Novel LILRB4 Antibodies and Their Uses

Abstract

The present invention provides antibodies that bind to LILRB4 and uses of the antibodies for detecting and treating cancer.

Description

[0001] Priority claim

[0002] This application claims priority to U.S. Provisional Application No. 62 / 730,715, filed on September 13, 2018, the entire contents of which are hereby incorporated by reference.

[0003] Sequence Listing

[0004] The Sequence Listing is contained in a file entitled "UTFH_P0349WO_ST25," which is 4 KB (as measured in Microsoft Windows) and was created on September 9, 2019, which is submitted electronically herewith and is incorporated by reference. Technical Field

[0005] The present disclosure relates generally to the fields of medicine, oncology, and immunology. More specifically, the present invention relates to antibodies that bind to LILRB and can treat cancers including leukemia. Background Art

[0006] Acute myeloid leukemia (AML) is the most common acute leukemia in adults and a common pediatric cancer. Current treatments for AML involve intensive cytotoxic chemotherapy, usually followed by myeloablative conditioning and stem cell transplantation. However, despite treatment, most patients relapse or die from the disease within 5 years. In order to effectively treat AML, new molecular targets and treatments must be identified. Recently, inhibitory leukocyte immunoglobulin-like receptors (LILRBs) and related receptors containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) LAIR1 have been shown to have tumor-promoting functions in various hematopoietic and solid cancer cells. Receptors containing ITIMs are expressed on a wide range of immune cells and transduce signals by recruiting phosphatases SHP-1, SHP-2 or SHIP, resulting in negative regulation of immune cell activation. Similar to CTLA4 and PD-1, LILRB is considered an immune checkpoint factor.

[0007] LILRBs inhibit the activity of multiple immune cell types that promote tumor immune escape. LILRB4 is expressed on monocytes, macrophages, and dendritic cells and can inhibit innate immunity in a cell-autonomous manner and inhibit T cell activation via an indirect mechanism. LILRB4 is a specific marker for monocytic AML (including refractory and relapsed disease). LILRB1-5 are primate and human specific, and there are two mouse orthologs: paired immunoglobulin-like receptor B (PirB) and gp49B1. The related receptor LAIR1, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM), has human and mouse forms of the protein. Due to the limited value of mouse models and the fact that the ligands of several LILRBs (including LILRB4) are unknown, the biological functions and clinical significance of these receptors remain poorly understood. Summary of the invention

[0008] Thus, in one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to LILRB4. In certain embodiments, the antibody or antigen-binding fragment, upon binding to LILRB4, modulates the activation of LILRB4. In certain embodiments, the antibody or antigen-binding fragment, upon binding to LILRB4, activates LILRB4. In certain embodiments, the antibody or antigen-binding fragment, upon binding to LILRB4, inhibits the activation of LILRB4. In certain embodiments, the antibody or antigen-binding fragment, upon binding to LILRB4, specifically blocks the binding of ApoE to LILRB4.

[0009] In certain embodiments, the antibody or antigen-binding fragment thereof comprises (a) a heavy chain (HC) variable region (VH) comprising the following complementarity determining regions (CDRs): a heavy chain CDR (HC-CDR) 1 which is a CDR1 in SEQ ID NO: 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 99, 223, 225, 226, 228, 229, 230, or 231; a HC-CDR2 which is a CDR2 in SEQ ID NO: 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 99, 223, 225, 226, 228, 229, 230, or 231; and a HC-CDR3 which is a CDR3 in SEQ ID NO: NO: 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 99, 223, 225, 226, 228, 229, 230 or 231 CDR3, and variants thereof, wherein one or more of the HC-CDRs have one, two or three amino acid substitutions, additions, deletions or a combination thereof; and (b) a light chain (LC) variable region (VL) comprising the following CDRs: a light chain CDR (LC-CDR) 1 which is a CDR1 in SEQ ID NO: 5, 12, 19, 26, 33, 40, 47, 54, 61, 68, 75, 82, 89, 96, 103, 232, 235, 236 or 237; a LC-CDR2 which is a CDR1 in SEQ ID NO: 5, 12, 19, 26, 33, 40, 47, 54, 61, 68, 75, 82, 89, 96, 103, 232, 235, 236 or 237; NO:5, 12, 19, 26, 33, 40, 47, 54, 61, 68, 75, 82, 89, 96, 103, 232, 235, 236 or 237; and LC-CDR3, which is CDR3 in SEQ ID NO:5, 12, 19, 26, 33, 40, 47, 54, 61, 68, 75, 82, 89, 96, 103, 232, 235, 236 or 237, and variants thereof, wherein one or more of the LC-CDRs have one, two or three amino acid substitutions, additions, deletions or a combination thereof. In certain embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the AbM definition, or a contact definition of a CDR.

[0010] In certain embodiments, the antibody comprises a heavy chain variable region comprising: a HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 2, 9, 16, 23, 30, 37, 44, 51, 58, 65, 72, 79, 86, 93 or 100; a HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 3, 10, 17, 24, 31, 38, 45, 52, 59, 66, 73, 80, 87, 94 or 101; and a HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 95, 102, 224 or 227.

[0011] In certain embodiments, the antibody comprises a light chain variable region comprising: a LC-CDR1 having the amino acid sequence shown in SEQ ID NO:6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76, 83, 90, 97 or 104; a LC-CDR2 having the amino acid sequence of SAS, KAS, GAS, ATS, DAS or AAS, or the amino acid sequence shown in SEQ ID NO:233; and a LC-CDR3 having the amino acid sequence shown in SEQ ID NO:7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105 or 234.

[0012] In certain embodiments, the antibodies are characterized in that the heavy and light chains of the cloned pair have Figures 28A to 28C and Figures 30A to 30C In certain embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 99, 223, 225, 226, 228, 229, 230, or 231. In certain embodiments, the antibody comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 5, 12, 19, 26, 33, 40, 47, 54, 61, 68, 75, 82, 89, 96, 103, 232, 235, 236, or 237. In certain embodiments, antibodies characterized by clonally paired heavy and light chains have CDRs that have 0, 1, or 2 amino acid differences from the CDRs in Tables 1 and 2.

[0013] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that competes for the same epitope as an antibody having the clone-paired heavy and light chain CDR sequences of Tables 1 and 2. In certain embodiments, the antibody competes for the same epitope as an antibody having Figures 28A to 28C and Figures 30A to 30C The cloned paired heavy and light chain variable regions of antibodies compete for the same epitope.

[0014] In certain embodiments, the epitope bound by the antibody or antigen-binding fragment is located in the connecting region between the D1 and D2 domains of human LILRB4. In certain embodiments, the epitope comprises at least one amino acid within one or more amino acid sequences of LILRB4 listed in Table 9. In certain embodiments, the epitope comprises at least one amino acid within one or more amino acid sequences selected from W18, G96, A97, Y98, S99, K100, Q122, S123, R124, S125, P126, H153 and Q154 of SEQ ID NO: 238 (D1 and D2 domains of human LILRB4 protein).

[0015] In certain embodiments, the isolated monoclonal antibodies described herein are chimeric antibodies, humanized antibodies or human antibodies. In certain embodiments, the isolated monoclonal antibodies described herein are of IgG1, IgG2, IgG3 or IgG4 type. In certain embodiments, the antigen-binding fragments described herein are recombinant ScFv (single-chain variable fragment) antibodies, Fab fragments, F(ab')2 fragments or Fv fragments.

[0016] In another aspect, a pharmaceutical composition is provided, comprising an isolated monoclonal antibody or antigen-binding fragment thereof as provided herein, and at least one pharmaceutically acceptable carrier.

[0017] In another aspect, an isolated nucleic acid is provided that encodes an isolated monoclonal antibody or antigen-binding fragment thereof as provided herein.

[0018] In another aspect, a vector is provided, comprising an isolated nucleic acid as provided herein.

[0019] In another aspect, a host cell is provided, comprising a vector as provided herein. The host cell may be a mammalian cell. The host cell may be a CHO cell.

[0020] In another aspect, a hybridoma is provided that encodes or produces an isolated monoclonal antibody as provided herein.

[0021] In another aspect, a method of producing an antibody is provided. The method may comprise culturing a host cell as provided herein under conditions suitable for expressing the antibody and recovering the antibody.

[0022] In another aspect, a chimeric antigen receptor (CAR) protein comprising an antigen binding fragment as provided herein is provided.

[0023] In another aspect, an isolated nucleic acid encoding a CAR protein as provided herein is provided.

[0024] In another aspect, an engineered cell comprising an isolated nucleic acid as provided herein is provided. In certain embodiments, the cell is a T cell, a NK cell, or a bone marrow cell.

[0025] In another aspect, a method for treating or improving the effects of cancer in a subject is provided. The method may include administering to the subject a therapeutically effective amount of an antibody or its Fab or an engineered cell as provided herein. In certain embodiments, the cancer is acute myeloid leukemia. In certain embodiments, the antibody or its Fab is administered intravenously, intra-arterially, intratumorally or subcutaneously. In certain embodiments, the antibody or its Fab comprises an anti-tumor drug (e.g., toxin, radioisotope, cytokine or enzyme) connected thereto. In certain embodiments, the separated monoclonal antibody or its Fab is conjugated with a liposome or nanoparticle.

[0026] In another aspect, a method for detecting cancer cells or cancer stem cells in a sample or subject is provided. In certain embodiments, the method comprises contacting a subject or a sample from a subject with an antibody or its Fab as provided herein and detecting the combination of the antibody with the cancer cells or cancer stem cells in the subject or sample. The sample can be a body fluid or a biological specimen. The sample can be blood, sputum, tears, saliva, mucus, serum, urine or feces. In certain embodiments, detection comprises immunohistochemistry, flow cytometry, FACS, ELISA, RIA or protein blotting. In certain embodiments, the separated monoclonal antibody or its Fab further comprises a label (e.g., a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule or a dye). In certain embodiments, the separated monoclonal antibody or its Fab is conjugated with a liposome or nanoparticle.

[0027] The term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one", but it also has the meaning of "one or more", "at least one" and "one or more than one". The term "about" means plus or minus 5% of the stated number.

[0028] It is contemplated that any method or composition described herein may be implemented according to any other method or composition described herein. Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating specific embodiments of the present invention, are given only by way of illustration, because those skilled in the art will readily appreciate various changes and modifications within the spirit and scope of the present invention from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following figures form part of the specification and are included to further demonstrate certain aspects of the 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] Figures 1A to 1B This illustrates the sequence conservation between LILRA and LILRB family members. Figure 1A Illustrate the percentage of amino acid sequence identity between the D1 domains of LILRB and LILRA family members. Figure 1B Phylogenetic tree illustrating the D1 domains of LILRB and LILRA family members.

[0031] Figures 2A to 2K This indicates that LILRB4 expressed on leukemia cells inhibits T cell proliferation. Figure 2A , LILRB4 surface expression was quantified by flow cytometric analysis of samples from 105 patients. Figure 2B , Comparison of LILRB4 surface expression on normal monocytes and neoplastic monocytes from the same AML patients (n=6). MFI: mean fluorescence intensity. Figure 2C Autologous T cells (pT, patient T cells) isolated from a patient with monocytic AML (AML#19) or allogeneic T cells (nT, normal T cells) isolated from a healthy donor were cultured with irradiated LILRB4+ or LILRB4- (B4+ or B4-) primary leukemic cells from patient AML#19 (n=3 biologically independent samples, mean ± SD). The absolute T cell numbers (CD3 + CD8 - CD4 - 、CD3 + CD8 + CD4 + 、CD3 + CD8 - CD4 + and CD3 + CD8 + CD4 - ) are displayed in different colors in a stacked bar chart. Figure 2D, T cells isolated from healthy donors (E: effector cells) were cultured with the indicated irradiated THP-1 cells (T: target cells) in cell-to-cell contact. The number of CD3+ T cells is shown at different E:T ratios. Figures 2E to 2F , human T cells and intravenously transplanted doxycycline (Dox)-inducible lilrb4 knockout THP-1 cells (GFP + ) were transplanted into NOD-SCID IL2Rγ-null (NSG) mice (n=5). LV, liver; BM, bone marrow. Figure 2G , Tumor growth of mouse AML C1498 cells expressing human LILRB4 (hlilrb4-C1498) implanted subcutaneously in C57BL / 6 mice treated with anti-LILRB4-N297A antibody or control antibody (n=5) in the absence or presence of anti-CD8. Figure 2H , Survival curves of mice bearing subcutaneous hlilrb4-C1498 tumors (n=12). Fig.2I , adoptive transplantation of spleen cells from control mice or tumor-bearing mice cured by anti-LILRB4-N297A treatment (n=5). Tumor size was monitored over time. Arrows indicate the day of re-challenge with 3-fold the number of AML cells in leukemia-free mice (n=4). Figures 2J to 2K CD45 expression in the bone marrow of mice xenografted with primary human monocytic AML cells after treatment with anti-LILRB4 antibody or control IgG (n = 8 biologically independent samples). + LILRB4 + Representative flow chart and quantification of cell percentages. The experiment was repeated on 16 independent patient samples with similar results.

[0032] Figures 3A to 3D LILRB4 was shown to promote AML cell migration and infiltration. WT and lilrb4-KO THP-1 cells (GFP+) were used with or without reconstitution of LILRB4 (wt) or LILRB4 lacking the intracellular domain (intΔ). The number of leukemic cells (GFP+) in the bone marrow (BM), liver (LV) and spleen (SP) was determined by flow cytometry and normalized to the number in peripheral blood. Figure 3A , Comparison of short-term (20 h) infiltration of the indicated WT cells or modified THP-1 cells in NSG mice (n=5). Figures 3B to 3D , Comparison of short-term (20 h) infiltration of human primary monocytic AML cells in NSG mice (n=5) after treatment with anti-LILRB4 antibody or IgG control. ns, not significant; p values ​​from two-tailed student t-test.

[0033] Figures 4A to 4M APOE was shown to be an extracellular binding protein of LILRB4. Figure 4A , activated indicated LILRB reporter cells (GFP + ) percentage. Figure 4B , Percentages of the indicated LILRB reporter cells activated by recombinant APOE (10 μg / ml). Figure 4C , Percentage of LILRB4 reporter cells activated by 10% mouse serum collected from wild-type or apoe-knockout KO mice or PBS control group. Figure 4D , Percentage of LILRB4 reporter cells activated by 10 μg / ml of APOE, APOE-POPC, APOA1, or APOA1-POPC. Figure 4E , Binding of His-tagged APOE to WT and lilrb4-KO THP-1 cells. Figure 4F , using microscale thermophoresis (MST) to measure the binding kinetics of human His-tagged APOE-3 to LILRB4-ECD. Top: Fluorescence intensity (Flu.Int.) plot and regression of binding; bottom: corresponding residual (Resi.) and fitted plot. Figure 4G , Percentage of LILRB4 reporter cells activated by WT and mutant APOE proteins. Mut-N, R142A / K143A / R145A / K146A / R147A / R150A; Mut-C1, deletion of residues 245-299; and Mut-C2, deletion of residues 279-299. Figure 4H , Percentage of cells reporting the indicated LILRB4 mutations activated by APOE protein. Data for mutants that interfere with binding are shown in red ( Figures 4G to 4H ) is highlighted. Fig. 4I T cells isolated from healthy donors were cultured with the indicated irradiated THP-1 cells with either lilrb4 or apoe knockout (WT) or knockout (lilrb4-KO, apoe-KO-1, apoe-KO-2). T cells were analyzed by flow cytometry after 7 days. Figures 4J to 4L , C57bl / 6 mouse splenocytes (as effector cells or E) were incubated with irradiated C1498 cells expressing human lilrb4 (GFP-hlilrb4) or control (GFP) (as target cells or T) at the indicated E:T ratios. The cells were supplemented with 5% serum collected from WT or apoe-KO mice, cultured with anti-CD3 / CD28 coated beads for 60 hours, and then stained with anti-CD3 antibody. A representative flow chart showing samples at E:T of 20:1 ( Figure 4J), CD3 + The percentage of T cells ( Figure 4K ) and APOE-KO serum rescue effect of APOE-POPC ( Figure 4L ). Figure 4M Forced expression of human lilrb4 in murine leukemic C1498 cells increased leukemic cell infiltration in WT recipient mice but not in apoe-KO recipient mice (n=5). ns, not significant; p values ​​are from two-tailed Student's t-test.

