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 including the antibodies, and methods of using the antibodies are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 334,365, filed on April 25, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Electronic Sequence Listing Reference

[0004] The contents of the electronic sequence listing (BDB-P-018-PCT SQL.xml; size: 89,038 bytes; and creation date: April 3, 2023) are incorporated herein by reference in its entirety. Technical Field

[0005] The present invention belongs to the field of immune checkpoint inhibition. Background Art

[0006] During cancer development, cancer cells can evade immune surveillance through various mechanisms. Understanding these escape mechanisms is critical to developing new cancer therapies. Cancer immunotherapy aims to eliminate cancer cells by reactivating the patient's immune system and is currently a highly active area of ​​cancer research, with unprecedented clinical results. Despite these advances, there is a constant need for new therapies that will achieve greater improvements in mortality and quality of life for patients, either as a single treatment or in combination with other agents.

[0007] Leukocyte immunoglobulin-like receptor subfamily B member 4 (ILT 3, CD85k, LILRB4) is expressed on various myeloid cells, specifically on myeloid cells with immunosuppressive activity, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and tolerogenic dendritic cells (DCtols). ILT3 contains two extracellular immunoglobulin-like domains and three intracellular tyrosine-based inhibitory motifs (ITIMs). It has been reported that in various malignancies, the expression of ILT3 in myeloid cells in a soluble form is associated with tumor immune escape, metastasis, and poor prognosis. ILT3 signaling on myeloid cells leads to reduced expression of co-stimulatory receptors and differentiation into an immunosuppressive phenotype. In addition to the regulation of myeloid cell maturation and function, ILT3 signaling also induces T cell anergy through unknown ligands and differentiation into T regulatory and T suppressive phenotypes.

[0008] For many years, ILT3 ligands remained undefined, but recently there has been increasing evidence that ILT3 binds proteins in the tumor microenvironment (TME) and tumor extracellular matrix (ECM). Proteins such as apolipoprotein E (APOE) and fibronectin (FN1) are present in the TME and are able to bind ILT3. Binding of ILT3 to its ligands induces an immunosuppressive phenotype in myeloid cells, mediates inhibition of T cell activity, and generates an immunosuppressive TME that supports tumor growth and proliferation.

[0009] Consistent with this reported data, inhibition of ILT3 using blocking antibodies is thought to increase the clearance of tumor cells. This may occur due to the inhibition of the immunosuppressive effects of tumor-resident myeloid cells, thereby inducing a proinflammatory phenotype of myeloid cells-which supports the activation of tumor-infiltrating T cells (TILs), thereby reshaping the TME from an immunosuppressive type to a proinflammatory TME. In short, ILT3 blockade increases the effector activity of various immune cells against malignant cells. Although anti-ILT3 antibodies are disclosed in international patent applications WO2006 / 138739, WO2018 / 089300, WO2019 / 099597, and WO2020 / 056077A, there is still an urgent need for new and excellent anti-ILT3 antibodies that target multiple pathways (ILT3 can create an immunosuppressive environment through these pathways) and have strong and lasting effects. Summary of the invention

[0010] The present invention provides antibodies that bind to leukocyte immunoglobulin-like receptor subfamily B member 4 (ILT3). Also provided are nucleic acid molecules encoding the antibodies, compositions comprising the antibodies, and methods of using the antibodies.

[0011] According to a first aspect, an antibody or an antigen-binding fragment thereof is provided, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0012] CDR-H1 includes the amino acid sequence set forth in SEQ ID NO:1 (GYSFX1GF), wherein X1 is S or T, CDR-H2 includes the amino acid sequence set forth in SEQ ID NO:2 (FPSX2GE), wherein X2 is S or N, CDR-H3 includes the amino acid sequence set forth in SEQ ID NO:3 (QAFYYFDX3), wherein X3 is S or Y, CDR-L1 includes the amino acid sequence set forth in SEQ ID NO:4 (KSSQSLLSSSNQKNYLA), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO:5 (WASTRES), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO:6 (QQYYSYPLT).

[0013] According to another aspect, an antibody or antigen-binding fragment thereof is provided, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0014] CDR-H1 includes the amino acid sequence set forth in SEQ ID NO:49 (SYAMS), CDR-H2 includes the amino acid sequence set forth in SEQ ID NO:50 (AITFGGGNTYYPDSVKG), CDR-H3 includes the amino acid sequence set forth in SEQ ID NO:51 (HGDGNYDFYAMDY), CDR-L1 includes the amino acid sequence set forth in SEQ ID NO:52 (KSSQSLLNSGNQKNYLT), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO:5 (WASTRES), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO:53 (QNDYSYPLT).

[0015] According to another aspect, an antibody or antigen-binding fragment thereof is provided, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0016] CDR-H1 includes the amino acid sequence set forth in SEQ ID NO:49 (SYAMS), CDR-H2 includes the amino acid sequence set forth in SEQ ID NO:56 (TISSDGGNTYYTDSVKG), CDR-H3 includes the amino acid sequence set forth in SEQ ID NO:57 (HDGRGALDY), CDR-L1 includes the amino acid sequence set forth in SEQ ID NO:58 (RASQDISNYLN), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO:59 (YTSRLHS), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO:60 (QQGNTLPWT).

[0017] According to another aspect, an antibody or antigen-binding fragment thereof is provided, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:

[0018] CDR-H1 includes the amino acid sequence set forth in SEQ ID NO:63 (NSAVH), CDR-H2 includes the amino acid sequence set forth in SEQ ID NO:64 (VIWAGGNTNYNSTLMS), CDR-H3 includes the amino acid sequence set forth in SEQ ID NO:65 (HETYGDSFDY), CDR-L1 includes the amino acid sequence set forth in SEQ ID NO:66 (RSSQSLLDSDGKTYLN), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO:67 (LVSKLDS), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO:68 (WQGTHFPFT).

[0019] According to another aspect, an antibody or an antigen-binding fragment thereof is provided, which competes with the antibody or antigen-binding fragment of the present invention for binding to ILT3.

[0020] According to another aspect, a nucleic acid molecule or a plurality of nucleic acid molecules is provided, which encodes the antibody or antigen-binding fragment of the invention.

[0021] According to another aspect, a pharmaceutical composition is provided, comprising the antibody or antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0022] According to another aspect, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject an antibody or antigen-binding fragment thereof of the present invention, thereby treating the cancer.

[0023] According to another aspect, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a pharmaceutical composition of the present invention, thereby treating the cancer.

[0024] According to some embodiments,

[0025] a.SEQ ID NO:1 is SEQ ID NO:15 (GYSFTGF), SEQ ID NO:2 is SEQ ID NO:16

[0026] (FPSNGE), and SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY);

[0027] b. SEQ ID NO: 1 is SEQ ID NO: 15 (GYSFTGF), SEQ ID NO: 2 is SEQ ID NO: 16

[0028] (FPSNGE), and SEQ ID NO:3 is SEQ ID NO:18 (QAFYYFDS);

[0029] c. SEQ ID NO: 1 is SEQ ID NO: 15 (GYSFTGF), SEQ ID NO: 2 is SEQ ID NO: 19

[0030] (FPSSGE), and SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY);

[0031] d. SEQ ID NO: 1 is SEQ ID NO: 20 (GYSFSGF), SEQ ID NO: 2 is SEQ ID NO: 16

[0032] (FPSNGE), and SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY);

[0033] e. SEQ ID NO: 1 is SEQ ID NO: 20 (GYSFSGF), SEQ ID NO: 2 is SEQ ID NO: 19

[0034] (FPSSGE), and SEQ ID NO:3 is SEQ ID NO:17 (QAFYYFDY); or

[0035] f. SEQ ID NO: 1 is SEQ ID NO: 20 (GYSFSGF), SEQ ID NO: 2 is SEQ ID NO: 19

[0036] (FPSSGE), and SEQ ID NO:3 is SEQ ID NO:18 (QAFYYFDS).

[0037] According to some embodiments, the antibody or antigen-binding fragment comprises at least one of the following:

[0038] 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;

[0039] 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;

[0040] 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;

[0041] 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;

[0042] 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;

[0043] 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;

[0044] 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;

[0045] 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;

[0046] 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;

[0047] 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;

[0048] 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

[0049] 1. 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] According to some embodiments, the heavy chain comprises an IgG4 constant region.

[0054] According to some embodiments, the IgG4 constant region comprises a sequence having at least 80% sequence identity to SEQ ID NO:21 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFSPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK).

[0055] 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.

[0056] According to some embodiments, the light chain comprises a kappa constant region, optionally wherein the kappa constant region comprises a sequence having at least 80% sequence identity to SEQ ID NO: 22 (RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0057] According to some embodiments, the antibody or antigen-binding fragment comprises at least one of the following

[0058] 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;

[0059] 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;

[0061] 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;

[0063] 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;

[0065] 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;

[0067] 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;

[0069] 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;

[0071] 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;

[0073] 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;

[0075] 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;

[0077] 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

[0078] Chain; and

[0079] 1. 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.

[0080] 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).

[0081] According to some embodiments, the ILT3 comprises the amino acid sequence provided in SEQ ID NO:71.

[0082] According to some embodiments, the ILT3 is on the surface of a myeloid cell, a dendritic cell, a macrophage, or a combination thereof.

[0083] 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, optionally wherein the macrophages are tumor-associated macrophages (TAMs), or a combination thereof.

[0084] According to some embodiments, said binding to ILT3 inhibits binding of ILT3 to an ILT3 ligand.

[0085] According to some embodiments, the binding inhibits the binding of ILT3 to both apolipoprotein E (APOE) and fibronectin (FN1).

[0086] According to some embodiments, the APOE, FN1, or both are present in a tumor microenvironment (TME).

[0087] According to some embodiments, said binding to ILT3 releases T cells, monocytes, macrophages, or a combination thereof from ILT3-mediated inhibition.

[0088] According to some embodiments, the release of the T cells comprises 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.

[0089] According to some embodiments, said binding to ILT3 increases dendritic cell activation.

[0090] According to some embodiments, increasing dendritic cell activation comprises restoring dendritic cell activation that is suppressed by FN1, APOE, or both.

[0091] According to some embodiments, increasing dendritic cell activation comprises increasing the secretion of pro-inflammatory cytokines or chemokines by said dendritic cells.

