Lilrb5 antibodies and uses thereof

Abstract

Provided herein are antibodies binding to LILRB5 and the uses of the antibodies in detecting and treating cancer and autoimmune diseases.

Description

DESCRIPTION LILRB5 ANTIBODIES AND USES THEREOF PRIORITY CLAIM This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 681,450, filed August 9, 2024, the entire contents of which are hereby incorporated by reference. SEQUENCE LISTING

[0001] The sequence listing that is contained in the file named “UTFHP0404WO_ST26”, which is 248,651 bytes in size and was created on July 28, 2025, is filed herewith by electronic submission and is incorporated by reference herein. FEDERAL GRANT SUPPORT This invention was made with government support under grant numbers CA263079 and CA248736 awarded by the National Institutes of Health. The government has certain rights inthe invention.BACKGROUND 1. Field

[0002] The present disclosure relates generally to the fields of medicine, oncology, immunology and immuno-oncology. More particularly, the disclosure relates to antibodies that bind to LILRB5 and can treat cancers, including leukemia and solid tumors. 2. Description of Related Art

[0003] Current immune checkpoint blockade strategies have been successful in treating certain types of solid cancer. However, most cancer patients do not respond to current checkpoint blockade or they relapse after treatment. Additionally, checkpoint blockade monotherapies have not been successful against most hematologic malignancies including multiple myeloma and leukemia.

[0004] It is believed that tumor microenvironment (TME) plays a critical role in regulating immune responses to tumors. Among the complex factors and components that constitute the tumor microenvironment, myeloid derived suppressor cells (MDSC), tumor 1 4919-0796-2971, v.1associated macrophages (TAMs) and extracellular matrix all play critical role in suppressing the immune responses to tumor.

[0005] Immunosuppressive myeloid cells, including myeloid derived suppressor cells (MDSCs), tumor-associated macrophages, and certain subsets of dendritic cells, neutrophils, and eosinophils, play crucial roles in the tumor microenvironment (TME) (Schuijs et al., 2020; Zhao et al., 2020; DeVito et al., 2019; Fleming et al., 2018; Mantovani et al., 2017; Salemme et al., 2021; Barry et al., 2023). These cells hinder anti-tumor immunity through mechanisms such as suppression of T cell activation, stimulation of cancer cell proliferation, induction of angiogenesis, and establishment of the pre-metastatic niche (Liu & Cao, 2016; Ya et al., 2022; Shojaei & Ferrara, 2008; Lee et al., 2005; Hao et al., 2021; Groth et al., 2019; Ouzounova et al., 2017; Srivastava et al., 2010; Condamine et al., 2015). Reprogramming these immunosuppressive myeloid cells to enhance their pro-inflammatory and antigen-presenting capabilities could reactivate anti-tumor immunity in patients unresponsive to current T cell- centric immune checkpoint therapies.

[0006] The members of the LILRB family of receptors are type I transmembrane proteins that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which are predominantly found on hematopoietic cells (Kang et al., 2016; Hirayasu & Arase, 2015; Trowsdale et al., 2015; Daeron et al., 2008; Takai et al., 2011; Katz 2006; Deng et al., 2021; van der Touw et al., 2017; Thomas et al., 2010; Zhang, 2022). When activated, the LILRBs recruit tyrosine phosphatases SHP-1 and SHP-2 or the inositol-phosphatase SHIP. Their ability to engage these phosphatases positions LILRBs as pivotal elements in immune checkpoint control (Kang et al., 2016; Hirayasu & Arase, 2015; Trowsdale et al., 2015; Daeron et al., 2008; Takai et al., 2011; Katz 2006; Deng et al., 2021; van der Touw et al., 2017; Thomas et al., 2010; Sharma et al., 2021; Chen et al., 2018; Suciu-Foca et al., 2007; Barkal et al., 2018). The inventors’ previous work demonstrated that LILRB2 is activated by the hormone Angptl2, whereas LILRB4 and several other LILRBs are activated by ApoE and various galectins (Zheng et al., 2012; Chen et al., 2020; Deng et al., 2014; Feng et al., 2019; Huang et al., 2024). Additionally, the inventors have shown that multiple LILRBs and related ITIM-receptor LAIR1 promote the development of acute myeloid leukemia (AML) and solid cancers (Wu et al., 2021; Anami et al., 2020; Kang et al., 2018; John et al., 2018; Deng et al., 2018; Kang et al., 2015). Blocking the signaling initiated by various LILRBs or LAIR1 in healthy or cancerous human myeloid or lymphoid cells triggers anti-tumor immune responses (Sharma et al., 2021; Chen et al., 2018; Barkal et al., 2018; Chen et al., 2020; Wu et al., 2021; Deng et 2 4919-0796-2971, v.1al., 2018; Singh et al., 2021; Xie et al., 2022; Ramos et al., 2021; Paavola et al., 2021; Kim et al., 2019; Gui et al., 2019). Consequently, the LILRB family members were suggested to be emerging myeloid immune checkpoint targets (Zhang, 2022; Paavola et al., 2021).

[0007] LILRB5, the least studied member of the LILRB family, is reported to be expressed by T cells, natural killer (NK) cells, and various myeloid cells, including monocytes, macrophages, osteoclasts, neutrophils, and is found in mast cell granules (Zhang et al., 2015; Jones et al., 2009; Hogan et al., 2016; Fan et al., 2021; Tedla et al., 2007). LILRB5 specifically binds to HLA-B7 and HLA-B27 heavy chains (Zhang et al., 2015). The existence of ITIMs in its cytoplasmic domain implies a role in the negative regulation of immune response, and LILRB5 is implicated in mast cell inflammatory response (Tedla et al., 2008), bacterial infection (Hogan et al., 2016), and muscle repair (Dube et al., 2014; Kristjansson et al., 2016; Siddiqui et al., 2017). Its expression pattern, binding proteins, and functions remain poorlycharacterized, however.

[0008] Thus, it is possible that antagonizing LILRB5, such as has been shown for other members of this family of receptors, may block the immunosuppressive function in various disease states, such as cancer. 3 4919-0796-2971, v.1SUMMARY

[0009] Thus, in one aspect, the present disclosure provides an isolated monoclonal antibody or an antigen-binding fragment thereof comprising clone paired heavy and light chain CDRs1-3 as set forth in Table 1 and Table 2, respectively. The isolated monoclonal antibody may be a murine, a rodent, a rabbit, a chimeric, humanized, or human antibody. The antigen- binding fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment. The antibody may be a human antibody or a humanized antibody. The antibody may be a chimeric antibody.

[0010] The isolated monoclonal antibody or an antigen-binding fragment thereof of claim may comprise VH and VL chains have amino acid sequences of at least 90%, 95% or 100% percent sequence identity to clone-paired VH and VL sequences of Table 10. The isolated monoclonal antibody or an antigen-binding fragment thereof of claim 1 may comprises VH and VL chains encoded by sequences of at least 80%, 90%, 95% or 100% percent sequence identity to clone-paired sequences of Tables 8 and 9, respectively. The isolated monoclonal antibody or an antigen binding fragment thereof may induce the activation of LILRB5 or may suppresses the activation of LILRB5.

[0011] Also provided is an isolated monoclonal antibody or an antigen-binding fragment thereof which competes for the same epitope with the isolated monoclonal antibody or an antigen-binding fragment comprising clone paired heavy and light chain CDRs1-3 as set forth in Table 1 and Table 2, comprising VH and VL chains having amino acid sequences of at least 90%, 95% or 100% percent sequence identity to clone-paired VH and VL sequences of Table 10, or comprising VH and VL chains encoded by sequences of at least 80%, 90%, 95% or 100% percent sequence identity to clone-paired sequences of Tables 8 and 9, respectively.

[0012] Further provide is a pharmaceutical composition comprising an isolated monoclonal antibody or an antigen-binding fragment thereof comprising clone paired heavy and light chain CDRs1-3 as set forth in Table 1 and Table 2, comprising VH and VL chains having amino acid sequences of at least 90%, 95% or 100% percent sequence identity to clone- paired VH and VL sequences of Table 10, or comprising VH and VL chains encoded by sequences of at least 80%, 90%, 95% or 100% percent sequence identity to clone-paired sequences of Tables 8 and 9, respectively, and a pharmaceutically acceptable carrier. 4 4919-0796-2971, v.1

[0013] Yet further provided is an isolated nucleic acid(s) that encodes an isolated monoclonal antibody as defined herein, a vector(s) comprising such the isolated nucleic acid(s), a host cell comprising such vector(s), such as a mammalian cell, in particular a CHO cell. Even further, there is provided a hybridoma or engineered cell encoding and / or producing the isolated monoclonal antibody as described herein.

[0014] In addition, there is provided a process of producing an antibody, comprising culturing a host cell engineered cell or hybridoma as disclosed herein under conditions suitable for expressing the antibody, and recovering the antibody.

[0015] Also provided is a chimeric antigen receptor (CAR) protein comprising an antigen-binding fragment as defined herein, an isolated nucleic acid that encodes such CAR protein, a vector comprising such isolated nucleic acid, an engineered cell comprising such isolated nucleic acid, such as a T cell, NK cell, or macrophage.

[0016] In another aspect, there is provided a method of treating or ameliorating the effect of a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or an antigen-binding fragment thereof as described here. The method may reduce or eradicate the tumor burden in the subject, may reduce the number of tumor cells and / or slows tumor growth rate, may reduce tumor size, may reduce or prevent tumor metastasis, or may eradicate the tumor in the subject.

[0017] The cancer may be a solid cancer, such as adrenal cancer, bile duct carcinoma, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, mesothelioma, melanoma, merkel cell cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pinealoma, prostate cancer, renal cell cancer, retinoblastoma, sarcoma, skin cancer, testicular cancer, thymic carcinoma, thyroid cancer, uterine cancer, and vaginal cancer. The method of claim 27, wherein monocytes, macrophages, dendritic cells, neutrophils and other myeloid cells, myeloid-derived suppressor cells, and tumor-associated macrophages are targeted.

[0018] The cancer may be a hematologic malignancy, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, chronic lymphoblastic leukemia (CLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic 5 4919-0796-2971, v.1myelomonocytic leukemia (CMML), chronic myelocytic leukemia (CML), pre-B acute lymphocytic leukemia (Pre-B ALL), diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, heavy chain disease, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Hodgkin’s lymphoma, non- Hodgkin’s lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma (MM), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, and polycythemia vera.

[0019] The antibody or an antigen-binding fragment thereof may be administered intravenously, intra-arterially, intra-tumorally, or subcutaneously. The method may further comprise administering to the subject one or more drugs selected from the group consisting of a topoisomerase inhibitor, an anthracycline topoisomerase inhibitor, an anthracycline, a daunorubicin, a nucleoside metabolic inhibitor, a cytarabine, a hypomethylating agent, a low dose cytarabine (LDAC), a combination of daunorubicin and cytarabine, a daunorubicin and cytarabine liposome for injection, Vyxeos®, an azacytidine, Vidaza®, a decitabine, an all- trans-retinoic acid (ATRA), an arsenic, an arsenic trioxide, a histamine dihydrochloride, Ceplene®, an interleukin-2, an aldesleukin, Proleukin®, a gemtuzumab ozogamicin, Mylotarg®, an FLT-3 inhibitor, a midostaurin, Rydapt®, a clofarabine, a farnesyl transferase inhibitor, a decitabine, an IDH1 inhibitor, an ivosidenib, Tibsovo®, an IDH2 inhibitor, an enasidenib, Idhifa®, a smoothened (SMO) inhibitor, a glasdegib, an arginase inhibitor, an IDO inhibitor, an epacadostat, a BCL-2 inhibitor, a venetoclax, Venclexta®, a platinum complex derivative, oxaliplatin, a kinase inhibitor, a tyrosine kinase inhibitor, a PI3 kinase inhibitor, a BTK inhibitor, an ibrutinib, IMBRUVICA®, an acalabrutinib, CALQUENCE®, a zanubrutinib, a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG3 antibody, an ICOS antibody, a TIGIT antibody, a TIM3 antibody, a CD40 antibody, a 4-1BB antibody, a CD47 antibody, a SIRP1α antibody or fusions protein, a CD70 antibody, and CLL1 antibody, a CD123 antibody, an antagonist of E-selectin, an antibody binding to a tumor antigen, an antibody binding to a T-cell surface marker, an antibody binding to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an alkaloid derived from a plant, a hormone therapy medicine, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor.

[0020] The isolated monoclonal antibody or an antigen binding fragment thereof may further comprise an antitumor drug linked thereto. The antitumor drug may be linked to said 6 4919-0796-2971, v.1antibody through a photolabile linker or through an enzymatically cleaved linker. The antitumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme.

[0021] In still another embodiment, there is provided method of detecting a cancer cell or cancer stem cell in a sample or subject comprising (a) contacting a subject or a sample from the subject with the antibody or an antigen-binding fragment as described herein; and (b) detecting binding of said antibody to a cancer cell or cancer stem cell in said subject or sample.

[0022] The sample may be a body fluid or biopsy, such as blood, bone marrow, sputum, tears, saliva, mucous, serum, urine or feces. Detection may comprise immunohistochemistry, flow cytometry, an immunoassay (including ELISA, RIA etc.) or Western blot. The method may further comprise performing steps (a) and (b) a second time and determining a change in detection levels as compared to the first time. The isolated monoclonal antibody or an antigen binding fragment thereof further may comprise a label, such as a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemo-luminescent molecule, or a dye. The isolated monoclonal antibody or an antigen binding fragment thereof may be conjugated to a liposome or nanoparticle.

[0023] Additionally, there is provided a method of treating or ameliorating the effect of an autoimmune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or an antigen-binding fragment thereof as described herein, engineered cell expressing such antibody or antigen-binding fragment thereof. In particular, monocytes, macrophages, dendritic cells, neutrophils and other myeloid cells may be targeted.

[0024] The antibody or an antigen-binding fragment thereof may be administered intravenously, intra-arterially, or subcutaneously. The method may further comprise administering to the subject one or more drugs selected from the group consisting of a steroid or an NSAID.

[0025] The autoimmune disease may be Guillain-Barre syndrome, Chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory disease, systemic lupus erythematosus, 7 4919-0796-2971, v.1Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile onset diabetes mellitus (also known as Type I diabetes mellitus), Wegener's granulomatosis, polymyositis, dermatomyositis, inclusion body myositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, Grave’s Disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult onset diabetes mellitus (also known as Type II diabetes mellitus), mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, anti-phospholipid syndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic disease, allergic encephalomyelitis, transfusion reaction, leprosy, malaria, leshmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, shistosomiasis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki's disease, endophthalmitis, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, tularemia, periodic fever syndromes, pyogenic arthritis, Familial Mediterranean Fever, TNF-receptor associated periodic syndrome (TRAPS), Muckle- Wells syndrome, or hyper-IgD syndrome.

[0026] In additional embodiments, there is provided (a) a method for enhancing T cell activation in a subject, the method comprising administering to the subject the antibody or an antigen-binding fragment thereof as described herein, or an engineered cell expressing such antibody or antigen-binding fragment thereof; or (b) a method for modulating M2a macrophage phenotype in a subject, the method comprising administering to the subject the antibody or an antigen-binding fragment thereof as described herein, or an engineered cell expressing such antibody or antigen-binding fragment thereof.

[0027] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent 8 4919-0796-2971, v.1with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0028] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. 9 4919-0796-2971, v.1BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0030] FIGS. 1A-H. Several Eph receptors bind to LILRB5. (FIG. 1A) Schematic of chimeric LILRB5 reporter cell. (FIG. 1B) Percent GFP+cells (indicative of reporter activation) upon incubation of LILRB5 reporter cells with 20 μg / mL of indicated immobilized Eph receptor proteins (Anti-LILRB5 (+) represents B5-2). (FIG. 1C) Percent GFP+cells (indicative of activation) upon incubation of LILRB2 reporter cells with 20 μg / mL of indicated immobilized Eph receptor proteins. (FIG. 1D) Schematic of LILRB5 constructs, and flow cytometry analyses of 293T cells expressing full-length LILRB5 (LILRB5-FL) or truncated LILRB5-d1 or LILRB5-d2 with 20 μg / mL of soluble EphA4-ECD-Fc, EphA7-ECD-Fc, EphA2-ECD-His, or EphB1-ECD-His proteins. MFIs of anti-Fc or anti-His signal of FLAG+and FLAG- cells are indicated. (FIG. 1E) Flow cytometry analyses of 293T cells transfected with EphA7 or EphB1 incubated with 20 μg / ml of LILRB5-Fc or LILRB2-Fc. MFIs of anti- Fc are indicated. (FIG. 1F) Schematic of EphA7 constructs, and flow cytometry analyses of 293T cells that express full-length EphA7 (EphA7-FL) or EphA7-d1, EphA7-d2, EphA7-d3, or EphA7-d4 incubated with LILRB5-Fc. MFIs of anti-Fc are indicated. (FIG.1G) Schematic of EphB1 constructs, and flow cytometry analyses of 293T cells that express full-length EphB1 (EphB1-FL) or EphB1-d1, EphB1-d2, or EphB1-d3 incubated with LILRB5-Fc. MFIs of anti- Fc are indicated. (FIG.1H) Upper panel shows bio-layer interferometry responses as a function of time of incubation of 100 nM LILRB5-Fc protein immobilized on protein A biosensor tips and dipped in various concentrations (1000 nM - 7000 nM) of soluble EphA7-His. Lower panel presents a table of calculated kinetic parameters for binding of LILRB5 to EphA7 and EphB1.