[0034] Figures 5A to 5M Demonstrating that LILRB4-mediated intracellular signaling controls AML cell migration and T cell suppression. Figure 5A , Expression and phosphorylation of the three phosphatases in wild-type and lilrb4-KO THP-1 cells. Figure 5B , primary T cells and irradiated indicated THP-1 cells were cultured in the lower and upper chambers, respectively. T cells were analyzed by flow cytometry after 7 days. Figures 5C to 5D , genetic knockout of shp-2 reduced both short-term and long-term infiltration of THP-1 cells in NSG mice (n=5). Figure 5E , Upstream transcription factor analysis of RNA-seq data generated from lilrb4-KO and WT THP-1 cells. Yellow dots highlight transcription factors involved in the JAK / STAT and NF-κB pathways. Fig. 5F , phosphorylation of IKKα / β was decreased in lilrb4-KO THP-1 cells. Figure 5G , NFκB was decreased in the nuclear fraction of lilrb4-KO THP-1 cells. Figures 5H to 5I NF-κB inhibitor reversed the inhibition of T cells by WT THP-1 cells ( Figure 5H ), and reduced the infiltration of MV4-11 cells in a LILRB4-dependent manner ( Fig.5I ). Figure 5J , T cells isolated from healthy donors were supplemented with 25% conditioned medium (CM) of WT or lilrb4-KO THP-1 cells. Representative cells were photographed (scale bar, 100 μm) and T cells were analyzed by flow cytometry. Figures 5K to 5L T cells were incubated with recombinant uPAR (uPA receptor, also known as urokinase receptor) supplemented with the indicated concentrations Figure 5K ) or ARG-1 (arginase-1) ( Figure 5L ) proteins were incubated with the indicated THP-1 cells for 7 days and analyzed by flow cytometry. Figure 5M, Overexpression of uPAR(plaur) or ARG1 rescues the invasion defect of lilrb4-KO MV4-11 cells (n=5). ns, not significant; p values ​​are from two-tailed Student's t-test.

[0035] Figures 6A to 6B LIRB4 expression in human AML patients was demonstrated and negatively correlated with overall patient survival. Fig. 6A , Correlation analysis between lilrb4 mRNA level and overall survival of AML patients (n=160) from TCGA database. Low, n=57; medium, n=48; high, n=55. Figure 6B , Multivariate Cox regression analysis to assess associations in the presence of adjustments for confounding factors including age, cytogenetics, and PML-RAR mutations in the TCGA database. The total sample size was 79. *, p<0.05 was considered significant.

[0036] Figures 7A to 7F Primary AML cells expressing LILRB4 were shown to inhibit T cell proliferation. Autologous T cells isolated from individual monocytic AML or B-ALL patients were cultured with irradiated lilrb4-positive (B4+) or lilrb4-negative (B4-) primary leukemia cells from the same patient. pT, patient T cells. Allogeneic T cells isolated from healthy donors were cultured with irradiated lilrb4-positive or lilrb4-negative primary leukemia cells from indicated AML or B-ALL patients at an E:T of 10:1. nT, normal T cells. After 14 days of culture with anti-CD3 / CD28 / CD137-coated beads and rhIL-2, T cells were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies and analyzed by flow cytometry. Black p values ​​indicate CD3 + CD8 + significance of cells; red p values ​​indicate CD3 + CD4 + Significance of cells. ns, not significant.

[0037] Figures 8A to 8O demonstrated that LILRB4 inhibits T cell proliferation in vitro. Fig. 8A, Schematic diagram of the preparation of lilrb4-regulated THP-1 cells and the detection of LILRB4 expression on the cell surface by flow cytometry. WT, THP-1 cells treated with scrambled control; lilrb4-KO, THP-1 cells with lilrb4 gene knockout; lilrb4-KO-wt, forced expression of wild-type lilrb4 on lilrb4-KO THP-1 cells; lilrb4-KO-intΔ, forced expression of intracellular domain deletion mutant lilrb4 on lilrb4-KO THP-1 cells. Figure 8B , LILRB4 expression on the cell surface of lilrb4-KO MV4-11 cells was detected by flow cytometry. Figures 8C to 8D , lilrb4 deletion on MV4-11 cells reduces T cell suppression. T cells isolated from healthy donors were cultured in the lower chamber of a 96-well transwell plate with irradiated MV4-11 cells (2:1 E:T) in the upper chamber separated by a membrane with 3 μm pores. After 7 days of culture with anti-CD3 / CD28 coated beads and rhIL-2, representative cells were photographed using an inverted microscope (scale bar, 100 μm) ( Figure 8C ), and T cells were stained with anti-CD3 and analyzed by flow cytometry ( Fig.8D ). n=3. Fig. 8E , loss of lilrb4 in MV4-11 cells did not affect cell proliferation. Figures 8F to 8G , T cells isolated from healthy donors (E: effector cells) were cultured with the indicated irradiated THP-1 cells (T: target cells) in transwells without direct contact for 2 days. E:T=2:1. T cells were treated with BrdU for 30 min, followed by BrdU and 7-AAD staining for flow cytometric analysis. Representative flow cytometric plots are shown in Fig.8F and cell cycle states are summarized in Figure 8G Middle. T control, T cells were cultured in the absence of THP-1 cells. Figures 8H to 8I , T cells isolated from healthy donors (E: effector cells) were stained with CFSE and cultured with the indicated irradiated THP-1 cells (T: target cells) in transwells without direct contact for 2 days. Representative flow cytometry plots are shown in Figure 8H The percentage of proliferating T cells indicated by low CFSE staining is shown in Figure 8I middle. Figure 8J, LILRB4 increases PD-1 expression on T cells in co-culture of leukemia cells and T cells. T cells isolated from healthy donors (E: effector cells) were cultured with indicated irradiated THP-1 cells (T: target cells) in a non-contact manner for 5 days. E:T=2:1. T cells were stained with anti-LAG-3, anti-TIM-3, anti-TIGIT, anti-PD-1 and anti-FasL antibodies for flow cytometry analysis. Representative flow cytometry plots are shown and the average values ​​of fluorescence intensity are calculated and displayed in the upper right corner (black, WT; red, KO). The experiment was performed three times with similar results. Figure 8K , anti-LILRB4 antibody had no effect on T cell proliferation. The number of human primary T cells after 5 days of in vitro treatment with IgG or anti-LILRB4 antibody is shown (n=3 biologically independent samples with mean and standard deviation). Figure 8L , primary T cells and irradiated THP-1 cells (E:T ratio, 2:1) were placed in the lower and upper chambers, respectively, and treated with 10 μg / ml control IgG or anti-LILRB4 antibody. T cells were stained with anti-CD3 and analyzed by flow cytometry. Figure 8M , Primary T cells stimulated with anti-CD3 / CD28 / CD137 coated beads were co-cultured with WT or lilrb4-KO-THP-1 cells at the indicated E:T ratios for 4 hours. The cytotoxicity of leukemic cells was determined by PI staining in flow cytometry analysis (n=3 biologically independent samples with mean and standard deviation). Figures 8N to 8O , CD8 stimulated with anti-CD3 / CD28 / CD137 coated beads + T cells (5×10 4 cells) and 5×10 cells stably expressing GFP 3 THP-1 cells were co-cultured and treated with 100 μg / ml anti-LILRB4 antibody or control IgG for 5 days. n=4. Display GFP + Quantification of leukemia cells ( Figure 8N , n = 4 biologically independent samples) and IFNγ secretion ( Fig.8O , n = 3 biologically independent samples with mean and standard deviation). ns, not significant.

[0038] Figures 9A to 9J demonstrated that inhibition of LILRB4 reduced leukemia development in humanized immunocompromised mice. Figures 9A to 9C WT or lilrb4-KO THP-1 cells (3×10 6cells / mouse) were implanted subcutaneously into hPBMC-bred NSG mice (WT, n=14 mice with mean and standard deviation; lilrb4-KO, n=10 mice with mean and standard deviation). Tumor size ( Fig. 9A ) and CD3 in peripheral blood of mice receiving the therapy on day 31 + Quantitative ( Fig. 9B ) and display CD4 + and CD8 + Representative flow chart of T cells ( Fig. 9C ). Figures 9D to 9E , LILRB4 increases PD-1 expression on tumor-infiltrating T cells. WT or lilrb4-KO THP-1 cells were implanted subcutaneously into hPBMC-bred NSG mice. Three weeks after implantation, 7 of 10 WT mice had large tumors and 3 of 10 KO mice had microtumors. These tumors were dissected for immunohistochemistry and flow cytometry by staining with anti-LILRB4, anti-CD3, anti-PD-1, or anti-arginase-1 antibodies. Fig.9D , Enlarged left corner image from the yellow highlighted area. In CD3 and PD-1 staining images, the orange dotted line indicates the tumor boundary. The black arrow indicates PD-1 positive cells. Scale bar, 100 μm. Fig.9E Tumors were dissected and cells in the tumor area were stained with anti-CD3 and anti-PD-1 antibodies for flow cytometric analysis. + The percentage of T cells (PD-1 + CD3 + Cells / CD3 + ratio of cells). Figures 9F to 9G , THP-1 cells were transplanted into hPBMC-bred NSG mice, and mice were treated with control IgG or anti-LILRB4 antibody 6 days later (10 mg / kg; n=5). T cell numbers in representative mice at day 26 are also shown. Figures 9H to 9I , Human T cells and intravenously transplanted doxycycline (Dox)-inducible lilrb4 knockout THP-1 cells (GFP) were expressed in NSG mice (n=5) 7 days before Dox administration. + ) transplantation. Figure 9J , Representative flow chart showing successful LILRB4 depletion in transplanted leukemic cells at the endpoint, in the bone marrow of Dox-fed mice. ns, not significant.

[0039] Figures 10A to 10D Display of anti-LILRB4 antibodies attenuated leukemia development in syngeneic mice. Fig. 10A Mouse AML C1498 cells (3×106 Cells / mouse) were subcutaneously implanted into C57bl / 6 mice. Anti-LILRB4-N297A antibody or control IgG was injected intravenously on days 6, 9, 12, 15, 18, and 21 after tumor cell implantation. Both groups of mice were treated with anti-CD8 antibody on days 3, 6, 9, and 12 after tumor cell implantation to achieve CD8 + Anti-LILRB4 antibodies reduce leukemic cell infiltration into, for example, the liver (LV, Fig. 10A Even in CD8 + When cells are cleared ( Fig. 10A ). Figures 10B to 10D C57bl / 6 mice were intravenously implanted with mouse AML C1498 cells expressing human LILRb4 (3×10 6 cells / mouse) expressing GFP. Anti-LILRB4-N297A antibody (n=9 mice) or control IgG (n=9 mice) was injected intravenously on days 6, 9, 12, 15, and 18 after tumor cell implantation. Anti-LILRB4 antibody reduced the percentage of leukemic cells in the bone marrow (BM) by Fig. 10B Anti-LILRB4 antibody increases CD8 in bone marrow + T cells ( Fig. 10C ). CD8 + The percentage of T cells was significantly negatively correlated with the percentage of leukemia cells ( Fig. 10D ). ns, not significant. All p values ​​(except those based on Pearson's correlation) Fig. 10D (External) from two-tailed Student's t-test.

[0040] Figures 11A to 11B Anti-LILRB4 antibodies were shown to attenuate leukemia development and restore autologous T cells in PDX mice. Fig.11A , from sixteen human patients (three presented in Figures 2J to 2K Primary mononuclear AML cells (5×10 6 Up to 1×10 7 Cells / mouse) were injected into NSG mice and then treated with IgG or anti-LILRB4 antibody (10 mg / kg, intravenously twice a week). Human CD45 cells collected from hematopoietic tissues including bone marrow, spleen, liver and peripheral blood at 2 to 4 months after transplantation were shown. + LILRB4 + The percentage of AML cells as determined by flow cytometry. Fig. 11B, showing the percentage of autologous human T cells collected from hematopoietic tissues including bone marrow, spleen, liver and peripheral blood at 2 to 4 months after transplantation, as determined by flow cytometry; and CD3 + CD8 + Representative flow diagram of T cells. ns, not significant. All PDXs had n=8 biologically independent samples except AML#11 which had n=20 biologically independent samples.

[0041] Figures 12A to 12J demonstrated that LILRB4 promoted the infiltration of AML cells. Fig. 12A and 12B , mouse AML cells C1498 ( Fig. 12A ) or WEHI-3 cells stably expressing lilrb4 ( Fig. 12B ) Detection of human LILRB4 expression on Fig. 12C , forced expression of LILRB4 did not affect cell proliferation of mouse AML cells such as WEHI-3 (n=3). Fig.12D , forced expression of human LILRB4 promotes transendothelial migration of mouse AML WEHI-3 cells (n=3). Fig.12E NSG mice (n=6) were injected with 1×10 6 THP-1 cells were immediately treated with IgG or anti-LILRB4 antibody and monitored by bioluminescence imaging. Figures 12F to 12G Anti-LILRB4 antibody reduced the infiltration of AML cells into the viscera. 6 The mice were sacrificed on day 21 after the inoculation of luciferase-expressing THP-1 cells for ex vivo bioluminescence imaging of the internal organs. Images showing the luminous flux (radiance) of representative mice ( Fig.12F ). 1: Gastrointestinal (GI) tract; 2: Leg; 3: Lung; 4: Spleen; 5: Liver; 6: Kidney; 7: Brain; 8: Heart. Infiltrating leukemic cells form tumor nodules in the liver ( Figure 12G ). Fig.12H , anti-LILRB4 antibody had no effect on LILRB4-negative cancer cells. LILRB4 is expressed on THP-1 and MV4-11 human AML cells, but not on U937 cells. NSG mice were injected with U937 human AML cells that do not express LILRB4 and then treated with anti-LILRB4 antibody ( Fig.12H IgG served as control antibody. Mice were sacrificed on day 25 after transplantation to analyze LV, BM, SP, and PB by flow cytometry. The presence of human AML cells was detected by staining with anti-human CD45 antibody (n=3). Fig.12IRepresentative flow cytometry plots show that NK cells (CD45 NK cells) in NSG mice depleted of each immune cell subset by treatment with anti-asialo GM1 antibody, clodronate liposomes, and anti-Ly6G antibody, respectively, compared with non-depleted (wild-type) NSG mice. + CD49b + ), macrophages (CD11b + F4 / 80 + ) and neutrophils (CD11b + CD11c - ) frequency was successfully reduced. Fig.12J CFSE-labeled MV4-11 cells (5×10 6 Figure 5. LV, SP, and BM numbers of leukemic cells (CFSE positive) in the LV, SP, and BM at 20 hours after injection, normalized to the number in the PB. ns, not significant.