[0092] According to some embodiments, said binding to ILT3 increases monocyte and / or macrophage activation.

[0093] According to some embodiments, increasing monocyte and / or macrophage activation comprises increasing the secretion of pro-inflammatory cytokines or chemokines by said dendritic cells.

[0094] 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.

[0095] According to some embodiments, the antigen binding fragment is selected from Fv, Fab, F(ab')2, scFV or scFV2 ​​fragments.

[0096] According to some embodiments, the antibody or antigen-binding fragment is humanized.

[0097] According to some embodiments, the antibody is a monoclonal antibody.

[0098] According to some embodiments, the agent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0099] According to some embodiments, the pharmaceutical composition is formulated for administration to a subject.

[0100] According to some embodiments, the pharmaceutical composition is formulated for systemic administration or intratumoral administration.

[0101] According to some embodiments, the cancer is a solid cancer.

[0102] 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.

[0103] According to some embodiments, the cancer is characterized by the presence of tumor-infiltrating immune cells expressing ILT3.

[0104] According to some embodiments, the cancer TME is characterized by expression of APOE, FN1, or both.

[0105] According to some embodiments, the expression is overexpression compared to non-cancerous tissue of the same tumor type.

[0106] According to some embodiments, the method further comprises administering an immune checkpoint inhibitor (ICI).

[0107] According to some embodiments, the ICI inhibits the PD-1 / PD-L1 / L2 checkpoint.

[0108] According to some embodiments, the ICI is selected from pembrolizumab, nivolumab, atezolizumab, cemiplimab, dostarlimab, durvalumab and avelumab.

[0109] Other embodiments and the entire scope of application of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given by way of illustration only, because through this detailed description, various changes and modifications falling within the spirit and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1A-Figure 1C : (1A) Histogram of ILT3 surface expression in monocytes and MDSCs derived from two healthy donors, (1B) immature dendritic cells (iDC), mature dendritic cells (mDC) and tolerogenic dendritic cells (DCtol) from two healthy donors, and (1C) monocytes differentiated into M0, M1 and M2 macrophages from two healthy donors. IC-isotype control, Mix-includes anti-ILT3 antibody. The black line shows control cells incubated with isotype control antibodies, while the gray line shows staining of cells with anti-ILT3 PE-conjugated antibodies.

[0111] Figure 2A-2B : (2A) Micrographs of tissue samples stained with ILT3 (red) and CD68 (blue) IHC. Arrows indicate double positive cells. (2B) Bar graph quantifying the percentage of all ILT3 positive cancers sampled for each cancer type.

[0112] Figure 3A-3B : (3A) Dot plot tracking relative MDSC enrichment of TCGA database cancers divided into 3 bins: low, medium, and high ILT3 expression. (3B) Bar plot of M2 fraction enrichment - relative to patients with different ILT3 levels in cancer within the same bin.

[0113] Figure 4A-4B: (4A) Histograms of exogenous hILT3 expression on the surface of HEK cells measured with a commercial antibody (ZM4.1) and an antibody of the invention. Isotype control histograms are shown in black, ILT3 binding is shown in grey. (4B) Histograms of antibody binding to cynomolgus monkey ILT3 (50 μg / ml). Isotype control histograms are shown in grey, ILT3 binding is shown in black.

[0114] Figure 5 : Line graph of ELISA detection of ILT3 in competition with naked ZM4.1 antibody. ZM4.1 competes with antibody AB#2 for binding to ILT3, while 5E5 binds to ILT3 with AB#3 at an epitope that does not cause competition with ZM4.1.

[0115] Figure 6A-6G : (6A) TNFa secretion by PBMC, (6B-6C) IFNg secretion by CD8 T cells in the presence of autologous MDSC, (6D-6F) IFNg secretion by CD4 T cells in the presence of different dendritic cells in the presence or absence of 5E5 antibody, (6G) IFNg secretion by CD4 T cells in the presence or absence of M2 macrophages in the presence or absence of 5E5 antibody, and (6C) bar graphs compared to Merck's m52B8 antibody. (6E-6F) (6E) The combination of 5E5 antibody and anti-PD-1 antibody was measured in the absence and (6F) of anti-CD3 stimulation of T cells. Although a single donor is shown in 6A-6D, 3-4 donors were examined and all showed comparable results. All donors are shown in 6E-6F and their respective increases are shown in the line graph on the right. *P<0.05; **P<0.01; unpaired Student's T test compared to mIgG1.

[0116] Figure 7A-7E : Line graph of blocking of ILT3 binding to (7A, 7C-7D) fibronectin and (7B, 7E) APOE in the presence of 5E5 antibody and (7A-7B, 7D) ZM4.1 antibody or (7C, 7E) m52B8 antibody or (7D) other antibodies of the present invention.

[0117] Figure 8A-8D: Bar graphs of (8A) IL-8 secretion by THP-1 cells, (8B) TNFa secretion by DCs, (8C) IL-8 secretion by DCs, and (8D) TNFa secretion by DCs after activation with Erbitux and culture with fibronectin in the presence or absence of various anti-ILT3 antibodies. h52B8 is a humanized Merck anti-ILT3 antibody, IO202 is a humanized Immune-Onc Therapeutics anti-ILT3 antibody, and h5A7 is a humanized NGM Bio anti-ILT3 antibody.

[0118] Figure 9A-9B : Bar graph of TNFa secretion by (9A) ILT3-expressing DCs and (9B) non-expressing DCs cultured in the presence of fibronectin in the presence and absence of 5E5 antibody. *P<0.05; unpaired Student's T-test compared to IgG.

[0119] Fig.10 : Line graph of various h5E5 antibodies binding to hILT3. EC was determined using GraphPad software. 50 level.

[0120] Figure 11A-11B : Line graph of % blocking of ILT3 binding to FN1 by (11A) various h5E5 antibodies and (11B) direct comparison of c5E5 and h5E5-133. IC was determined using GraphPad software 50 level.

[0121] Figures 12A-12D : Bar graphs of (12A) IFNg secretion by CD8 T cells in the presence of autologous MDSCs, (12B) TNFa secretion by DCs in the presence of fibronectin and rituximab, and (12C-12D) IL-8 secretion by THP-1 cells treated with (12C) c5E5 or h5E5 or (12D) m5E5 or alternative antibodies of the invention in the presence of fibronectin and rituximab. 12A includes an inset of the 11 donor samples tested, showing the levels after administration of isotype control and h5E5 at a concentration of 16 μg / ml.

[0122] Figure 13A-13B : Bar graph of TNFa secretion from monocytes differentiated into M1 activated macrophages (MF) in the presence of (13A) gastric cancer cells or (13B) colon cancer cells in the presence and absence of h5E5 antibody. *P<0.05; unpaired Student's T test.

[0123] Fig.14: Bar graph of TNFa secretion by immature DCs stimulated with IL-10 and LPS to generate DCtol when cultured with or without anti-ILT3 antibody or isotype control. Samples from 2 donors are shown.

[0124] Figure 15A-15B : Bar graphs of (15A) IFNg secretion by CD8 T cells incubated with autologous MDSCs and (15B) IFNg secretion by CD4 T cells co-cultured with the indicated dendritic cells treated with anti-PD1 antibody, h5E5, or a combination of both.

[0125] Figures 16A-16D : (16A-16B) Bar graphs of IFNg production in activated (16A) sarcoma tumoroids and (16B) breast cancer tumoroids in the presence of h5E5 or isotype control. (16C-16D) Bar graphs of CCL3, CCL4 and IL-8 production in activated (16C) breast cancer tumoroids and (16D) CRC tumoroids 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.

[0126] Fig.17 : Line graph of MC38 tumor size over time in syngeneic transgenic hILT3 mice treated with c5E5 or isotype control. DETAILED DESCRIPTION

[0127] The present invention, in some embodiments, provides antibodies that bind to leukocyte immunoglobulin-like receptor subfamily B, member 4 (ILT3). Also provided are nucleic acid molecules encoding the antibodies, compositions comprising the antibodies, and methods of using the antibodies.

[0128] The present invention is based on the generation of several ILT3 blocking antibodies using hybridoma technology. The lead antibody 5E5 exhibits the desired binding profile, selectivity, cross-reactivity, blocking activity and activity in functional assays. This antibody was humanized using germline humanization. Humanized 5E5 is able to block the interaction of ILT3 with various ligands (e.g., APOE and FN1) and inhibit the immunosuppressive effects mediated by ILT3 signaling. By blocking and neutralizing ILT3, 5E5 re-regulates the immunosuppressive TME, inducing a pro-inflammatory phenotype in tumor-resident myeloid cells, thereby enhancing the activation of tumor-infiltrating T cells and tumor growth inhibition.

[0129] In a first aspect, an antibody or an antigen-binding fragment thereof that binds to ILT3 is provided.

[0130] Leukocyte immunoglobulin-like receptor subfamily B member 4 is known as LILRB4, ILT3, and CD85K, among others. The Entrez gene identifier for human ILT3 is 11006 and the mouse identifier is 14727. The protein sequence for human ILT3 can be found under the Uniprot identifier Q8NHJ6 and the mouse protein sequence can be found under the identifier Q61450. The human mRNA sequence can be found in RefSeq sequences NM_001081438, NM_001278426, NM_001278427, NM_001278428, and NM_001278429. The mouse mRNA sequence can be found in 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.

[0131] As used herein, the term "antibody" refers to a polypeptide or polypeptide group comprising at least one binding domain, which is formed by folding a polypeptide chain having a three-dimensional binding space, the internal surface shape and charge distribution of which are complementary to the characteristics of the antigenic determinant of the antigen. Antibodies generally have a tetrameric form, including two pairs of identical polypeptide chains, each pair having a "light" chain and a "heavy" chain. The variable region of each light / heavy chain pair forms an antibody binding site. Antibodies can be oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, camelid antibodies, CDR-grafted antibodies, multispecific antibodies, bispecific antibodies, catalytic antibodies, humanized antibodies, fully human antibodies, anti-idiotypic antibodies, and antibodies and fragments that can be labeled in soluble form or in a combined form, including epitope binding fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies can be from any species. The term antibody also includes binding fragments, including but not limited to Fv, Fab, Fab', F(ab')2 single-chain antibodies (svFC), dimer variable regions (diabodies) and disulfide-linked variable regions (dsFv). Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. Antibody fragments may or may not be fused to another immunoglobulin domain, including but not limited to the Fc region or a fragment thereof. The skilled person will further understand that other fusion products can be generated, including but not limited to scFv-Fc fusions, variable regions (e.g., VL and VH) to Fc fusions and scFv-scFv-Fc fusions.