[0031] FIGS. 2A-G. Development of LILRB5 blocking antibodies. (FIGS. 2A-C) Bio-layer interferometry responses as a function of time of incubation of anti-LILRB5 #1 (B5- 1) in FIG.2A, anti-LILRB5 #27 (B5-27) in FIG.2B, or anti-LILRB5 #29 (B5-29) in FIG.2C immobilized on Protein A biosensors and dipped in various concentrations (3.1 nM - 200 nM) of soluble LILRB5. (FIG. 2D) ELISA between LILRB5 anti-LILRB5 #1 (B5-1) antibodies. (FIG.2E) ELISA between LILRB5 and anti-LILRB5 #27 (B5-27) antibodies. (FIG.2F) Flow cytometry diagrams of reporter cells stained with anti-LILRB5 #1 (B5-1) antibodies conjugated 10 4919-0796-2971, v.1with R-PE. (FIG. 2G) Percent GFP+cells (indicative of reporter activation) upon culture of LILRB5 reporter cells on plates coated with Eph receptor proteins in the presence of human IgG or anti-LILRB5 (B5-2) antibody.

[0032] FIGS. 3A-H. Eph receptor binding to LILRB5 induces bi-directional signaling. (FIG.3A) Western blot analysis of phosphorylation of LILRB5-FLAG precipitated from 293T cells transfected with LILRB5-FLAG and individual Src kinases (Src, Yes, Fyn, Lck, Blk, Lyn, and Frk). (FIG. 3B) Western blot analysis of phosphorylation of full-length LILRB5 (LILRB5FL) and ITIM mutants (ITIM1 YF, ITIM2 YF, and ITIM1&2 YYFF) co- expressed with Src in 293T cells. (FIG. 3C) Western blot analysis of phosphorylation of LILRB5-FLAG co-expressed with Src in 293T cells that were co-cultured with 293T cells that express EphA7. (FIG. 3D) Western blot analysis of phosphorylation of LILRB5-FLAG co- expressed with Src in 293T cells that were co-cultured with 293T cells that express EphB1. (FIG. 3E) Western blot analysis of phosphorylation of LILRB5 expressed in THP-1 cells incubated with immobilized CD64 and EphA7 with or without anti-LILRB5 (B5-2) antibody. (FIG. 3F) Western blot analysis of phosphorylation of LILRB5 expressed in THP-1 cells incubated with immobilized CD64 and EphB1 with or without anti-LILRB5 (B5-2) antibody. (FIG.3G) Western blot analysis of phosphorylation of EphA7-FLAG expressed in 293T cells incubated with soluble EphrinA5, LILRB5-ECD, or LILRB2-ECD. (FIG. 3H) Western blot analysis of phosphorylation of EphB1-FLAG expressed in 293T cells incubated with soluble EphrinB2, LILRB5-ECD, or LILRB2-ECD.

[0033] FIGS.4A-B. LILRB5 is expressed on monocytes and neutrophils. (FIG.4A) Flow cytometry analysis of expression of LILRB5 on different subsets of lymphocytes, monocytes, and granulocytes in the peripheral blood of a representative human patient with lung cancer. Dark grey = FMO control, light grey = anti-LILRB5 (B5-27) antibody (FIG.4B) Flow cytometry analysis of expression of LILRB5 expression patterns in peripheral blood of a healthy donor, a patient with melanoma, kidney cancer patients, a lung cancer patient, and a breast cancer patient.

[0034] FIGS.5A-D. Eph receptors activate LILRB5 in vitro and promote immune suppressive functions. (FIG.5A) IL-8 secretion by THP-1 cells that overexpressing LILRB5 upon incubation with immobilized human IgG, BSA, EphA7, or EphB1 and soluble anti- LILRB5 antibodies (left panel using B5-29 mAb and right panel using B5-27 mAb). (FIG.5B) CD163 and CD206 MFI levels measured via flow cytometry in MDSCs isolated from a renal 11 4919-0796-2971, v.1cancer patient cultured with BSA or EphA7 and treated with human IgG or anti-LILRB5 (B5- 1) antibody. (FIG.5C) CD163 and CD206 MFI levels measured via flow cytometry in MDSCs isolated from a prostate cancer patient cultured with T cells and BSA or EphB1 and treated with human IgG or anti-LILRB5 (B5-1) antibody. (FIG.5D) CD80, CD86, CD163 and CD206 MFI levels on MDSCs isolated from a prostate cancer patient that underwent in vitro M2 macrophage differentiation and cultured with BSA or EphB1 and treated with human IgG or anti-LILRB5 (B5-2) antibody.

[0035] FIGS. 6A-I. EphA7 overexpression supports tumor growth in myeloid- specific LILRB5 transgenic mice and LILRB5 blockade suppresses tumor growth. (FIG. 6A) Proliferation of MC38 cells transduced with empty vector or vector for expression of EphA7 as measured using an MTS proliferation assay monitored at 490 nm. (FIG. 6B) Flow cytometry analysis of binding of control or LILRB5-ECD-Fc to MC38 cells that express EphA7 cells. (FIGS.6C-D) WT and LILRB5-transgenic mice were injected with MC38 cells that express empty vector or EphA7. Tumor volumes as a function of time are shown in FIG. 6C. Tumor weights at day 20 are presented in FIG.6D. (FIG.6E) Immune cell populations in TMEs of WT and LILRB5-transgenic mice with tumors resulting from injection of MC38 cells that express EphA7; percentages were normalized to total CD45+immune cells. (FIG. 6F) CD206 MFI on myeloid cells and effector CD8+T cell populations in TMEs of WT and LILRB5-transgenic mice with tumors that express EphA7. (FIGS.6G-H) LILRB5-transgenic mice with tumors that express EphA7 were treated with human IgG or anti LILRB5 (B5-27) antibody. Tumor volumes over time are shown in FIG. 6G.Tumor weights at day 18 are presented in FIG. 6H. (FIG. 6I) CD206 MFI on myeloid cells and effector CD8+T cell populations in the TMEs of tumors that express EphA7 from LILRB5-transgenic mice treated with human IgG or anti-LILRB5 (B5-27) antibody.

[0036] FIGS. 7A-H. EphB1 overexpression supports tumor growth in myeloid- specific LILRB5 transgenic mice and LILRB5 blockade suppresses tumor growth. (FIG. 7A) Proliferation of MC38 cells transduced with empty vector or vector for expression of EphB1 measured using an MTS proliferation assay monitored at 490 nm. (FIG. 7B) Flow cytometry analysis of binding of control or LILRB5-Fc to MC38 cells that express EphB1. (FIGS. 7C-D) WT and LILRB5-transgenic mice were injected with MC38 cells that express empty vector or EphB1. Tumor volumes measured over time are shown in FIG. 7C. Tumor weights at day 18 are demonstrated in FIG. 7D. (FIG.7E) CD206 MFI on myeloid cells and 12 4919-0796-2971, v.1effector CD8+T cell populations in WT and LILRB5 transgenic mice with tumors that express EphB1 TME. (FIGS. 7F-G) LILRB5-transgenic mice with tumors that express EphB1 were treated with human IgG or anti LILRB5 (B5-2) antibody. Tumor volumes as a function of time are presented in FIG.7F. Tumor weights on day 19 are shown in FIG.7G. (FIG.7H) CD206 MFI on myeloid cells and effector CD8+T cell populations in the TMEs of MC38 tumors that express EphB1 from LILRB5-transgenic mice treated with human IgG or anti-LILRB5 (B5-2) antibody.

[0037] FIG. 8. Determination of binding affinity of LILRB5 antibodies using ELISA. LILRB5 mAb label and estimated EC50 values are shown on each graph. X-axis indicates antibody concentrations used and Y-axis indicates binding signals measured in ELISA as absorbance at wavelength of 450nm. EC50s are determined using concentration titration curves and GraphPad prism software using 4-parameter fitting model. LILRB5 #1 represents B5-1 mAb, LILRB5 #2 for B5-2 mAb, LILRB5 #4 for B5-4 mAb, LILRB5 #8 for B5-8 mAb, LILRB5 #11 for B5-11 mAb, LILRB5 #6 for B5-6 mAb, LILRB5 #14 for B5-14 mAb, LILRB5 #16 for B5-16 mAb, LILRB5 #17 for B5-17 mAb, LILRB5 #20 for B5-20 mAb, LILRB5 #22 for B5-22 mAb, LILRB5 #25 for B5-25 mAb, LILRB5 #26 for B5-26 mAb, LILRB5 #27 for B5-27 mAb, LILRB5 #28 for B5-28 mAb, and LILRB5 #29 for B5-29 mAb.

[0038] FIG. 9. Determination of kinetic binding affinity using Octet 96-Red instrument. LILRB5 antibody (30 µg / mL) was loaded onto the protein A biosensors for 4 min. Following incubation in kinetics buffer to establish baseline. Antibody loaded biosensors were exposed to a series of concentrations (starting 200 nM) of LILRB5 protein (HIS tag at C terminus). Background subtraction was used to correct sensor drifting. LILRB5 #1 represents B5-1 mAb, LILRB5 #6 for B5-6 mAb, LILRB5 #11 for B5-11 mAb, LILRB5 #14 for B5-14 mAb, LILRB5 #16 for B5-16 mAb, LILRB5 #20 for B5-20 mAb, LILRB5 #27 for B5-27 mAb, and LILRB5 #29 for B5-29 mAb.

[0039] FIGS. 10A-D. Cross reactivity to cynomolgus monkey LILRB5. LILRB5 mAb label is shown on the top of each graph (FIGS. 10A-C). X-axis indicates antibody concentrations used and Y-axis indicates binding signals measured in ELISA as absorbance at wavelength of 450nm. EC50s in (FIG.10D) for human LILRB5 (Human B5) and cynomolgus (Cyno-B5) are determined using concentration titration curves and GraphPad prism software using 4-parameter fitting model. 13 4919-0796-2971, v.1

[0040] FIG.11. LILRB5 antibody binding to LILR family members using ELISA. Extracellular proteins (ECD) of all members (LILRA1-6 and LILRB1-5) and LAIR1 were recombinantly expressed in HEK293 cells with a human Fc tag for purification. Purified ECDs at 2µg / ml in PBS were used to coat on high-binding 96-well plate for ELISA detection of LILRB5 mAb binding at concentration of 1 µg / ml. Control (the first bar next to Y-axis) is an isotype human monoclonal antibody and LILR members are labeled on X-axis and Y-axis shows the binding signal. LILRB5 #1 represents B5-1 mAb, LILRB5 #2 for B5-2 mAb, LILRB5 #4 for B5-4 mAb, LILRB5 #6 for B5-6 mAb, LILRB5 #8 for B5-8 mAb, LILRB5 #11 for B5-11 mAb, LILRB5 #14 for B5-14 mAb, LILRB5 #16 for B5-16 mAb, LILRB5 #17 for B5-17 mAb, LILRB5 #20 for B5-20 mAb, LILRB5 #22 for B5-22 mAb, LILRB5 #25 for B5-25 mAb, LILRB5 #26 for B5-26 mAb, LILRB5 #27 for B5-27 mAb, LILRB5 #28 for B5- 28 mAb, and LILRB5 #29 for B5-29 mAb.

[0041] FIG. 12. Extracellular domains (ECDs) of human LILRB5 protein construct.

[0042] FIG. 13. LILRB5 antibodies selectively bind to LILRB5 expressed on cell surface. Binding of LILRB5 mAbs on LILRB family members expressed on cell surface was detected using flow cytometry. Y-axis (% of GFP cells) shows engagement of LILLRB5 and LILRB5 mAbs did not show binding to other members in LILRB family. B5#1 stands for B5- 1 mAb, B5#2 for B5-2 mAb, B5#4 for B5-4 mAb, B5#6 for B5-6 mAb, B5#8 for B5-8 mAb, B5#11 for B5-11 mAb, B5#14 for B5-14 mAb, B5#16 for B5-16 mAb, B5#17 for B5-17 mAb, B5#20 for B5-20 mAb, B5#22 for B5-22 mAb, B5#25 for B5-25 mAb, B5#26 for B5-26 mAb, B5#27 for B5-27 mAb, B5#28 for B5-28 mAb, and B5#29 for B5-29 mAb.

[0043] FIGS. 14A-D. Eph receptors do not bind to LILRB1, LILRB3, LILRB4 and LAIR1. Activation of LILRB1 reporter cells (FIG. 14A), LILRB3 reporter cells (FIG. 14B), LILRB4 reporter cells (FIG. 14C), and LAIR1 reporter cells (FIG. 14D) by 20 μg / mL immobilized Eph receptor proteins, quantified by GFP+(%). 14 4919-0796-2971, v.1DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0044] Here, the inventors demonstrated that LILRB5 is functionally expressed on the immunosuppressive myeloid cells from patients with cancer. Blockade of LILRB5 with specific antibodies significantly reduced immunosuppressive marker expression on human MDSCs from patients with cancer in vitro. They discovered that several Eph receptors, including EphA2, EphA4, EphA7, and EphB1, activate LILRB5 and LILRB2. Eph receptors, the largest family of receptor tyrosine kinases in mammals, bind to ephrin ligands and are aberrantly expressed in cancer and have tumor-promoting or tumor-suppressing effects (Kou et al., 2018; Hafner et al., 2004; Pasquale, 2010; Pasquale, 2024). The interactions with LILRBs led to the phosphorylation of EphA7 and EphB1. Furthermore, using EphA7 and EphB1 as representatives, the inventors showed that the expression of Eph on tumor cells led to elevated tumor development, increased numbers of immunosuppressive myeloid cells, and decreased anti-tumor T cells in the TMEs of myeloid-specific LILRB5-transgenic syngeneic mice; anti-LILRB5 blocking antibody inhibited these effects. These results suggest that the Eph / LILRB functional interplay promotes cancer development and that LILRB5 blockade may be an attractive option to treat certain patients with cancer.

[0045] The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety. I. Definition

[0046] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components 15 4919-0796-2971, v.1that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term “portion” can include part of a moiety or the entire moiety.

[0047] As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0048] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosed subject matter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0049] The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An “antibody” is a species of an antigen binding protein. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies as described further below. The antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two 16 4919-0796-2971, v.1full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0050] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length “light” (in certain embodiments, about 25 kDa) and one full-length “heavy” chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within full- length light and heavy chains, typically, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form the antigen binding site.

[0051] The term “variable region” or “variable domain” refers to a portion of the light and / or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target.

[0052] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called 17 4919-0796-2971, v.1complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987) or Chothia et al., Nature, 342:878-883 (1989).

[0053] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0054] In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition.

[0055] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268- 9272 (1989); “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an 18 4919-0796-2971, v.1antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0056] By convention, the CDR regions in the heavy chain are typically referred to as H1, H2, and H3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as L1, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

[0057] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0058] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxyl-terminus, with the CH3 being closest to the carboxy-terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

[0059] A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0060] The term “antigen” refers to a substance capable of inducing adaptive immune responses. Specifically, an antigen is a substance which serves as a target for the receptors of an adaptive immune response. Typically, an antigen is a molecule that binds to antigen-specific receptors but cannot induce an immune response in the body by itsself. Antigens are usually 19 4919-0796-2971, v.1proteins and polysaccharides, less frequently also lipids. Suitable antigens include without limitation parts of bacteria (coats, capsules, cell walls, flagella, fimbrai, and toxins), viruses, and other microorganisms. Antigens also include tumor antigens, e.g., antigens generated by mutations in tumors. As used herein, antigens also include immunogens and haptens.

[0061] An “antigen binding protein” (“ABP”) as used herein means any protein that binds a specified target antigen. In the instant application, the specified target antigen is the LILRB protein or fragment thereof. “Antigen binding protein” includes but is not limited to antibodies and antigen-binding fragment thereof. Peptibodies are another example of antigen binding proteins.

[0062] The term “antigen-binding fragment” as used herein refers to a portion of a protein which is capable of binding specifically to an antigen. In certain embodiment, the antigen-binding fragment is derived from an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. In certain embodiments, the antigen-binding fragment is not derived from an antibody but rather is derived from a receptor. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody, a single domain antibody (sdAb), a camelid antibody or a nanobody, a domain antibody, and a bivalent domain antibody. In certain embodiments, an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies. In certain embodiments, the antigen-binding fragment is derived from a receptor and contains one or more mutations. In certain embodiments, the antigen-binding fragment does not bind to the natural ligand of the receptor from which the antigen-binding fragment is derived.

[0063] A “Fab fragment” comprises one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. 20 4919-0796-2971, v.1

[0064] A “Fab′ fragment” comprises one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form an F(ab′)2molecule.

[0065] A “F(ab′)2fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab′)2fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains.

[0066] An “Fc” region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0067] The “Fv region” comprises the variable regions from both the heavy and light chains but lacks the constant regions.

[0068] “Single-chain antibodies” are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. No. 4,946,778 and No.5,260,203, the disclosures of which are incorporated by reference.

[0069] A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0070] A “bivalent antigen binding protein” or “bivalent antibody” comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. Bivalent antigen binding proteins and bivalent antibodies can be bispecific, see, infra. A bivalent antibody other than a “multispecific” or “multifunctional” antibody, in certain embodiments, typically is understood to have each of its binding sites identical. 21 4919-0796-2971, v.1

[0071] A “multispecific antigen binding protein” or “multispecific antibody” is one that targets more than one antigen or epitope.

[0072] A “bispecific,” “dual-specific” or “bifunctional” antigen binding protein or antibody is a hybrid antigen binding protein or antibody, respectively, having two different antigen binding sites. Bispecific antigen binding proteins and antibodies are a species of multispecific antigen binding protein antibody and can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol.79:315-321; Kostelny et al., 1992, J. Immunol.148:1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind to two different epitopes, which can reside on the same or different protein targets.

[0073] “Binding affinity” generally refers to the strength of the sum total of non- covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

[0074] An antibody that “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. For example, the LILRB5 specific antibodies of the present invention are specific to LILRB5. In some embodiments, the antibody that binds to LILRB5 has a dissociation constant (Kd) of ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10−8M or less, e.g., from 10−8M to 10−13M, e.g., from 10−9M to 10−13M). The dissociation constant Kd used herein refers to the ratio of the dissociation rate to the association rate (koff / kon), which may be determined by using any conventional method known in the art, 22 4919-0796-2971, v.1including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method. In certain embodiments, the Kd value can be appropriately determined by using flow cytometry.