[0042] Figures 13A to 13C Demonstration of anti-LILRB4 inhibition of infiltration of primary AML cells. Comparison of infiltration of human primary monocytic AML cells in NSG mice (n=5) after treatment with anti-LILRB4 antibody or IgG control. Figures 13A to 13B , injected with primary human peripheral blood monocytes from patients with monocytic AML. Fig. 13C , injected with xenografted primary human monocytic AML cells (human CD45 + LILRB4 + cells). ns, not significant. All p values ​​are from two-tailed Student's t-test.

[0043] Figures 14A to 14G The results showed that the loss of APOE in AML cells restored T cell proliferation and inhibited AML cell migration in vitro. APOE expression in apoe-knockout THP-1 and MV4-11 cells was detected by immunoblotting ( Fig.14A and 14C ). Primary T cells and irradiated THP-1 or MV4-11 cells (E:T=2:1) ​​were cultured in the lower and upper chambers, respectively. Fig. 14B and 14D , scale bar, 100 μm) and quantified by flow cytometry after 7 days ( Fig. 4I and 14E ). Figures 14F to 14G, APOE deficiency inhibits transendothelial migration of human AML THP-1 and MV4-11 cells (n=4 biologically independent samples with mean and standard deviation).

[0044] Figures 15A to 15D LILRB4 was shown to upregulate phosphorylation of SHP-2 and NF-kB signaling. Fig.15A , after lilrb4 knockout (KO) in MV4-11 cells, phosphorylated SHP-2, phosphorylated IKB, uPAR, and ARG1 were downregulated. Fig. 15B , co-immunoprecipitation confirmed that LILRB4 interacts with SHP-2 in THP-1 cells. Figures 15C to 15D Two different NF-κB inhibitors restored T cell proliferation inhibited by THP-1 cells in a LILRB4-dependent manner. THP-1 cells were pretreated with various doses of NF-κB inhibitors for 1 hour. Primary T cells and radiation-pretreated THP-1 cells (E:T=2:1) ​​were cultured in the lower and upper chambers, respectively. T cells ( Fig. 15C , scale bar, 100 μm) and analyzed by flow cytometry 7 days later ( Fig.15D ). ns, not significant.

[0045] Figures 16A to 16H LILRB4 was shown to upregulate uPAR and arginase-1 to inhibit T cell activity and promote leukemia migration. Fig.16A , surface uPAR was downregulated in lilrb4 knockout THP-1 and MV4-11 AML cells. Fig. 16B , T cells isolated from healthy donors were cultured with anti-CD3 / CD28 coated beads and rhIL-2 and supplemented with indicated concentrations of uPAR protein for 3 days (n=4 biologically independent samples). Representative cells were photographed using an inverted microscope and T cells were analyzed by flow cytometry. Fig. 16C , the expression of uPAR and arginase-1 (ARG1) was downregulated in lilrb4 gene knockout THP-1 and MV4-11 AML cells. Fig.16D Arginase activity, as determined by a colorimetric method (DARG-100, BioAssay Systems), was reduced in the conditioned medium of lilrb4-KO THP-1 and MV4-11 cells. Fig.16E , primary T cells and irradiated THP-1 cells (E:T=2:1) ​​were cultured in the lower and upper chambers, respectively, and supplemented with 0.002 U / L recombinant ARG1 protein for 7 days. T cells were photographed. Fig.16F, T cells isolated from healthy donors were cultured with anti-CD3 / CD28 coated beads and rhIL-2 and supplemented with the indicated concentrations of ARG1 protein for 3 days (n=4 biologically independent samples). Representative cells were photographed using an inverted microscope and T cells were analyzed by flow cytometry. Figure 16G , autologous T cells isolated from individual monocytic AML patients were cultured with irradiated lilrb4-positive or lilrb4-negative primary leukemia cells from the same patient at an E:T ratio of 10:1, supplemented with recombinant anti-LILRB4 antibody, APOE-VLDL, uPAR or ARG1. pT, patient T cells. After 14 days of culture with anti-CD3 / CD28 / CD137-coated beads and rhIL-2, T cells were stained with anti-CD3, anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Fig.16H , Supplementation of culture medium with recombinant uPAR or ARG1 rescues the reduced transendothelial transmigration ability of lilrb4-KO THP-1 or lilrb4-KO MV4-11 cells (n=3). Scale bar, 100 μm. ns, not significant. All p values ​​are from two-tailed Student's t test.

[0046] Figures 17A to 17B Demonstrated is the detection of SHP-2 / NF-κB signaling and uPAR and arginase-1 expression in primary human monocytic AML cells. Fig.17A , for LILRB4-positive or high CD33 + AML cells (red boxes) and LILRB4 negative or low CD33 + AML cells (blue boxes) were gated for further intracellular staining of SHP-2 phosphorylated at Y580, IKKα / β phosphorylated at S176 / S180, NF-κB phosphorylated at S529, uPAR, and arginase-1 (ARG1). Isotype IgG was used as a negative control. Red numbers indicate LILRB4 positivity or high CD33 + MFI (mean fluorescence intensity) of AML cells; blue numbers indicate LILRB4 negativity or low CD33 + MFI of AML cells. Fig. 17B , LILRB4 positive or high CD33 + AML cells vs LILRB4 negative or low CD33 + Quantification of individual staining in AML cells.

[0047] Fig.18 Schematic diagram showing the mechanism by which LILRB4 inhibits T cells and promotes leukemic infiltration.

[0048] Figures 19A to 19DA comparison of LILRB4-mediated intracellular signaling in leukemic cells and normal hematopoietic cells is shown. Figures 19A to 19B , APOE activates LILRB4 intracellular signaling in leukemia cells. The indicated THP-1 cells and primary AML (M5) cells were serum starved overnight and then treated with the indicated concentrations of human recombinant APOE protein for the indicated times. Phosphorylated SHP-2, phosphorylated NFκB, and arginase-1 were detected by Western blotting. Fig.19C , the effect of APOE on normal monocytes or in vitro differentiated macrophages. Normal monocytes were isolated from healthy donors and macrophages were derived from these monocytes after one week of in vitro differentiation. Cells were serum starved overnight and then treated with human recombinant APOE protein at the indicated concentrations for the indicated time. Phosphorylated SHP-2, phosphorylated NFκB and arginase-1 were detected by Western blotting. Fig.19D , APOE induces uPAR upregulation on AML cells but not in normal monocytes. Normal monocytes were isolated from healthy donors. The indicated primary AML cells and normal monocytes were serum starved overnight and then treated with 20 μg / ml human recombinant APOE protein for eight hours. Surface uPAR was detected by flow cytometry. Representative flow charts are shown and the mean fluorescence intensity is shown in the upper right corner (black, PBS control; red, APOE treatment). The experiment was performed three times with similar results. The p value is from a two-tailed Student's t test.

[0049] Figures 20A to 20B Demonstrated that anti-LILRB4 does not affect engraftment of normal hematopoietic cells. Fig. 20A , Comparison of LILRB4 surface expression on normal monocytes from two healthy donors and on WT and lilrb4-KO THP-1 cells. Fig. 20B , anti-LILRB4 antibody does not affect the homing ability of normal monocytes. Fig. 20A ) were isolated via CD14 positive selection. These isolated monocytes were combined and stained with CFSE. After staining, monocytes (5×10 per mouse) were 6 CFSE in liver, spleen and bone marrow + Cell numbers were normalized to those in peripheral blood as determined by flow cytometry.

[0050] Figures 21A to 21C show that LILRB4 expressed on MDSCs inhibits T cells. Figure 21A, Peripheral blood mononuclear cells were isolated from blood samples of 11 solid cancer patients from the UTSW group by ficoll density gradient centrifugation. LILRB4 expression in MDSCs (CD14 + HLA-DR low / - 21B, autologous T cells were cultured with LILRB4-positive or -negative MDSCs at the indicated E:S ratios (E, effector T cells; S, MDSC suppressor cells) in T cell culture medium (RPMI-1640 medium supplemented with 10% FBS, 30U / ml human IL-2 at a 1:1 bead to cell ratio, and anti-CD3 / CD28 Dynabeads) for 5 days. Representative photos of T cells are shown. Fig. 21C In myeloid-derived suppressor cell (MDSC) / T cell co-cultures in vitro, anti-LILRB4 increased IFNγ secretion by T cells. T cells (E: effector cells; CD3+) and MDSC (S: suppressor cells; HLA-DR low / - CD14 + ) were isolated from peripheral blood of melanoma patients. T cells were co-cultured with MDSC for 5 days. E:S=2:1. The supernatant of the culture medium was collected and the content of IFNγ was determined by ELISA. The experiment was repeated three times.

[0051] Fig. 22 Exemplary LILRB4 monoclonal antibodies (mAbs) binding to human LILRB4 ECD were assayed by ELISA using concentration titration (0-10 μg / ml). The X-axis indicates antibody concentration and the Y-axis is binding signal in OD (450 nm). LILRB4 ECD recombinant protein was coated on a high absorbance 96-well plate. Serial dilutions (3-fold) of LILRB4 mAb were added to the coated / blocked plate and stained with goat anti-rabbit IgG F(ab) 2 Conjugated HRP was used as secondary antibody for detection. Titration curves were fitted using a 4-parameter curve fit and EC50 was estimated using GraphPad software.

[0052] Figures 23A to 23B . Fig.23A Schematic diagram of the LILRB4 extracellular domain (ECD). The mutations of the two residues (W106 and Y121) that significantly reduce APOE activation of LILRB4 are located in the first Ig domain and in the linker between the two Ig domains, respectively. Fig. 23BDetermination of exemplary LILRB4 mAbs binding to Ig domain-1 (D1 domain) of human LILRB4 is illustrated. The X-axis indicates antibody concentration and the Y-axis is binding signal in OD (450 nm). LILRB4D1 recombinant protein was coated on a high absorbance 96-well plate. Serial dilutions (3-fold) of LILRB4 mAb were added to the coated / blocked plate and stained using goat anti-rabbit IgG F(ab) 2 HRP conjugated was used as secondary antibody for detection. In this assay, only the D1 domain of LILRB4 was sufficient for binding to seven mAbs, whereas D1 alone was insufficient for binding to B4-193 (open blue triangles).

[0053] Figures 24A to 24B . Fig.24A It is a schematic diagram of the LILRB4 membrane protein, showing the Ig domain-1 (D1), Ig domain-2 (D2) and stalk region (SR) in the extracellular domain, and an illustration of an antibody that recognizes the D1 domain. Fig. 24B Determination of B4-193 binding domains is described. The LILRB4 D1 domain (D1), D2 domain (D2), stalk region (SR), D1+D2, D2+SR, and full-length ECD (D1+D2+SR) of human LILRB4 recombinant protein were coated on a high-absorbance 96-well plate. Serial dilutions (3-fold) of B4-193 were added to the coated / blocked plate and stained with goat anti-rabbit IgG F(ab) 2 HRP-conjugated was used as a secondary antibody for detection. In this assay, B4-193 only bound to the full-length ECD of human LILRB4.

[0054] Fig.25 The contribution of amino acid Y121 (tyrosine at position 121. Note that the numbering of Y at this position 121 is in the additional N-terminal sequence preceding D1; this position is identical to Y98 in SEQ ID No: 238, which begins at D1 without preceding N-terminal sequence) on LILRB4 to B4-193 binding is illustrated. Wild-type and Y121A mutant human LILRB4 ECD recombinant proteins were coated on high absorbance 96-well plates. Serial dilutions (3-fold) of mAb B4-193 were added to the coated / blocked plates and stained with goat anti-rabbit IgG F(ab) 2 HRP conjugated was used as secondary antibody for detection. The Y121A mutation of LILRB4 significantly reduced the binding of B4-193 to LILRB4.

[0055] Fig.26Illustrate kinetic binding measurements (sensing profiles) of an exemplary LILRB4 antibody assayed using Octet. 30 μg / mL of antibody was loaded onto a protein A sensor for 4 minutes. Following a short baseline of kinetic buffer, the loaded sensor was exposed to a range of recombinant human LILRB4 concentrations (0.1-200 nM) and sensor drift was corrected using background subtraction. All experiments were performed with shaking at 1,000 rpm. Background wavelength shift was measured using a reference sensor loaded with antibody only. ForteBio's data analysis software was used to fit the data to a 1:1 binding model to extract association and dissociation rates. KD was calculated based on the ratio koff / kon using ForteBio's data analysis software 7.0.

[0056] Fig. 27 Epitope binding of an exemplary LILRB4 mAb is illustrated. A classic sandwich epitope binning experiment was performed in an 8-channel Red96. The primary antibody (40 μg / mL) was loaded onto the protein A sensor for 4 minutes and the remaining Fc binding sites on the sensor were blocked with an irrelevant rabbit antibody (20 μg / ml) for 4 minutes, followed by soaking the sensor in kinetic buffer for 10 seconds. The sensor was then exposed to recombinant LILRB4 (25 μg / mL) for 4 minutes. Finally, the sensor was exposed to the secondary antibody (40 μg / mL) for 4 minutes to check for binding. The raw data was processed using ForteBio's Data Analysis Software 7.0 and the competitive binding of the antibody pairs was evaluated. Additional binding of the second antibody indicates an unoccupied epitope (non-competitor "-"), while no binding indicates epitope blocking (competitor "+").

[0057] Figures 28A to 28C The amino acid sequence of the heavy chain variable region of an exemplary LILRB4 antibody is illustrated.

[0058] Figures 29A to 29C The nucleic acid sequence of the heavy chain variable region of an exemplary LILRB4 antibody is illustrated.

[0059] Figures 30A to 30C The amino acid sequence of the light chain variable region of an exemplary LILRB4 antibody is illustrated.

[0060] Figures 31A to 31C The nucleic acid sequence of the light chain variable region of an exemplary LILRB4 antibody is illustrated.

[0061] Fig.32 Illustrated is APOE competition of exemplary anti-LILRB4 antibodies at different concentrations (0.1 μg / ml, 1 μg / ml and 10 μg / ml). Effective blocking of APOE activity is demonstrated by reduced GFP positivity (%) compared to PBS control.

[0062] Fig.33 Co-culture of LILRB4 reporter cells / K562 with exemplary anti-LILRB4 antibodies at different concentrations (0.1 μg / ml, 1 μg / ml and 10 μg / ml) is illustrated. Due to potential cross-linking of antibodies by Fc receptors on K562 cells, GFP positivity (%) caused by potential agonistic antibodies can be relatively higher than PBS controls in this assay.

[0063] Fig.34 Illustrated are LILRB4 reporter cells treated with different concentrations (0.1 μg / ml, 1 μg / ml and 10 μg / ml) of coated anti-LILRB4 antibodies. Since the antibodies are immobilized on the plastic surface in this assay, antibodies recognizing the extracellular domain of LILRB4 tend to record positive GFP (%) signals compared to PBS controls.

[0064] Fig.35 Illustrated are LILRB4 reporter cells treated with different concentrations (0.1 μg / ml, 1 μg / ml and 10 μg / ml) of soluble anti-LILRB4 antibody.