[0132] The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0133] In some embodiments, the antibody is a murine antibody. In some embodiments, the antibody is 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 lacking an Fc domain.

[0134] In some embodiments, the peptide is selected from antibodies, antigen-binding fragments of antibodies, Fab fragments, single-chain antibodies, single-domain antibodies, nanobodies, VHH antibodies, and antibody mimetics. As used herein, the term "antibody mimetics" refers to organic compounds that can specifically bind to a target antigen. In some embodiments, antibody mimetics are structurally unrelated to antibodies. Examples of antibody mimetics include, but are not limited to, affilins, affimers, affitins, alpha antibodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and nano-CLAMPS. In some embodiments, the antibody mimetics are DARPins. All of these agents are known in the art and are known to block interactions between receptors and their ligands. Small molecules and proteins that can bind to mCD28 can block cleavage sites or can cause access to proteases that hinder or impair them. In some embodiments, the protein is an antibody mimetics. As used herein, the term "DARPin" refers to a designed ankyrin repeat protein. DARPins are genetically engineered antibody mimetic proteins that are generally highly specific for their protein targets. Thus, a DARPin for CD28 may be an example of an agent.

[0135] The basic unit of naturally occurring antibody structure is a heterotetrameric glycoprotein complex of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains, which are linked together by non-covalent association and by 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 independent units, and the differences in chain length and sequence). The class and subclass of an antibody are its isotype.

[0136] The amino terminal regions of the heavy and light chains are more diverse in sequence than the carboxyl terminal regions, and are therefore referred to as variable domains. 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 a single antigen binding site, and therefore, the basic immunoglobulin unit has two antigen binding sites. It is believed that specific amino acid residues form an interface between the light chain 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 824592-4596).

[0137] The carboxyl-terminal portions of the heavy and light chains form the constant domains, CH1, CH2, CH3, CL. Although these domains are much less diverse, they vary from one animal species to another, and furthermore, within the same individual, there are several different antibody isotypes, each with different functions.

[0138] The term "framework region" or "FR" refers to the amino acid residues in the variable domain of an antibody that are different from the hypervariable region amino acid residues defined herein. As used herein, the term "hypervariable region" refers to the amino acid residues in the variable domain of an antibody that are responsible for antigen binding. The hypervariable region includes amino acid residues from the "complementarity determining region" or "CDR". The CDR is primarily responsible for binding to the epitope of the antigen. The range of FR and CDR has been precisely defined (see Kabat et al.).

[0139] Immunoglobulin variable domains can also be obtained using the IMGT information system (www: / / imgt.cines.fr / ) ( / V-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. J6: W503-508 (2008).

[0140] Chothia et al. also defined a numbering system applicable to the variable domain sequence of any antibody. Those skilled in the art can clearly 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 proposed 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).

[0141] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase similarity to a human antibody. Humanized antibodies can be produced by recombinant DNA that produces CDRs of non-human antibodies that are surrounded by sequences similar to human antibodies. In some embodiments, humanized antibodies are chimeric antibodies. In some embodiments, humanization includes inserting CDRs of the present invention into a human antibody support or skeleton. Humanized antibodies are well known in the art, and any method for producing humanized antibodies that retain CDRs of the present invention can be used.

[0142] In some embodiments, antibody is a monoclonal antibody. As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homotypic antibody population, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for variants that may occur during the production of the monoclonal antibody, which are usually present in small amounts. Contrary to the polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" represents the characteristics of the antibody obtained from a substantially homotypic antibody population, and should not be construed as produced by any specific preparation method. The monoclonal antibody used according to the method provided herein can be prepared by the hybridoma method first described by Kohler et al, Nature 256:495 (1975), or can be prepared by a recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described by Clackson et al, Nature 352:624-628 (1991) and Marks et al, J. Mol. Biol. 222:581-597 (1991).

[0143] The mAb of the present invention can be any immunoglobulin class, including IgG, IgM, IgD, IgE or IgA. The hybridoma producing mAb can be cultured in vitro or in vivo. High titer mAb can be obtained when produced in vivo, wherein cells from a single hybridoma are injected intraperitoneally into pristine-primed Balb / c mice to produce ascites containing high concentrations of the desired mAb. mAbs of isotype IgM or IgG can be purified from these ascites fluids or culture supernatants using column chromatography well known to those skilled in the art.

[0144] "Antibody fragments" include a portion of a complete antibody, preferably including its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al, Protein Eng. 8(10): 1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, mini antibodies, single-chain antibody molecules; multispecific antibodies formed by antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, double scFv, double scFv or tandem (double, three)-scFv); and bispecific T cell engagers (BiTE).

[0145] 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 its ability to crystallize readily. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0146] "Fv" is the smallest antibody fragment containing a complete antigen recognition and antigen binding site. This region consists of a dimer of a heavy chain and a light chain variable domain in tight, non-covalent association. The three surfaces of the VH-VL dimer are in this configuration. In general, these six hypervariable regions give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv that includes only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0147] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' herein, in which the cysteine ​​residue(s) of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0148] The "light chains" of antibodies (immunoglobulins) from 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.

[0149] According to the amino acid sequence of the constant domain of its heavy chain, antibodies can be assigned to different classes. There are five major classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are respectively referred to as α, δ, ε, γ and μ (micro). The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

[0150] "Single-chain Fv" or "scFv" antibody fragments include 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 includes a polypeptide connector between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun's The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0151] The term "diabody" refers to a small antibody fragment with two antigen binding sites, which includes 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, these domains are forced to pair with the complementary domains of another chain and generate 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).

[0152] The term "multispecific antibody" is used in the broadest sense and specifically encompasses 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) wherein the VHVL unit has polyepitopic specificity, antibodies having two or more VL and VH domains wherein each VHVL unit binds to a different epitope, antibodies having two or more single variable domains wherein each single variable domain binds to a different epitope, full length antibodies, antibody fragments (e.g., Fab, Fv, dsFv, scFv), diabodies, bispecific diabodies, triabodies, trifunctional antibodies, covalently or non-covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0153] The monoclonal antibodies of the present invention can be prepared using methods known in the art. Examples include various techniques such as those described in Kohler, G. and Milstein, C, Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4:72 (1983); Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985) (pp. 77-96).

[0154] In addition to the conventional methods of producing antibodies in vivo, phage display technology can also be used to produce antibodies in vitro. Compared with conventional antibody production, the production of this recombinant antibody is much faster, and recombinant antibodies can be produced for a large number of antigens. In addition, when using conventional methods, many antigens are shown to be non-immunogenic or highly toxic, and therefore cannot be used to produce antibodies in animals. In addition, the affinity maturation (i.e., increasing affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, a large number of different antibodies for specific antigens can be produced in a selection program. In order to produce recombinant monoclonal antibodies, people can use various methods based on display libraries to produce a large number of antibody libraries with different antigen recognition sites. This library can be constructed in several ways: a synthetic repertoire can be generated by cloning a synthetic CDR3 region in the heavy chain germline gene library, thereby generating a large antibody repertoire, from which recombinant antibody fragments with various specificities can be selected. People can use human lymphocyte libraries as the starting material for constructing antibody libraries. The original repertoire of human IgM antibodies can be constructed, thereby creating a human library with a wide variety of diversity. This method has been widely and successfully used to select large numbers of antibodies against different antigens. Protocols for phage library construction and recombinant antibody selection are provided in the well-known reference text Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

[0155] Non-human antibodies can be humanized by any method known in the art. In one method, non-human complementary determining regions (CDRs) are inserted into human antibodies or consensus antibody framework sequences. Further changes can then be introduced into the antibody framework to adjust affinity or immunogenicity.

[0156] In some embodiments, antibodies and parts 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 Fv, Fab, F(ab')2, scFV or scFV2 ​​fragments.

[0157] In some embodiments, the invention provides nucleic acid sequences encoding an antibody or antigen-binding portion of the invention.

[0158] For example, a polynucleotide can encode a complete immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (VH), but also a heavy chain constant region (CH), which generally includes three constant domains: CH1, CH2, and CH3; and a "hinge" region. In some cases, the presence of a constant region is desirable.

[0159] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments such as single domain antibodies ("dAb"), Fv, scFv, Fab' and CHI, and CK or CL domains have been removed. Since mini antibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical / diagnostic use, but they should retain higher binding affinity than monovalent antibody fragments (such as dAb) for bivalent use. Therefore, unless the context otherwise provides, the term "antibody" as used herein includes not only whole antibody molecules, but also antigen-binding antibody fragments of the types discussed above. Each framework region present in the encoded polypeptide may include at least one amino acid substitution relative to the corresponding human receptor framework. Therefore, for example, the framework region may include a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions relative to the receptor framework region. In view of the characteristics of the individual amino acids constituting the disclosed protein product, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions, ie, "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.

[0160] Suitably, the polynucleotides described herein may be isolated and / or purified.In some embodiments, the polynucleotide is an isolated polynucleotide.

[0161] As used herein, the term "non-naturally occurring" matter, composition, entity and / or any combination of matter, composition or entity, or any grammatical variation thereof, is a conditional term that expressly excludes but only those forms of matter, composition, entity and / or combination of matter, composition or entity that are known to one of ordinary skill in the art as "naturally occurring" or that may at any time be determined or interpreted by a judge or administrative or judicial body to be "naturally occurring".

[0162] 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), wherein X1 is S or T, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2 (FPSX2GE), wherein X2 is S or N, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3 (QAFYYFDX3), wherein 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 by the Chothia numbering system and the CDRs comprise SEQ ID NOs: 1-6.