[0075] The term “compete” when used in the context of antigen binding proteins (e.g., antibody or antigen-binding fragment thereof) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or antigen-binding fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., LILRB or a fragment thereof). Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol.32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. 23 4919-0796-2971, v.1

[0076] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. The epitope can be either linear epitope or a conformational epitope. A linear epitope is formed by a continuous sequence of amino acids from the antigen and interacts with an antibody based on their primary structure. A conformational epitope, on the other hand, is composed of discontinuous sections of the antigen’s amino acid sequence and interacts with the antibody based on the 3D structure of the antigen. In general, an epitope is approximately five or six amino acid in length. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0077] A “cell”, as used herein, can be prokaryotic or eukaryotic. A prokaryotic cell includes, for example, bacteria. A eukaryotic cell includes, for example, a fungus, a plant cell, and an animal cell. The types of an animal cell (e.g., a mammalian cell or a human cell) includes, for example, a cell from circulatory / immune system or organ, e.g., a B cell, a T cell (cytotoxic T cell, natural killer T cell, regulatory T cell, T helper cell), a natural killer cell, a granulocyte (e.g., basophil granulocyte, an eosinophil granulocyte, a neutrophil granulocyte and a hypersegmented neutrophil), a monocyte or macrophage, a red blood cell (e.g., reticulocyte), a mast cell, a thrombocyte or megakaryocyte, and a dendritic cell; a cell from an endocrine system or organ, e.g., a thyroid cell (e.g., thyroid epithelial cell, parafollicular cell), a parathyroid cell (e.g., parathyroid chief cell, oxyphil cell), an adrenal cell (e.g., chromaffin cell), and a pineal cell (e.g., pinealocyte); a cell from a nervous system or organ, e.g., a glioblast (e.g., astrocyte and oligodendrocyte), a microglia, a magnocellular neurosecretory cell, a stellate cell, a boettcher cell, and a pituitary cell (e.g., gonadotrope, corticotrope, thyrotrope, somatotrope, and lactotroph); a cell from a respiratory system or organ, e.g., a pneumocyte (a type I pneumocyte and a type II pneumocyte), a clara cell, a goblet cell, and an alveolar macrophage; a cell from circular system or organ (e.g., myocardiocyte and pericyte); a cell from digestive system or organ, e.g., a gastric chief cell, a parietal cell, a goblet cell, a paneth cell, a G cell, a D cell, an ECL cell, an I cell, a K cell, an S cell, an enteroendocrine cell, an enterochromaffin cell, an APUD cell, and a liver cell (e.g., a hepatocyte and Kupffer cell); a cell from integumentary system or organ, e.g., a bone cell (e.g., an osteoblast, an osteocyte, and an osteoclast), a teeth cell (e.g., a cementoblast, and an ameloblast), a cartilage cell (e.g., a chondroblast and a chondrocyte), a skin / hair cell (e.g., a trichocyte, a keratinocyte, and a melanocyte (Nevus cell), a muscle cell (e.g., myocyte), an adipocyte, a fibroblast, and a tendon cell; a cell from urinary system or organ (e.g., a podocyte, a juxtaglomerular cell, an intraglomerular mesangial cell, an extraglomerular mesangial cell, a kidney proximal tubule 24 4919-0796-2971, v.1brush border cell, and a macula densa cell); and a cell from reproductive system or organ (e.g., a spermatozoon, a Sertoli cell, a leydig cell, an ovum, an oocyte). A cell can be normal, healthy cell; or a diseased or unhealthy cell (e.g., a cancer cell). A cell further includes a mammalian zygote or a stem cell which include an embryonic stem cell, a fetal stem cell, an induced pluripotent stem cell, and an adult stem cell. A stem cell is a cell that is capable of undergoing cycles of cell division while maintaining an undifferentiated state and differentiating into specialized cell types. A stem cell can be an omnipotent stem cell, a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell and a unipotent stem cell, any of which may be induced from a somatic cell. A stem cell may also include a cancer stem cell. A mammalian cell can be a rodent cell, e.g., a mouse, rat, hamster cell. A mammalian cell can be a lagomorpha cell, e.g., a rabbit cell. A mammalian cell can also be a primate cell, e.g., a human cell.

[0078] The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificially constructed hybrid protein or polypeptide containing an antigen binding domain of an antibody (e.g., a single chain variable fragment (scFv)) linked to a domain or signaling, e.g., T-cell signaling or T-cell activation domains, that activates an immune cell, e.g., a T cell or a NK cell (see, e.g., Kershaw et al., supra, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720- 724 (1993), and Sadelain et al., Curr. Opin. Immunol. 21(2): 215-223 (2009)). CARs are capable of redirecting the immune cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, taking advantage of the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition confers immune cells expressing CARs on the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. In addition, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T-cell receptor (TCR) alpha and beta chains.

[0079] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0080] The term “host cell” means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in 25 4919-0796-2971, v.1morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

[0081] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math.48:1073.

[0082] In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res.12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3× the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; 26 4919-0796-2971, v.1Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[0083] Examples of parameters that can be employed in determining percent identity for polypeptides or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol.48:443-453.

[0084] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 or other number of contiguous amino acids of the target polypeptide.

[0085] The term “link” as used herein refers to the association via intramolecular interaction, e.g., covalent bonds, metallic bonds, and / or ionic bonding, or inter-molecular interaction, e.g., hydrogen bond or noncovalent bonds.

[0086] The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter that is operably linked to a coding sequence will direct the expression of the coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0087] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0088] The term “polynucleotide” or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said 27 4919-0796-2971, v.1modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2′,3′-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

[0089] The terms “polypeptide” or “protein” means a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally-occurring and non- recombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers. The terms “polypeptide” and “protein” specifically encompass LILRB antigen binding proteins, antibodies, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acid of antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein. In certain embodiments, fragments are about five to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of a LILRB-binding antibody, useful fragments include but are not limited to a CDR region, a variable domain of a heavy and / or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

[0090] The pharmaceutically acceptable carriers useful in this invention are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms) , conventional non-toxic solid carriers can include, for example, 28 4919-0796-2971, v.1pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically- neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0091] As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0092] The term “therapeutically effective amount” or “effective dosage” as used herein refers to the dosage or concentration of a drug effective to treat a disease or condition. For example, with regard to the use of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein to treat cancer, a therapeutically effective amount is the dosage or concentration of the monoclonal antibody or antigen-binding fragment thereof capable of reducing the tumor volume, eradicating all or part of a tumor, inhibiting or slowing tumor growth or cancer cell infiltration into other organs, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting or slowing tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination thereof.

[0093] “Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.

[0094] As used herein, a “vector” refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A vector may also include one or more therapeutic genes and / or selectable marker genes and other 29 4919-0796-2971, v.1genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like. II. LILRB5

[0095] Leukocyte immunoglobulin-like receptor subfamily B member 5, or LILRB5 (also known as CD85c and LIR-8), is encoded by the LILRB5 gene, found in a gene cluster at chromosomal region 19q13.4. It contains 2 copies of a cytoplasmic motif that is referred to as the immunoreceptor tyrosine-based inhibitor motif (ITIM). This motif is involved in modulation of cellular responses. The phosphorylated ITIM motif can bind the SH2 domain of several SH2-containing phosphatases. Several other LIR subfamily B receptors are expressed on immune cells where they bind to MHC class I molecules on antigen-presenting cells and inhibit stimulation of an immune response. Multiple transcript variants encoding different isoforms have been found for this gene.

[0096] In accordance with the present disclosure, the inventors propose the use of LILRB5 antibodies to block LILRB5’s immunosuppressive function in the treatment of diseases, such as hyperoliferative disease. While hyperproliferative diseases can be associated with any disease which causes a cell to begin to reproduce uncontrollably, the prototypical example is cancer. Examples of cancers can be generally categorized into solid tumors and hematologic malignancies. Solid tumors include but are not limited to, adrenal cancer, bile duct carcinoma, bone cancer, brain cancer (e.g., astrocytoma, brain stem glioma, craniopharyngioma, ependymoma, hemangioblastoma, medulloblastoma, meningioma, oligodendroglioma, spinal axis tumor), breast cancer (including acoustic neuroma, basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer (including Wilms tumor), liver cancer (including hepatocellular carcinoma (HCC)), lung cancer (including bronchogenic carcinoma, non-small cell lung cancer (squamous / non-squamous), bronchioloalveolar cell lung cancer, papillary adenocarcinomas), mesothelioma, melanoma, merkel cell cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pinealoma, prostate cancer, renal cell cancer, retinoblastoma, sarcoma (including chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, leiomyosarcoma, liposarcoma, myxosarcoma, osteogenic 30 4919-0796-2971, v.1sarcoma, rhabdomyosarcoma, synovial sarcoma), skin cancer (including basal cell carcinoma, sebaceous gland carcinoma, squamous cell carcinoma), testicular cancer (including seminoma), thymic carcinoma, thyroid cancer (e.g., medullary thyroid carcinoma, papillary thyroid carcinoma), uterine cancer, and vaginal cancer.

[0097] Hematologic malignancies include but are not limited to blastic plasmacytoid dendritic cell neoplasm (BPDCN), heavy chain disease, leukemias (including but not limited to acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML) (including but not limited to acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML), B-cell leukemia, chronic lymphoblastic leukemia (CLL), chronic myelomonocytic leukemia (CMML), chronic myelocytic leukemia (CML), pre-B acute lymphocytic leukemia (Pre-B ALL), diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma), lymphomas (including but not limited to Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Waldenstrom's macroglobulinemia), multiple myeloma (MM), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, and polycythemia vera.

[0098] Immunotherapy holds great promise to achieve long-lasting anti-tumor effects. Immune checkpoint PD-1 and CTLA-4 blockade therapies have been successful in treating some types of cancers but not others. These immunotherapies target inhibitory molecules on T cells to reactivate dysfunctional T cells within the tumor microenvironment (TME). Other populations of immune cells, including monocytic cells, are present in the TME in even larger numbers than T cells. In fact, monocyte-derived macrophages are the most abundant immune cell population in tumor tissues. While these innate cells possess the capacity to kill tumor cells and to prime or reactivate T cells, they become dysfunctional in TME and turn into MDSCs and tumor-associated macrophages (TAMs) that support tumor development and suppress immune surveillance and attack. MDSCs, including monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs), represent a heterogeneous population of immature myeloid cells that fail to terminally differentiate. TAMs are a mixed macrophage population in TME. They are anti-inflammatory and correlated with a poor prognosis. Despite their phenotypic plasticity, MDSCs and TAMs are defined by their immunosuppressive 31 4919-0796-2971, v.1function. Removing, reprogramming, or blocking trafficking of these immune-suppressive monocytic cells is becoming an attractive anti-cancer therapeutic strategy.

[0099] Autoimmune or inflammatory diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, systemic scleroderma, progressive systemic sclerosis (PSS), systemic sclerosis (SS), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus (SLE), Takayasu arteritis, temporal arteritis / giant cell arteritis, inflammatory bowel disease (IBD), ulcerative colitis, Cohn's disease, intestinal mucosal inflammation, wasting disease associated with colitis, uveitis, vitiligo and Wegener's granulomatosis, Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy, ventilator induced lung injury, viral infections, autoimmune diabetes and the like. Inflammatory disorders include, for example, chronic and acute inflammatory disorders. III. Monoclonal Antibodies and Production Thereof

[0100] The monoclonal antibodies described herein can be prepared using standard methods, followed by screening, characterization and functional assessment. Variable 32 4919-0796-2971, v.1regions can be sequenced and then subcloned into a human expression vector to produce the chimeric antibody genes, which are then expressed and purified. These chimeric antibodies can be tested for antigen binding, signaling blocking, and in xenograft experiments. The monoclonal antibodies described herein can also be prepared using phage display method, in which a large library of phage displayed human scFv is panned against the target protein. The human scFv selected to specifically binding to the target protein can be sequenced and then subcloned into a human expression vector to produce the desired human antibody. A. General Methods

[0101] It will be understood that monoclonal antibodies binding to LILRB5 will have several applications. These include the production of diagnostic kits for use in detecting and diagnosing cancer, as well as for cancer therapies. In these contexts, one may link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265).

[0102] The classical methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host. As is well known in the art, a given composition for immunization may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis- biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants and aluminum hydroxide adjuvant. 33 4919-0796-2971, v.1

[0103] The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and / or the animal can be used to generate MAbs.

[0104] Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human / mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp.65-66, 1986; Campbell, pp.75-83, 1984).

[0105] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v / v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp. 71-74, 1986). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10-6to 1 x 10-8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of 34 4919-0796-2971, v.1nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. Ouabain is added if the B cell source is an Epstein Barr virus (EBV) transformed human B cell line, in order to eliminate EBV transformed lines that have not fused to the myeloma.

[0106] The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain is also used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.

[0107] Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by 35 4919-0796-2971, v.1the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.

[0108] MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0109] It also is contemplated that a molecular cloning approach may be used to generate monoclonals. For this, RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 104times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

[0110] Recently, additional methods for generating mAb, such as scFv phage display, have been developed (see CM Hammers and JR Stanley, Antibody phage display: technique and applications, J Invest Dermatol (2014) 134: e17). Generally, a panel of human mAbs that bind to a target protein, e.g., human LILRB5, are generated by panning a large diversity of human scFv phage displayed antibody library.

[0111] To generate the human scFv phage displayed antibody library, RNA is extracted from the chosen cell source, e.g., peripheral blood mononuclear cells. The RNA is then reversed-transcribed into cDNA, which is used for PCR of the VH and VL chains of the 36 4919-0796-2971, v.1encoded antibodies. Defined sets of primers specific for the different VH and VL chain region gene families allow the amplification of all transcribed rearranged variable regions within a given immunoglobulin repertoire, reflecting all antibody specificities in a particular individual.

[0112] The VH and VL PCR products that represent the antibody repertoire are ligated into a phage display vector that is engineered to express the VH and VL as an scFv fused to the pIII minor capsid protein of a filamentous bacteriophage of E. coli that was originally derived from the M13 bacteriophage. This generates a library of phages, each of which expresses on its surface a scFv and harbors the vector with the respective nucleotide sequence within.

[0113] The library is then screened for phage binding to a target antigen through its expressed surface scFv by a technique called bio-panning. In short, the target protein is coated on solid phase for incubation with phage libraries. After washing and elution, antigen enriched phages are recovered and used for next rounds of phage panning. After at least three rounds of phage panning, single bacterial colonies are picked for phage ELISA and other functional / genetic analysis.

[0114] The positive hits are sequenced for the scFv region and are converted to full human IgG heavy and light chain constructs, which are used to generate the mAb of interest using the methods disclosed supra. For example, the IgG expressing plasmids are cotransfected into Expi293 cells using transfection reagent PEI. After 7 days of expression, supernatants are harvested, and antibodies are purified by affinity chromatography using protein A resin.

[0115] Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973 which describes antibody-therapeutic agent conjugates. B. Antibodies of the Present Disclosure 1. Antibodies to LILRB5

[0116] Antibodies or antigen-binding fragments thereof according to the present disclosure may be defined, in the first instance, by their binding specificity, which in this case is for LILRB5. Those of skill in the art, by assessing the binding specificity / affinity 37 4919-0796-2971, v.1of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims.

[0117] In one aspect, there are provided antibodies and antigen-binding fragments specifically bind to LILRB5. In some embodiments, when bound to LILRB5, such antibodies modulate the activation of LILRB5. In certain embodiments, the antibody or antigen-binding fragment, when bound to LILRB5, activates LILRB5. In certain embodiments, the antibody or antigen-binding fragment, when bound to LILRB5, suppresses activation of LILRB5. In certain embodiments, the antibody or antigen-binding fragment, when bound to LILRB5, can specifically interfere with, block or reduce the interaction between LILRB5 and its binding partners. In certain embodiments, the antibody or antigen-binding fragment provided herein is capable of inhibiting the immunosuppressive activity of MDSCs and other solid tumor-infiltrating myeloid cells, such as tumor-associated macrophages (TAMs) and tolerogenic dendritic cells (DCs). In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically or selectively bind to human LILRB5.

[0118] In some embodiments, the antibodies or antigen-binding fragments bind specifically to human LILRB5 and / or substantially inhibits binding of human LILRB5 to HLA- G, ANGPTLs, SEMA4A by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by an assay as disclosed in the Example). In some embodiments, the antibody or antigen-binding fragment has a Kd of less (binding more tightly) than 10−6, 10−7, 10−8, 10−9, 10−10, 10−11, 10−12, 10−13M. In some embodiments, the antibody or antigen-binding fragment has an IC50 for blocking the binding of HLA-G, ANGPTLs, SEMA4A to LILRB5 of less than 10 uM, 10 uM to 1 uM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM.

[0119] In some embodiments, the antibodies or antigen-binding fragments provided herein having the clone-paired CDRs illustrated in Table 2.

[0120] In certain embodiments, the antibodies may be defined by their variable sequence, which include additional “framework” regions. The antibody is characterized by clone-paired heavy chain and light chain amino acid sequences from Appendices I and III. Furthermore, the antibodies sequences may vary from these sequences, particularly in regions outside the CDRs. For example, the amino acids may vary from those set out above by a given 38 4919-0796-2971, v.1percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or the amino acids may vary from those set out above by permitting conservative substitutions (discussed below). Each of the foregoing apply to the amino acid sequences of Appendices I and III. In another embodiment, the antibody derivatives of the present disclosure comprise VL and VH domains having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions, while still exhibiting the desired binding and functional properties.

[0121] While the antibodies of the present disclosure were generated as IgG’s, it may be useful to modify the constant regions to alter their function. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Thus, the term “antibody” includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda. Within light and heavy chains, the variable and constant regions are joined by a 35 "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch.7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989).