[0065] Fig.36 An exemplary anti-LILRB4 antibody binding to CHO cells expressing cynomolgus monkey LILRB4 (cynob4) as determined by flow cytometry is illustrated.

[0066] Fig.37 Illustrated are cross-reactivity of exemplary anti-LILRB4 antibodies with LILRB family members, LILRA family members, and cynomolgus monkey LILRB4 (cynoB4). "+" indicates binding of the antibody to a particular recombinant protein.

[0067] Fig.38 Demonstration of exemplary anti-LILRB4 antibody rabbit #B4-193 rescuing T cells suppressed by THP-1 cells.

[0068] Figures 39A to 39C It is demonstrated that exemplary anti-LILRB4 antibodies Rab-#128-3 and Rab-#193 inhibit leukemia development in THP-1 xenograft mice.

[0069] Fig.40 Illustrate the binding of h193 antibody to human LILRB4 protein as determined by flow cytometry. The numbers under each histogram line refer to the specific recombinant humanized antibody shown in Table 8. Antibody 39 is an irrelevant antibody. Antibody 39 and PBS both served as negative controls.

[0070] Figures 41A to 41M This indicates that h193 antibody specifically binds to LILRB4 protein. Figures 41A to 41LThe specificity of the h193 antibody as determined by the LILR reporter assay is illustrated, in which LILR reporter cells are added to plates coated with the h193 antibody. Binding of the h193 antibody to the ECD of the LILR member on the surface of the reporter cells induces a GFP signal. Figure 41M Specificity of exemplary h193 antibodies as determined by ELISA is illustrated. LILRA and LILRB recombinant proteins were coated on high absorbance 96-well plates. Serial dilutions (5-fold) of mAb h193 were added to the coated / blocked plates and detected using goat anti-human IgG F(ab)2 conjugated HRP as secondary antibody.

[0071] Fig.42 Illustrate the binding of h193 antibody to cynomolgus monkey LILRB4 determined by flow cytometry. The numbers under each histogram line refer to the specific recombinant humanized antibody shown in Table 8. Antibody 39 is an irrelevant antibody. Antibody 39 and PBS both served as negative controls.

[0072] Fig.43A and 43B Instructions for use of ApoE competition assay ( Fig.43A ) and K562 co-culture analysis ( Fig.43B ) The numbers under each histogram line refer to the specific recombinant humanized antibody shown in Table 8. Antibody 39 is an irrelevant antibody. Antibody 39 and PBS both served as negative controls.

[0073] Figures 44A to 44D Illustrated are the results of a cytokine array analysis measuring the effect of the h193 antibody in modulating cytokine secretion in PBMCs (peripheral blood mononuclear cells).

[0074] Figures 45A to 45D Illustrated are the results of a cytokine array analysis measuring the effect of the h193 antibody in modulating cytokine secretion in PBMC cells co-cultured with THP-1 cells.

[0075] Figures 46A to 46D Illustrated are the results of a cytokine array analysis comparing cytokine secretion in human IgG-treated PBMCs and PBMCs co-cultured with human IgG-treated THP-1 cells.

[0076] Fig.47 Exemplary images (in duplicate) of the cytokine array analysis of Figures 44 to 46 are shown.

[0077] Fig.48 The exemplary h193 antibody is shown to inhibit leukemia development in a xenograft mouse model.

[0078] Figures 49A to 49DThe crystal structure of LILRB4 in complex with h193 is illustrated. Fig.49A , the overall structure of the LILRB4 / h193 scFv complex is shown in a sketch. h193 (heavy chain, orange; light chain, yellow) binds to the D1D2 hinge loop and BC and C'E loops of the D2 domain of the LILRB4 molecule (magenta). The heavy chain ( Fig.49B ) and light chain ( Fig.49C ) Atomic interaction details of the binding to the LILRB4 moiety. The residues involved in the interaction are shown as sticks and are labeled. Hydrogen bonds are shown as red dashes. Fig.49D , The binding surface of LILRB4 contacted by h193. Residues contacting the h193 heavy or light chain are colored orange or yellow, respectively, and overlapping residues bound by both the heavy and light chains are colored green. DETAILED DESCRIPTION

[0079] The inventors have isolated a group of novel monoclonal antibodies that recognize the LILRB4 protein (ITIM-containing receptor) that can be used for cancer treatment. LILRB4 is upregulated on some tumor cells (especially leukemia cells) and promotes tumor growth. The identified anti-human ILIRB4 antibodies block LILRB4 signaling and can modulate immunity to fight cancer.

[0080] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various equivalents, changes and modifications can be achieved without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments will be included herein. All documents cited herein (including disclosures, patents and patent applications) are incorporated herein by reference in their entirety.

[0081] I. Definitions

[0082] It should be understood that the foregoing general description and the following detailed description are merely illustrative and explanatory and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless expressly stated otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. In addition, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. In addition, unless expressly stated otherwise, terms such as "element" or "component" encompass elements and components comprising one unit as well as elements and components comprising more than one subunit. In addition, the use of the term "portion" can include a portion of a portion or the entire portion.

[0083] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0084] As used herein, when referring to measurable values ​​such as amount, time interval, etc., the term "about" means to cover a variation of up to ±10% of the specified value. Unless otherwise indicated, all numbers used in the specification and claims to represent the amount of components, such as molecular weight, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters given in the following specification and the attached claims are all approximate values, which can be changed according to the desired characteristics to be obtained by the disclosed subject. At least, and without attempting to limit the application of the theory of equivalents to the scope of the claims, each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques. Although the numerical ranges and parameters describing the wide range of the present invention are approximate values, the numerical values ​​described in the specific examples should be reported as accurately as possible. However, any numerical value inherently contains certain errors, which are inevitably caused by the standard deviation present in its respective test measurements.

[0085] The term "antibody" refers to a complete immunoglobulin of any isotype or a fragment that can compete with a complete antibody for specific binding to a target antigen, and includes, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. "Antibody" is an antigen binding protein species. A complete antibody will generally include at least two full-length heavy chains and two full-length light chains, but may include fewer chains in some cases, such as naturally occurring antibodies in camels that may include only heavy chains. An antibody may be derived from a single source only, or may be "chimeric", i.e., different parts of an antibody may be derived from two different antibodies as further described below. Antigen binding proteins, antibodies, or binding fragments may be produced in hybridomas by recombinant DNA technology or by enzyme or chemical cleavage of a complete antibody. Unless otherwise indicated, the term "antibody" includes derivatives, variants, fragments, and mutant proteins thereof except antibodies comprising two full-length heavy chains and two full-length light chains, examples of which are described below. Furthermore, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, miniantibodies, 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.

[0086] Naturally occurring antibody structural units typically comprise tetramers. Each such tetramer is typically composed of two pairs of identical polypeptide chains, each pair having one full-length "light" chain (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, which is typically responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant region that may be responsible for effector function. Human light chains are typically divided into κ light chains and λ light chains. Heavy chains are typically divided into μ, δ, γ, α, or ε, and the isotype of the antibody is defined 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. In full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about more than 10 amino acids. See, e.g., Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (incorporated herein by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form an antigen binding site.

[0087] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chain of an antibody, which generally includes approximately 120 to 130 amino-terminal amino acids in the heavy chain and about 100 to 110 amino-terminal amino acids in the light chain. In certain embodiments, the variable regions of different antibodies vary widely in amino acid sequence, even among antibodies of the same species. The variable region of an antibody generally determines the specificity of a particular antibody for its target.

[0088] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions (also called complementarity determining regions or CDRs). The CDRs from the two chains of each pair are typically aligned by the framework regions, which enables binding to specific epitopes. The light and heavy chain variable regions typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The amino acid assignments to the various domains are typically based on the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) or Chothia et al., Nature, 342: 878-883 (1989).

[0089] In certain embodiments, the antibody heavy chain binds to the antigen in the absence of an antibody light chain. In certain embodiments, the antibody light chain binds to the antigen in the absence of an antibody heavy chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of an antibody light chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of an antibody heavy chain. In certain embodiments, the single variable region specifically binds to the antigen in the absence of other variable regions.

[0090] In certain embodiments, the unambiguous delineation of the CDRs and the identification of the residues comprising the binding site of the antibody are achieved by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. In certain embodiments, it can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be used 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.

[0091] The Kabat definition is a standard for numbering residues in antibodies and is often used to identify CDR regions. See, e.g., Johnson and Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the location of certain structural loop regions. See, e.g., Chothia et al., Journal of Molecular Biology, 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated suite of computer programs for antibody structure modeling produced by the Oxford Molecular Group. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbM TM , a computer program for modeling antibody variable regions", Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of a knowledge database and an ab initio method to model the tertiary structure of an antibody from the primary sequence, such as those described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", PROTEINS, Structure, Function and Genetics. Suppl., 3:194-198 (1999). Contact definitions are based on analysis of available composite crystal structures. See, e.g., MacCallum et al., J. Molecular Biology, 5:732-45 (1996).

[0092] By convention, the CDR regions in the heavy chain are usually referred to as H1, H2 and H3 and are numbered consecutively from the amino terminus to the carboxyl terminus. The CDR regions in the light chain are usually referred to as L1, L2 and L3 and are numbered consecutively from the amino terminus to the carboxyl terminus.

[0093] The term "light chain" includes full-length light chains and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length light chain includes a variable region VL and a constant region CL. The variable region of the light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0094] The term "heavy chain" includes full-length heavy chains and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length heavy chain includes a variable region VH and three constant regions CH1, CH2, and CH3. The VH domain is located at the amino terminus of the polypeptide, and the CH domain is located at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0095] Bispecific or bifunctional antibodies are generally artificial hybrid antibodies with two different heavy chain / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including but not limited to, hybridoma fusion 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).

[0096] The term "antigen" refers to a substance that can induce an adaptive immune response. Specifically, an antigen is a substance that acts as a target of a receptor for an adaptive immune response. Typically, an antigen is a molecule that binds to an antigen-specific receptor, but cannot induce an immune response in vivo by itself. Antigens are usually proteins and polysaccharides, and less commonly lipids. Suitable antigens include, but are not limited to, parts of bacteria (coatings, capsules, cell walls, flagella, cilia, and toxins), viruses, and other microorganisms. Antigens also include tumor antigens, for example, antigens produced by mutations in tumors. As used herein, antigens also include immunogens and haptens.

[0097] As used herein, "antigen binding protein" ("ABP") means any protein that binds to a specified target antigen. In the present application, the specified target antigen is a LILRB protein or a fragment thereof. "Antigen binding protein" includes, but is not limited to, antibodies and antigen-binding fragments thereof. Peptibodies are another example of antigen binding proteins.

[0098] As used herein, the term "antigen binding fragment" refers to a portion of a protein that is capable of specifically binding to an antigen. In certain embodiments, an 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 contain a complete native antibody structure. In certain embodiments, an antigen binding fragment is not derived from an antibody but is actually derived from a receptor. Examples of antigen binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab') 2 , Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibodies, single domain antibodies (sdAb), camel antibodies or nanobodies, domain antibodies and bivalent domain antibodies. In certain embodiments, the antigen binding fragment is capable of binding to the same antigen that the parent antibody binds. In certain embodiments, the antigen binding fragment may comprise one or more CDRs from a specific human antibody that is transplanted to the framework region of 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.

[0099] "Fab fragment" contains one light chain and the CH1 and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0100] A "Fab' fragment" comprises one light chain and a portion of one heavy chain, which contains the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab') 2 molecular.

[0101] “F(ab') 2 A "fragment" contains two light chains and two heavy chains, wherein the heavy chain contains a portion of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. Thus, F(ab') 2 The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0102] The "Fc" region comprises two heavy chain fragments, which comprise 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.

[0103] The "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0104] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been linked by a flexible linker to form a single polypeptide chain, thereby forming an antigen binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference.

[0105] "Domain antibodies" are immunologically functional immunoglobulin fragments containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently joined by a peptide linker to produce a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0106] A "bivalent antigen binding protein" or "bivalent antibody" comprises two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. Bivalent antigen binding proteins and bivalent antibodies can be bispecific, see below. In certain embodiments, bivalent antibodies other than "multispecific" or "multifunctional" antibodies are generally understood to have each of their binding sites being identical.

[0107] A "multispecific antigen-binding protein" or "multispecific antibody" is an antigen-binding protein or antibody that targets more than one antigen or epitope.

[0108] "Bispecific," "dual specific," or "bifunctional" antigen binding proteins or antibodies are hybrid antigen binding proteins or antibodies, respectively, that have two different antigen binding sites. Bispecific antigen binding proteins and antibodies are a class of multispecific antigen binding protein antibodies and can be produced by various 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 may be present on the same or different protein targets.

[0109] "Binding affinity" generally refers to the strength of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art (including methods described herein). Low-affinity antibodies generally bind antigen slowly and tend to decompose easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound for a longer time. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0110] An antibody that "specifically binds to a specific polypeptide or an epitope on a specific polypeptide" or "is specific for a specific polypeptide or an epitope on a specific polypeptide" is an antibody that binds to a specific polypeptide or an epitope on a specific polypeptide and does not substantially bind to any other polypeptide or polypeptide epitope. For example, the LILRB4-specific antibody of the present invention is specific for LILRB4. In some embodiments, the dissociation constant (Kd) of the antibody that binds to LILRB4 is ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or lower; e.g. 10 -8 M to 10 -13 M; e.g. 10 -9 M to 10 -13 M).

[0111] The term "compete" when used in the context of antigen binding proteins (e.g., antibodies or antigen binding fragments thereof) competing for the same epitope, refers to competition between the antigen binding proteins as determined by an assay in which the test antigen binding protein (e.g., antibody or antigen binding fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., LILRB or fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen binding protein competes with another antigen binding protein, 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, 1996); Press); solid phase direct labeling RIA using 1-125 labeling (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 directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such analyses involve the use of purified antigen bound to a solid surface or cell carrying either an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cell in the presence of a test antigen binding protein. Typically, there is an excess of the test antigen binding protein. Antigen binding proteins identified by competition analysis (complete antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding protein and antigen binding proteins that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antigen binding protein so that steric hindrance exists. Additional details on methods for determining competitive binding are provided in the Examples herein.Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen binding protein to the 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 cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0112] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. An epitope can be a linear epitope or a conformational epitope. A linear epitope is formed by a continuous amino acid sequence from an antigen and interacts with an antibody based on its primary structure. On the other hand, a conformational epitope is composed of a non-continuous portion of the amino acid sequence of the antigen and interacts with an antibody based on the 3D structure of the antigen. In general, an epitope is approximately five or six amino acids in length. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0113] As used herein, "cell" can be prokaryotic or eukaryotic. Prokaryotic cells include, for example, bacteria. Eukaryotic cells include, for example, fungi and animal cells. Types of animal cells (e.g., mammalian cells or human cells) include, for example, cells from the circulating / immune system or organs, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, granulocytes (e.g., basophils, eosinophils, neutrophils and multisegmented neutrophils), monocytes or macrophages, red blood cells (e.g., reticulocytes), mast cells, thrombocytes or megakaryocytes and dendritic cells; cells from the endocrine system or organs, such as Such as thyroid cells (e.g., thyroid epithelial cells, parafollicular cells), parathyroid cells (e.g., parathyroid chief cells, eosinophils), adrenal cells (e.g., chromaffin cells) and pineal cells (e.g., pinealocytes); cells from the nervous system or organs, such as glial cells (e.g., astrocytes and oligodendritic glial cells), microglial cells, magnocellular neurosecretory cells, stellate cells, Boettcher cells and pituitary cells (e.g., gonadotropins, adrenocorticotropic hormone cells from the respiratory system or organs, such as parietal cells (type I pneumocytes and type II pneumocytes), clara cells, goblet cells, and alveolar macrophages; cells from the circulatory system or organs (such as myocardial cells and coat cells); cells from the digestive system or organs, such as chief cells, parietal cells, goblet cells, Paneth cells, and gastric cells, such as paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells, enteroendocrine cells, enterochromaffin cells, APUD cells and liver cells (e.g., hepatocytes and Kupffer cells); cells from the epidermal system or organs, such as bone cells (e.g., osteoblasts, osteocytes and osteoclasts), tooth cells (e.g., chondroblasts and ameloblasts), cartilage cells (e.g., chondroblasts and chondrocytes), skin / hair cells (e.g., hair cells, keratinocytes and melanocytes (nevus cells (Nevus cells (Nevus cells (Nevus cells (Nevus cells cell))), muscle cells (e.g., myocytes), adipocytes, fibroblasts, and tendon cells; cells from the urinary system or organs (e.g., podocytes, juxtaglomerular cells, mesangial cells, adventitial cells, renal proximal tubule brush border cells, and macula densa cells); and cells from the reproductive system or organs (e.g., sperm, Sertoli cells, Leydig cells, oocytes, and ovum).The cell can be a normal healthy cell; or a diseased or unhealthy cell (e.g., a cancer cell). The mammalian cell can be a rodent cell, such as a mouse cell, a rat cell, or a hamster cell. The mammalian cell can be a rabbit cell, such as a rabbit cell. The mammalian cell can also be a primate cell, such as a human cell.