[0163] In some embodiments, the antibody or antigen-binding portion thereof includes three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes the amino acid sequence set forth in SEQ ID NO: 13 (GFYID), CDR-H2 includes the amino acid sequence set forth in SEQ ID NO: 14 (YIFPSX2GETSYNQKFKG), wherein X2 is S or N, CDR-H3 includes the amino acid sequence set forth in SEQ ID NO: 3 (QAFYYFDX33), wherein X3 is S or Y, CDR-L1 includes the amino acid sequence set forth in SEQ ID NO: 4 (KSSQSLLSSSNQKNYLA), CDR-L2 includes the amino acid sequence set forth in SEQ ID NO: 5 (WASTRES), and CDR-L3 includes the amino acid sequence set forth in SEQ ID NO: 6 (QQYYSYPLT). In some examples, the CDRs are numbered by the Kabat numbering system and the CDRs include SEQ ID NOs: 13-14 and 3-6. Those skilled in the art will appreciate that the Chothia and Kabat systems are interchangeable and that CDRs from either system can be converted into CDRs from the other system.

[0164] 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).

[0165] 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. It is understood that an antibody or antigen binding domain comprising SEQ ID NO:16 according to the Chothia system will comprise SEQ ID NO:32 according to the Kabat system. Similarly, an antibody or antigen binding domain comprising SEQ ID NO:19 according to the Chothia system will comprise SEQ ID NO:33 according to the Kabat system.

[0166] 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 comprises ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFSPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:21) has at least 70%, 75%, 80%, 85%, 90%, 95%, 93%, 95%, 97%, 99% or 100% identity. Each possibility represents a separate embodiment of the present invention. In some embodiments, the IgG4 constant region includes a sequence with at least 80% identity to SEQ ID NO:21. In some embodiments, the IgG4 constant region includes a sequence with 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 includes a mutation of serine 124 to proline (S124P). In some embodiments, the numbering is about SEQ ID NO:21. In some embodiments, S124P is S241P, and the numbering is about the complete heavy chain. In some embodiments, the IgG4 constant region includes a mutation of leucine 131 to glutamic acid (L131E). In some embodiments, the numbering is about SEQ ID NO:21. In some embodiments, L131E is L248E and the numbering is with respect to the entire heavy chain. It should be understood that the numbering provided for these two mutations may be slightly shifted depending on the deletion or insertion in the entire heavy chain.

[0167] 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 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22). Each possibility represents a separate embodiment of the present 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.

[0168] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIDWVKQSPGKSLEWIGYIFPSNGETSY NQKFKGKATLTVDKSSSTVNMQLNSLTSEDSAVYYCARQAFYYFDYWGQGTTLTVSS (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 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLSSSNQKNYLAWYQQKPGQSPKLLIYWAST RESGVPDRFAGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 35). In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 34; and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment is a mouse antibody. In some embodiments, the antibody or antigen-binding fragment is 5E5.

[0169] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIDWVKQSPGKSLEWIGYIFPSNGETSY NQKFKGKATLTVDKSSSTVNMQLNSLTSEDSAVYYCARQAFYYFDYWGQGTTLTVSS (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 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLSSSNQKNYLAWYQQKPGQSPKLLIYWAST RESGVPDRFAGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 37). In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 36; and a light chain comprising a variable region comprising or consisting 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.

[0170] 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 DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQAPRLLIYWASTR ESGVPDRFAGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIK (SEQ ID NO: 39). In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 38; 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. In some embodiments, the antibody or antigen-binding fragment is 5E5.

[0171] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising or consisting of the amino acid sequence of evql 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 DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQPPKLLIYWASTR ESGVPDRFAGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIK (SEQ ID NO: 41). In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 40; 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.

[0172] 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.

[0173] 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: 39. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0174] 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.

[0175] 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 DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIK (SEQ ID NO: 45). In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 44; 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 comprises: a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 44; and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 45. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0176] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSSGETS YNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSS (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.

[0177] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising or consisting of the amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETS YNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSS (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.

[0178] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising or consisting of the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETS YNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDSWGQGTTVTVSS (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.

[0179] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain constant region comprising EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIDWVKQSPGKSLEWIGYIFPSNGETSYNQKFKGKATLTVDKSSSTVNMQLNSLTSEDSAVYYCARQAFYYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASS The amino acid sequence of or consisting of TKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFIYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 7). In some embodiments, the antibody or antigen-binding fragment comprises: a light chain comprising or consisting of the amino acid sequence of DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLSSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 8). In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising or consisting of SEQ ID NO: 7; and a light chain comprising or consisting of SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment is a mouse antibody.

[0180] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIDWVKQSPGKSLEWIGYIFPSNGETSYNQKFKGKATLTVDKSSSTVNMQLNSLTSEDSAVYYCARQAFYYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:9) or consisting of the amino acid sequence. In some embodiments, the antibody or antigen-binding fragment comprises: a light chain comprising or consisting of the amino acid sequence of DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLSSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (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.

[0181] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 11) or consisting of the amino acid sequence thereof. 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 the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0182] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising EVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:23) or consisting of the amino acid sequence thereof. In some embodiments, the antibody or antigen-binding fragment comprises: a light chain comprising or consisting of the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLSSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFAGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24). In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising or consisting of SEQ ID NO: 23; 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.

[0183] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 25) or consisting thereof. 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.

[0184] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising a variable region comprising or consisting of SEQ ID NO: 25; 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.

[0185] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising QVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDSWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 26) or consisting thereof. 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.

[0186] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising EVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSNGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:27) or consisting of the amino acid sequence thereof. 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 comprising or consisting of SEQ ID NO: 27; and a light chain comprising a variable region comprising or consisting of SEQ ID NO: 28. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody.

[0187] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising EVQLVQSGAEVKKPGASVKVSCKASGYSFTGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 29) or consisting thereof. 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.

[0188] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising EVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 30) or consisting thereof. 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.

[0189] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising QVQLVQSGAEVKKPGASVKVSCKASGYSFSGFYIDWVKQPPGKGLEWIGYIFPSSGETSYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARQAFYYFDSWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 31) or consisting thereof. 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.

[0190] In some embodiments, the antibody or antigen-binding fragment 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: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 the CDRs of SEQ ID NOs:49-53 and 5 is 3F6.

[0191] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPAKRLEWVAAITFGGGNTY YPDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCARHGDGNYDFYAMDYWGQGTSVTVSS (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 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWAST RESGVPDRFTGSGSRTDFTLTISRVQAEDLAVYYCQNDYSYPLTFGAGTKLELK (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 thereof comprises: a heavy chain variable region comprising or consisting of SEQ ID NO: 54; and a light chain variable region comprising or consisting of SEQ ID NO: 55. In some embodiments, antibody 3F6 comprises: a heavy chain variable region comprising or consisting of SEQ ID NO: 54; and a light chain variable region comprising or consisting of SEQ ID NO: 55.

[0192] In some embodiments, the antibody or antigen-binding fragment 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: 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.

[0193] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPAKRLEWVATISSDGGNTY YTDSVKGRFTISRDNARNTLDLQMSSLRSEDTAMYYCARHDGRGALDYWGQGTSVTV SS (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 thereof comprises: a heavy chain variable region comprising or consisting of SEQ ID NO: 61; and a light chain variable region comprising or consisting of SEQ ID NO: 62. In some embodiments, antibody 7A5 comprises: a heavy chain variable region comprising or consisting of SEQ ID NO: 61; and a light chain variable region comprising or consisting of SEQ ID NO: 62.

[0194] In some embodiments, the antibody or antigen-binding fragment 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: 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.

[0195] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence QVQLKESGPVLVAPSQSLSITCTVSGFSLTNSAVHWVRQPPGKGLEWLGVIWAGGNTNY NSTLMSRLTINKDNSKSQVFLRMNSLQTDDTAIYYCAKHETYGDSFDYWGQGTTLTVSS (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 thereof comprises: a heavy chain variable region comprising or consisting of SEQ ID NO: 69; and a light chain variable region comprising or consisting of SEQ ID NO: 70. In some embodiments, antibody 10G12 comprises: a heavy chain variable region comprising or consisting of SEQ ID NO: 69; and a light chain variable region comprising or consisting of SEQ ID NO: 70.

[0196] 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, ILT3 is membrane ILT3 (mILT3). In some embodiments, ILT3 is soluble ILT3 (sILT3). "Anti-ILT3 antibodies," "antibodies that recognize ILT3," or "antibodies against ILT3" are antibodies that bind to ILT3 with sufficient affinity and specificity. In some embodiments, the binding of the antibody to ILT3 is increased. In some embodiments, the binding of the antibody to ILT3 is increased compared to commercially available ILT3 antibodies. In some embodiments, commercially available ILT3 antibodies are 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, ILT3 is mammalian ILT3. In some embodiments, ILT3 is human ILT3. In some embodiments, the mammal is a monkey. In some embodiments, the monkey is selected from rhesus monkeys and cynomolgus monkeys. 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.

[0197] 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 the membrane of an immune cell. In some embodiments, the immune cell is selected from myeloid cells, dendritic cells and macrophages. 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 an inhibitory macrophage. In some embodiments, the macrophage is an M2 macrophage. In some embodiments, the M2 macrophage is an inhibitory 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).

[0198] As used herein, the terms "increased binding affinity" and "greater binding affinity" are interchangeable. In some embodiments, the antibodies or antigen-binding portions thereof of the invention have a greater binding affinity for sCD28 than for mCD28. In one embodiment, the greater affinity as used herein is 10% greater. In one embodiment, the greater affinity as used herein is 30% greater. In one embodiment, the greater affinity as used herein is 50% greater. In one embodiment, the greater affinity as used herein is 75% greater. In one embodiment, the greater affinity as used herein is 100% greater. In one embodiment, the greater affinity as used herein is 150% greater. In one embodiment, the greater affinity as used herein is 250% greater. In one embodiment, the greater affinity as used herein is 500% greater. In one embodiment, the greater affinity as used herein is 1,000% greater. In one embodiment, the greater affinity as used herein is 1.5 times greater. In one embodiment, the greater affinity as used herein is 2 times greater. In one embodiment, the greater affinity as used herein is 5 times greater. In one embodiment, the greater affinity as used herein is 10 times greater. In one embodiment, the greater affinity as used herein is 50 times greater. In one embodiment, the greater affinity as used herein is 100 times greater. In one embodiment, the greater affinity as used herein is 500 times greater. In one embodiment, the greater affinity as used herein is 1,000 times greater.

