino acid sequence of SEQ ID NO: 7 and a light chain containing the amino acid sequence of SEQ ID NO: 8; b. A heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10; c. A heavy chain containing the amino acid sequence of SEQ ID NO: 11 and a light chain containing the amino acid sequence of SEQ ID NO: 12; d. A heavy chain containing the amino acid sequence of SEQ ID NO: 23 and a light chain containing the amino acid sequence of SEQ ID NO: 24; e. A heavy chain containing the amino acid sequence of SEQ ID NO: 25 and a light chain containing the amino acid sequence of SEQ ID NO: 24; f. A heavy chain containing the amino acid sequence of SEQ ID NO: 25 and a light chain containing the amino acid sequence of SEQ ID NO: 12; g. A heavy chain containing the amino acid sequence of SEQ ID NO: 26 and a light chain containing the amino acid sequence of SEQ ID NO: 12; h. A heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 12; i. A heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 28; j. A heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 12; k. A heavy chain containing the amino acid sequence of SEQ ID NO: 30 and a light chain containing the amino acid sequence of SEQ ID NO: 12; and l. A heavy chain containing the amino acid sequence of SEQ ID NO: 31 and a light chain containing the amino acid sequence of SEQ ID NO: 12; The antibody or antigen-binding fragment according to claim 8, comprising at least one of the above.

10. a. The ILT3 comprises the amino acid sequence provided in SEQ ID NO: 71; b. The ILT3 is located on the surface of bone marrow cells, dendritic cells, macrophages, or a combination thereof; c. The ILT3 is located on the surface of a myeloid cell, the myeloid cell is a myeloid-derived suppressor cell (MDSC), the dendritic cell is a tolerogenic dendritic cell, the macrophage is an inhibitory macrophage, and optionally the macrophage is a tumor-associated macrophage (TAM), or a combination of tumor-associated macrophages (TAM); d. The binding of the antibody or its antigen-binding fragment to ILT3 inhibits the binding of ILT3 to its ligand, which may optionally be apolipoprotein E (APOE), fibronectin (FN1), or both; e. The binding of the antibody or its antigen-binding fragment to ILT3 releases T cells, monocytes, macrophages, or combinations thereof from ILT3-mediated inhibition; f. Binding to ILT3 releases T cells from ILT3-mediated inhibition, and this release of T cells includes increased T cell proliferation, increased secretion of pro-inflammatory cytokines by the T cells, increased secretion of pro-inflammatory chemokines by the T cells, or a combination thereof; g. Binding to ILT3 releases T cells from ILT3-mediated inhibition, and the release of T cells includes increased secretion of pro-inflammatory cytokines by the T cells, wherein the pro-inflammatory cytokines are selected from interferon-gamma (IFNG), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α), and the pro-inflammatory chemokines are selected from C-C motif chemokine ligand 3 (CCL3) and CCL4 or both; h. Binding to ILT3 increases dendritic cell activation; i. Binding to ILT3 increases dendritic cell activation, and this increased dendritic cell activation restores dendritic cell activation that is suppressed by FN1, APOE, or both; j. Binding to ILT3 increases dendritic cell activation, and this increased dendritic cell activation increases the secretion of pro-inflammatory cytokines or chemokines by the dendritic cells; k. Binding to ILT3 increases the activation of monocytes and / or macrophages; and l. Binding to ILT3 increases the activation of monocytes and / or macrophages, and this increased activation of monocytes and / or macrophages increases the secretion of pro-inflammatory cytokines or chemokines by the dendritic cells; An antibody or antigen-binding fragment according to any one of claims 1 to 2 and 6 to 7, which is at least one of the following.

11. a. The antigen-binding fragment is Fv, Fab, F(ab') ２ , scFV, or scFV ２ Selected from a group consisting of fragments; b. The antibody or antigen-binding fragment is humanized; c. The antibody is a monoclonal antibody; and d. The antibody or its antigen-binding fragment does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC); An antibody or antigen-binding fragment according to any one of claims 1 to 2 and 6 to 7, which is at least one of the following.

12. A nucleic acid molecule or a plurality of nucleic acid molecules encoding an antibody or antigen-binding fragment according to any one of claims 1 to 2 and 6 to 7.

13. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 2 and 6 to 7, and a pharmaceutically acceptable carrier, excipient, or adjuvant, which may optionally be formulated for administration to a subject.

14. The pharmaceutical composition according to claim 13, formulated for systemic administration or intratumor administration.

15. The pharmaceutical composition according to claim 13, for use in treating cancer in a subject that requires it.

16. The aforementioned cancer, a. Solid tumors; b. Cancer selected from breast cancer, kidney cancer, head and neck cancer, lung cancer, sarcoma, stomach cancer, colorectal cancer, and ovarian cancer; c. Cancer characterized by the presence of ILT3-expressing tumor-infiltrating immune cells; and d. Cancers containing a tumor microenvironment (TME) characterized by the expression of APOE, FN1, or both; The pharmaceutical composition according to claim 15, wherein at least one of the above.

17. The pharmaceutical composition according to claim 13, for use in combination with an immune checkpoint inhibitor (ICI) for treating cancer in a subject that requires it.

18. The cancer is a. Solid tumors; b. Cancer selected from breast cancer, kidney cancer, head and neck cancer, lung cancer, sarcoma, stomach cancer, colorectal cancer, and ovarian cancer; c. Cancer characterized by the presence of ILT3-expressing tumor-infiltrating immune cells; and d. Cancers containing a tumor microenvironment (TME) characterized by the expression of APOE, FN1, or both; The pharmaceutical composition according to claim 17, wherein at least one of the following is the pharmaceutical composition according to claim 17.

19. A pharmaceutical composition according to claim 17, wherein the ICI inhibits the PD-1 / PD-L1 / L2 checkpoint, wherein the ICI may optionally be selected from pembrolizumab, nivolumab, atezolizumab, semiprimab, dostallimab, durvalumab, and avelumab.

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