[0114] As used herein, the term "chimeric antigen receptor" or "CAR" 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)) connected to a domain or signaling (e.g., T cell signaling or T cell activation domain) of an activated immune cell (e.g., T cell or NK cell) (see, e.g., Kershaw et al., 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)). CAR is able to utilize the antigen binding properties of monoclonal antibodies to direct immune cell specificity and reactivity to a selected target in a non-MHC restricted manner. Non-MHC restricted antigen recognition gives CAR-expressing immune cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main tumor escape mechanism. Additionally, when expressed in T cells, it is advantageous that the CAR does not dimerize with the endogenous T cell receptor (TCR) α and β chains.

[0115] As used herein, "substantially free" with respect to a specified component is used herein to mean that none of the specified components are purposefully formulated into the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the specified component resulting from any unintended contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the specified component cannot be detected by standard analytical methods.

[0116] The term "host cell" means a cell that has been or can be transformed with a nucleic acid sequence and thereby expresses the gene of interest. The term includes progeny of a parent cell, regardless of whether the progeny is identical to the original parent cell in morphology or genetic makeup, as long as the gene of interest is present.

[0117] The term "identity" refers to the 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. "Identity percentage" means the percentage of identical residues between the amino acids or nucleotides in the compared molecules, and is calculated based on the smallest size in the compared molecules. For these calculations, gaps in the alignment, if any, are preferably handled by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primers, 1993, New York: Academic Press; Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM Journal of Applied Mathematics, 48:1073.

[0118] When calculating the percent identity, the compared sequences are usually aligned in a manner that obtains the maximum match between the sequences. An example of a computer program that can be used to determine the percent identity is the GCG program package, which includes GAP (Devereux et al., (1984) Nucl. Acid Res. 12: 387; Genetics Computer Group, Wisconsin, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to compare two polypeptides or polynucleotides to be determined for the percent identity of the sequence. The sequences are aligned to obtain the best match ("match span" as determined by the algorithm) of their respective amino acids or nucleotides. A gap opening penalty (which is calculated as 3× average diagonal, where “average diagonal” is the average of the diagonals of the comparison matrix used; “diagonal” is the score or value assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (which is typically 1 / 10 times the gap opening penalty) are used in conjunction with the algorithm, as well as a comparison matrix such as PAM 250 or BLOSUM 62. In certain embodiments, the algorithm also uses a standard comparison matrix (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0119] Examples of parameters that can be used to determine percent identity of polypeptide or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol. 48:443-453.

[0120] Certain alignment schemes used to align two amino acid sequences can produce matches of only shorter regions of the two sequences, and this smaller alignment region can have very high sequence identity, even if there is no significant relationship between the two full-length sequences. Therefore, if so desired, the selected alignment method (GAP program) can be adjusted to produce an alignment spanning at least 50 or other numbers of consecutive amino acids of the target polypeptide.

[0121] As used herein, the term "linked" refers to association via intramolecular interactions (eg, covalent bonds, metallic bonds, and / or ionic bonds) or intermolecular interactions (eg, hydrogen bonds or non-covalent bonds).

[0122] Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) is a protein encoded by the LILRB4 gene in humans. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family, which is found in the gene cluster of chromosome region 19q13.4. The encoded protein belongs to the subfamily B class of LIR receptors, which contain two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The receptor is expressed on immune cells, where it binds to class I MHC molecules on antigen-presenting cells and transduces negative signals that inhibit immune response stimulation. The receptor may also play a role in antigen capture and presentation. It is believed that controlling inflammatory responses and cytotoxicity helps to focus the immune response and limit autoreactivity. LILRB4 is also expressed in human gastric cancer cells and can promote tumor growth. Multiple transcript variants encoding different isoforms of this gene have been found. LILRB4 has been shown to interact with PTPN6.

[0123] The term "operably linked" refers to an arrangement of elements in which the elements so described are configured to perform their common functions. Thus, a given signal peptide operably linked to a polypeptide directs secretion of the polypeptide from a cell. In the case of a promoter, a promoter operably linked to a coding sequence will direct the expression of the coding sequence. A promoter or other control element need not be adjacent to a coding sequence, as long as it functions to direct its expression. For example, an intervening sequence that is not translated but transcribed may be present between a promoter sequence and a coding sequence and the promoter sequence may still be considered to be "operably linked" to the coding sequence.

[0124] Although the present disclosure supports definitions referring to alternatives only and "and / or," the use of the term "or" in the claims is intended to refer to "and / or" unless explicitly indicated as referring to alternatives only or the alternatives are mutually exclusive. As used herein, "another" may mean at least a second or more.

[0125] The term "polynucleotide" or "nucleic acid" includes single-stranded and double-stranded nucleotide polymers. Nucleotides, including polynucleotides, can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. Such modifications include base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and internucleotide linkage modifications, such as phosphorothioates, phosphorodithioates, selenophosphates, diselenophosphates, aniline phosphorothioates, aniline phosphates, and phosphoamino esters.

[0126] The term "polypeptide" or "protein" means a macromolecule having the amino acid sequence of a native protein, i.e., a protein produced by a naturally occurring and non-recombinant cell; or a molecule produced by a genetically engineered or recombinant cell and comprising a molecule having the amino acid sequence of a native protein, or having one or more amino acids deleted, added and / or substituted from the native sequence. The term also includes amino acid polymers and polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids. The terms "polypeptide" and "protein" specifically encompass LILRB antigen binding proteins, antibodies, or sequences having one or more amino acids deleted, added and / or substituted from the antigen binding protein. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion and / or an internal deletion compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. In certain embodiments, the fragment is about five to 500 amino acids long. For example, a fragment 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. Suitable polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of LILRB binding antibodies, suitable fragments include (but are not limited to) CDR regions, variable domains of heavy and / or light chains, a portion of an antibody chain or just the variable region of two CDRs, etc.

[0127] The pharmaceutically acceptable carriers used in the present invention are conventional. "Remington's Pharmaceutical Sciences", co-published by EW Martin, Mack, Easton, PA, 15th edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins disclosed herein. In general, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral formulations typically include injectable fluids, including pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. as vehicles. For solid compositions (such as powders, pills, tablets or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0128] As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject can be a patient, which refers to a human being presented to a medical provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". A subject may suffer from or be susceptible to a disease or condition, but may or may not display symptoms of the disease or condition.

[0129] As used herein, the term "therapeutically effective amount" or "effective dose" refers to a dose or drug concentration that is effective for treating a disease or condition. For example, with respect to the use of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein for treating cancer, a therapeutically effective amount is a dose or concentration of a monoclonal antibody or antigen-binding fragment thereof that is capable of reducing tumor volume, eradicating all or part of a tumor, inhibiting or slowing tumor growth or cancer cell infiltration into other organs, inhibiting the growth or proliferation of cells that mediate cancerous conditions, inhibiting or slowing tumor cell metastasis, improving any symptoms or markers associated with a tumor or cancerous condition, preventing or delaying the progression of a tumor or cancerous condition, or a combination thereof.

[0130] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the onset or rate of worsening of the condition, reducing the risk of developing the condition, preventing or delaying worsening of symptoms associated with the condition, reducing or stopping symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0131] As used herein, "vector" refers to a nucleic acid molecule introduced into a host cell, thereby producing a transformed host cell. A vector may include a nucleic acid sequence that permits it to replicate in a 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 genetic elements known in the art. A vector is capable of transducing, transforming or infecting a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. The vector optionally includes materials that facilitate entry of nucleic acids into the cell, such as viral particles, liposomes, protein coatings, and the like.

[0132] II. Cancer

[0133] A. Cancer

[0134] Although hyperproliferative diseases can be associated with any disease that causes cells to begin to regenerate uncontrollably, the archetypal example is cancer. One of the key elements of cancer is that the normal apoptotic cycle of cells is interrupted and therefore agents that interrupt cell growth are important as therapeutic agents for treating these diseases. In the present invention, the tubulysin analogs described herein can be used to cause a decrease in cell counts and therefore can potentially be used to treat various types of cancer lines. In some aspects, it is expected that the tubulysin analogs described herein can be used to treat almost any malignant tumor. Here, the only requirement is the presence of LILRB on the surface of cancer cells, and particularly on the surface of cancer stem cells.

[0135] Cancer cells that may be treated according to the present disclosure include, but are not limited to, cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, stomach, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. In addition, cancers may specifically be of the following histological types, but are not limited to these: malignant tumor; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid; bronchioalveolar adenocarcinoma ; Papillary adenocarcinoma; Chromophobe cell carcinoma; Oncophilic carcinoma; Apophilic adenocarcinoma; Basophilic carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Unencapsulated sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Carcinoma of skin appendages; Apocrine adenocarcinoma; Sebaceous adenocarcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Ring cell carcinoma; Infiltrating ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast disease); acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma cell tumor; malignant granulosa cell tumor; malignant testicular blastoma; sertolicell cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrohistiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Mullerian mixed tumor; Wilms' tumor ; hepatoblastoma; carcinosarcoma; malignant stromal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian thyroid tumor; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma;malignant odontogenic tumors; ameloblastic odontoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; myofibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neuroma; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease dgkin's disease); granulomatous; small lymphocytic malignant lymphoma;, large cell diffuse malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphatic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In certain aspects, the tumor may comprise osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, neuroblastoma, or leukemia. ;

[0136] B. Acute myeloid leukemia

[0137] Acute myeloid leukemia (AML), also known as acute myeloid leukemia or acute non-lymphocytic leukemia (ANLL), is a cancer of the myeloid lineage of blood cells characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. AML is the most common acute leukemia affecting adults, and its incidence increases with age. In the United States, although AML is a relatively rare disease, it accounts for approximately 1.2% of cancer deaths, and its incidence is expected to increase as the population ages.

[0138] Symptoms of AML are caused by leukemic cells replacing normal bone marrow, which results in a decrease in red blood cells, platelets, and normal white blood cells. These symptoms include fatigue, shortness of breath, easy bruising and bleeding, and an increased risk of infection. Several risk factors and chromosomal abnormalities have been identified, but the specific cause is not known. As an acute leukemia, AML progresses rapidly and is usually fatal within weeks or months if untreated.

[0139] AML has several subtypes; treatment and prognosis vary with subtype. Five-year survival rates vary from 15-70%, and relapse rates vary from 33-78%, depending on the subtype. AML is initially treated with chemotherapy aimed at inducing remission; patients may go on to receive additional chemotherapy or a hematopoietic stem cell transplant. Recent studies of AML genetics have led to the availability of tests that can predict which drugs will work best for a particular patient, and how long the patient is likely to survive.

[0140] Most signs and symptoms of AML are caused by leukemia cells replacing normal blood cells. The lack of normal white blood cell production makes the patient susceptible to infection; although the leukemia cells themselves are derived from white blood cell precursors, they have no ability to fight infection. A low red blood cell count (anemia) can cause fatigue, paleness, and shortness of breath. A lack of platelets can cause easy bruising or bleeding after minor injuries.

[0141] Early signs of AML are often vague and nonspecific and may resemble those of the flu or other common illnesses. Some general symptoms include fever, fatigue, weight loss or loss of appetite, shortness of breath, anemia, easy bruising or bleeding, ecchymoses (flat, pin-sized spots under the skin caused by bleeding), bone and joint pain, and persistent or frequent infections.

[0142] Spleen enlargement may occur in AML, but it is usually mild and asymptomatic. Lymph node swelling is rare in AML compared to acute lymphoblastic leukemia. Skin involvement takes the form of leukemic skin about 10% of the time. AML may rarely be associated with Sweet's syndrome, a paraneoplastic inflammatory disease of the skin.

[0143] Some patients with AML may suffer from swelling of the gums due to infiltration of leukemic cells into the gum tissue. Rarely, the first sign of leukemia may be a solid leukemic mass or worsening of a tumor outside the bone marrow, called a chloroma. Occasionally, a person may not show symptoms and leukemia may be discovered accidentally during a routine blood test.

[0144] Several risk factors for the progression of AML have been identified, including other blood disorders, chemical exposure, ionizing radiation, and genetics.

[0145] "Preleukemic" blood disorders, such as myelodysplastic syndrome or myeloproliferative disease, can evolve into AML; the exact risk depends on the type of MDS / MPS. Exposure to anticancer chemotherapy, specifically alkylating agents, can increase the risk of subsequent progression of AML. The risk is highest about three to five years after chemotherapy. Other chemotherapy agents, specifically epipodophyllotoxins and anthracyclines, have also been associated with treatment-related leukemias. These treatment-related leukemias are often associated with specific chromosomal abnormalities in the leukemic cells. Occupational chemical exposure to benzene and other aromatic organic solvents is controversial as a cause of AML. Benzene and many of its derivatives are known to be carcinogenic in vitro. Although some studies have shown an association between occupational exposure to benzene and an increased risk of AML, other studies have shown that the attributable risk, if any, is mild. Exposure to large amounts of ionizing radiation can increase the risk of AML. There appears to be a genetic risk for AML. Multiple cases of AML have been reported in families with progression rates higher than would be predicted by chance alone. Several congenital conditions can increase the risk of leukemia; perhaps the most common is Down syndrome, which is associated with a 10- to 18-fold increased risk of AML.

[0146] The first clue to the diagnosis of AML is usually an abnormal result on a complete blood count. Although an abnormal excess of white blood cells (leukocytosis) is a common finding, and leukemic blasts are sometimes found, AML can also present with an isolated decrease in platelets, red blood cells, or even a low white blood cell count (leukopenia). Although a presumptive diagnosis of AML can be made by examining a peripheral blood smear in the presence of circulating leukemic blasts, a definitive diagnosis usually requires an adequate bone marrow aspiration and biopsy.