[0122] The present disclosure further comprises nucleic acids which hybridize to nucleic acids encoding the antibodies disclosed herein. In general, the nucleic acids hybridize under moderate or high stringency conditions to nucleic acids that encode antibodies disclosed herein and also encode antibodies that maintain the ability to specifically bind to an LILRB5. A first nucleic acid molecule is “hybridizable” to a second nucleic acid molecule when a single stranded form of the first nucleic acid molecule can anneal to the second nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., MOLECULARCLONING: A LABORATORYMANUAL, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Typical moderate stringency hybridization conditions are 40% formamide, with 5X or 6X SSC and 0.1% SDS at 42°C. High stringency hybridization conditions are 50% formamide, 5X or 6X SSC (0.15M NaC1 and 0.015M Na-citrate) at 42°C or, optionally, at a higher temperature (e.g., 57°C, 59°C, 60°C, 62°C, 63°C, 65°C or 68°C). Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible. The 39 4919-0796-2971, v.1appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency under which the nucleic acids may hybridize. For hybrids of greater than 100 nucleotides in length, equations for calculating the melting temperature have been derived (see Sambrook et al., supra). For hybridization with shorter nucleic acids, e.g., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra). 2. Exemplary Epitopes and Competing Antigen Binding Proteins

[0123] In another aspect, the present disclosure provides epitopes to which anti- LILRB5 antibodies bind. In some embodiments, epitopes that are bound by the antibodies described herein are useful. In certain embodiments, an epitope provided herein can be utilized to isolate antibodies or antigen binding proteins that bind to LILRB5. In certain embodiments, an epitope provided herein can be utilized to generate antibodies or antigen binding proteins which bind to LILRB5. In certain embodiments, an epitope or a sequence comprising an epitope provided herein can be utilized as an immunogen to generate antibodies or antigen binding proteins that bind to LILRB5. In certain embodiments, an epitope described herein or a sequence comprising an epitope described herein can be utilized to interfere with biological activity of LILRB5.

[0124] In some embodiments, antibodies or antigen-binding fragments thereof that bind to any of the epitopes are particularly useful. In some embodiments, an epitope provided herein, when bound by an antibody, modulates the biological activity of LILRB5. In some embodiments, an epitope provided herein, when bound by an antibody, activates LILRB5. In some embodiments, an epitope provided herein, when bound by an antibody, suppress the activation of LILRB5. In some embodiments, an epitope provided herein, when bound by an antibody, block the interaction between LILRB5 and its binding partners.

[0125] In some embodiments, the domain(s) / region(s) containing residues that are in contact with or are buried by an antibody can be identified by mutating specific residues in LILRB5 and determining whether the antibody can bind the mutated LILRB5 protein. By making a number of individual mutations, residues that play a direct role in binding or that are in sufficiently close proximity to the antibody such that a mutation can affect binding between the antibody and antigen can be identified. From knowledge of these amino acids, the 40 4919-0796-2971, v.1domain(s) or region(s) of the antigen that contain residues in contact with the antigen binding protein or covered by the antibody can be elucidated. Such a domain can include the binding epitope of an antigen binding protein.

[0126] In another aspect, the present disclosure provides antigen-binding proteins that compete with one of the exemplified antibodies or antigen-binding fragment binding to the epitope described herein for specific binding to LILRB5. Such antigen binding proteins can also bind to the same epitope as one of the herein exemplified antibodies or the antigen-binding fragment, or an overlapping epitope. Antigen-binding proteins that compete with or bind to the same epitope as the exemplified antibodies are expected to show similar functional properties. The exemplified antibodies include those described above, including those with the heavy and light chain variable regions and CDRs included in Table 2, heavy and light chains as shown in Appendices I and III, and heavy and light chain coding regions as shown in Appendices II and IV. C. Engineering of Antibody Sequences

[0127] In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross- reactivity or diminished off-target binding. The following is a general discussion of relevant techniques for antibody engineering.

[0128] Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns. Recombinant full-length IgG antibodies may be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies collected a purified from the 293 or CHO cell supernatant.

[0129] The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using 41 4919-0796-2971, v.1pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g / L was achieved within 9 weeks of transfection.

[0130] Antibody molecules will comprise fragments (such as F(ab’), F(ab’)2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form “chimeric” binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule. 1. Antigen Binding Modifications

[0131] In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0132] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine 42 4919-0796-2971, v.1(+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0133] It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0134] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0135] The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.

[0136] Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. 2. Fc Region Modifications

[0137] The antibodies disclosed herein can also be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or effector function (e.g., antigen-dependent cellular cytotoxicity). Furthermore, the antibodies disclosed herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional 43 4919-0796-2971, v.1properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat. The antibodies disclosed herein also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Patent 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702. Such modification can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region.

[0138] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Patent 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent 6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patents 5,869,046 and 6,121,022. In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibodies. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patents 5,624,821 and 5,648,260.

[0139] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94 / 29351. In yet another example, 44 4919-0796-2971, v.1the Fc region is modified to increase or decrease the ability of the antibodies to mediate antibody dependent cellular cytotoxicity (ADCC) and / or to increase or decrease the affinity of the antibodies for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00 / 42072. Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described. Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcγRIII. Additionally, the following combination mutants were shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A.

[0140] In one embodiment, the Fc region is modified to decrease the ability of the antibodies to mediate effector function and / or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody to mediate effector function and / or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.

[0141] In one embodiment, the Fc region is modified to abolish the ability of the antibodies to mediate effector function by modifying residues 234, 235 and 329 to alanine or glycine (L234A-L235A-P329G).

[0142] In still another embodiment, the antibody comprises a particular glycosylation pattern. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). The glycosylation pattern of an antibody may be altered to, for example, increase the affinity or avidity of the antibody for an antigen. Such modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity or avidity of the antibody for antigen. See, e.g., U.S. Patents 5,714,350 and 6,350,861. 45 4919-0796-2971, v.1

[0143] An antibody may also be made in which the glycosylation pattern includes hypofucosylated or afucosylated glycans, such as a hypofucosylated antibodies or afucosylated antibodies have reduced amounts of fucosyl residues on the glycan. The antibodies may also include glycans having an increased amount of bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such modifications can be accomplished by, for example, expressing the antibodies in a host cell in which the glycosylation pathway was been genetically engineered to produce glycoproteins with particular glycosylation patterns. These cells have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α (1,6)- fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were created by the targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704. As another example, EP 1 176 195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the α-1,6 bond-related enzyme. EP 1176195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell. Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna (US Patent 7,632,983). Methods for production of antibodies in a plant system are disclosed in the U.S. Patents 6,998,267 and 7,388,081. PCT Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies.

[0144] Alternatively, the fucose residues of the antibodies can be cleaved off using a fucosidase enzyme; e.g., the fucosidase α-L-fucosidase removes fucosyl residues from 46 4919-0796-2971, v.1antibodies. Antibodies disclosed herein further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian- or human-like glycosylation patterns. A particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular N-glycan structure predominates (see, e.g., U.S. Patents 7,029,872 and 7,449,308). These genetically modified host cells have been used to produce antibodies that have predominantly particular N-glycan structures.

[0145] In addition, since fungi such as yeast or filamentous fungi lack the ability to produce fucosylated glycoproteins, antibodies produced in such cells will lack fucose unless the cells are further modified to include the enzymatic pathway for producing fucosylated glycoproteins (see for example, PCT Publication WO2008112092). In particular embodiments, the antibodies disclosed herein further include those produced in lower eukaryotic host cells and which comprise fucosylated and nonfucosylated hybrid and complex N-glycans, including bisected and multiantennary species, including but not limited to N-glycans such as GlcNAc(1- 4)Man3GlcNAc2; Gal(1-4)GlcNAc(1-4)Man3GlcNAc2; NANA(1-4)Gal(1-4)GlcNAc(1- 4)Man3GlcNAc2. In particular embodiments, the antibody compositions provided herein may comprise antibodies having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In particular aspects, the hybrid N-glycan is the predominant N-glycan species in the composition. In further aspects, the hybrid N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the hybrid N-glycans in the composition.

[0146] In particular embodiments, the antibody compositions provided herein comprise antibodies having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In particular aspects, the complex N-glycan is the predominant N-glycan species in the composition. In further aspects, the complex N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N- 47 4919-0796-2971, v.1glycans in the composition. In particular embodiments, the N-glycan is fusosylated. In general, the fucose is in an α1,3-linkage with the GlcNAc at the reducing end of the N-glycan, an α1,6- linkage with the GlcNAc at the reducing end of the N-glycan, an α1,2-linkage with the Gal at the non-reducing end of the N-glycan, an α1,3-linkage with the GlcNac at the non-reducing end of the N-glycan, or an α1,4-linkage with a GlcNAc at the non-reducing end of the N-glycan.

[0147] Therefore, in particular aspects of the above the glycoprotein compositions, the glycoform is in an α1,3-linkage or α1,6-linkage fucose to produce a glycoform selected from the group consisting of Man5GlcNAc2(Fuc), GlcNAcMan5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc); in an α1,3-linkage or α1,4-linkage fucose to produce a glycoform selected from the group consisting of GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fuc1-2)Man3GlcNAc2, GalGlcNAc2(Fuc1- 2)Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc1- 2)Man3GlcNAc2, and NANA2Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2; or in an α1,2-linkage fucose to produce a glycoform selected from the group consisting of Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2, NANAGal2(Fuc1- 2)GlcNAc2Man3GlcNAc2, and NANA2Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2.

[0148] In further aspects, the antibodies comprise high mannose N-glycans, including but not limited to, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or N-glycans that consist of the Man3GlcNAc2 N-glycan structure. In further aspects of the above, the complex N-glycans further include fucosylated and non-fucosylated bisected and multiantennary species. As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to an N-linked oligosaccharide, for example, one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein. D. Single Chain Antibodies

[0149] A Single Chain Variable Fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original 48 4919-0796-2971, v.1immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

[0150] Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 × 106different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VHC terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.

[0151] The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin / avidin, which permit the combination of two antibodies.

[0152] In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked 49 4919-0796-2971, v.1together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).

[0153] Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

[0154] An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross- linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).

[0155] It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic / preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.

[0156] Another cross-linking reagent is SMPT, which is a bifunctional cross- linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.

[0157] The SMPT cross-linking reagent, as with many other known cross- linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide 50 4919-0796-2971, v.1bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3'-dithiopropionate. The N- hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

[0158] In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0159] U.S. Patent 4,680,338 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and / or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.

[0160] U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques. E. Purification

[0161] In certain embodiments, the antibodies of the present disclosure may be purified. The term “purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally- obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition. 51 4919-0796-2971, v.1

[0162] Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.

[0163] In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[0164] Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies is bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.).

[0165] Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the 52 4919-0796-2971, v.1particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

[0166] It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS / PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. V. Treatment of Cancer A. Formulation and Administration

[0167] The present disclosure provides pharmaceutical compositions comprising anti-LILRB antibodies and antigens for generating the same. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0168] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified 53 4919-0796-2971, v.1form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

[0169] Antibodies of the present disclosure, as described herein, can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intra-arterial, intramuscular, subcutaneous, intra-tumoral or even intraperitoneal routes. The antibodies could alternatively be administered by a topical route directly to the mucosa, for example by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0170] Passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.

[0171] Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. 54 4919-0796-2971, v.1

[0172] The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. B. Cell Therapies

[0173] In another aspect, the present disclosure provides immune cells which express a chimeric antigen receptor (CAR). In some embodiment, The CAR comprises an antigen-binding fragment provided herein. In an embodiment, the CAR protein includes from the N-terminus to the C-terminus: a leader peptide, an anti-LILRB5 heavy chain variable domain, a linker domain, an anti-LILRB5 light chain variable domain, a human IgG1–CH2- CH3 domain, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular co- stimulatory signaling and a CD3 ζ intracellular T cell signaling domain.

[0174] Also provided are methods for immunotherapy comprising administering an effective amount of the immune cells of the present disclosure. In one embodiment, a medical disease or disorder is treated by transfer of an immune cell population that elicits an immune response. In certain embodiments of the present disclosure, cancer or infection is treated by transfer of an immune cell population that elicits an immune response. Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of an antigen-specific cell therapy.

[0175] The immune cells may be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, NKT cells, or macrophages. Also provided herein are methods of producing and engineering the immune cells as well as methods of using and administering the cells for adoptive cell therapy, in which case the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, such as to target cancer cells.

[0176] The immune cells may be isolated from subjects, particularly human subjects. The immune cells can be obtained from healthy human subjects, healthy volunteers, or healthy donors. The immune cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a 55 4919-0796-2971, v.1predisposition to a particular disease or condition, or a subject who is undergoing therapy for a particular disease or condition. Immune cells can be collected from any location in which they reside in the subject including, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells may be used directly, or they can be stored for a period of time, such as by freezing.

[0177] The immune cells may be enriched / purified from any tissue where they reside including, but not limited to, blood (including blood collected by blood banks or cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. Tissues / organs from which the immune cells are enriched, isolated, and / or purified may be isolated from both living and non- living subjects, wherein the non-living subjects are organ donors. In particular embodiments, the immune cells are isolated from blood, such as peripheral blood or cord blood. In some aspects, immune cells isolated from cord blood have enhanced immunomodulation capacity, such as measured by CD4- or CD8-positive T cell suppression. In specific aspects, the immune cells are isolated from pooled blood, particularly pooled cord blood, for enhanced immunomodulation capacity. The pooled blood may be from 2 or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0178] The population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject in need of therapy. Alternatively, the population of immune cells can be obtained from a donor, preferably a histocompatibility matched donor. The immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells reside in said subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, such as from pooled cord blood.

[0179] When the population of immune cells is obtained from a donor distinct from the subject, the donor is preferably allogeneic, provided that the cells obtained are subject- compatible in that they can be introduced into the subject. Allogeneic donor cells may or may not be human-leukocyte-antigen (HLA)-compatible. To be rendered subject-compatible, allogeneic cells can be treated to reduce immunogenicity. 56 4919-0796-2971, v.1

[0180] The immune cells can be genetically engineered to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, the host cells (e.g., autologous or allogeneic T-cells) are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen. In particular embodiments, NK cells are engineered to express a TCR. The NK cells may be further engineered to express a CAR. Multiple CARs and / or TCRs, such as to different antigens, may be added to a single cell type, such as T cells or NK cells.

[0181] Suitable methods of modification are known in the art. See, for instance, Sambrook et al., supra; and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and John Wiley & Sons, NY, 1994. For example, the cells may be transduced to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen using transduction techniques described in Heemskerk et al. (2008) and Johnson et al. (2009).

[0182] In some embodiments, the cells comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric). C. Combination Therapies

[0183] It may also be desirable to provide combination treatments using antibodies of the present disclosure in conjunction with additional anti-cancer therapies. These therapies would be provided in a combined amount effective to achieve a reduction in one or more disease parameter. This process may involve contacting the cells / subjects with the both agents / therapies at the same time, e.g., using a single composition or pharmacological formulation that includes both agents, or by contacting the cell / subject with two distinct 57 4919-0796-2971, v.1compositions or formulations, at the same time, wherein one composition includes the antibody and the other includes the other agent.

[0184] Alternatively, the antibody may precede or follow the other treatment by intervals ranging from minutes to weeks. One would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapies would still be able to exert an advantageously combined effect on the cell / subject. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay time of only about 12 hours. In some situations, it may be desirable to extend the time period for treatment significantly; however, where several 10 days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0185] It also is conceivable that more than one administration of either the anti- LILRB5 antibody or the other therapy will be desired. Various combinations may be employed, where the antibody is “A,” and the other therapy is “B,” as exemplified below: A / B / A B / A / B B / B / A A / A / B B / A / A A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B B / B / B / A A / A / A / B B / A / A / A A / B / A / A A / A / B / A A / B / B / B B / A / B / B B / B / A / B

[0186] Other combinations are contemplated. To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present invention, one may contact a target cell or site with an antibody and at least one other therapy. These therapies would be provided in a combined amount effective to kill or inhibit proliferation of cancer cells. This process may involve contacting the cells / site / subject with the agents / therapies at the same time.

[0187] Particular agents contemplated for combination therapy with antibodies of the present disclosure include chemotherapy and hematopoietic stem cell transplantation. Chemotherapy may include cytarabine (ara-C) and an anthracycline (most often daunorubicin), high-dose cytarabine alone, all-trans-retinoic acid (ATRA) in addition to induction chemotherapy, usually an anthracycline, histamine dihydrochloride (Ceplene) and interleukin 2 (Proleukin) after the completion of consolidation therapy, gemtuzumab ozogamicin (Mylotarg) for patients aged more than 60 years with relapsed AML who are not candidates for high-dose chemotherapy, clofarabine, as well as targeted therapies, such as kinase 58 4919-0796-2971, v.1inhibitors, farnesyl transferase inhibitors, decitabine, and inhibitors of MDR1 (multidrug- resistance protein), or arsenic trioxide or relapsed acute promyelocytic leukemia (APL).

[0188] In certain embodiments, the agents for combination therapy are one or more drugs selected from the group consisting of a topoisomerase inhibitor, an anthracycline topoisomerase inhibitor, an anthracycline, a daunorubicin, a nucleoside metabolic inhibitor, a cytarabine, a hypomethylating agent, a low dose cytarabine (LDAC), a combination of daunorubicin and cytarabine, a daunorubicin and cytarabine liposome for injection, Vyxeos®, an azacytidine, Vidaza®, a decitabine, an all-trans-retinoic acid (ATRA), an arsenic, an arsenic trioxide, a histamine dihydrochloride, Ceplene®, an interleukin-2, an aldesleukin, Proleukin®, a gemtuzumab ozogamicin, Mylotarg®, an FLT-3 inhibitor, a midostaurin, Rydapt®, a clofarabine, a farnesyl transferase inhibitor, a decitabine, an IDH1 inhibitor, an ivosidenib, Tibsovo®, an IDH2 inhibitor, an enasidenib, Idhifa®, a smoothened (SMO) inhibitor, a glasdegib, an arginase inhibitor, an IDO inhibitor, an epacadostat, a BCL-2 inihbitor, a venetoclax, Venclexta®, a platinum complex derivative, oxaliplatin, a kinase inhibitor, a tyrosine kinase inhibitor, a PI3 kinase inhibitor, a BTK inhibitor, an ibrutinib, IMBRUVICA®, an acalabrutinib, CALQUENCE®, a zanubrutinib, a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG3 antibody, an ICOS antibody, a TIGIT antibody, a TIM3 antibody, a CD40 antibody, a 4-1BB antibody, a CD47 antibody, a SIRP1α antibody or fusions protein, a CD70 antibody, and CLL1 antibody, a CD123 antibody, an antagonist of E-selectin, an antibody binding to a tumor antigen, an antibody binding to a T-cell surface marker, an antibody binding to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an alkaloid derived from a plant, a hormone therapy medicine, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor. VI. Antibody Conjugates

[0189] Antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or 59 4919-0796-2971, v.1polynucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

[0190] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Antibody–drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in “armed” MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013 by FDA validated the approach. There are currently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization are becoming more and more mature, the discovery and development of new ADCs are increasingly dependent on the identification and validation of new targets that are suitable to this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated / high levels of expression in tumor cells and robust internalization.