[0199] 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 its binding antigen.

[0200] "Antigen" is a molecule or part of a molecule that can elicit the formation of antibodies and be bound by antibodies. An antigen may have one or more epitopes. The specific reaction mentioned above is meant to indicate that an antigen will react in a highly selective manner with its corresponding antibody rather than with a large number of other antibodies that may be elicited by other antigens.

[0201] According to the present invention, the term "antigenic determinant" or "epitope" refers to the region of an antigen molecule that reacts specifically with a particular antibody. The peptide sequence derived from the epitope can be used alone or in combination with a carrier moiety to immunize an animal and generate additional polyclonal or monoclonal antibodies using methods known in the art. Immunoglobulin variable domains can also be obtained using the IMGT information system (www: / / imgt.cines.fr / ) ( / V-Quest) analysis to identify variable segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. J6: W503-508 (2008).

[0202] Kabat et al. also defined a variable domain sequence numbering system applicable to any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable domain sequence, independent of any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al, U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0203] 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 lacking an Fc domain. In some embodiments, the agent is a single domain antibody. In some embodiments, the agent is a camelid, a shark, or a nanobody. In some embodiments, the antibody or fragment is fused to another protein or a fragment of a protein. In some embodiments, the second protein or fragment increases half-life, particularly half-life in serum. In some embodiments, the half-life extending protein is human serum albumin. In some embodiments, the agent is modified by producing a modified chemical that increases half-life. In some embodiments, the modification is PEGylation and the chemical is polyethylene glycol. Those skilled in the art will appreciate that any half-life extending protein or chemical agent or modification known in the art may be used.

[0204] In some embodiments, the combination of an antibody or antigen binding domain with a cell does not kill the cell. In some embodiments, the combination of an antibody or antigen binding domain with a cell does not cause 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 includes an IgG2 or IgG4 domain. In some embodiments, the antibody or antigen binding domain includes an IgG2 domain. In some embodiments, the antibody or antigen binding domain includes an IgG4 domain. In some embodiments, the antibody or antigen binding domain includes an IgG1 or IgG3 that is mutated to reduce cell death mediated by antibody binding. In some embodiments, 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 any mutation or amino acid change known to reduce antibody-mediated cell killing may be used.

[0205] In some embodiments, the antibody or antigen binding domain does not include 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 includes an IgG1 or IgG3 comprising a mutation that reduces ADCC, CDC or both induced by antibody binding. In some embodiments, ADCC, CDC or both are reduced to nothing by mutation. ADCC and CDC are well characterized and the antibody sequences that achieve induction of these cytotoxic pathways are known. Mutation, such as non-limiting examples, is known to IgG1 or IgG3 to IgG2 or IgG4. Any such mutation can be used in the backbone of the antibody of the present invention.

[0206] In some embodiments, binding to ILT3 inhibits the binding of ILT3 to a ligand. In some embodiments, binding to ILT3 inhibits the 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 the binding of ILT3 to both APOE and FN1. In some embodiments, APOE is present in a tumor microenvironment (TME). In some embodiments, FN1 is present in the TME. In some embodiments, APOE is present in an extracellular matrix. In some embodiments, FN1 is present in an extracellular matrix. In some embodiments, the extracellular matrix is ​​a tumor extracellular matrix.

[0207] 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, the binding of the antibody or antigen binding fragment produces ILT3 blocking. In some embodiments, the binding to ILT3 releases the 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, the binding of ILT3 releases the T cell from ILT3-mediated inhibition. In some embodiments, the binding of ILT3 releases monocytes from ILT3-mediated inhibition. In some embodiments, the binding of ILT3 releases macrophages from ILT3-mediated inhibition. In some embodiments, the binding of ILT3 releases dendritic cells 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 an inhibitory 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.

[0208] In some embodiments, release is reduction. In some embodiments, reduction is reduction by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. Each possibility represents a separate embodiment of the present invention. In some embodiments, release comprises immune cell activation. In some embodiments, activation is increased activation. In some embodiments, the increase is an increase of at least 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%. 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 includes restoring T cell activation suppressed by MDSC. In some embodiments, increasing T cell activation includes restoring T cell activation suppressed by tolerogenic dendritic cells (DCtol). In some embodiments, the activation is Fc receptor-mediated activation. In some embodiments, increasing DC activation comprises restoring DC activation inhibited by fibronectin or APOE. In some embodiments, increasing monocyte activation comprises restoring monocyte activation inhibited by fibronectin or APOE.

[0209] In some embodiments, release comprises increased proliferation. In some embodiments, activation comprises increased proliferation. In some embodiments, release comprises increased secretion of proinflammatory cytokines. In some embodiments, release comprises increased secretion of proinflammatory chemokines. In some embodiments, activation comprises increased secretion of proinflammatory cytokines. In some embodiments, activation comprises increased secretion of proinflammatory chemokines. In some embodiments, activation comprises increased secretion of proinflammatory chemokines. In some embodiments, the proinflammatory cytokines are selected from TNFa, IFNg and IL-8. In some embodiments, the proinflammatory cytokine is TNFa. 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.

[0210] In another aspect, an antibody or antigen-binding fragment is provided that competes with the antibody or antigen-binding fragment of the present invention for binding to ILT3.

[0211] In some embodiments, competitive binding includes 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 within a ligand binding domain. In some embodiments, the epitope is close enough to the ligand binding domain to occlude, block or change the ligand binding domain of the antibody or antigen binding domain. In some embodiments, the epitope has low homology to the corresponding mouse ILT3 sequence.

[0212] In another aspect, a nucleic acid molecule is provided that encodes an antibody or antigen-binding fragment of the invention.

[0213] In some embodiments, nucleic acid molecules are multiple nucleic acid molecules. In some embodiments, nucleic acid molecules include open reading frames encoding antibodies or Fabs of the present invention. In some embodiments, open reading frames are multiple open reading frames. In some embodiments, nucleic acid molecules encode heavy chains. In some embodiments, nucleic acid molecules encode light chains. In some embodiments, the same nucleic acid molecules encode heavy chains and light chains. In some embodiments, different nucleic acid molecules encode heavy chains and light chains.

[0214] 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 to be expressed in a target cell. In some embodiments, the nucleic acid molecule comprises an open reading frame.

[0215] For example, a polynucleotide can encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (VH), but also a heavy chain constant region (CH), which generally includes three constant regions: CH1, CH2, and CH3; and a "hinge" region. In some cases, it is desirable to have a constant region.

[0216] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments such as single domain antibodies ("dAb"), Fv, scFv, Fab' and CHI, and CK or CL domains have been removed. Since mini antibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical / diagnostic use, but they should retain higher binding affinity than monovalent antibody fragments (such as dAb) for bivalent use. Therefore, unless the context otherwise provides, the term "antibody" as used herein includes not only whole antibody molecules, but also antigen-binding antibody fragments of the types discussed above. Each framework region present in the encoded polypeptide may include at least one amino acid substitution relative to the corresponding human receptor framework. Therefore, for example, the framework region may include a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions relative to the receptor framework region. In view of the characteristics of the individual amino acids constituting the disclosed protein product, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions, ie, "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.

[0217] Suitably, the polynucleotides described herein may be isolated and / or purified.In some embodiments, the polynucleotide is an isolated polynucleotide.

[0218] As used herein, the term "non-naturally occurring" matter, composition, entity and / or any combination of matter, composition or entity, or any grammatical variation thereof, is a conditional term that expressly excludes but only those forms of matter, composition, entity and / or combination of matter, composition or entity that are known to one of ordinary skill in the art as "naturally occurring" or that may at any time be determined or interpreted by a judge or administrative or judicial body to be "naturally occurring".

[0219] The term "nucleic acid" is well known in the art. As used herein, "nucleic acid" will generally refer to a molecule (i.e., a strand) of DNA, RNA, or derivatives or analogs thereof that include a nucleobase. Nucleobases include, for example, naturally occurring purine or pyrimidine bases present in DNA (e.g., adenine "A", guanine "G", thymine "T", or cytosine "C") or RNA (e.g., A, G, uracil "U", or C).

[0220] 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 and other short interfering nucleic acids), snoRNA, snRNA, tRNA, piRNA, tnRNA, small rRNA, hnRNA, lnc nucleic acid, circulating nucleic acid, genomic DNA or RNA fragments, degraded nucleic acid, ribozyme, viral RNA or DNA, infectious source nucleic acid, amplification product, modified nucleic acid, cytoplasmic or organellar nucleic acid and artificial nucleic acid (such as oligonucleotide).

[0221] As used herein, the term "oligonucleotide" refers to a short (eg, no longer than 100 bases), 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 (eg, by ligation).

[0222] As used herein, the term "expression" refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. Thus, the expression of a nucleic acid molecule may refer to the transcription of a nucleic acid fragment (e.g., the transcription of an mRNA or other functional RNA) and / or the translation of an RNA into a precursor or mature protein (polypeptide).

[0223] The expression of genes in cells is well known to those skilled in the art, and its delivery can be carried out by the methods of the present invention or using the compositions 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 adenoviral 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.

[0224] In some embodiments, a gene or open reading frame is operably linked to a promoter or other regulatory element. The term "operably linked" means that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that enables expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell by the methods 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.

[0225] As used herein, the term "promoter" refers to a group of transcriptional control modules that are clustered around the start site of RNA polymerase, RNA polymerase II. A promoter is composed of discrete functional modules, each of which consists of about 7-20 bp of DNA and contains one or more recognition sites for transcriptional activators or repressors.

[0226] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAs and microRNAs.

[0227] 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, which can be purchased from Invitrogen; pCI, which can be purchased from Promega; pMbac, pPbac, pBK-RSV and pBK-CMV, which can be purchased from Strategene; pTRES, which can be purchased from Clontech, and derivatives thereof.

[0228] In some embodiments, the present invention uses expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses. 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 vectors that allow protein expression under the guidance of 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 show effective expression in eukaryotic cells.