[0147] Bone marrow or blood is examined via optical microscopy and flow cytometry to diagnose the presence of leukemia, distinguish AML from other types of leukemia (e.g., acute lymphoblastic leukemia-ALL), and classify the subtypes of the disease (see below). Bone marrow or blood samples are also typically tested for chromosomal abnormalities by conventional cytogenetics or fluorescent in situ hybridization. Genetic studies can also be performed to look for specific mutations in genes, such as FMS-like tyrosine kinase 3 (FLT3), nucleophosmin, and KIT, which can affect the outcome of the disease.

[0148] Cytochemical stains on blood and bone marrow smears help distinguish AML from ALL and help in the subdivision of AML. The combination of myeloperoxidase or Sudan black staining and nonspecific esterase staining will provide the information needed in most cases. Myeloperoxidase or Sudan black reactions are most useful for establishing the identification of AML and distinguishing it from ALL. Nonspecific esterase staining is used to identify the mononuclear cell component in AML and to distinguish poorly differentiated monocytic leukemia from ALL.

[0149] The diagnosis and classification of AML can be challenging and should be performed by a qualified hematopathologist or hematologist. In simple cases, the presence of certain morphological features (e.g., Auer rods) or specific flow cytometric results can distinguish AML from other leukemias; however, in the absence of such features, diagnosis can be difficult.

[0150] According to the widely used WHO criteria, the diagnosis of AML is established by demonstrating involvement of more than 20% of the blood and / or bone marrow with leukemic myeloid blasts. The French-American-British (FAB) classification is more stringent, requiring the presence of at least 30% blast percentages in the bone marrow (BM) or peripheral blood (PB) for the diagnosis of AML. AML must be carefully distinguished from "preleukemic" conditions (e.g., myelodysplasia or myeloproliferative syndromes), which are treated differently.

[0151] Because acute promyelocytic leukemia (APL) has the highest curability and requires unique treatment modalities, it is important to quickly confirm or exclude the diagnosis of this leukemia subtype. Fluorescence in situ hybridization of blood or bone marrow is commonly used for this purpose because it readily identifies the chromosomal translocation [t(15;17)(q22;q12);] that characterizes APL. It is also desirable to detect the presence of the PML / RARA fusion protein, which is the oncogenic product of the translocation, in molecular form.

[0152] First-line treatment for AML consists primarily of chemotherapy and is divided into two phases: induction and post-remission (or consolidation) therapy. The goal of induction therapy is to achieve complete remission by reducing the number of leukemic cells to undetectable levels; the goal of consolidation therapy is to eliminate any residual undetectable disease and achieve a cure. If induction chemotherapy fails or the patient relapses, hematopoietic stem cell transplantation is usually considered, but transplantation is sometimes also used as a first-line therapy for patients with high-risk disease.

[0153] All FAB subtypes except M3 are usually given induction chemotherapy with cytarabine (ara-C) and anthracycline (most often daunomycin). This induction chemotherapy regimen is called "7+3" (or "3+7") because cytarabine is administered as a continuous IV infusion for seven consecutive days, while anthracycline is administered as an IV push for three consecutive days. Up to 70% of patients will achieve remission with this regimen. Other alternative induction regimens, including high-dose cytarabine alone, FLAG-like regimens, or investigational agents, can also be used. Due to the toxic effects of the therapy (including bone marrow suppression and increased risk of infection), induction chemotherapy may not be offered to the elderly, and options may include less intensive chemotherapy or palliative care.

[0154] The M3 subtype of AML, also known as acute promyelocytic leukemia (APL), is almost universally treated with the drug all-trans retinoic acid (ATRA) in addition to induction chemotherapy (usually an anthracycline). Care must be taken to prevent disseminated intravascular coagulation (DIC), which complicates the treatment of APL when promyelocytes release the contents of their granules into the peripheral circulation. APL is very curable, following well-documented treatment regimens.

[0155] The goal of the induction phase should be to achieve a complete remission. A complete remission does not mean that the disease is cured; rather, it means that the disease cannot be detected by available diagnostic methods. About 50%-75% of newly diagnosed adults achieve a complete remission, although this can vary based on the prognostic factors listed above. The length of remission depends on the prognostic characteristics of the initial leukemia. In general, all remissions will fail without additional consolidation therapy.

[0156] Even after achieving complete remission, the number of leukemic cells may still be too small to detect with current diagnostic techniques. Without further post-remission or consolidation therapy, almost all patients will eventually relapse. Therefore, more therapy is needed to eliminate undetectable disease and prevent relapse, i.e., achieve a cure.

[0157] The specific type of post-remission therapy is individualized based on the patient's prognostic factors (see above) and general health. For leukemias with a good prognosis (i.e., inv(16), t(8;21), and t(15;17)), patients will typically undergo an additional three to five courses of intensive chemotherapy, called consolidation chemotherapy. For patients at high risk of relapse (e.g., patients with high-risk cytogenetics, underlying MDS, or therapy-related AML), allogeneic stem cell transplantation is usually recommended if the patient can tolerate the transplant and has a suitable donor. The optimal post-remission therapy for intermediate-risk AML (normal cytogenetics or cytogenetic changes that do not fall into the good-risk or high-risk groups) is less clear and depends on the specific circumstances, including the patient's age and overall health, the patient's personal values, and whether a suitable stem cell donor is available.

[0158] For patients who are not eligible for stem cell transplantation, immunotherapy with a combination of histamine dihydrochloride (Ceplene) and interleukin 2 (Proleukin) after completion of consolidation has demonstrated a 14% reduction in the absolute risk of relapse, translating into a 50% increase in the likelihood of maintaining remission.

[0159] For patients with relapsed AML, the only proven potential cure is hematopoietic stem cell transplantation (if hematopoietic stem cell transplantation has not already been performed). In 2000, gemtuzumab ozogamicin (Mylotarg), a cytotoxic agent linked to a monoclonal antibody, was approved in the United States for patients older than 60 years of age with relapsed AML who were not candidates for high-dose chemotherapy. This drug was voluntarily withdrawn from the market by its manufacturer, Pfizer, in 2010. Because treatment options for relapsed AML are very limited, palliative care can be provided.

[0160] Relapsed AML patients who are not candidates for stem cell transplantation or have relapsed after stem cell transplantation can be offered treatment in clinical trials because conventional treatment options are limited. Agents being studied include cytotoxic drugs, such as clofarabine, and targeted therapies, such as farnesyl transferase inhibitors, decitabine, and MDR1 (multidrug resistance protein) inhibitors. For relapsed acute promyelocytic leukemia (APL), arsenic trioxide has been tested in trials and approved by the U.S. FDA. Like ATRA, arsenic trioxide does not work for other subtypes of AML.

[0161] Although acute myeloid leukemia is a curable disease, the chance of a particular patient being cured depends on a variety of prognostic factors. The single most important prognostic factor in AML is the cytogenetics, or chromosomal structure of the leukemic cells. Certain cytogenetic abnormalities are associated with excellent outcomes (e.g., the (15:17) translocation in acute promyelocytic leukemia). About half of AML patients have "normal" cytogenetics; they belong to the intermediate-risk group. A variety of other cytogenetic abnormalities are known to be associated with poor prognosis and a high risk of relapse after treatment.

[0162] AML arising from a pre-existing myelodysplastic syndrome (MDS) or myeloproliferative disorder (so-called secondary AML) has a worse prognosis, as does treatment-related AML arising after chemotherapy for another preceding malignancy. These entities are all associated with a high rate of unfavorable cytogenetic abnormalities.

[0163] In some studies, age >60 years and elevated lactate dehydrogenase levels have also been associated with poor outcomes. As with most forms of cancer, functional status (ie, the patient's general physical condition and activity level) also plays an important role in prognosis.

[0164] FLT3 internal tandem duplication (ITD) has been shown to confer a poor prognosis in AML. Treating these patients with more aggressive therapies, such as stem cell transplantation in first remission, has not been shown to improve long-term survival. ITD of FLT3 can be associated with leukostasis. In 2012, the FLT3 inhibitor quizartinib showed positive Phase II trial results in AML patients with FLT3-ITD mutations. In 2017, the FLT3 inhibitor quizartinib, approved by the FDA, was approved by the FDA for Phase II therapy. (midostaurin, formerly PKC412) is indicated for the treatment of patients with newly diagnosed FLT3 mutation-positive (FLT3+) AML as detected by an FDA-approved test in combination with chemotherapy.

[0165] Researchers are studying the clinical significance of c-KIT mutations in AML. Since tyrosine kinase inhibitors (such as imatinib and sunitinib) can be used to pharmacologically block the activity of c-KIT, these are common and clinically relevant. Other genes being studied as prognostic factors or therapeutic targets include CEBPA, BAALC, ERG, and NPM1.

[0166] B. Chronic myelomonocytic leukemia (CMML)

[0167] CMML is a malignant hematopoietic stem cell disorder with clinical and pathological features of myeloproliferative neoplasms and myelodysplastic syndromes. Patients may experience symptoms or complications caused by previously unrecognized cytopenias (e.g., infection, fatigue, dyspnea, ecchymosis, bleeding), skin lesions, or splenomegaly-related symptoms (e.g., early satiety, abdominal distension). CMML is characterized by peripheral blood mononucleosis accompanied by bone marrow dysplasia. In the United States, approximately 2,000 new CMML cases are diagnosed each year. 15 to 30% of cases deteriorate into AML (Swerdlow et al., 2017), and the median survival rate of the entire WHO prognostic group is discouraging within the range of 10 to 48 months after initial diagnosis. Only HSCT has improved the disease of CMML patients to a clinically significant degree, and other therapies including cell reduction and hypomethylating agents provide symptom relief (Schuler et al., 2014). Therefore, new therapies are urgently needed to reduce the morbidity and prolong survival of CMML patients.

[0168] C. Acute lymphoblastic leukemia (ALL)

[0169] Acute lymphoblastic leukemia (ALL) or acute lymphoblastic leukemia is an acute form of leukemia or white blood cell cancer, characterized by excessive production of cancerous immature white blood cells (called lymphoblasts). In individuals suffering from ALL, lymphoblasts are overproduced and multiply in the bone marrow, causing damage and death by suppressing the production of normal cells (such as red blood cells and white blood cells and platelets) in the bone marrow and by infiltrating other organs. ALL is most common in childhood, with a peak incidence at 2-5 years of age and another peak at old age.

[0170] Symptoms of ALL indicate a decreased production of functional blood cells because the leukemia wastes the resources of the bone marrow, which are normally used to produce new functional blood cells. These symptoms can include fever, increased risk of infection (especially bacterial infections such as pneumonia, due to neutropenia; symptoms of such infections include shortness of breath, chest pain, cough, vomiting, changes in bowel or bladder habits), increased bleeding tendency (due to thrombocytopenia), and signs indicative of anemia, including pallor, tachycardia (high heart rate), fatigue, and headache.

[0171] About 6,000 cases are reported annually in the United States; statistics for other countries are difficult to obtain, but it is known to be more common in the United States, Italy, and Costa Rica. Cure is a realistic goal and is achieved in more than 80% of infected children, but only 20-40% of adults can be cured. "Acute" refers to the relatively short course of the disease as distinguished from chronic lymphocytic leukemia, which has an insidious course of many years.

[0172] Symptoms are not specific to ALL but worsen to the point of seeking medical help. It is caused by a lack of normal and healthy blood cells because they are crowded out by malignant and immature leukocytes (white blood cells). Therefore, people with ALL experience symptoms caused by dysfunction of their erythrocytes (red blood cells), white blood cells, platelets. Laboratory tests that may show abnormalities include blood count tests, kidney function tests, electrolyte tests, and liver enzyme tests.

[0173] Signs and symptoms of ALL are variable but result from bone marrow replacement and / or organ infiltration and include general weakness and fatigue, anemia, dizziness, frequent or unexplained fevers and infections, weight loss and / or loss of appetite, excessive and unexplained bruising, bone pain, joint pain (caused by the spread of "blast" cells into the joints on the surface of bones or in the bone marrow cavity), difficulty breathing, enlarged lymph nodes, liver and / or spleen, pressure edema (swelling) of the lower extremities and / or abdomen, and ecchymoses, which appear as tiny red spots or lines in the skin due to low platelet levels.

[0174] In general, cancer is caused by DNA damage, which leads to uncontrollable cell growth and spreads throughout the body by increasing chemical signals that cause growth or by interrupting chemical signals that control growth. Damage can be caused by the formation of fusion genes, and abnormal regulation of proto-oncogenes through their juxtaposition with the promoter of another gene (such as a T cell receptor gene). This damage can be caused by environmental factors such as chemicals, drugs, or radiation, and occurs naturally during mitosis or other normal processes (although cells have a variety of DNA repair mechanisms that help reduce this damage).

[0175] ALL is associated with exposure to radiation and chemicals in animals and humans. High-level radiation exposure is a known risk factor for leukemia, as found in studies of survivors of the atomic bombings of Hiroshima and Nagasaki. In animals, exposure to benzene and other chemicals can cause leukemia. Epidemiological studies have linked leukemia to exposure to chemicals in the workplace, but these studies are not conclusive. Some evidence suggests that in individuals treated with radiation and chemotherapy for other cancers, secondary leukemias may worsen as a result of the treatment.

[0176] Diagnosing ALL begins with a medical history, physical examination, complete blood count, and blood smear. Because the symptoms are so common, many other diseases with similar symptoms must be ruled out. Generally, the higher the white blood cell count, the worse the prognosis. In most cases, blasts are found on the blood smear (blasts are the precursors (stem cells) of all immune cell lines). A bone marrow biopsy is conclusive evidence of ALL. A lumbar puncture (also called a spinal tap) will indicate whether the spine and brain have been affected.

[0177] Pathological examination, cytogenetics (specifically, the presence of the Philadelphia chromosome), and immunophenotyping determine whether there is a problem with the bone marrow blasts (neutrophils, eosinophils, or basophils) or lymphoblasts (B lymphocytes or T lymphocytes) cells. RNA testing can determine how aggressive the disease is; different mutations have been associated with shorter or longer survival. Immunohistochemistry testing can reveal TdT or CALLA antigens on the surface of leukemic cells. TdT is a protein expressed early in the development of pre-T cells and pre-B cells, while CALLA is an antigen found in 80% of ALL cases, as well as in the "blast crisis" of CML. Medical imaging (such as ultrasound or CT scans) can reveal invasion of other organs, usually the lungs, liver, spleen, lymph nodes, brain, kidneys, and reproductive organs.

[0178] The earlier ALL is detected, the more effective treatment is. The goal is to induce a sustained remission, defined as the absence of detectable cancer cells in the body (usually less than 5% blasts in the bone marrow). Treatment of ALL may include chemotherapy, steroids, radiation therapy, intensive combination therapy (including bone marrow or stem cell transplant), and growth factors.

[0179] Chemotherapy is the initial treatment of choice. Most patients with ALL will receive a combination of different therapies. Surgery is not an option due to the systemic distribution of the malignant cells. In general, cytotoxic chemotherapy for ALL combines multiple anti-leukemic drugs in various combinations. Chemotherapy for ALL consists of three phases: remission induction therapy, intensification therapy, and maintenance therapy.