[0191] Antibody conjugates are also preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging." Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.

[0192] In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III), with gadolinium being particularly 60 4919-0796-2971, v.1preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).

[0193] In the case of radioactive isotopes for therapeutic and / or diagnostic application, one might mention astatine211,14carbon,51chromium,36chlorine,57cobalt,58cobalt, copper67,152Eu, gallium67,3hydrogen, iodine123, iodine125, iodine131, indium111,59iron,32phosphorus, rhenium186, rhenium188,75selenium,35sulphur, technicium99mand / or yttrium90.125I is often being preferred for use in certain embodiments, and technicium99mand / or indium111are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present disclosure may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and / or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the disclosure may be labeled with technetium99mby ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).

[0194] Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red.

[0195] Another type of antibody conjugate contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and / or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such 61 4919-0796-2971, v.1labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.

[0196] Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.

[0197] Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and may be used as antibody binding agents.

[0198] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4- hydroxyphenyl)propionate.

[0199] In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and 62 4919-0796-2971, v.1sensitivity (U.S. Patent 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O’Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation. VII. Immunodetection Methods

[0200] In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting LILRB-related cancers. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine and other virus stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of H1 antigens in viruses. Alternatively, the methods may be used to screen various antibodies for appropriate / desired reactivity profiles.

[0201] Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of LILRBs also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample suspected of containing LILRB-related cancers and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0202] These methods include methods for detecting or purifying LILRBs or LILRB-related cancer cells from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the LILRB-related cancer cells will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the LILRB-expressing cells immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column. 63 4919-0796-2971, v.1

[0203] The immunobinding methods also include methods for detecting and quantifying the amount of LILRB-related cancer cells or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing LILRB-related cancer cells and contact the sample with an antibody that binds LILRBs or components thereof, followed by detecting and quantifying the amounts of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing LILRB-related cancers, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid, including blood and serum, or a secretion, such as feces or urine.

[0204] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to LILRBs. After this time, the sample- antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0205] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art.

[0206] The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an 64 4919-0796-2971, v.1antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0207] Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[0208] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0209] Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody 65 4919-0796-2971, v.1complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule. 1. ELISAs

[0210] Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

[0211] In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the LILRB-related cancer cells is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-LILRB antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection may also be achieved by the addition of a second anti-LILRB5 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0212] In another exemplary ELISA, the samples suspected of containing the LILRB5-related cancer cells are immobilized onto the well surface and then contacted with the anti- LILRB5 antibodies of the disclosure. After binding and washing to remove non- specifically bound immune complexes, the bound anti-LILRB5 antibodies are detected. Where the initial anti-LILRB5 antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-LILRB5 antibody, with the second antibody being linked to a detectable label.

[0213] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below. 66 4919-0796-2971, v.1

[0214] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0215] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[0216] “Under conditions effective to allow immune complex (antigen / antibody) formation” means that the conditions preferably include diluting the antigens and / or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0217] The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C or may be overnight at about 4°C or so.

[0218] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined. 67 4919-0796-2971, v.1

[0219] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0220] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer. 2. Western Blot

[0221] The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native / non- denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0222] Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing. 68 4919-0796-2971, v.1

[0223] The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

[0224] In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies. 3. Immunohistochemistry

[0225] The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic 69 4919-0796-2971, v.1factors and is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0226] Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and / or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings.

[0227] Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted. 4. Immunodetection Kits

[0228] In even further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect LILRB-related cancer cells, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to an LILRB, and optionally an immunodetection reagent.

[0229] In certain embodiments, the antibody may be pre-bound to a solid support, such as a column matrix and / or well of a microtitre plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0230] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second 70 4919-0796-2971, v.1antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

[0231] The kits may further comprise a suitably aliquoted composition of LILRBs, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

[0232] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. 5. Flow Cytometry and FACS

[0233] The antibodies of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser- or impedance-based technology employed in many detection assays, including cell counting, cell sorting, biomarker detection and protein engineering. The technology suspends cells in a stream of fluid and passing them through an electronic detection apparatus, which allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis disorders, especially blood cancers, but has many other applications in basic research, clinical practice and clinical trials.

[0234] Fluorescence-activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogenous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescent characteristics of each cell. In general, the technology involves a cell suspension entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescence of each cell is 71 4919-0796-2971, v.1measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based immediately prior to fluorescence intensity being measured, and the opposite charge is trapped on the droplet as it breaks form the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge.

[0235] In certain embodiments, to be used in flow cytometry or FACS, the antibodies of the present disclosure are labeled with fluorophores and then allowed to bind to the cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine. VIII. Examples

[0236] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 Materials & Methods

[0237] Mice. Wild-type (WT) C57BL / 6J mice were purchased from and maintained at the animal core facility of the University of Texas Southwestern (UTSW) Medical Center. LILRB5 complementary DNA was inserted into the pR26 expression plasmid via AsiSI / MluI. The plasmid was purified, and the absence of endotoxin was confirmed. Cas9 mRNA, guide RNA targeting the mouse Rosa26 locus and the LILRB5 plasmid were co- injected into mouse oocytes at the Transgenic Core Facility of UTSW. The LILRB5-positive mice were identified using LILRB5-specific primers and crossbred with LysM-Cre mice (JAX, 004781). Co-expression of LILRB5, CD11b, Ly6C, and Ly6G was confirmed in peripheral blood and tissue. Mice in each experiment were sex and age matched. All work in this study was approved by the UTSW Institutional Animal Care and Use Committee. 72 4919-0796-2971, v.1

[0238] Cell culture. 293T cells and MC38 mouse colon cancer cells were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. THP- 1 cells, a human AML cell line, and LILRB reporter cells were cultured in RPMI-1640 with 10% FBS and 1% penicillin / streptomycin. Primary human monocytes and T cells were isolated by autoMACS or magnetic bead-based separation (Miltenyi Biotec, 130-097-043 and 130-050- 201, respectively) and cultured in RPMI-1640 with 10% FBS ang 1% penicillin / streptomycin. Mycoplasma testing was performed using the Lookout Mycoplasma PCR Detection Kit (Sigma, MP0035-1KT).

[0239] Primary human samples. The primary human solid cancer peripheral blood samples were obtained through UTSW Tissue Management Shared Resource. Informed consent was obtained, and experiments were approved by the Institutional Review Board of the UTSW Medical Center (IRB STU 122013-023). The samples were distributed to the laboratory in a de-identified fashion, and the study was not considered human research.

[0240] Plasmids. LILRB5 cDNA (Uniprot Entry O75023) was cloned into the pR26 expression vector and the pLVX lentivirus expression vector. The EphA7 sequence (Uniprot Entry Q15375) was codon optimized for expression and synthesized (Genewiz), and EphB1 (Accession NP_004432) was cloned from a cDNA plasmid (R&D Systems, RDC0501) into pLVX lentivirus expression vector under the regulation of an Ef1α promoter. Src, Lck, and Frk cDNAs were purchased from UTSW Center for Human Genetics and cloned into an expression vector with a HA tag. Fyn (HG12346-UT), Lyn (HG10829 CY), Yes (HG16953- CY), and Blk (HG10782-CY) cDNAs were purchased from Sino Biologicals and cloned into an expression vector with a HA tag.

[0241] Generation of LILRB5 antibody. A complete human scFv phage library was generated in-house and used for LILRB5 antibody panning. Briefly, human LILRB5 extracellular domain protein was coated onto wells of a 96-well plate, a pre-blocked phage library was added to LILRB5-coated wells, and samples were incubated for 2 hours at room temperature. Wells were washed to remove unbound phage, and bound phages were eluted and used to infect TG1 bacteria for amplification. The panning process was repeated to enrich for high-affinity binders. Sequences of phage bound to LILRB5 were analyzed using GeneBank IgBLAST1.10.0 to identify germline V(D)J gene segments. Individual VHand VL genes were mapped to the germline of major IGL and IGH loci. Framework and CDR sequences were annotated according to IMGT (imgt.org) nomenclature. The VH- and VL- 73 4919-0796-2971, v.1encoding genes from the phage plasmids were cloned into a human IgG-expressing vector. DNA fragments encoding VHand VLwere amplified by PCR using family-leader, region- specific primers. The PCR product of VH and VL genes, of around 400 bp, was collected and purified, and infusion PCR was carried out using the In-Fusion HD Cloning kit (Clontech). Human anti-LILRB5 antibodies were expressed in HEK293F cells and purified using affinity chromatography with Protein A resin. Equal molar amounts of heavy-chain and light-chain plasmids were co-transfected into HEK293F cells for transient expression of antibodies. Supernatants were harvested after 7 days in culture, and IgGs were purified with Protein A resin (GE Healthcare). Antibody affinities were determined as described previously (Gui et al., 2019) using the Octet RED96 instrument. Antibody (30 mg / mL) was loaded on to the Protein A biosensors, then exposed to a series of concentrations of recombinant LILRB5 (3.1-200 nM), and background subtraction was used to correct for sensor drift. FortéBio’s data analysis software was used to extract association and dissociation rates assuming a 1:1 binding model. The dissociation constant KD was calculated as the ratio of kd:ka. Four anti-LILRB5 antibodies were used in this study, anti-LILRB5 #1 (B5-1), #2 (B5-2), #27 (B5-27), and #29 (B5-29).

[0242] Virus production and infection. For lentivirus production, plasmids were mixed with psPAX2 and pMD2G at a ratio of 4:3:1 and transfected into 293T cells using Polyjet (SignaGen). After 72 hours, the viral supernatant was filtered through 0.45-μm filters and centrifuged onto cancer cell lines in the presence of 8 μg / mL polybrene. Clones were selected using 10 μg / mL puromycin, and cells were sorted using a FACS Melody.

[0243] 293T reconstituted signaling. A total of 1 x 106293T cells were plated into 6-well tissue-culture treated plates. After 24 hours, 1.4 μg of plasmid DNA (1 μg LILRB5- FLAG or LILRB5-FLAG ITIM mutant and 400 ng Src) were transfected into one set of cells, and 1 μg of EphA7 or EphB1 plasmid were transfected into another set of cells using Polyjet (SignaGen). After 48 hours, the 293T-LILRB5 cells were co-cultured with the 293T- EphA7 / EphB1 cells for 30 minutes. Cells were washed with PBS. Ice-cold IP Lysis Buffer (1% NP-40, 0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA) containing protease inhibitor (Sigma, 11836153001) and phosphatase inhibitor (Sigma, 4906837001) was added, and the samples analyzed by western blot.

[0244] THP1-LILRB5 signaling. THP-1 cells that express LILRB5 were serum-starved (RPMI-1640 media only) for 20 hours prior to signaling experiments. Cells were collected by centrifugation and resuspended in ice-cold PBS. Equivalent numbers of cells (8 x 74 4919-0796-2971, v.1106) were added to flat-bottom plates coated with anti-CD64 (R&D Systems, MAB1257), recombinant EphA7 (Sino Biological, 11657-H08H), or recombinant EphB1 (Sino Biological, 11963-H08H). Cells were centrifuged onto the plate at 300x g for 1 minute at 4 °C. Plates were placed into a humidified 37 °C tissue-culture incubator. After 10 minutes, ice-cold IP Lysis Buffer containing protease inhibitor (Sigma, 11836153001) and phosphatase inhibitor (Sigma, 4906837001) was added, and the samples analyzed by western blot.

[0245] Western blot and co-immunoprecipitation. Cells were lysed with IP Lysis Buffer containing protease inhibitor (Sigma, 11836153001) and phosphatase inhibitor (Sigma, 4906837001). The LILRB5-Flag complex was immunoprecipitated with anti-FLAG M2 magnetic agarose (Thermo Fisher Scientific, A36797) per the manufacturer’s protocol. Samples were mixed with 4x LDS loading buffer (Thermo Fisher Scientific, NP0007) containing 5% BME, boiled at 95 °C for 10 minutes, and separated by reducing SDS-PAGE. After transfer to nitrocellulose membranes (Biorad Trans-Blot Transfer System), membranes were blocked with 5% dry milk diluted in TBST. Proteins were detected with specific primary antibodies and HRP-conjugated secondary antibodies.

[0246] Flow cytometry. Cells were washed in FACS medium (PBS containing 0.1% BSA, 2 mM EDTA, 1% penicillin / streptomycin), blocked with human IgG for 15 minutes at 4 °C and stained with flow cytometry antibodies for at least 30 minutes at 4 °C. Data were collected using a FACS Calibur, a FACS Melody, or a Cytek Northern Lights. Zombie yellow or propidium iodide staining was used to exclude dead cells. Flow data were analyzed using Flowjo v10. Anti-LILRB5 antibody was labeled using a site-specific PE conjugation kit (ThermoFisher Scientific, S10467) for use in flow cytometry.

[0247] Octet binding kinetics. The binding kinetics of LILRB5 to EphA7 and to EphB1 were analyzed using surface interferometry (Octet Red 384, FortéBio). The inventors immobilized 100 nM of LILRB5-ECD-Fc on pre-equilibrated Protein-A biosensors (Sartorius, 18-5010) in 1x kinetics buffer (Sartorius, 18-1105). The biosensors were then dipped in varying concentrations of EphA7-His or EphB1-His proteins. The association and dissociation steps for EphA7-His binding were 420 seconds, whereas for EphB1-His binding these steps were 100 seconds. The kinetic curves were fitted to a 1:1 binding model to calculate the KD, ka, and kdusing the Octet System Data Analysis Software (FortéBio). Double referencing, which used both the reference well and reference sensor, was used to account for non-specific binding. 75 4919-0796-2971, v.1

[0248] Chimeric reporter cell assay. Briefly, proteins were plated on 96-well plates at 37 °C overnight. After removing the remaining soluble protein, wells were washed with PBS at least twice. Next, 5 x 104LILRB reporter cells developed as the inventors previously described (Deng et al., 2018) were seeded into each well. When anti-LILRB5 was used to block reporter cell activation, 20 μg / mL human IgG control antibody or anti-LILRB5 antibody was added into culture media. After culture for at least 16 hours, the percentage of GFP+reporter cells was determined by flow cytometry.

[0249] M2 macrophage polarization. The protocol for M2 macrophage differentiation was performed essentially as described with minor modifications (Zhang et al., 2013; Buchacher et al., 2015). CD14+cells (1 x 105per well of a 96-well plate) were cultured in DMEM with 10% FBS supplemented with 100 ng / mL M-CSF (Sino Biological, 11792- H08H-20) for 6 days. Medium was replaced every 3 days, and 20 ng / mL human IL-4 (Sino Biological, 11846-HNAE-1) was added on day 3. Polarization started on day 6 by supplementation of the medium with 100 ng / mL lipopolysaccharide (Sigma, L4516) and lasted for another 48 hours. Antibody and recombinant protein were added each time medium was replaced.

[0250] ELISA. The inventors followed the manufacturer’s protocols for the Human IL-8 ELISA MAXTMDeluxe Set (431504) in the experiments. Briefly, one day prior to running the ELISA, human IL-8 capture antibodies were diluted in 1x coating buffer A and added to 96 well plate. Plates were washed with wash buffer between each step and incubated subsequently with 1x assay diluent A, undiluted cell culture medium, detection antibody, avidin-HRP, substrate solution C, and stop solution. Absorbance at 450 nm and 570 nm were measured using a 96 well plate reader (BioTek Synergy LX). Absorbance at 570 nm was subtracted from absorbance at 450 nm and human IL-8 concentration was calculated based on a standard curve.

[0251] MTS proliferation assay. 1 x 104MC38 cells in 100 μL media were seeded onto individual wells of a 96 well flat bottom culture plate. Cells were cultured in DMEM medium with 10% FBS and 1% penicillin / streptomycin. At each timepoint, 20 μL CellTiter 96®AQueous One Solution Reagent (Promega, G3582) were added to each well. The cells were incubated at 37 °C for 1 hour and the absorbance at 490 nm was recorded using a 96 well plate reader (BioTek Synergy LX). 76 4919-0796-2971, v.1

[0252] Tumor experiments in LILRB5-transgenic mice. Tumor cells (5 x 105MC38-Vector, MC38-EphA7, or MC38-EphB1) were injected subcutaneously into the right flank of each mouse (WT C57BL / 6 or LILRB5-transgenic) at day 0. Starting from day 3, 200 μg human IgG control antibody or anti-LILRB5 antibody was injected intraperitoneal twice weekly. Tumor sizes were measured twice per week. Mice were euthanized when tumors reached maximum allowed limits (2000 mm3). Tumors were removed and digested in 200 ug / mL DNase and 1 mg / mL collagenase in 37 °C for 1 hour. Samples were filtered through a 70-μm cell strainer (Fisherbrand, 22363548). Spleens were mechanically ground and filtered through a 70-μm cell strainer. Cells were subjected to zombie yellow staining, human IgG blocking, staining with appropriate antibodies, and flow cytometry.