[0229] In some embodiments, the recombinant viral vector that provides advantages such as lateral infection and targeting specificity is used for in vivo expression.In one embodiment, lateral infection is intrinsic in the life cycle of for example retrovirus, and is the process that single infected cell produces many progeny virions, and this progeny virion sprouts and infects adjacent cells.In one embodiment, the result is that large tracts of land are infected rapidly, and wherein most of them are not infected by original virus particles at first.In one embodiment, produce the viral vector that can not spread horizontally.In one embodiment, if required purpose is only to import specific gene into the targeted cell of local quantity, then this characteristic can be useful.

[0230] In another aspect, a composition is provided comprising an antibody or antigen-binding fragment of the invention.

[0231] 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 preventive composition. In some embodiments, the composition includes a therapeutically effective amount of an antibody or antigen binding domain. In some embodiments, the composition includes a therapeutically acceptable carrier, excipient, or adjuvant.

[0232] The term "therapeutically effective amount" refers to an amount of a drug that is effective to treat a disease or disorder in a mammal. The term "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic or preventive result at the necessary dosage and for the necessary period of time. The exact dosage form and regimen will be determined by the physician based on the patient's condition.

[0233] As used herein, the term "carrier", "excipient" or "adjuvant" refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid, liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffered antibodies or antigen-binding fragments thereof such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol solutions and phosphate buffered saline solutions, and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can be used as carriers herein include sugars, starches, 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 buffered solution, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible materials used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavoring agents, excipients, stabilizers, antioxidants and preservatives may also be present. Any non-toxic, inert and effective carrier can be used to formulate the compositions contemplated herein. In this regard, suitable pharmaceutically acceptable carriers, excipients and diluents are well known to those skilled in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); 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 (which are incorporated herein by reference in their entirety).Examples of pharmaceutically acceptable excipients, carriers, and diluents for use in the present compositions include distilled water, saline, Ringer's solution, glucose solution, Hank's solution, and DMSO. These additional inactive components and effective preparations and administration procedures are well known in the art and are described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th edition, Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st edition, 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, slow-release particles, and other media that increase 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 used with the peptides described herein are formed by standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids typically depends on considerations such as liposome size and stability in blood. For example, Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, summarizes various methods that can be used to prepare liposomes, and also see U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0234] Carriers may comprise in total from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0235] In some embodiments, the composition is used in the methods of the present invention. In some embodiments, the antibody or antigen binding domain is used in the methods of the present invention. In some embodiments, the composition is used to enhance immune function. In some embodiments, the antibody or antigen binding domain is used to enhance immune function. In some embodiments, enhancing immune function is to reduce immunosuppression. In some embodiments, the composition is used to treat cancer. In some embodiments, the antibody or antigen binding domain is used to treat cancer.

[0236] In some embodiments, the composition is formulated for administration to an object. 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 "administering", "administration" and similar terms refer to any method of delivering a composition containing an active agent to an object in a reasonable medical practice in a manner that provides a therapeutic effect. One aspect of the subject matter of the present invention provides for intravenous administration of a therapeutically effective amount of the composition of the subject matter to a patient in need. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular or intraperitoneal.

[0237] The dosage administered 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.

[0238] According to another aspect, a method of increasing the immune function of a subject in need thereof is provided, the method comprising administering to the subject a pharmaceutical composition of the present invention or an antibody or antigen-binding fragment of the present invention, thereby increasing the immune function of the subject.

[0239] In some embodiments, increasing immune function comprises increasing immune surveillance. In some embodiments, increasing immune function comprises increasing immune response. In some embodiments, increasing immune function comprises reducing immunosuppression. In some embodiments, immune function comprises immune-mediated cell killing. In some embodiments, the killing is the killing of diseased cells. In some embodiments, the immune response is directed against diseased cells. In some embodiments, the cell is a target cell.

[0240] In some embodiments, the disease is a disease that can be treated by killing target cells. In some embodiments, the disease is a disease that can be treated by inducing an immune response against target cells. In some embodiments, the target cells are diseased cells. In some embodiments, the disease is characterized by the presence of immune cells expressing ILT3.

[0241] 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 the 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, expression is overexpression. In some embodiments, overexpression is compared to non-disease tissue. In some embodiments, non-disease tissue is 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 as the tumor. In some embodiments, the tissue type is identical and is derived from the same tissue type.

[0242] As used herein, "cancer" or "pre-malignancy" is a disease associated with cell proliferation. Non-limiting types of cancer include carcinomas, sarcomas, lymphomas, leukemias, blastomas, and germ cell tumors.

[0243] In some embodiments, cancer is solid cancer. In some embodiments, cancer is a tumor. In some embodiments, cancer is selected from hepatobiliary cancer, cervical cancer, urogenital cancer (e.g., urothelial carcinoma), testicular cancer, prostate cancer, thyroid cancer, ovarian cancer, nervous system cancer, eye cancer, lung cancer, soft tissue cancer, bone cancer, pancreatic cancer, bladder cancer, skin cancer, intestinal cancer, liver cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, breast cancer (e.g., triple negative breast cancer), renal cancer (renal cancer) (e.g., renal cancer (renal carcinoma)), skin cancer, head and neck cancer, leukemia and lymphoma. In some embodiments, cancer is selected from breast cancer, renal cancer (kidney cancer), head and neck cancer, lung cancer, sarcoma, gastric cancer and ovarian cancer. In some embodiments, cancer is gastric cancer. In some embodiments, cancer is colon cancer.

[0244] In some embodiments, the method further comprises administering an immune checkpoint inhibitor (ICI). In some embodiments, inhibition is blocking. In some embodiments, the method further comprises administering an immunostimulant. 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 immunostimulant is specific for a target selected from CTLA-4, PD-1, PD-LI, PD-L2, CD40, OX40, CD137, GFTR, ILT2, or LAG3, or a ligand 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 agent 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, cemiplizumab, dotalimumab, durvalumab, and avelumab.

[0245] In some embodiments, the antibodies or antigen binding fragments of the present invention are used in combination with an immunostimulant. In some embodiments, the compositions of the present invention are used in combination with an immunostimulant. In some embodiments, the use is a method of the present invention. In some embodiments, the antibodies or antigen binding fragments of the present invention are used to produce a drug for treating a disease. In some embodiments, the compositions of the present invention are used to produce a drug for treating a disease.

[0246] As used herein, the term "about" when combined with a 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.

[0247] Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes a plurality of such polynucleotides, and reference to a "polypeptide" includes one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0248] In those cases where a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended to be in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, having A and B together, having A and C together, having B and C together, and / or having A, B, and C together, etc.). One skilled in the art would also understand that, in practice, any antonymous conjunctions and / or phrases presenting two or more optional terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of these terms, two of these terms, or both of these terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0249] It should be understood that certain features of the invention 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 described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the invention are specifically encompassed by the invention and disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed by the invention and disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0250] After examining the following examples, other objects, advantages and novel features of the present invention will become apparent to those skilled in the art, and these examples are not intended to be limiting. In addition, as described above and as claimed in the following claims, each of the various embodiments and aspects of the present invention is experimentally supported in the following examples.

[0251] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0252] Example

[0253] Generally, 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 fully in the literature.See, e.g., "Molecular Cloning: A laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Vol. to Molecular Cloning", John Wiley&Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Volumes 1-4, ColdSpring Harbor Laboratory Press, New York (1998); 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", Vols. I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology" Vols. 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 document.

[0254] Example 1: Breadth of ILT3 expression

[0255] As a starting point, the breadth of ILT3 expression in various immune cells, cancer-associated immune cells, and cancers was examined. First, PBMCs from two healthy donors were stained using a commercially available anti-ILT3 antibody conjugated to PE (ZM4.1-PE, Biolegend Cat. #333008) and then analyzed by FACS. Monocytes isolated based on CD14 expression were low 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. MDSCs were found to have upregulated ILT3 surface expression compared to monocytes ( Figure 1A ).

[0256] Immature dendritic cells (iDC) were generated from monocytes isolated from two other healthy donors by culturing for 6 days in the presence of GM-CSF (50 ng / ml) and IL-4 (20 ng / ml). Mature dendritic cells (mDC) were generated by further culturing for 48 hours in the presence of LPS (100 ng / ml), and tolerogenic dendritic cells (DCtol) were generated by culturing iDC for 48 hours with IL-10 (100 U / ml) and IFNa-2b (1000 U / ml). For cells from both donors, DCtol showed the highest level of surface ILT3 ( Figure 1B ).

[0257] Monocytes were again isolated from healthy donors and differentiated into various types of macrophages. First, the isolated monocytes were cultured with M-CSF (50ng / ml) for 6 days to produce M0 macrophages. M0 macrophages were then cultured with IFNg (20ng / ml) and LPS (100ng / ml) for 48 hours to produce M1 macrophages. M2 macrophages were produced by culturing M0 macrophages with M-CSF (10ng / ml) and IL4 (20ng / ml) for 48 hours. After differentiation, the obtained population was verified by the expression of CD80, CD206, CD14, and HLA-DR. The levels of ILT3 in these cells were evaluated by FACS. Compared with control monocytes, all macrophages (M0, M1 and M2) showed increased ILT3 expression, of which M1 macrophages showed an increase of more than 10 times ( Figure 1C ).

[0258] Next, sections of various tumors were subjected to immunohistochemistry to examine ILT3 expression. Sections were double stained for CD68 (a marker of tumor-associated macrophages) and ILT3 ( Figure 2A If more than 200 ILT3-positive cells were identified, the sample was considered ILT3-positive. Among the breast cancer, kidney cancer, head and neck cancer, lung cancer, sarcoma, gastric cancer, and ovarian cancer samples, 30% to 90% were ILT3-positive, i.e., contained ILT3-positive infiltrating immune cells ( Figure 2B ).

[0259] When the enrichment of MDSCs and M2 (suppressive) macrophages in cancers was compared with the average ILT3 expression in these cancers, it was found that patients with high ILT3 levels were also enriched for MDSCs ( Figure 3A ) and M2 macrophages ( Figure 3B All these results highlight the need for an antibody that can effectively block ILT3 on tumor-associated immune cells and transform the immunosuppressive TME into a TME with active immune effector functions.

[0260] Example 2: Relief of immune cell suppression

[0261] Anti-ILT3 antibodies were generated by hybridoma technology. These antibodies were screened for human ILT3 specificity and non-human primate (NHP) ILT3 cross-reactivity. The screening revealed functional activity of antibodies specific for ILT3.