[0180] Because chemotherapy regimens can be intensive and prolonged (usually about 2 years with the GMALL UKALL, HyperCVAD, or CALGB regimens; about 3 years for ALL with the 2-month COG regimen in men; 2 years for ALL in women - longer in men because the testicles are a potential reservoir), many patients have an intravenous catheter (called a central venous catheter or Hickman line) inserted into a large vein, or a central venous port (Portacath), a cone-shaped port with a silicone nose that is surgically implanted under the skin (usually near the clavicle) and allows for the most effective use of drugs because of the low risk of infection and the long-term durability of the central venous port.

[0181] Radiation therapy (or radiotherapy) is used for painful bone areas with high disease burden, or as part of preparation for bone marrow transplantation (whole body radiation). Radiation in the form of whole brain radiation is also used for central nervous system prevention to prevent the recurrence of leukemia in the brain. Whole brain prophylactic radiation is a common method for treating childhood ALL. Recent studies have shown that CNS chemotherapy provides favorable results, while exacerbating side effects are less. Therefore, the use of whole brain radiation is more limited. Most experts who are good at adult leukemia have given up using radiation therapy for CNS prevention and use intrathecal chemotherapy.

[0182] For some subtypes of relapsed ALL, targeting biological targets such as the proteasome, combined with chemotherapy, has given promising results in clinical trials. Selecting biological targets based on their combined effects on leukemic lymphoblasts can improve the therapeutic effect of ALL in clinical trials. In ongoing clinical trials, the CD19-CD3 bispecific monoclonal murine antibody, Blinatumomab, shows great promise.

[0183] Chimeric antigen receptor (CAR) has been developed as a promising therapy for ALL. This technology uses a single-chain variable fragment (scFv) designed to recognize the cell surface marker CD19 as a method for treating ALL. CD19 is a molecule found on all B cells and can be used as a method to distinguish potential malignant B cell populations in patients. In this therapy, mice are immunized with CD19 antigens and produce anti-CD19 antibodies. Hybridomas produced from mouse spleen cells fused with myeloma cell lines can be developed as sources of cDNA encoding CD19-specific antibodies. The cDNA is sequenced and the sequences encoding the variable heavy and variable light chains of these antibodies are cloned together using a small peptide linker. This resulting sequence encodes scFv. This sequence can be cloned into a transgenic, which encodes the intracellular domain of the CAR. There are different arrangements of subunits used as intracellular domains, but they are usually composed of a hinge region connected to the scFv, a transmembrane region, an intracellular region of a costimulatory molecule such as CD28, and an intracellular domain of CD3-ζ containing ITAM repeats. Other sequences often included are 4-1bb and OX40. The final transgenic sequence containing scFv and intracellular domain sequence is then inserted into the immune effector cells obtained from the patient and amplified in vitro. In the aforementioned experiments, these are cytotoxic T cell types. Inserting DNA into effector cells can be achieved by several methods. Most commonly, this is done using a lentivirus encoding a transgenic. Pseudotypes, self-inactivating lentiviruses have been shown to be effective methods for stably inserting the desired transgene into the target cell genomic DNA. Other methods include electroporation and transfection, but the efficacy of these methods is limited because transgenic expression will weaken over time. Genetically modified effector cells are then transplanted back into the patient. Typically, this process is carried out together with conditioning regimens such as cyclophosphamide, which have been shown to enhance the effect of infused T cells. This effect has been attributed to the formation of an immune space ecological niche. The whole process produces effector cells, which are generally T cells that can recognize tumor cell antigens and initiate cytotoxic reactions in a major tissue-compatible complex-independent manner.

[0184] D. Chronic lymphoblastic leukemia (CLL)

[0185] B-cell chronic lymphocytic leukemia (B-CLL), also known as chronic lymphatic leukemia (CLL), is the most common type of leukemia (a type of white blood cell cancer) in adults. CLL affects B-cell lymphocytes that originate in the bone marrow, develop in the lymph nodes, and usually fight infection by producing antibodies. In CLL, B cells grow out of control and accumulate in the bone marrow and blood, where they crowd out healthy blood cells. CLL is at the stage of small lymphocytic lymphoma (SLL), a type of B-cell lymphoma that mainly appears in the lymph nodes. CLL and SLL are considered to be the same underlying disease with only different manifestations. CLL is an adult disease. Most (>75%) newly diagnosed people with CLL are over 50 years old, and most are men. However, in rare cases, it can occur in teenagers and occasionally in children. Some of them may be related to genetic predisposition.

[0186] Most people are asymptomatic at diagnosis as a result of routine blood tests reporting high white blood cell counts, but as it worsens, CLL causes swelling of the lymph nodes, spleen, and liver, and eventually causes anemia and infection. Early-stage CLL is not treated, while late-stage CLL is treated with chemotherapy and monoclonal antibodies.

[0187] DNA analysis has distinguished two main types of CLL with different survival times. CLL that is positive for the marker ZAP-70 has an average survival of 8 years, while CLL that is negative for ZAP-70 has an average survival of more than 25 years. Many patients, especially the elderly, whose disease progresses slowly, can be reassured and may not require any treatment during their lifetime.

[0188] Most people have no symptoms at diagnosis as a result of a routine blood test reporting a high white blood cell count. Less commonly, CLL can present with swollen lymph nodes but with a high white blood cell count in the blood or no signs of the disease. This is called small lymphocytic lymphoma. In some individuals, the disease becomes apparent only after the neoplastic cells fill up the bone marrow, causing anemia that produces fatigue or weakness.

[0189] CLL is usually first suspected with the presence of lymphocytosis, an increase in one type of white blood cell, on a complete blood count (CBC) test. This is usually discovered incidentally during a routine physician visit. The most common lymphocyte count is greater than 4000 cells per microliter (μl) of blood, but can be much higher. The presence of lymphocytosis in an elderly individual raises a strong suspicion of CLL, and confirmatory diagnostic testing, specifically flow cytometry, should be performed unless clinically unnecessary.

[0190] The diagnosis of CLL is based on the demonstration of the presence of abnormal B lymphocyte populations in the blood, bone marrow or tissues, which display abnormal but characteristic molecular patterns on the cell surface of the B lymphocyte population. This atypical molecular pattern includes the co-expression of the cell surface markers cluster of differentiation 5 (CD5) and cluster of differentiation 23 (CD23). In addition, all CLL cells in an individual are cloned, i.e., genetically identical. In fact, this is inferred by detecting only one of the mutually exclusive antibody light chains κ or λ in all abnormal B cell populations. Normal B lymphocytes are composed of different antibody-producing cells, producing a mixture of κ-expressing cells and λ-expressing cells. The lack of normal distribution of B cells that produce κ and B cells that produce λ is a basis for demonstrating clonality, which is a key element for diagnosing any B cell malignancy (B cell non-Hodgkin's lymphoma).

[0191] The diagnosis of CLL requires the combination of peripheral blood microscopy and flow cytometry analysis of lymphocytes to confirm clonality and marker molecule expression. Both are easily completed with a small amount of blood. Flow cytometry is an instrument that can examine the expression of molecules on individual cells in body fluids. This requires the use of specific antibodies to mark molecules with fluorescent tags recognized by the instrument. In CLL, lymphocytes are genetic clones, belonging to the B cell lineage (expressing marker molecules differentiation cluster 19 (CD19) and CD20), and typically express marker molecules CD5 and CD23. Under the microscope, these B cells are similar to normal lymphocytes, although slightly smaller, but are fragile when applied to a glass slide, thereby producing many broken cells, which are called "smear" or "smear" cells.

[0192] Matutes' CLL scoring allows the identification of homogeneous subgroups of classical CLL, which are different from atypical / mixed CLL for the expression of five markers (CD5, CD23, FMC7, CD22 and immunoglobulin light chains). Matutes' CLL scoring system is very helpful for the differential diagnosis between classical CLL and other B-cell chronic lymphoproliferative disorders, but is unhelpful for the immune distinction between mixed / atypical CLL and mantle cell lymphoma (MCL malignant B cells). The discrimination between CLL and MCL can be improved by adding unconventional markers such as CD54 and CD200. In conventional markers, most of the distinguishing features are the CD20 / CD23 mean fluorescence intensity ratio. In contrast, FMC7 expression can be unexpectedly misleading for marginal cases.

[0193] Staging to determine the extent of the disease is done using the Rai staging system or the Binet classification (see details) and is based primarily on the presence of low platelet or red blood cell counts. Early stage disease does not require treatment.

[0194] CLL treatment focuses on controlling the disease and its symptoms rather than on a complete cure. CLL is treated with chemotherapy, radiation therapy, biological therapy, or a bone marrow transplant. Symptoms are sometimes treated surgically (splenectomy to remove the enlarged spleen) or with radiation therapy (to "de-inflate" the swollen lymph nodes).

[0195] Initial CLL treatment varies depending on the exact diagnosis and progression of the disease, and even on the preferences and experience of the healthcare practitioner. Dozens of agents are used for CLL therapy. An initial treatment regimen containing fludarabine, cyclophosphamide, and rituximab (referred to as FCR) has demonstrated high overall and complete response rates.

[0196] The study, conducted by researchers at the University of Pennsylvania, used genetically modified T cells to attack cells expressing the CD19 protein to fight the disease. In 2013, the researchers announced that 26 of 59 patients had achieved complete remission and remained tumor-free.

[0197] Leukemia is rarely associated with pregnancy, affecting only about 1 in 10,000 pregnant women. Treatment of chronic lymphocytic leukemia can usually be delayed until after the pregnancy is over. If treatment is necessary, chemotherapy given during the second or third trimester is less likely to result in pregnancy loss or birth defects than treatment during the first trimester.

[0198] Although CLL is generally considered incurable, it deteriorates slowly in most cases. Many people with CLL live normal and active lives for many years - in some cases for decades. Due to its slow onset, early CLL is generally not treated because it is believed that early CLL intervention does not improve survival time or quality of life. Instead, symptoms are monitored over time to detect any changes in disease patterns.

[0199] The decision to start CLL treatment is made when the patient's clinical symptoms or blood counts indicate that the disease has worsened to a point where it may affect the patient's quality of life. Clinical "staging systems" such as the Rai 4 platform system and the Binet classification can help determine when and how to treat patients. Determining when to start treatment and by what method is often difficult; studies have shown that treating the disease too early does not have a survival advantage. The National Cancer Institute Working Group has issued treatment guidelines in which specific markers should be met before it is started.

[0200] Combination chemotherapy regimens are effective for both newly diagnosed and relapsed CLL. The combination of fludarabine and an alkylating agent (cyclophosphamide) produces higher response rates and longer progression-free survival than single agents: FC (fludarabine and cyclophosphamide); FR (fludarabine and rituximab); FCR (fludarabine, cyclophosphamide, and rituximab); and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone).

[0201] Although the purine analog fludarabine demonstrated superior response rates to chlorambucil as primary therapy, there is no evidence that early use of fludarabine improves overall survival, and some clinicians prefer to reserve fludarabine for recurrent disease.

[0202] In a large randomized trial of CLL patients who were selected to be in good health, chemoimmunotherapy with FCR has been shown to improve response rate, progression-free survival, and overall survival. This is the first clinical trial showing that the selection of first-line therapy can improve overall survival in CLL patients. Alkylating agents approved for CLL include bendamustine and cyclophosphamide.

[0203] Targeted therapy attacks specific targets on cancer cells with the goal of not harming normal cells. Monoclonal antibodies such as alemtuzumab (targeting CD52) and rituximab and ofatumumab (targeting CD20) are used in CLL. Tyrosine kinase inhibitor therapy can also be used in CLL. In February 2014, the FDA approved ibrutinib for the treatment of chronic lymphocytic leukemia. Ibrutinib is a Bruton's tyrosine kinase (BTK) inhibitor. In July 2014, the FDA and EMA approved idelalisib for the treatment of different types of leukemia. Idelalisib is a PI3K inhibitor that targets the PI3Kδ pathway. It is taken orally.

[0204] Autologous stem cell transplantation, which uses the recipient's own cells, is not curative. Younger individuals who are at high risk of dying from CLL may consider allogeneic hematopoietic stem cell transplantation (HSCT). Myeloablative (bone marrow-killing) forms of allogeneic stem cell transplantation, a high-risk treatment using blood cells from healthy donors, can be curative, but treatment-related toxicity is significant. An intermediate level, called a conditioned allogeneic stem cell transplant of reduced intensity, may be better tolerated by elderly or frail patients.

[0205] "Refractory" CLL is disease that no longer responds well to treatment. In this case, more aggressive therapies are considered, including lenalidomide, flavopiridol, and a bone marrow (stem cell) transplant. The monoclonal antibody, alemtuzumab (directed against CD52), can be used in patients with refractory bone marrow-based disease.

[0206] Complications include Richter's syndrome, hypogammaglobulinemia leading to recurrent infections, warm autoimmune hemolytic anemia in 10-15% of patients, and transformation to high-grade lymphomas. Chronic lymphocytic leukemia can transform to Richter's syndrome, which is a deterioration in patients with chronic lymphocytic leukemia to rapidly growing diffuse large B-cell lymphoma, prolymphocytic leukemia, Hodgkin's lymphoma, or acute leukemia. The incidence in patients with CLL is estimated to be approximately 5%.

[0207] Chronic lymphocytic leukemia can rarely involve the gastrointestinal (GI) tract. Some clinical manifestations that have been reported include intussusception, bacterial contamination of the small intestine, colitis, and others. Usually, GI complications of CLL develop after Richter transformation. To date, there are two case reports of GI involvement in chronic lymphocytic leukemia without Richter transformation.

[0208] III. Monoclonal Antibodies and Their Production

[0209] The monoclonal antibodies described herein can be prepared using standard methods, followed by screening, characterization, and functional evaluation. The variable regions can be sequenced and then subcloned into human expression vectors to produce chimeric antibody genes, which are then expressed and purified. These chimeric antibodies can be tested in antigen binding, signal transduction blocking, and xenotransplantation experiments.

[0210] A. General Methods

[0211] It will be appreciated that monoclonal antibodies that bind to LILRB4 will have several applications. These applications include the manufacture of diagnostic kits for the detection and diagnosis of cancer, and for use in cancer therapy. In these contexts, such antibodies may be combined with diagnostic or therapeutic agents, used as capture agents or competitors in competitive assays, or used independently without attachment to additional agents. Antibodies may be mutated or modified, as further discussed 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. Pat. No. 4,196,265).

[0212] Methods for producing monoclonal antibodies (MAbs) generally begin along the same production lines as those used to prepare polyclonal antibodies. The first step in these methods is immunization of an appropriate host. As is well known in the art, the immunogenicity of a given immunization composition may vary. Therefore, it is generally necessary to enhance the host immune system, which can be achieved by coupling the 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. The means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-nitrobenzidine. As is also well known in the art, the immunogenicity of a particular immunogenic composition can be increased by using a nonspecific immune response stimulator (called an adjuvant). Exemplary and preferred adjuvants include complete Freund's adjuvant (a nonspecific immune response stimulator containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0213] The amount of the immunogenic composition used to produce polyclonal antibodies varies according to the nature of the immunogen and the animal used for immunization. Immunogens can be administered using a variety of approaches (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies can be monitored by obtaining blood samples of the immunized animals at different time points after immunization. A second booster injection can also be given. The process of booster immunization and titration is repeated until a suitable titer is reached. When the desired immunogenicity level is obtained, blood can be drawn from the immunized animals and serum can be separated and stored, and / or animals can be used to produce MAbs.