[0253] Statistics. Statistical significance of differences was assessed using the two-tailed Student’s t-test. A p value of 0.05 or less was considered significant, and significant differences are labeled with asterisks. Values are reported as means ± standard deviations. Results

[0254] Several Eph receptors bind to LILRB5. A genome-wide screen of membrane proteins for those that bind to LILRB5 indicated that more than one Eph receptor interacts with LILRB5. The inventors employed multiple assays to confirm this finding and to determine whether Eph receptors activate LILRB5. First, they developed chimeric LILRB5 receptor reporter cells as a sensitive system to detect functional interactions of candidate binding proteins with LILRB5 as the inventors described for other LILRB and LAIR1 reporter cells (Chen et al., 2020; Deng et al., 2014; Deng et al., 2018; Kang et al., 2015; Gui et al., 2019). The inventors found that EphA2, EphA4, EphA7, and EphB1 activated LILRB2 and LILRB5 reporter cells (Figs. 1A-B) but not LILRB1, LILRB3, LILRB4, or LAIR1 reporter cells (Figs.14A-D). Next, the inventors expressed EphA2, EphA4, EphA7, or EphB1 and full- length LILRB5 on 293T cells. They co-cultured the Eph or control cells with LILRB5- expressing cells and evaluated interactions using flow cytometry. When co-cultured with control cells that do not express an Eph, the FLAG+ / FLAG- ratio was 1.23. In co-cultures with cells that express EphA4, EphA7, EphA2, and EphB1, the ratios were 18.03, 9.05, 1.79, and 2.82, respectively (Fig.1C). Cells that expressed EphA2 and EphA4 did not bind to cells that expressed LILRB5-d1, which had deletions of D1 and D2 domains of the extracellular domain (ECD), whereas EphA7 and EphB1 had weaker binding to LILRB5-d1. No binding was detected to cells that expressed LILRB5-d2, which lacks D1-4 domains of the ECD.293T cells 77 4919-0796-2971, v.1that expressed EphA7 or EphB1 robustly bound to cells that expressed either LILRB2 or LILRB5 (Fig. 1D). Given that LILRB5 displayed strong binding to EphA7 and EphB1 and given that the functions of LILRB5 are not as well characterized as those of LILRB2, the inventors focused on LILRB5 in subsequent experiments. Truncations of the ECDs of EphB1 and EphA7 showed that these domains were essential for LILRB5 binding (Figs.1E-F). Using bio-layer interferometry, they determined that the KDvalues of the interactions of LILRB5 with EphA7 and with EphB1 were 564 nM and 61 nM, respectively (Fig. 1G). Together, these results indicate that Eph receptors EphA7 and EphB1 bind to LILRB5.

[0255] LILRB5 antibodies blocked ligand interaction with LILRB5. To develop fully human anti-LILRB5 blocking antibodies, the inventors screened a highly diverse naive scFv phage library through multiple panning rounds with increased stringency to select phages that bound to the LILRB5 ECD. Unique scFv sequences were converted to fully humanized IgG format. Bio-layer interferometry was employed to assess the affinity between anti-LILRB5 antibodies and LILRB5. Anti-LILRB5 antibodies #1 (B5-1), #27 (B5-27), and #29 (B5-29) had KD values of 6.02 x 10-9M, 3.22 x 10-9M, and 5.44 x 10-10M, respectively (Fig.2A-C). Anti-LILRB5 antibodies #1 (B5-1) and #27 (B5-27) had equivalent efficacies in ELISA assays against LILRB5 with half-maximal effective concentrations (EC50s) of 0.03 µg / mL (Fig. 2D-E). The inventors conjugated anti-LILRB5 #1 (B5-1) with R-phycoerythrin (R-PE) and confirmed through flow cytometry that this antibody conjugate specifically stains LILRB5 reporter cells but not other LILRB / relative reporter cells (Fig. 2F). To determine whether these antibodies inhibit the function of LILRB5, the inventors evaluated the effects of anti-LILRB5 antibodies on the activation of LILRB5 reporter cells by Eph receptors. The anti- LILRB5 antibodies (B5-2 mAb) completely inhibited the activation induced by EphA2, EphA4, EphA7, and EphB1, whereas human IgG had no effect (Fig.2G). Thus, the inventors identified high-affinity, specific antibodies capable of blocking LILRB5 function.

[0256] Eph receptors and LILRB5 binding induces bi-directional signaling. The inventors next sought to determine whether the interaction of Eph receptors and LILRB5 leads to downstream signaling. In 293T cells transfected with LILRB5-FLAG and individual Src family kinases, they found that Src, Yes, Blk, and Lyn can phosphorylate LILRB5 at tyrosine residues in the ITIMs (Fig. 3A). Mutating the tyrosines in the ITIM domains of LILRB5 to phenylalanines singly or simultaneously completely abolished the phosphorylation signal, demonstrating the functional importance of both ITIM domains of 78 4919-0796-2971, v.1LILRB5 in mediating downstream signaling (Fig. 3B). To study whether Eph receptors can induce LILRB5 signaling, the inventors co-cultured 293T cells that express LILRB5-FLAG and Src kinase with 293T cells that express EphA7 or EphB1. Co-immunoprecipitation using FLAG specific beads demonstrated that after 30 minutes of co-culture, expression of either EphA7 or EphB1 increased LILRB5 phosphorylation (Figs.3C-D). In addition, the inventors constructed THP-1 cells that stably express LILRB5-FLAG and showed that immobilized EphA7 or EphB1 stimulated the phosphorylation of LILRB5; this phosphorylation was blocked by anti-LILRB5 antibody (B5-2 mAb) (Figs.3E-F).

[0257] To investigate whether the Eph receptor-LILRB5 interaction can induce bi-directional signaling, the inventors treated 293T cells that express EphA7-FLAG or EphB1- FLAG with the recombinant ECD of LILRB5 (LILRB5-ECD). LILRB5-ECD induced phosphorylation of EphA7 and of EphB1, as did incubation with the positive control EphrinA5- ECD (Figs. 3G-H). In sum, these results show that EphA7 or EphB1 bind to LILRB5 and induce its phosphorylation and vice versa. Thus, there is bi-directional signaling between these Eph receptors and LILRB5.

[0258] LILRB5 is expressed on monocytes and neutrophils. To study the functions of LILRB5 in humans, the inventors sought to clarify its expression pattern. They conjugated LILRB5-specific antibodies (B5-27 mAb) with R-PE for use in flow cytometry. Peripheral blood samples were collected from both healthy donors and cancer patients. The inventors observed that LILRB5 was expressed on CD14+monocytes and CD66a+neutrophils, but it was absent on lymphocytes including CD3+T cells, CD19+B cells, and CD56+NK cells (Fig. 4A). Further classification of monocytes into CD14+ / CD16- classical monocytes, CD14+ / CD16+intermediate monocytes, and CD14dim / CD16+non-classical monocytes or CD14+ / HLA-DR+monocytes and CD14+ / HLA-DRdim / -monocytes revealed uniform LILRB5 expression across these subcategories. Similarly, CD66a+ / CD177- and CD66a+ / CD177+populations of neutrophils showed comparable levels of LILRB5 expression. The pattern of LILRB5 expression was consistent among all human peripheral blood specimens (healthy, melanoma, kidney cancer, lung cancer, breast cancer) evaluated (Fig.4B).

[0259] Eph receptors activate LILRB5 to promote immune suppression in vitro. The inventors used several in vitro functional assays to investigate whether the interaction between Eph receptors and LILRB5 regulates the activity of immunosuppressive myeloid cells. Using THP-1 cells that stably express LILRB5, the inventors demonstrated that 79 4919-0796-2971, v.1the addition of recombinant EphA7 and EphB1 decreased IL-8 production by 23.74% (SD = 1.82) and 9% (SD = 2.42), respectively. Notably, treatment with anti-LILRB5 antibody (B5- 29 or B5-27 mAb) reversed this suppression, restoring IL-8 to control levels (Fig. 5A). Next, the inventors collected enriched MDSCs from peripheral blood of human cancer patients. EphA7 or EphB1 addition promoted the immunosuppressive phenotype of MDSCs as shown by the upregulation of immunosuppressive markers CD163 and CD206, and anti-LILRB5 antibody (B5-1 mAb) treatment decreased the expression of CD163 and CD206 in cells treated with EphA7 or EphB1 (Figs.5B-C). The inventors further studied the functional significance of Eph receptors in macrophage polarization. During in vitro M2 macrophage differentiation, EphB1 reduced the expression of activation markers CD80 and CD86, whereas blocking LILRB5 function with anti-LILRB5 antibody (B5-2 mAb) not only restored the expression of these markers but also diminished the expression of immunosuppressive markers CD163 and CD206 (Fig.5D). Thus, LILRB5 is functionally expressed on MDSCs enriched from patients with cancer, and Eph receptors promote the activity of the immunosuppressive myeloid cells through an interaction with LILRB5.

[0260] Eph receptors activate LILRB5 on myeloid cells to support cancer development in vivo. To study the function of human LILRB5 in vivo, the inventors developed transgenic C57BL / 6 mice, in which human LILRB5 is expressed on myeloid cells as previously described for other LILRBs (Wu et al., 2021; Xie et al., 2022). To model the aberrant expression of Eph receptors seen in cancer, they engineered MC38 cancer cells to stably express human EphA7. Expression of EphA7 did not alter proliferation of these cells, but expression of EphA7 resulted in binding to LILRB5-ECD-Fc (Figs. 6A-B). The inventors implanted control MC38 cells and MC38 cells that expressed EphA7 subcutaneously into WT C57BL / 6 mice or the LILRB5-transgenic mice. They found that tumors induced by injection of control MC38 cells did not exhibit any growth differences in WT and LILRB5-transgenic mice (Figs. 6C-D). However, tumors that originated from MC38 cells that expressed EphA7 grew significantly faster in LILRB5-transgenic mice than in WT mice. In tumors induced by MC38 cells that expressed EphA7, the presence of LILRB5 on myeloid cells in the LILRB5- transgenic mice led to increased numbers of CD206+immunosuppressive myeloid cells, increased CD206 median fluorescence intensity (MFI), and decreased effector CD8+T cell infiltration compared to WT mice (Figs. 6E-F). Compared to control human IgG antibodies, anti-LILRB5 blocking antibodies (B5-27 mAb) suppressed growth of tumors induced by 80 4919-0796-2971, v.1MC38 cells that expressed EphA7 (Fig.6G-H) and increased effector CD8+T cell infiltration in the TME (Fig.6I).

[0261] As did EphA7, EphB1 had tumor-promoting effects in LILRB5- transgenic mice when expressed on MC38 cells (Figs.7A-D). The tumors induced by injection of MC38 cells that expressed EphB1 grew significantly larger in LILRB5-transgenic mice compared to WT mice. The interaction between EphB1 and LILRB5 led to increased activity of immunosuppressive myeloid cells and decreased numbers of effector CD8+T cells in TME (Fig. 7E). LILRB5 blockade (via B5-2 mAb) suppressed tumor growth (Fig. 7F-G) and decreased the MFI of immunosuppressive marker CD206, and increased effector CD8+T cell numbers in the TME (Fig.7H). In sum, the expression of Eph receptors on MC38 cancer cells increased tumor growth, increased numbers of immunosuppressive myeloid cells, and decreased numbers of functional T cells when myeloid cells of mice express LILRB5. Further, LILRB5 blockade suppressed tumor growth, reduced immunosuppressive myeloid cells, and rescued anti-tumor T cell infiltration.

[0262] Leukocyte Immunoglobulin Like Receptor B family (LILRBs) is a family of immune inhibitory receptors mainly expressed on myeloid cells. LILRB5 is one of the LILRB family members and is currently the least studied member in the LILRB family. LILRB5 is expressed on human myeloid cells with the existence of ITIMs in its cytoplasmic domain suggesting that this receptor also contributes to negative regulation of immune response. This invention reports discovery of a panel of LILRB5 specific binding monoclonal antibodies (mAbs) and the mAbs showed blocking functional properties of LILRB5 with potential for therapeutic development. This discovery uses assays with newly established ligands for LILRB5 in signaling. The inventors used several sources of myeloid cells including myeloid derived suppressor cells (MDSCs) derived from cancer patients. The ligand induced LILRB5 reporter cells were used to screen anti-LILRB5 blocking antibodies. These LILRB5 mAbs showed blocking activities of LILRB5 signaling and can reverse both ligand-induced signaling activation of LILRB5 and immunosuppressive activity of myeloid cells induced by LILRB5 ligands. The inventors also showed that the anti-LILRB5 blocking antibodies can suppress tumor development in the mouse tumor model with human LILRB5 gene expression. See FIGS.8-13 and Tables 1-7.

[0263] Selection and sequence analysis of anti-LILRB5 monoclonal antibodies. LILRB5 extracellular (ECD) protein (Sino Biological) was used to pan the 81 4919-0796-2971, v.1inventors’ in-house made phage display scFv library (diversity of 5 x 1011). Binders were selected using phage ELISA by coating LILRB5 protein on 96-well plates (max-sorb plates, Nunc) and detected using an anti-M13 phage antibody conjugated with horseradish peroxidase (HRP) and TMB substrate (cell signaling). Antibody variable DNA sequences (scFv) in selected E. coli clones were isolated using a plasmid preparation kit (Qiagen) and sequenced. CDR sequences are analyzed using the IMGT online software (world-wide-web at imgt.org) and amino acid sequences of heavy chain and light chain of each antibody are listed in Tables 1 & 2, respectively. CDR sequences of DNA for heavy chain and light chain of each antibody are shown in (Tables 3 & 4), respectively. Variable region of heavy chain and light chain DNA sequences are shown in Sequence Appendices 1 and 2. Amino acid sequences for variable heavy and light chains of LILRB5 mAbs are shown in Sequence Appendix 3.

[0264] Expression and purification of LILRB5 mAbs. Full length IgGs are expressed using an expression vector system in human embryonic kidney (HEK293) cells. Selected LILRB5 binding antibodies were expressed as human IgG1 and / or Fc engineered for null Fc mediated effector function with a mammalian expression vector system in HEK293 cells using a shaker flask CO2incubator. Antibodies were purified using a column with protein A resin (GenScript) using a fast protein liquid chromatography (FPLC) separation unit. Purified LILRB5 binding antibodies were used for characterization of their biochemical and biological properties.

[0265] Binding affinity of anti-LILRB5 monoclonal antibodies. ELISA titration was used to determine the binding affinity of a panel of monoclonal antibodies to LILRB5 antigen (Fig. 8). Binding affinities of the LILRB5 monoclonal antibodies were measured as EC50 using 4-parameter curve fitting of concentration titration graphs with GraphPad Prism program.

[0266] For kinetic binding affinity measurements, antibody (30 µg / mL) was loaded onto the protein A biosensors for 4 min. Following incubation in kinetics buffer to establish baseline. Antibody loaded biosensors were exposed to a series of concentrations (0.1- 100 nM) of LILRB5 protein (HIS tag at C terminus). Background subtraction was used to correct sensor drifting. All experiments were performed with shaking at 1,000 rpm according to the manufacturer’s suggestion. Kinetic sensorgrams for antibodies are shown in Fig. 9. ForteBio’s data analysis software was used to determine association rate (kon) and dissociation 82 4919-0796-2971, v.1rate (koff) and KDwas calculated using the ratio of koff / kon.The binding affinities of mAbs are shown in Table 5.

[0267] Cross reactivity to cynomolgus monkey LILRB5 analog protein. ELISA titration was used to determine the binding affinity of LILRB5 monoclonal antibodies to human LILRB5 (Human-B5) and cynomolgus monkey LILRB5 (cybo-B5) in Figs.10A-D.

[0268] Determine binding specificity to LILR family members in ELISA. Extracellular proteins (ECD) of all currently known family members (LILR-A and -B) and a closely related Lair1 were recombinantly expressed in HEK293 cells with a human Fc tag for purification. Purified ECDs were used to coat on high-binding 96-well plate for ELISA. LILRB5 monoclonal antibody at 1µg / ml concentration was added to the ECD coated plate for binding by incubating at room temperature for 1 hour. Binding signals were detected using an anti-human Fab2 fragment antibody with HRP (Jackson ImmunoResearch) with TMB substrate for colormetric reading at A450nm. All LILRB5 mAbs showed specific to LILRB5 (Fig.11).

[0269] Antibody binning based on domain binding using ELISA. LILRB5 has 4 ECD domains and the inventors produced domain 1-3 (amino acids 27-313) and full length ECD as shown in Fig. 12. Based on binding to domain 1-3 and full ECD protein in ELISA, they grouped the LILRB5 mAbs into two bins (Table 6).