[0262] HEK cells exogenously overexpressing recombinant human ILT3 were generated and then used to evaluate antibody binding to membrane ILT3. Hybridoma culture medium was used as a negative control, and a commercial anti-ILT3 antibody (ZM4.1) was used as a positive control. The lead antibody, designated 5E5, was found to produce an ILT3 expression signal as good as (if not better than) the commercial antibody (mean fluorescence 125 vs. commercial antibody 118) ( Figure 4A ). It was found that the other three antibodies (10G12, 3F6 and 7A5) also produced positive signals and their levels were comparable to the control antibody (10G12 showed a slightly reduced signal; 79 vs. 118). Since these antibodies bind to recombinant ILT3, they may also bind to soluble ILT3 (sILT3). Interestingly, although 5E5 strongly binds to cynomolgus monkey ILT3 ( Figure 4B), but it did not bind to mouse recombinant ILT3 at all (data not shown). Three other antibodies comparable to ZM4.1 also showed some binding to cynomolgus monkey ILT3, but the level of binding was significantly lower than 5E5. Several other antibodies generated did not bind to cynomolgus monkeys, and this was also true for the commercial antibody tested (ZM4.1). 5E5 was also evaluated for binding to other ILT proteins by ELISA, and was not found to bind to ILT5, ILT11, or ILT8, but several of the other antibodies generated from hybridomas did show cross-reactivity.

[0263] A competition assay was performed to examine whether the 5E5 antibody binds to the same epitope as the commercial antibody ZM4.1. The ILT3 antibody was biotinylated and used at a constant concentration in the ILT3 binding ELISA (1 μg / mL). The competing antibody was added at different dilutions and the OD was measured. As a positive control, the same "naked" antibody as 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 of these hybridoma antibodies (AB#2) did compete with ZM4.1.

[0264] Next, the ability of the 5E5 antibody to enhance PBMC activation was evaluated. PBMCs were isolated from healthy donors and activated with LPS at 37°C for 6 hours in the presence or absence of the 5E5 antibody. TNF-α secretion levels were quantified by ELISA as a measure of immune cell activation. Culture medium alone as well as naive PBMCs were used as negative controls, as was mouse IgG1. 5E5 was able to more than double TNF-α secretion levels compared to LPS stimulation without antibody ( Fig. 6A ), indicating that this antibody is indeed able to enhance immune cell activation. It is worth noting that several other antibodies generated, although specific for ILT3, did not enhance activation (data not shown).

[0265] T cell activation was next examined. As described above, MDSCs were generated from PBMCs of healthy donors. 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. Figure 6B As shown, activated T cells alone secrete very high levels of IFNg, which is essentially completely abolished by co-culture with MDSC (media or mIgG1 control). However, the 5E5 antibody almost completely abolished the suppressive effect of MDSC, maintaining IFNg levels at more than 70% of the levels observed from activated T cells alone.

[0266] The 5E5 antibody was now compared to the m52B8 antibody (Merck). While 52B8 did restore some IFNg secretion, indicating blockade of MDSC effects, the 5E5 antibody was found to be superior ( Figure 6C The level of IFNg increase when using the 5E5 antibody compared to the negative control (medium / mIgG1) was almost twice that of the m52B8 antibody.

[0267] iDCs were generated from healthy donor monocytes and converted to DCtol as described above. Allogeneic CD4 T cells were activated with soluble anti-CD3 antibody and then incubated with DCtol in the presence of 5E5 or mIgG1 control. CD4 T cells were also incubated with iDC positive control. Fig.6D As can be seen in the results, DCtol reduced IFNg secretion by T cells, but when the 5E5 antibody was added, its level returned to the control (iDC) level. Therefore, the antibody was able to completely eliminate the immunosuppressive effect of DCtol on CD4 T cells. This experiment was repeated with iDCs and their allogeneic CD4 T cells generated from 5 different healthy donors. The increase in IFNg secretion produced by the 5E5 antibody was again observed and found to be statistically significant ( Fig. 6E ). This experiment was also performed in combination with anti-PD-1 checkpoint blockade. As expected, anti-PD-1 antibodies administered alone (along with isotype control antibodies) produced robust IFNg secretion, and interestingly, the h5E5 antibody produced an additive effect on top of the PD-1 effect, suggesting 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 antibodies, but overall IFNg secretion was much lower ( Fig. 6F ). Unexpectedly, in the absence of anti-CD3 activation, the combination of anti-PD-1 and 5E5 no longer produced only an additive increase, but rather a synergistic increase, with IFNg secretion levels increased by much more than expected based on the effects of the 5E5 antibody itself.

[0268] In parallel experiments, M2c macrophages were generated by monocytes isolated from healthy donors. In brief, monocytes were cultured in complete RPMI medium supplemented with M-CSF (50ng / ml) for 5-6 days to generate M-0 macrophages. M-0 macrophages were stimulated with IL10 (20ng / ml) for 48 hours to generate M2c macrophages. Then M2c macrophages were incubated with allogeneic CD4T cells for 120 hours and IFNg secretion levels were measured by ELISA. As expected, M2c macrophages greatly inhibited IFNg secretion of T cells. However, when the M2c generation protocol was performed in the presence of 5E5, the inhibitory nature of M2c cells was lower, with T cells producing higher levels of IFNg ( Figure 6G ). Isotype controls produced similar results when no antibody was added and M1 macrophages were used as a positive control.

[0269] Example 3: ILT3 ligand blocking

[0270] 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. The binding of recombinant ILT3-His to fibronectin or APOE was determined by OD analysis using anti-His conjugated to HRP by ELISA. The blocking percentage was determined by normalization 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 significantly higher in FN1 ( Fig. 7A ) and APOE( Figure 7B ) were comparable. 5E5 produced a higher level of blockade at the same concentration. m52B8 was found to be a very poor blocker of the ILT3-FN1 interaction, producing no blockade except at the highest concentration tested, while 5E5 was clearly superior ( Figure 7C Several of the other antibodies generated also blocked FN1 binding, but 10G12 again showed slightly inferior function ( Fig.7D ). Both antibodies are almost identical in blocking APOE, but the m52B8 antibody may be slightly better ( Fig. 7E ).

[0271] To functionally test the blockade of ligand binding to ILT3, THP-1 monocytes were activated via the Fc receptor signaling pathway using immobilized rituximab. Activation was monitored by measuring the secretion of the proinflammatory cytokine IL-8. When fibronectin was added to the culture, it inhibited IL-8 secretion by approximately 67% ( Fig. 8A ). Various anti-ILT3 antibodies were then tested for their ability to restore IL-8 secretion. As expected, m52B8 had no effect on IL-8 secretion because it does not block the binding of fibronectin to ILT3. Antibody IO202 (Immune-Onc Therapeutics) had a modest effect, increasing IL-8 secretion but not to the levels observed without fibronectin. In contrast, 5E5 and ZM4.1 not only restored IL-8 levels, but actually increased them to levels not observed when activated with rituximab alone. Both antibodies increased IL-8 secretion by about one-fold. Similar results were observed with APOE.

[0272] Similar experiments were performed on DCs. DCs were activated via Fc receptor signaling using immobilized rituximab. The addition of fibronectin modestly reduced TNFa ( Figure 8B ) and IL-8( Figure 8C ) secretion, which was restored by the 5E5 antibody as well as m52B8 and IO202. ILT3 blockade increased the level of proinflammatory cytokine secretion to levels far above those produced by rituximab alone. Again, the increase was more than doubled, and 5E5 was slightly superior to both antibodies. To induce Fc receptor signaling-mediated activation, the same experiment was also performed using immobilized Erbitux. Both c5E5 and h5E5 antibodies (see Example 4 below) restored TNFa secretion that was inhibited by fibronectin ( Fig.8D ). In fact, the antibodies of the present invention are slightly better than the known antibody ZM4.1, and are much better than the humanized form of the known m52B8 antibody (h52B8) and the known humanized h5A7 antibody (NGM Biopharmaceuticals Inc., from U.S. patent application US2021 / 0221887). Similar results were observed with APOE instead of fibronectin.

[0273] To demonstrate that the enhanced activation was ILT3-dependent, DCs expressing and not expressing ILT3 were generated. DCs were incubated with fibronectin in the presence of 5E5 antibody or IgG control. Fibronectin only reduced the activation of DCs expressing ILT3 ( Fig. 9A ) but not TNFa secretion by DCs that do not express ( Fig. 9B ). Similar results were observed with APOE. The 5E5 antibody had no effect on non-expressing DCs and partially restored TNFa secretion by ILT3-positive DCs. Therefore, the inhibition and restoration of proinflammatory cytokine secretion are clearly ILT3-dependent.

[0274] Example 4: Generation and validation of humanized 5E5 antibodies

[0275] To generate the humanized 5E5 antibody, the mouse antibody (heavy chain SEQ ID NO: 7, light chain SEQ ID NO: 8) was sequenced and the CDRs were identified (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 generate chimeric antibodies (c5E5, heavy chain SEQ ID NO: 9, light chain SEQ ID NO: 10). In addition, 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 examined for their ability to bind to human ILT3 ( Fig.10). All antibodies were also found to bind to cynomolgus and rhesus monkey ILT3. 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 subsequent experiments. Table 1 summarizes the various human antibodies.

[0276] Table 1: Human antibodies identified by sequence ID number (CDRs identified using Chothia)

[0277]

[0278]

[0279] Confirm that various h5E5 antibodies blocked the interaction between ILT3 and FN1, with c5E5 used as a positive control ( Fig.11A ). It is noteworthy that h5E5-133 is a superior blocker compared to c5E5 ( Fig. 11B ).

[0280] Next, h5E5 was functionally examined. CD8 T cell activation after MDSC inhibition was examined as described above. c5E5 increased IFNg secretion by T cells, but the h5E5 antibody was even better at alleviating MDSC inhibition compared to the IgG control and produced a statistically significant increase ( Fig. 12A As described above, DC activation in the presence of rituximab and fibronectin was examined. In this assay, h5E5-133 was again slightly superior to c5E5, with a stronger effect at lower concentrations ( Fig. 12B Similar results were observed when THP1 activation was restored ( Fig. 12C Several other antibodies were also found to be slightly superior to murine 5E5 ( Fig.12D ). Similar results were observed when APOE was used instead of fibronectin.