[0270] Flow cytometry assay on cell surface binding. The inventors expressed LILRB family members 5 on cell surface for reporter cell assay. LILRB5 mAbs showed specific binding to LILRB5 expressed on cell surface and did not show binding to other members in LILRB family (Fig.13). Table 1. Amino acid sequences of heavy chain CDRs of LILRB5 antibodies mAb CDR1 SEQ CDR2 SEQ CDR3 SEQ :83 4919-0796-2971, v.1B5-2 GDSVSSNSAA 5 TYYRSKWYN 24 AREGQLLDAFDI 43 B5-4 GFTFSSFG 6 ISHDGSKK 25 ARDLYDSNYGYFQH 44Table 2: Amino acid sequences of light chain CDRs for each LILRB5 antibodies mAb CDR1 SEQ ID CDR2 CDR3 SEQ ID Names NO: NO:84 4919-0796-2971, v.1Table 3. LILRB5 mAb DNA sequences of heavy chain CDRs mAb CDR1 SEQ CDR2 SEQ CDR3 SEQ Names ID ID ID85 4919-0796-2971, v.1B5-29 ggatacaccttcacc 114 atcaaccctaacagt 133 gcgagagaggtagaactgg 152 ggctactat ggtggcaca attggcccccctactactactmAb CDR1 SEQ ID CDR2 CDR3 SEQ ID Names NO: NO:86 4919-0796-2971, v.1Table 5. Binding affinities of anti-LILRB5 antibodies determined using Octet 96-Red instrument LILRB5- KD (M) KD Error kon(1 / Ms) kon(1 / Ms) kdis(1 / s) Kdis Full R^2 mAb Error ErrorTable 6. LILRB5 antibody binning based on ELISA domain bindingAntibody Binding to Mabs groups domainsLILRB5 mAbs Gal-4 activation blocking Gal-7 activation blocking87 4919-0796-2971, v.1#22 + +88 4919-0796-2971, v.1TABLE 8 - VARIABLE HEAVY CHAIN DNA SEQUENCES OF ANTI-LILRB5 ANTIBODIES >B5Cm-T1 (SEQ ID NO: 191) caggtgcaggtggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttc agcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaa actacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag atctgaggacacggccgtgtattactgtgcgattagtagtggttattattataacccatttgactactggggccagggaaccctggtcact gtctcctca >B5Cm-T6 (SEQ ID NO: 192) caagtgcagctggtgcagtctgggnctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttc agcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaa actacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag atctgaggacacggccgtgtattactgtgcgagagggggataccagccactggattactggggccagggaaccctggtcagcgtctc ctca >B5Cm-L1 (SEQ ID NO: 193) caggtccagctggtgcaatctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttc agcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaa actacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag atctgaggacacggccgtgtattactgtgcgagagagggttatcttgatgcttttgatatctggggccaagggaccacggtcaccgtctc ctca >B5-1 (SEQ ID NO: 194) caggtgcagctggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcaccttta gcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacat actacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagag ccgaggacacggccgtatattactgtgcgaaagattccagcagctggtacccccactttgactactggggccagggaaccctggtca gcgtctcctcag >B5-#2 (SEQ ID NO: 195) caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtct ctagcaacagtgctgcttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagt ggtataatgattatgcagtatctgtgaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactctg tgactcccgaggacacggctgtgtattactgtgcaagagaggggcagcttttggatgcttttgatatctggggccaagggacaatggtc accgtctcttcag >B5-#4 (SEQ ID NO: 196) caggtgcagatggtggagtctgggggaggcgtggtccagcctgcgaggtccctgagactctcctgtgcagcctctggattcaccttca gtagctttggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagctatctcacatgatggaagtaaaaaag actatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgcatctgcaaatgaacagcctgagaa gtgaggacacggctgtgtattactgtgcgagagatttgtatgatagtaattatggatacttccaacactggggccaagggacaatggtca ccgtctcttcag >B5-#6 (SEQ ID NO: 197) Caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttc agcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaa actacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag atctgaggacacggccgtgtattactgtgcgactgggggggcttactatgatagtagttccaatgcttttgatatctggggccaagggac aatggtcaccgtctcttcag >B5-#8 (SEQ ID NO: 198) caggtgcagatggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcaccttta gcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacat actacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagag 89 4919-0796-2971, v.1ccgaggacacggccgtatattactgtgcgaaatccaactactacgatgcttttgatatctggggccaagggaccacggtcaccgtctctt cag >B5-#11 (SEQ ID NO: 199) caggtgcagctggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcaccttta gcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacat actacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagag ccgaggacacggccgtatattactgtgcgaaagattccagcagctggtacccccactttgactactggggccagggaaccctggtca gcgtctcctcag >B5-#14 (SEQ ID NO: 200) Gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagtatctggattccccttc agttcttacaccatgaattgggtccgccaggctccagggaaggggctggagtgggtctcatccattagttttactggtgaccatctatact acgcagactcagtgaggggccgcttcaccatctccagagacaacgccaagaactcgctgtatctgcaaatggacagcctgagagtcg aagatacggctatttattactgtgcgagcggcttccgtggggtggctggtactttccagcactggggcccgggcaccctggtcactgtct cctcag >B5-#16 (SEQ ID NO: 201) gaggtgcagctggtggagactgggggaggcttggtacagccagggcggtccctgagactctcctgtacagattctggattcacctttg gtgattatgctatgagctgggtccgccaggctccagggaaggggctggagtgggtaggtttcattagaagcaaagcttatggtgggac aacagaatacgccgcgtctgtgaaaggcagattcaccatctcaagagacaacgccaagaacacactatatctgcagatggacagcct gagggtcgaggacacagctatttactactgtgcgagatggcaagaattcatgggggcatttgacatctggggccacgggacaatggtc accgtctcttcag >B5-#17 (SEQ ID NO: 202) caggtgcagctggtgcagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctctggattcaccttca atagctatgctatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatcatatgatggaagcaataaata ctacgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagct gaggacacggctgtgtattactgtgcgagagattatggttcggggagcttagactactggggccagggaaccctggtcaccgtctcttc ag >B5-#20 (SEQ ID NO: 203) caggtgcagatggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcaccttta gcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacat actacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagag ccgaggacacggccgtatattactgtgcgaaaaatgacccacgacaatggacttggtacttcgatctctggggccgtggcaccctggt caccgtctcctcag >B5-#22 (SEQ ID NO: 204) gaggtgcagctggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcaccttta gcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacat actacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagag ccgaggacacggccgtatattactgtg >B5-#25 (SEQ ID NO: 205) caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcaactgcgctgtctatggtgggcccttca ggggttactactggtactggatccgccagtccccagagaaggggctcgagtggattggggaaatcaattttgatggaagaaccaacta caacccgtccctcaagagtcgggtcaccatgtccgtggacatgtccaagaatcagttttctctgaaactgacctctgtgaccgccgcgg acacggctgtgtattactgtgcgagaggcagggactggtttgggggcttctttgactactggggccagggaatcctggtcactgtctcct cag cgaaagtagggtatagcagtggctggcatgagtactactggggccagggaaccctggtcaccgtctcctcag >B5-#26 (SEQ ID NO: 206) caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcagcttc aacaattatggtatcaattgggtgcgacaggccccgggacaagggcttgagtggatgggcgggatcatcccgtcctttggtacagcaa attacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgtacacagtttacatggagctgagcagcctgacat ctgaggacacggccgtatactactgcgcgagaggggtggttatccccaacgatgctctcgatatgtggggccaagggacaatggtca ccgtctcctcag >B5-#27 (SEQ ID NO: 207) 90 4919-0796-2971, v.1cagctgcagctgcaggagtcgggcccaggactggtgaagccttcggggaccctgtccctcacctgcgctgtctctggtggctccatc agcagtagtaactggtggagttgggtccgccagcccccagggaaggggctggagtggattggggaaatctatcatagtgggagcac caactacaacccgtccctcaagagtcgagtcaccatatcagtagacaagtccaagaaccagttctccctgaagctgagctctgtgacc gccgcggacacggccgtgtattactgtgcgagagcggcggcgacggaaggggatgcttttgatatctggggccaagggacaatggt cactgtctcctcag >B5-#28 (SEQ ID NO: 208) caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggggaccctgtccctcacctgcgctgtctctggtggctccatc agcagtagtaactggtggagttgggtccgccagcccccagggaaggggctggagtggattggggaaatctatcatagtgggagcac caactacaacccgtccctcaagagtcgagtcaccatatcagtagacaagtccaagaaccagttctccctgaagctgagctctgtgacc gccgcggacacggccgtgtattactgtgcgagagcccagccgggttctgatgcttttgatatctggggccaagggacaatggtcaccg tctcttcag >B5-#29 (SEQ ID NO: 209) caggtgcaggtggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttc accggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcaca aactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgag atctgacgacacggccgtgtattactgtgcgagagaggtagaactggattggcccccctactactactacggtatggacgtctggggc caagggacaatggtcaccgtctcctcag TABLE 9 - VARIABLE LIGHT CHAIN DNA SEQUENCES OF ANTI-LILRB5 ANTIBODIES >B5Cm-T1 (SEQ ID NO: 210) gaaattgtaatgacacagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagc agctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatca aggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacaga gttacagtaccccaatcactttcggcgganggaccaagctggagatcaaa >B5Cm-T6 (SEQ ID NO: 211) gacatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattag cagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatc aaggttcannggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagatttttgcaactttactactgtcaac agagttacagtaccccattcacttttcggccctgggaccaaggtggaaatcaaa >B5Cm-L1 (SEQ ID NO: 212) cagtctgtgctgactcagccaccctcgctgtctgcgtcccccgggcagagggtcaccatctcctgttctggaagcagctccaacatccc ggttaatgctgtaaactggtaccagcaactcccaggagaggctcccaaactcatcatcttttatgatgatctgctgccctcgggggtccct gaccgattctctgcctccaaggctggcacctcagcctccctggccatcagtgggctccagtctgcggatgaggctgactattactgtgc agcctgggataacaaccggaatggtctggtattcggcgcagggaccaaggtcaccgtccta >B5-#1 (SEQ ID NO: 213) cagtctgtgttgacgcagccaccctcactgtctgggactcccgggcagagggtcaccatctcttgttctggaagcggctccaatatcgg aagtaatgctgttaactggtaccagcaattcccaggagcggcccccaaactcctcatatatggtactaatgagcggccctcaggggtcc ctgaccgattctctggctccaagtctggcgcctcagcctccctggccatcagtgggctccagtctgcggatgaggctgattatttttgtga ggcatgggatgacaccctgaatgctccgtttttcggcggagggaccacggtgaccgtcctgg >B5-#2 (SEQ ID NO: 214) cagtctgtgctgactcagccaccctcggtgtctgaagccccccggcagagggtcaccatctcctgctctggaagcagctccaacatcg gaaataatgctgtaaactggtaccagcagctcccaggaaaggctcccaaactcctcatctattatgatgatctgctgccctcaggggtct ctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctggggatgaggctgattattactgtg cagcatgggatgacagcctgaataattatgtcttcggaactgggaccacggtgaccgtcctgg >B5-#4 (SEQ ID NO: 215) gacatccagatgacccagtctccatcctccctgtctgcatctgtcggagacagagtcaccctcacttgccgggcaagtcagagcattat cacctatttaaattggtatcagcagaaaccagggaaagcccctaatctcctcatctatgatggtaacattttgcaaagtggggtcccatca 91 4919-0796-2971, v.1aggttcagtggcagtggatctgggacagatttcgctctcaccatcaacagtctgcaacctgaagattttgcaacttactactgtcaacaga gttacggtaccccgaccttcggccaagggacacgactggacattaaag >B5-#6 (SEQ ID NO: 216) cagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatc ggggcaggttatgatgtacattggtaccagcagcttccaggaacagcccccaaactcctcatctatggtaacaccaatcggccctcag gggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcactgggctccaggctgaggatgaggctgattat tactgccagtcctatgacagtaacctgaggggtgtagtattcggcggagggaccaaggtcaccgtcctag >B5-#8 (SEQ ID NO: 217) caggctgtgctgactcagccaccctcaacgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcg gaagtaatactgtaaactggtaccagcagctcccaggaacggcccccaaactcctcatctatagtaataatcagcggccctcaggggt ccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactg tgcagcatgggatgacagcctgaatggtccggtgttcggcggagggaccaaggtcaccgtcctag >B5-#11 (SEQ ID NO: 218) cagtctgtgttgacgcagccaccctcactgtctgggactcccgggcagagggtcaccatctcttgttctggaagcggctccaatatcgg aagtaatgctgttaactggtaccagcaattcccaggagcggcccccaaactcctcatatatggtactaatgagcggccctcaggggtcc ctgaccgattctctggctccaagtctggcgcctcagcctccctggccatcagtgggctccagtctgcggatgaggctgattatttttgtga ggcatgggatgacaccctgaatgctccgtttttcggcggagggaccacggtgaccgtcctgg >B5-#14 (SEQ ID NO: 219) ctgcctgtgctgactcagccaccctcggtgtcagtggccccaggaaagacggccattgtcacctgtgggggaaacaacattgggagt aaaagtgtccactggtaccagcagaagccaggccaggcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccct gagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgt caggtgtgggatagtagtagtgatcattatgtcttcggaagtgggaccaaggtcaccgtcctag >B5-#16 (SEQ ID NO: 220) tcctatgagctgatgcagccaccctcagtgtccgtgtccccaggacagacagccaccatcacctgctctggagataaattggggaata gatatgcttcctggtatcagcagaagccaggccagtcccctctgttggtcatctatctagataccaagcggccctcaggaatccctgag cgattctctggctccaactctgggaacacagccactctgaccatcagcgggacacagcctatggatgaggctgactattactgtcaggc gtgggacagcagcaccagagaatttgtcttcggaactgggaccaaggtcaccgtcctag >B5-#17 (SEQ ID NO: 221) aattttatgctgactcagccccactctgtgtcggagtctccgggagagacggtaaccatctcctgcaccggcagcagtggcagcattgc cagcaactatgtgcagtggtaccagcagcgcccgggcagtgcccccactattgtgatctatgaagatgaccgaagaccttctggggtc cctgatcgcttcagcggctccatcgacccctcctccaactctgcctccctcaccgtctctggactgaagactgaggacgaggctgacta ctactgtcagtcttatgatagcaccaaacattgggtgttcggcggagggaccaaggtcaccgtcctag >B5-#20 (SEQ ID NO: 222) agtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcgg aactaatactgtaaactggtaccagcagctcccaggaacggcccccaaactgctcatctatagtaatgataagcggccctcaggggtc cctgaccgagtctctggctccaagtctggcacctcagcctccctggccatcagtggcctccagtctgaggatgaggctgatttttattgtg cagcatgggatgacagcctgaatggtccggtattcggcggagggaccaaggtcaccgtcctag >B5-#22 (SEQ ID NO: 223) ctgcctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcg gaagtaatactgtaaactggtaccagcagctcccaggaacggcccccaaactcctcatctatagtaataatcagcggccctcaggggt ccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactg tgcagcatgggatgacagcctgaatggtccggtgttcggcggagggaccacggtgaccgtcctgg >B5-#25 (SEQ ID NO: 224) cagtctgccctgaatcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaacccgccgtgacgttggt gcttatgactatgtctcctggtaccaacagcacccaggcgaggcccccaaactcatcatttatgatgtcactacacggccctcaggggtt cctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgc agctcatatgcaggcagcagcacgatattcggcggagggacccagctgaccgtcctag >B5-#26 (SEQ ID NO: 225) cagtctcccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttggt ggttataactatgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttatgaggtcagtaatcggccctcaggggtt 92 4919-0796-2971, v.1tctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgc agctcatatacaagcagcagcactctggtgttcggcggagggaccacggtgaccgtcctgg >B5-#27 (SEQ ID NO: 226) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattag cagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatc aaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacag agttacagtaccccgctcactttcggcggagggaccaaagtggatatcaaag >B5-#28 (SEQ ID NO: 227) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattag cagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatc aaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacag agttacagtaccccactcactttcggcggagggaccaaagtggaaatcaaag >B5-#29 (SEQ ID NO: 228) cagtctgccctgaatcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatttcctgcactggaactagcagtgacgttggt gattataattatgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttatgatgtcagtattcggccctcaggggttt ctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgca gctcatggacaagcagcagcacccccattgtcttcggaactgggaccaaggtcaccgtcctag TABLE 10 - VARIABLE HEAVY CHAIN (-H) AND VARIABLE LIGHT CHAIN (-L) AMINO ACID SEQUENCES OF ANTI-LILRB5 ANTIBODIES B5-#1_H (SEQ ID NO: 229) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSSWYPHFDYWG QGTLVSVSS B5-#1_L (SEQ ID NO: 230) QSVLTQPPSLSGTPGQRVTISCSGSGSNIGSNAVNWYQQFPGAAPKLLIYGTNERPSG VPDRFSGSKSGASASLAISGLQSADEADYFCEAWDDTLNAPFFGGGTTVTVL B5-#2_H (SEQ ID NO: 231) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREGQLLDAFDIWG QGTMVTVSS B5-#2_L (SEQ ID NO: 232) QSVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSG VSDRFSGSKSGTSASLAISGLQSGDEADYYCAAWDDSLNNYVFGTGTTVTVL B5-#4_H (SEQ ID NO: 233) QVQMVESGGGVVQPARSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAAISHDG SKKDYADSVKGRFTISRDNSKNTLHLQMNSLRSEDTAVYYCARDLYDSNYGYFQH WGQGTMVTVSS B5-#4_L (SEQ ID NO: 234) DIQMTQSPSSLSASVGDRVTLTCRASQSIITYLNWYQQKPGKAPNLLIYDGNILQSGV PSRFSGSGSGTDFALTINSLQPEDFATYYCQQSYGTPTFGQGTRLDIK B5-#6_H (SEQ ID NO: 235) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGT ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATGGAYYDSSSNAFDIW GQGTMVTVSS B5-#6_L (SEQ ID NO: 236) QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSNLRGVVFGGGTKVTVL 93 4919-0796-2971, v.1B5-#8_H (SEQ ID NO: 237) QVQMVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSNYYDAFDIWGQGT TVTVSS B5-#8_L (SEQ ID NO: 238) QAVLTQPPSTSGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSG VPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKVTVL B5-#11_H (SEQ ID NO: 239) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSSWYPHFDYWG QGTLVSVSS B5-#11_L (SEQ ID NO: 240) QSVLTQPPSLSGTPGQRVTISCSGSGSNIGSNAVNWYQQFPGAAPKLLIYGTNERPSG VPDRFSGSKSGASASLAISGLQSADEADYFCEAWDDTLNAPFFGGGTTVTVL B5-#14_H (SEQ ID NO: 241) EVQLVESGGGLVKPGGSLRLSCAVSGFPFSSYTMNWVRQAPGKGLEWVSSISFTGD HLYYADSVRGRFTISRDNAKNSLYLQMDSLRVEDTAIYYCASGFRGVAGTFQHWGP GTLVTVSS B5-#14_L (SEQ ID NO: 242) LPVLTQPPSVSVAPGKTAIVTCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGI PERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGSGTKVTVL B5-#16_H (SEQ ID NO: 243) EVQLVETGGGLVQPGRSLRLSCTDSGFTFGDYAMSWVRQAPGKGLEWVGFIRSKAY GGTTEYAASVKGRFTISRDNAKNTLYLQMDSLRVEDTAIYYCARWQEFMGAFDIWG HGTMVTVSS B5-#16_L (SEQ ID NO: 244) SYELMQPPSVSVSPGQTATITCSGDKLGNRYASWYQQKPGQSPLLVIYLDTKRPSGIP ERFSGSNSGNTATLTISGTQPMDEADYYCQAWDSSTREFVFGTGTKVTVL B5-#17_H (SEQ ID NO: 245) QVQLVQSGGGVVQPGRSLRLSCAASGFTFNSYAMHWVRQAPGKGLEWVAVISYDG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGSGSLDYWGQ GTLVTVSS B5-#17_L (SEQ ID NO: 246) NFMLTQPHSVSESPGETVTISCTGSSGSIASNYVQWYQQRPGSAPTIVIYEDDRRPSGV PDRFSGSIDPSSNSASLTVSGLKTEDEADYYCQSYDSTKHWVFGGGTKVTVL B5-#20_H (SEQ ID NO: 247) QVQMVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNDPRQWTWYFDLW GRGTLVTVSS B5-#20_L (SEQ ID NO: 248) QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNTVNWYQQLPGTAPKLLIYSNDKRPSG VPDRVSGSKSGTSASLAISGLQSEDEADFYCAAWDDSLNGPVFGGGTKVTVL B5-#22_H (SEQ ID NO: 249) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGYSSGWHEYYWG QGTLVTVSS B5-#22_L (SEQ ID NO: 250) LPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGV PDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTTVTVL B5-#25_H (SEQ ID NO: 251) 94 4919-0796-2971, v.1QVQLQQWGAGLLKPSETLSLNCAVYGGPFRGYYWYWIRQSPEKGLEWIGEINFDGR TNYNPSLKSRVTMSVDMSKNQFSLKLTSVTAADTAVYYCARGRDWFGGFFDYWGQ GILVTVSS B5-#25_L (SEQ ID NO: 252) QSALNQPASVSGSPGQSITISCTGTRRDVGAYDYVSWYQQHPGEAPKLIIYDVTTRPS GVPNRFSGSKSGNTASLTISGLQAEDEADYYCSSYAGSSTIFGGGTQLTVL B5-#26_H (SEQ ID NO: 253) QVQLVQSGAEVKKPGSSVKVSCKASGGSFNNYGINWVRQAPGQGLEWMGGIIPSFG TANYAQKFQGRVTITADESTYTVYMELSSLTSEDTAVYYCARGVVIPNDALDMWG QGTMVTVSS B5-#26_L (SEQ ID NO: 254) QSPLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPS GVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGGGTTVTVL B5-#27_H (SEQ ID NO: 255) QLQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGS TNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARAAATEGDAFDIWGQG TMVTVSS B5-#27_L (SEQ ID NO: 256) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIK B5-#28_H (SEQ ID NO: 257) QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGS TNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARAQPGSDAFDIWGQGT MVTVSS B5-#28_L (SEQ ID NO: 258) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK B5-#29_H (SEQ ID NO: 259) QVQVVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINP NSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREVELDWPPYY YYGMDVWGQGTMVTVSS B5-#29_L (SEQ ID NO: 260) QSALNQPASVSGSPGQSITISCTGTSSDVGDYNYVSWYQQHPGKAPKLMIYDVSIRPS GVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSWTSSSTPIVFGTGTKVTVL >B5Cm-T1_H (SEQ ID NO: 261) QVQVVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGT ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAISSGYYYNPFDYWGQG TLVTVSS >B5Cm-T1_L (SEQ ID NO: 262) EIVMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGGXTKLEIK >B5Cm-T6_H (SEQ ID NO: 263) QVQLVQSGXEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGT ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGYQPLDYWGQGTL VSVSS >B5Cm-T6_L (SEQ ID NO: 264) DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFXGSGSGTDFTLTISSLQPEDFCNFTTVNRVTVPHSLFGPGTKVEIK B5Cm-L1_H (SEQ ID NO: 265) 95 4919-0796-2971, v.1QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGT ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGYLDAFDIWGQGTT VTVSS >B5Cm-L1_L (SEQ ID NO: 266) QSVLTQPPSLSASPGQRVTISCSGSSSNIPVNAVNWYQQLPGEAPKLIIFYDDLLPSGV PDRFSASKAGTSASLAISGLQSADEADYYCAAWDNNRNGLVFGAGTKVTVL * * * * * * * * * * * * *

[0271] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 96 4919-0796-2971, v.1REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Anami et al., Mol Cancer Ther, 2020. Barkal et al., Nat Immunol, 2018.19(1): p.76-84. Barry et al., Nature Reviews Cancer, 2023.23(4): p.216-237. Brantley-Sieders et al., PloS one, 2011.6(9): p. e24426. Brantley-Sieders et al., The Journal of clinical investigation, 2008.118(1): p.64-78. Buchacher et al., PloS one, 2015.10(11): p. e0143593. Chen et al., J Clin Invest, 2018.128(12): p.5647-5662. Chen et al., J Immunother Cancer, 2020.8(2). Condamine et al., Annual review of medicine, 2015.66: p.97-110. Daeron et al., Immunol Rev, 2008.224: p.11-43. 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Pasquale, Nature Reviews Cancer, 2010.10(3): p.165-180. Pasquale, Nature Reviews Cancer, 2024.24(1): p.5-27. Ramos et al., Elife, 2021.10. Salemme et al., Frontiers in oncology, 2021.11: p.610303. Schuijs et al., Nature immunology, 2020.21(9): p.998-1009. Sharma et al., J Exp Med, 2021.218(7). Sheng et al., Pathobiology, 2008.75(5): p.274-280. Shojaei & Ferrara, Cancer research, 2008.68(14): p.5501-5504. Siddiqui et al., Eur Heart J, 2017.38(48): p.3569-3575. Singh et al., Mol Cancer Res, 2021.19(4): p.702-716. Srivastava et al., Cancer Res, 2010.70(1): p.68-77. Suciu-Foca et al., J Immunol, 2007.178(11): p.7432-41. Takai et al., J Biomed Biotechnol, 2011.2011: p.275302. Tedla et al., Journal of Leukocyte Biology, 2007.83(2): p.334-343. Tedla et al., J Leukoc Biol, 2008.83(2): p.334-43. 984919-0796-2971, v. 1Teng et al., Neuro-oncology, 2013.15(12): p.1710-1720. Thomas, et al., Clin Rev Allergy Immunol, 2010.38(2-3): p.159-62. Trowsdale et al., Immunol Rev, 2015.267(1): p.117-36. van der Touw et al., Cancer Immunol Immunother, 2017.66(8): p.1079-1087. Vreeken et al., Journal of Leucocyte Biology, 2020.108(3): p.999-1011. Wang et al., Oncology, 2008.73(3-4): p.238-245. Wang et al., Journal of Cancer, 2020.11(14): p.4123. Wimmer-Kleikamp et al., The Journal of cell biology, 2004.164(5): p.661-666. Wu et al., Nat Cancer, 2021.2(11): p.1170-1184. Xie et al., Front Immunol, 2022.13: p.996026. Ya et al., Front Oncol, 2022.12: p.975261. Zhang et al., Zhonghua wai ke za zhi [Chinese journal of surgery], 2010.48(1): p.53-56. Zhang et al., Cell research, 2013.23(7): p.898-914. Zhang et al., PloS one, 2015.10(6): p. e0129063. Zhang, Biochem Biophys Res Commun, 2022.633: p.64-67. Zhao et al., Pharmacology & Therapeutics, 2020.212: p.107556. Zheng et al., Nature, 2012.485(7400): p.656-60. 994919-0796-2971, v. 1

Claims

WHAT IS CLAIMED IS:

1. An isolated monoclonal antibody or an antigen-binding fragment thereof comprising clone paired heavy and light chain CDRs1-3 as set forth in Table 1 and Table 2, respectively.

2. The isolated monoclonal antibody or the antigen binding fragment thereof of claim 1, wherein the isolated monoclonal antibody is a murine, a rodent, a rabbit, a chimeric, humanized, or human antibody.

3. The isolated monoclonal antibody or the antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment.

4. The isolated monoclonal antibody or the antigen binding fragment thereof of claim 1, wherein the isolated monoclonal antibody is a human antibody.

5. The isolated monoclonal antibody or the antigen-binding fragment thereof of claim 1, wherein the VH and VL chains have amino acid sequences of at least 90% or 95% percent sequence identity to clone-paired VH and VL sequences of Table 10.

6. The isolated monoclonal antibody or the antigen-binding fragment thereof of claim 1, wherein the VH and VL chains are encoded by sequences of at least 80% or 90% percent sequence identity to clone-paired sequences of Tables 8 and 9, respectively.

7. The isolated monoclonal antibody or the antigen-binding fragment thereof of claim 5, wherein the VH and VL chains have amino acid sequences identical to clone-paired VH and VL sequences of Table 10.

8. The isolated monoclonal antibody or the antigen binding fragment thereof of claim 5, wherein the VH and VL chains are encoded by nucleic acid sequences identical to clone-paired sequences of Tables 8 and 9, respectively.

9. The isolated monoclonal antibody or the antigen binding fragment thereof of claims 1- 8, wherein the isolated monoclonal antibody is a humanized antibody. 100 4919-0796-2971, v.

110. The isolated monoclonal antibody or the antigen binding fragment thereof of claim 1, wherein the antibody is a chimeric antibody.

11. The isolated monoclonal antibody or the antigen binding fragment thereof of claim 1, which blocks the phosphorylation of LILRB5.

12. The isolated monoclonal antibody or an antigen binding fragment thereof of claim 1, which suppresses the activation of LILRB5.

13. An isolated monoclonal antibody or an antigen-binding fragment thereof, which competes for the same epitope with the isolated monoclonal antibody or an antigen- binding fragment thereof according to any of claims 1-12.

14. A pharmaceutical composition comprising the isolated monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-13, and a pharmaceutically acceptable carrier.

15. An isolated nucleic acid that encodes the isolated monoclonal antibody or the antigen- binding fragment thereof according to any one of claims 1-13.

16. A vector comprising the isolated nucleic acid of claim 15.

17. A host cell comprising the vector of claim 16.

18. The host cell of claim 17, wherein the host cell is a mammalian cell.

19. The host cell of claim 17, wherein the host cell is a CHO cell.

20. A hybridoma or engineered cell encoding and / or producing the isolated monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-13.

21. A process of producing an antibody, comprising culturing the host cell of claim 17 or the hybridoma or engineered cell of claim 20 under conditions suitable for expressing the antibody, and recovering the antibody.

22. A chimeric antigen receptor (CAR) protein comprising the antigen-binding fragment according to any one of claims 1-13. 101 4919-0796-2971, v.

123. An isolated nucleic acid that encodes a CAR protein of claim 22.

24. A vector comprising the isolated nucleic acid of claim 23.

25. An engineered cell comprising the isolated nucleic acid of claim 23.

26. The engineered cell of claim 25, wherein the cell is a T cell, NK cell, or macrophage.

27. A method of treating or ameliorating the effect of a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-13 or the engineered cell of claims 25 or 26.

28. The method of claim 27, wherein the method reduces or eradicates the tumor burden in the subject.

29. The method of claim 27, wherein the method reduces the number of tumor cells and / or slows tumor growth rate.

30. The method of claim 27, wherein the method reduces tumor size.

31. The method of claim 27, wherein the method reduces or prevents tumor metastasis.

32. The method of claim 27, wherein the method eradicates the tumor in the subject.

33. The method of claim 27, wherein the cancer is a solid cancer.

34. The method of claim 33, wherein the solid cancer is selected from the group consisting of adrenal cancer, bile duct carcinoma, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, mesothelioma, melanoma, merkel cell cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pinealoma, prostate cancer, renal cell cancer, retinoblastoma, sarcoma, skin cancer, testicular cancer, thymic carcinoma, thyroid cancer, uterine cancer, and vaginal cancer. 102 4919-0796-2971, v.

135. The method of claim 27, wherein one or more of monocytes, macrophages, dendritic cells, neutrophils other myeloid cells, myeloid-derived suppressor cells, and tumor- associated macrophages are targeted.

36. The method of claim 27, wherein the cancer is a hematologic malignancy.

37. The method of claim 36, wherein the hematologic malignancy is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, chronic lymphoblastic leukemia (CLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myelomonocytic leukemia (CMML), chronic myelocytic leukemia (CML), pre-B acute lymphocytic leukemia (Pre-B ALL), diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, heavy chain disease, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Hodgkin’s lymphoma, non- Hodgkin’s lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma (MM), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, and polycythemia vera.

38. The method of claim 27, wherein the antibody or the antigen-binding fragment thereof is administered intravenously, intra-arterially, intra-tumorally, or subcutaneously.

39. The method of claim 27, further comprising administering to the subject one or more drugs selected from the group consisting of a topoisomerase inhibitor, an anthracycline topoisomerase inhibitor, an anthracycline, a daunorubicin, a nucleoside metabolic inhibitor, a cytarabine, a hypomethylating agent, a low dose cytarabine (LDAC), a combination of daunorubicin and cytarabine, a daunorubicin and cytarabine liposome for injection, Vyxeos®, an azacytidine, Vidaza®, a decitabine, an all-trans-retinoic acid (ATRA), an arsenic, an arsenic trioxide, a histamine dihydrochloride, Ceplene®, an interleukin-2, an aldesleukin, Proleukin®, a gemtuzumab ozogamicin, Mylotarg®, an FLT-3 inhibitor, a midostaurin, Rydapt®, a clofarabine, a farnesyl transferase inhibitor, a decitabine, an IDH1 inhibitor, an ivosidenib, Tibsovo®, an IDH2 inhibitor, an enasidenib, Idhifa®, a smoothened (SMO) inhibitor, a glasdegib, an arginase inhibitor, an IDO inhibitor, an epacadostat, a BCL-2 inhibitor, a venetoclax, Venclexta®, a platinum complex derivative, oxaliplatin, a kinase inhibitor, a tyrosine kinase inhibitor, 103 4919-0796-2971, v.1a PI3 kinase inhibitor, a BTK inhibitor, an ibrutinib, IMBRUVICA®, an acalabrutinib, CALQUENCE®, a zanubrutinib, a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG3 antibody, an ICOS antibody, a TIGIT antibody, a TIM3 antibody, a CD40 antibody, a 4-1BB antibody, a CD47 antibody, a SIRP1α antibody or fusions protein, a CD70 antibody, and CLL1 antibody, a CD123 antibody, an antagonist of E- selectin, an antibody binding to a tumor antigen, an antibody binding to a T-cell surface marker, an antibody binding to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an alkaloid derived from a plant, a hormone therapy medicine, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor.

40. The method according to any of claims 27-39, wherein said isolated monoclonal antibody or said antigen binding fragment thereof further comprises an antitumor drug linked thereto.

41. The method of claim 40, wherein said antitumor drug is linked to said antibody or said antigen binding fragment thereof through a photolabile linker.

42. The method of claim 40, wherein said antitumor drug is linked to said antibody or said antigen binding fragment thereof through an enzymatically cleaved linker.

43. The method of claim 40, wherein said antitumor drug is a toxin, a radioisotope, a cytokine, or an enzyme.

44. A method of detecting a cancer cell or cancer stem cell in a sample or subject comprising: (a) contacting a subject or a sample from the subject with the isolated monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-13; and (b) detecting binding of said antibody or the antigen-binding fragment thereof to a cancer cell or cancer stem cell in said subject or sample.

45. The method of claim 44, wherein the sample is a body fluid or biopsy. 104 4919-0796-2971, v.

146. The method of claim 44, wherein the sample is blood, bone marrow, sputum, tears, saliva, mucous, serum, urine or feces.

47. The method of claim 44, wherein detection comprises immunohistochemistry, flow cytometry, an immunoassay (including ELISA, RIA, etc.) or Western blot.

48. The method of claim 44, further comprising performing steps (a) and (b) a second time and determining a change in detection levels as compared to the first time.

49. The method of claim 44, wherein said isolated monoclonal antibody or said antigen binding fragment thereof further comprises a label.

50. The method of claim 49, wherein said label is a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemo-luminescent molecule, or a dye.

51. The method according to any of claims 27-50, wherein said isolated monoclonal antibody or said antigen binding fragment thereof is conjugated to a liposome or nanoparticle.

52. A method of treating or ameliorating the effect of an autoimmune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or the antigen-binding fragment thereof according to any one of claims 1-13 or the engineered cell of claims 25 or 26.

53. The method of claim 52, wherein one or more of monocytes, macrophages, dendritic cells, neutrophils and other myeloid cells are targeted.

54. The method of claim 52, wherein the antibody or the antigen-binding fragment thereof is administered intravenously, intra-arterially, or subcutaneously.

55. The method of claim 52, further comprising administering to the subject one or more drugs selected from the group consisting of a steroid or an NSAID.

56. The method of claim 52, wherein the autoimmune disease is Guillain-Barre syndrome, Chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, 105 4919-0796-2971, v.1juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory disease, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile onset diabetes mellitus (also known as Type I diabetes mellitus), Wegener's granulomatosis, polymyositis, dermatomyositis, inclusion body telangiectasia myositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, Grave’s Disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and), adult onset diabetes mellitus (also known as Type II diabetes mellitus), mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, anti- phospholipidsyndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic disease, allergic encephalomyelitis, transfusion reaction, leprosy, malaria, leshmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, shistosomiasis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki's disease, endophthalmitis, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, tularemia, periodic fever syndromes, pyogenic arthritis, Familial Mediterranean Fever, TNF-receptor associated periodic syndrome (TRAPS), Muckle-Wells syndrome, or hyper-IgD syndrome.

57. A method for enhancing T cell activation in a subject, the method comprising administering to the subject the antibody or the antigen-binding fragment thereof according to any one of claims 1-13 or the engineered cell of claims 25 or 26. 106 4919-0796-2971, v.

158. A method for modulating M2a macrophage phenotype in a subject, the method comprising administering to the subject the antibody or the antigen-binding fragment thereof according to any one of claims 1-13 or the engineered cell of claims 25 or 26. 107 4919-0796-2971, v.1

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