[0281] To further evaluate h5E5 function, 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. Then, M0 macrophages underwent differentiation into M1 macrophages and were activated by LPS stimulation in the presence or absence of cancer cells and h5E5 antibody. M1 macrophages secreted high levels of TNFa, but when the cells were combined with autologous gastric cancer cells (GA-04) ( Fig.13A ) or colon cells ( Fig. 13B) was abolished when co-cultured with 5E5 cells. This inhibition of cancer cells is mediated by ILT3, as the addition of h5E5 was able to relieve the inhibition and restore TNFa secretion. The addition of hIgG4 was used as a negative control. Thus, human antibodies are able to relieve cancer-induced immunosuppression of M1 macrophages and highlight the importance of ILT3 in myeloid maturation.

[0282] Next, monocytes were cultured in ImmunoCult differentiation medium for 5 days to generate immature DC (iDC). DCtol was generated by culturing iDC for 2 days with IL-10 and LPS. iDC stimulation was performed in the presence of h5E5 antibody, commercially available humanized h52B8 antibody, isotype control, or in the absence of any addition (medium alone). TNFa secretion levels were determined by ELISA. The TNFa levels produced by DCtol were very low, and this was also the case when isotype controls were used. However, stimulation in the presence of h5E5 antibody produced significantly elevated cytokine secretion levels, which were superior to the levels produced in the presence of h52B8 antibody ( Fig.14 ).

[0283] It is known that ILT3 blockade can be combined with other immune checkpoint inhibitors (ICIs). To test this, T cell activation in the presence of MDSCs was used to monitor the effects of h5E5, anti-PD1, and h5E5+PD-1 combination treatments. In fact, it was found that the two blockades had a combined effect on T cell activation and produced a more robust effect than either antibody on its own ( Fig.15A Indeed, the combined treatment was able to increase IFNg secretion levels above those observed in control activated CD8 T cells. A similar combined effect was observed when IFNg secretion by CD4 cells co-cultured with DCtol was examined ( Fig. 15B Similar results were observed in the DCtol and CD4 T cell co-culture assay ( Figure 6E-6F ).

[0284] Example 5: In vitro and in vivo evaluation of the therapeutic effect of humanized 5E5 antibody

[0285] To test the therapeutic efficacy of the antibodies of the invention, tissues were processed using TissueGrinder to process primary tumor samples into tumor-like particles. Tumor-like particles were activated for 48 hours with PMA and LPS (ionomycin was added for sarcoma-like tumors), and control tumor-like particles were not stimulated. Subsequently, h5E5 antibody or commercially available anti-ILT3 antibody was added, and cytokine secretion was measured by ELISA. Initial experiments found that h5E5 had a significant effect on sarcoma-like tumors ( Fig.16A ) and breast cancer tumors ( Fig. 16B) enhanced IFNg production in both breast cancer and breast cancer tumors. When various commercially available antibodies were applied to breast cancer tumors, no increase in pro-inflammatory chemokine (CCL3, CLL4) or cytokine (IL-8) production was observed, although h5E5 produced robust dose-dependent increases in all molecules tested ( Fig. 16C Results were similar in colorectal cancer (CRC) tumors, but the effect of h5E5 antibody was less pronounced, while h52B8 antibody produced a significant increase in cytokine / chemokine production ( Fig.16D Surprisingly, incubation with the anti-PD-1 antibody pembrolizumab had no significant effect on cytokine / chemokine secretion, however, when pembrolizumab was combined with h5E5, the increase was very significant and indicated a synergistic increase compared to each agent alone ( Fig.16D ).

[0286] The therapeutic efficacy of 5E5 antibody was evaluated using hILT3 transgenic mice. Mice were subcutaneously inoculated with MC-38 cancer cells (a syngeneic mouse colon cancer cell line). MC-38 transplanted mice were then treated with c5E5 or hIgG4 control (20 mg / kg / dose) twice a week starting from day 0 (treatment on days 0, 3, 7, 10, and 14). Tumor volume was recorded 3 times a week. Fig.17 As shown, anti-ILT3 antibodies are very effective, reducing tumor growth rate and overall tumor size. Therefore, the antibodies of the present invention are effective therapeutic agents for treating cancer. Pembrolizumab alone or in combination with h5E5 was also administered to mice vaccinated with tumor cells that had a moderate or poor response to anti-PD-1 therapy and a synergistic reduction in tumor size was observed.

[0287] Although the present invention has been described in conjunction with the specific embodiments of the present invention, it is apparent that various alternatives, modifications and variations are apparent to those skilled in the art. Therefore, the present invention is intended to include all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

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

2. The antibody or antigen-binding fragment according to claim 1, wherein: 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).

3. The antibody or antigen-binding fragment according to claim 1 or 2, comprising 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 1. 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.

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

5. The antibody or antigen-binding fragment according to claim 4, comprising: 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.

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

7. The antibody or antigen-binding fragment according to claim 4, comprising: 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.

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

9. The antibody or antigen-binding fragment according to claim 4, comprising: 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.

10. The antibody or antigen-binding fragment of any one of claims 1 to 9, wherein the heavy chain comprises an IgG4 constant region, optionally wherein the IgG4 constant region comprises the amino acid sequence of SEQ ID NO: 21 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG [0143] The invention relates to a sequence having at least 80% sequence identity to SEQ ID NO: 21, wherein said SEQ ID NO: 21 comprises S124P and L131E mutations.

11. The antibody or antigen-binding fragment of any one of claims 1 to 10, wherein the light chain comprises a kappa constant region, optionally wherein the kappa constant region comprises a sequence having at least 80% sequence identity to SEQ ID NO:22 (RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

12. The antibody or antigen-binding fragment according to claim 9 or 11, comprising 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 1. 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.

13. The antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment is capable of binding to leukocyte immunoglobulin-like receptor subfamily B, member 4 (LILRB4 or ILT3), optionally wherein the ILT3 comprises the amino acid sequence provided in SEQ ID NO:

71.

14. The antibody or antigen-binding fragment of claim 13, wherein the ILT3 is on the surface of a myeloid cell, a dendritic cell, a macrophage, or a combination thereof.

15. The antibody or antigen-binding fragment of claim 14, wherein the myeloid cell is a myeloid-derived suppressor cell (MDSC), the dendritic cell is a tolerogenic dendritic cell, the macrophage is a suppressive macrophage, optionally wherein the macrophage is a tumor-associated macrophage (TAM), or a combination thereof.

16. The antibody or antigen-binding fragment of any one of claims 13 to 15, wherein the binding to ILT3 inhibits binding of ILT3 to an ILT3 ligand.

17. The antibody or antigen-binding fragment of claim 16, wherein the binding inhibits the binding of ILT3 to both apolipoprotein E (APOE) and fibronectin (FN1).

18. The antibody or antigen-binding fragment of claim 17, wherein the APOE, FN1, or both are present in a tumor microenvironment (TME).

19. The antibody or antigen-binding fragment of any one of claims 13 to 18, wherein said binding to ILT3 releases T cells, monocytes, macrophages, or a combination thereof from ILT3-mediated inhibition.

20. The antibody or antigen-binding fragment of claim 19, wherein the release of the T cells comprises 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.

21. The antibody or antigen-binding fragment of any one of claims 13 to 20, wherein said binding to ILT3 increases dendritic cell activation.

22. The antibody or antigen-binding fragment of claim 21, wherein increasing dendritic cell activation comprises restoring dendritic cell activation that is inhibited by FN1, APOE, or both.

23. The antibody or antigen-binding fragment of claim 21 or 22, wherein increasing dendritic cell activation comprises increasing secretion of pro-inflammatory cytokines or chemokines by the dendritic cells.

24. The antibody or antigen-binding fragment of any one of claims 13 to 23, wherein said binding to ILT3 increases monocyte and / or macrophage activation.

25. The antibody or antigen-binding fragment of claim 24, wherein increasing monocyte and / or macrophage activation comprises increasing secretion of pro-inflammatory cytokines or chemokines by the dendritic cells.

26. The antibody or antigen-binding fragment according to any one of claims 20 to 25, wherein 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.

27. The antibody or antigen-binding fragment of any one of claims 1 to 26, wherein the antigen-binding fragment is selected from a Fv, Fab, F(ab')2, scFV or scFV2 ​​fragment.

28. The antibody or antigen-binding fragment of any one of claims 1 to 27, wherein the antibody or antigen-binding fragment is humanized.

29. The antibody or antigen-binding fragment of any one of claims 1 to 28, wherein the antibody is a monoclonal antibody.

30. The antibody or antigen-binding fragment of any one of claims 1 to 29, wherein the agent does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

31. A nucleic acid molecule or molecules encoding the antibody or antigen-binding fragment of any one of claims 1 to 30.

32. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 30 and a pharmaceutically acceptable carrier, excipient or adjuvant.

33. The pharmaceutical composition of claim 32, formulated for administration to a subject.

34. The pharmaceutical composition of claim 32 or 33, formulated for systemic or intratumoral administration.

35. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1 to 30 or the pharmaceutical composition of any one of claims 32 to 34, thereby treating the cancer.

36. The method of claim 35, wherein the cancer is a solid cancer.

37. The method of claim 35 or 36, wherein the cancer is selected from breast cancer, renal cancer, head and neck cancer, lung cancer, sarcoma, gastric cancer, colorectal cancer, and ovarian cancer.

38. The method of any one of claims 35 to 37, wherein the cancer is characterized by the presence of tumor-infiltrating immune cells expressing ILT3.

39. The method of any one of claims 35 to 38, wherein the cancer TME is characterized by expression of APOE, FN1, or both.

40. The method of claim 39, wherein expression is overexpression compared to non-cancerous tissue of the same tumor type.

41. The method of any one of claims 35 to 40, further comprising administering an immune checkpoint inhibitor (ICI).

42. The method of claim 41, wherein the ICI inhibits the PD-1 / PD-L1 / L2 checkpoint.

43. The method of claim 42, wherein the ICI is selected from pembrolizumab, nivolumab, atezolizumab, cemiplizumab, dotarizumab, durvalumab, and avelumab.

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