[0214] After immunization, somatic cells that may produce antibodies (specifically, B lymphocytes (B cells)) are selected for use in MAb production schemes. These cells can be obtained from the spleen or lymph nodes of a biopsy, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of immortal myeloma cells (generally immortal myeloma cells of an animal of the same species as the immunized animal) or human or human / mouse chimeric cells. Myeloma cells suitable for use in fusion procedures for producing hybridomas preferably do not produce antibodies, have high fusion efficiency, and are insufficient in enzymes to prevent them from growing in certain selective culture media that only support the growth of the desired fusion cells (hybridomas). It is known to those skilled in the art that any of a variety of myeloma cells can be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984).

[0215] Methods for producing hybrids of antibody-producing spleen or lymph node cells and myeloma cells generally comprise mixing somatic cells with myeloma cells in a 2:1 ratio in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion, but the ratio may vary from about 20:1 to about 1:1 individually. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and fusion methods using polyethylene glycol (PEG), such as 37% (v / v) PEG, have been described by Gefter et al. (1977). Fusion methods using electrical induction are also suitable (Goding, pp. 71-74, 1986). The fusion procedure is generally performed at about 1×10 -6 Up to 1×10 -8 The lower frequency of producing viable hybrids. However, this will not cause problems, because the viable fusion hybrids are differentiated by culturing in a selective medium by infusion of the parental cells (specifically, the infusion myeloma cells that usually continue to divide indefinitely). The selective medium is generally a medium containing a medicament that prevents nucleotides from being resynthesized in tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate and azoserine. Aminopterin and methotrexate prevent the resynthesis of purines and pyrimidines, while azoserine only prevents purine synthesis. In the case of using aminopterin or methotrexate, the culture medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT culture medium). In the case of using azoserine, the culture medium is supplemented with hypoxanthine. If the B cell source is a human B cell line transformed by Epstein Barr virus (EBV), then in order to remove the EBV transformed line that is not fused with myeloma, ouabain is added.

[0216] The preferred selection medium is HAT or HAT containing ouabain. Only cells that can operate the nucleotide salvage path can survive in the HAT medium. Myeloma cells lack the key enzymes of the salvage path, such as hypoxanthine phosphoribosyltransferase (HPRT), and they cannot survive. B cells can operate this path, but they have a limited lifespan when cultivated and generally die within about two weeks. Therefore, the only cells that can survive in the selection medium are those hybrids formed by myeloma and B cells. When the B cell source for fusion is a B cell line transformed by EBV, as here, ouabain is also used for drug selection of hybrids, because the B cells transformed by EBV are easily killed by drugs, and the myeloma partner used should select ouabain resistance.

[0217] Cultivation provides a group of hybridomas from which specific hybridomas are selected. Typically, the selection of hybridomas is carried out by culturing cells with a dilution of a single clone in a microtiter plate, followed by testing the desired reactivity of individual clone supernatants (after about two to three weeks). The analytical method should be sensitive, simple and rapid, such as radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, spot immunobinding assay, etc. The selected hybridoma is then serially diluted or single cell sorted by flow cytometry sorting, and cloned into individual antibody-producing cell lines, which can then be infinitely propagated to provide MAbs. MAbs can be produced in two basic ways using cell lines. Hybridoma samples can be injected into animals (e.g., mice) (usually injected into the peritoneal cavity). Optionally, before injection, animals are presensitized with hydrocarbons (especially oils, such as pristane (tetramethylpentadecane)). When using human hybridomas in this way, it is best to inject into immunocompromised mice (e.g., SCID mice) to prevent tumor rejection. The injected animal produces a tumor that secretes the specific monoclonal antibody produced by the fused cell hybrid. The animal's body fluids, such as serum or ascites, can then be extracted to provide high concentrations of MAbs. Individual cell lines can also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which high concentrations of MAbs can be easily obtained. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in cell supernatants. The cell line can be adapted to grow in serum-free medium to optimize the ability to recover highly purified human monoclonal immunoglobulins.

[0218] If necessary, MAbs produced by either method can be further purified using filtration, centrifugation, and various chromatographic methods (e.g., FPLC or affinity chromatography). Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes (e.g., pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0219] It is also contemplated that monoclonal antibodies can be produced using molecular cloning methods. To this end, RNA can be isolated from hybridoma lines and antibody genes obtained by RT-PCR and cloned into immunoglobulin expression vectors. Alternatively, a combinatorial immunoglobulin phage library is prepared from RNA isolated from a cell line and phages expressing appropriate antibodies are selected by panning using viral antigens. The advantage of this method over conventional hybridoma technology is that approximately 10 phages can be generated and screened in one round. 4 The H chain and L chain can generate new specificities, thereby further increasing the chance of discovering appropriate antibodies.

[0220] Other U.S. patents (each incorporated herein by reference) that teach the manufacture of antibodies suitable for use in the present disclosure include U.S. Patent 5,565,332, which describes the use of combinatorial methods to produce chimeric antibodies; U.S. Patent 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973, which describes antibody-therapeutic agent conjugates.

[0221] B. Antibodies of the Disclosure

[0222] 1. Antibodies against LILRB4

[0223] The antibodies or antigen-binding fragments thereof according to the present disclosure can in the first instance be defined by their binding specificity (in this case to LILRB4). One skilled in the art can determine whether such an antibody falls within the scope of the present claims by assessing the binding specificity / affinity of a given antibody using techniques well known to those skilled in the art.

[0224] In one aspect, antibodies and antigen-binding fragments that specifically bind to LILRB4 are provided. In some embodiments, when bound to LILRB4, such antibodies regulate the activation of LILRB4. In certain embodiments, the antibody or antigen-binding fragment activates LILRB4 when bound to LILRB4. In certain embodiments, the antibody or antigen-binding fragment inhibits the activation of LILRB4 when bound to LILRB4. In certain embodiments, the antibody or antigen-binding fragment can specifically interfere with, block or reduce the interaction between ApoE and LILRB4 when bound to LILRB4. In certain embodiments, the antibodies or antigen-binding fragments provided herein can inhibit ApoE-mediated LILRB4 activity. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically or selectively bind to human LILRB4.

[0225] In some embodiments, the antibody or antigen-binding fragment specifically binds to human LILRB4 and / or substantially inhibits the binding of human LILRB4 to ApoE by at least about 20%-40%, 40-60%, 60-80%, 80-85% or more (e.g., according to an assay as disclosed in the Examples). In some embodiments, the antibody or antigen-binding fragment has a Kd of less than (binds more tightly) 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13In some embodiments, the antibody or antigen-binding fragment blocks the binding of ApoE to LILRB4 with an IC50 of less than 1 μM, 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.

[0226] In some embodiments, the antibodies or antigen-binding fragments provided herein have Figures 28A to 28C The heavy chain and Figures 30A to 30C Such antibodies can be produced using the methods described herein from clones discussed in the Examples section below. In certain embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the AbM definition, or a contact definition of the CDR. In certain embodiments, the antibodies or antigen-binding fragments are characterized by the heavy and light chain CDR sequences of the cloned pairs of Tables 1 and 2.

[0227] In certain embodiments, antibodies can be defined by their variable sequences, which include additional "framework" regions. Figures 28A to 28C and Figures 30A to 30B The heavy and light chain amino acid sequences of the cloned pairings of the antibody may be different from these sequences, in particular, regions outside of the CDRs. For example, the amino acids may differ from those set forth above by a certain percentage, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or the amino acids may differ from those set forth above by allowing conservative substitutions (discussed below). Each of the foregoing is applicable to Figures 28A to 28C and Figures 30A to 30C 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.

[0228] Although the antibodies disclosed herein are produced in the form of IgG, it may be applicable to modify the constant region to change its function. The constant region of an antibody generally mediates the binding of the antibody to a host tissue or factor, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Therefore, the term "antibody" includes complete immunoglobulins of IgA, IgG, IgE, IgD, IgM types (and their subtypes), wherein the light chain of the immunoglobulin may be a κ or λ type. Within the light chain and the heavy chain, the variable region and the constant region are joined by 35 "J" regions with about 12 or more amino acids, wherein the heavy chain also includes a "D" region with about 10 or more amino acids. Generally, see Chapter 7 of "Fundamental Immunology" (Paul, W., ed., 2nd edition Raven Press, NY (1989)).

[0229] The present disclosure further includes nucleic acids that hybridize to nucleic acids encoding antibodies disclosed herein. In general, nucleic acids hybridize to nucleic acids encoding antibodies disclosed herein and also encoding antibodies that retain the ability to specifically bind to LILRB4 under medium or high stringency conditions. A first nucleic acid molecule is "hybridizable" to a second nucleic acid molecule when the single-stranded form of the first nucleic acid molecule is able to anneal to pair with the second nucleic acid molecule under appropriate conditions of temperature and solution ionic strength (see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001). The conditions of temperature and ionic strength determine the "stringency" of hybridization. Typical medium stringency hybridization conditions are 40% formamide, 5× or 6× SSC and 0.1% SDS at 42°C. High stringency hybridization conditions are 50% formamide, 5× or 6× SSC (0.15 M NaCl and 0.015 M sodium citrate) at 42° C. or, optionally, at higher temperatures (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, but depending on the stringency of the hybridization, mismatches between bases are possible. The stringency applicable to the hybridization of nucleic acids depends on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency with which the nucleic acids can hybridize. For hybrids of more than 100 nucleotides in length, equations for calculating melting temperatures have been derived (see Sambrook et al., supra). For hybridization of shorter nucleic acids (e.g., oligonucleotides), the position of the mismatch becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra).

[0230] Table 1. Heavy chain CDR sequence, amino acid

[0231]

[0232] Table 2. κ light chain CDR, amino acid sequence

[0233]

[0234]

[0235] Table 3. Sequence ID numbers of LILRB4 antibodies

[0236]

[0237] Table 4. Sequence ID numbers of humanized 193 (h193) antibody heavy chain

[0238] Heavy chain Variable region (CDR1, CDR2, CDR3) AASEQ ID NO. h193-H1 223(100,101,224) h193-H2 225(100,101,224) h193-H3 226(100,101,227) h193-H4 228(100,101,227) h193-H5 229(100,101,227) h193-H6 230(100,101,227) h193-H7 231(100,101,227)

[0239] Table 5. Sequence ID numbers of humanized 193 (h193) antibody light chain

[0240] Light chain Variable region (CDR1, CDR2, CDR3) AASEQ ID NO. h193-K1 232(104,233,234) h193-K2 235(104,233,234) h193-K3 236(104,233,234) h193-K4 237(104,233,234)

[0241] 2. Exemplary Epitopes and Competing Antigen Binding Proteins

[0242] In another aspect, the present disclosure provides epitopes to which anti-LILRB4 antibodies bind.

[0243] In some embodiments, the epitopes bound by the antibodies described herein are useful. In certain embodiments, the epitopes provided herein can be used to isolate antibodies or antigen binding proteins that bind to LILRB4. In certain embodiments, the epitopes provided herein can be used to generate antibodies or antigen binding proteins that bind to LILRB4. In certain embodiments, the epitopes or sequences comprising the epitopes provided herein can be used as immunogens to generate antibodies or antigen binding proteins that bind to LILRB4. In certain embodiments, the epitopes described herein or sequences comprising the epitopes described herein can be used to interfere with the biological activity of LILRB4.

[0244] In some embodiments, antibodies or antigen-binding fragments thereof that bind to any epitope are particularly useful. In some embodiments, the epitopes provided herein modulate the biological activity of LILRB4 when bound to an antibody. In some embodiments, the epitopes provided herein activate LILRB4 when bound to an antibody. In some embodiments, the epitopes provided herein inhibit the activation of LILRB4 when bound to an antibody. In some embodiments, the epitopes provided herein block the interaction between ApoE and LILRB4 when bound to an antibody.

[0245] In some embodiments, domains / regions containing residues that are in contact with or buried by an antibody can be identified by mutating specific residues in LILRB4 and determining whether the antibody can bind to the mutated LILRB4 protein. By making multiple individual mutations, residues that play a direct role in binding or are sufficiently close to the antibody that the mutation can affect the binding between the antibody and the antigen can be identified. Based on the knowledge of these amino acids, antigenic domains or regions containing residues that are in contact with or buried by the antibody can be elucidated. Such domains can include binding epitopes of antigen binding proteins.

[0246] In another aspect, the present invention provides an antigen binding protein that competes with one of the exemplary antibodies or antigen binding fragments that bind to an epitope described herein for specific binding to LILRB4. Such antigen binding proteins may also bind to the same epitope or overlapping epitopes as one of the antibodies or antigen binding fragments exemplified herein. Antigen binding proteins that compete with or bind to the same epitope as the exemplary antibodies are expected to exhibit similar functional properties. Exemplary antibodies include the antibodies described above, including antibodies comprising heavy and light chain variable regions and CDRs included in Tables 1 and 2. In certain embodiments, the epitope is located in the linker region between the D1 and D2 domains of human LILRB4. In certain embodiments, the epitope comprises at least one amino acid within one or more amino acid sequences listed in Table 9. In certain embodiments, the epitope comprises at least one amino acid within one or more amino acid sequences selected from W18, G96, A97, Y98, S99, K100, Q122, S123, R124, S125, P126, H153 and Q154 of SEQ ID NO: 238 (D1 and D2 domains of human LILRB4 protein).

[0247] C. Engineering of Antibody Sequences

[0248] In various embodiments, the sequence of the identified antibody may be chosen to be engineered for various reasons, such as improved expression, improved cross-reactivity, or reduced off-target binding. The following generally discusses relevant techniques for antibody engineering.

[0249] Hybridomas can be cultivated, cells are then lysed and total RNA is extracted. Random hexamers can be used at RT to produce cDNA copies of RNA, and then PCR is performed using a multivariate mixture of PCR primers that are expected to amplify all human variable gene sequences. PCR products can be cloned into pGEM-T Easy vectors, and then sequenced using standard vector primers by automatic DNA sequencing. Analysis of binding and neutralization can be performed using antibodies, which are collected from hybridoma supernatants and purified by FPLC using protein G columns. Recombinant full-length IgG antibodies can be produced by subcloning the heavy chain and light chain Fv DNA from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies and purification are collected from 293 or CHO cell supernatants.

[0250] The rapid availability of antibodies produced in the same host cells and cell culture methods as the final cGMP manufacturing process is likely to shorten the duration of the method development program. Lonza has developed a universal method that uses pooled transfections grown in CDACF culture media for rapid production of small amounts (up to 50 g) of antibodies in CHO cells. Although slightly slower than the actual transient system, the advantages include higher product concentrations and the use of the same host and method as the production cell line. An example of the growth and productivity of the GS-CHO pool expressing a model antibody in a disposable bioreactor: In a disposable bag bioreactor culture (5L working volume) operated in a batch feed mode, a collection antibody concentration of 2 g / L was reached within 9 weeks of transfection.

[0251] Antibody molecules will include fragments (e.g., F(ab'), F(ab') and F(ab')) generated by, for example, proteolytic cleavage of mAbs. 2 ), or single chain immunoglobulins that can be produced, for example, via recombinant methods. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with each other, or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Obviously, such chimeric molecules can contain substituents that can bind to different epitopes of the same molecule.

[0252] 1. Antigen Binding Modification

[0253] In related embodiments, the antibody is a derivative of the disclosed antibody, such as an antibody containing CDR sequences that are consistent with the CDR sequences in the disclosed antibody (e.g., a chimeric antibody or a CDR-grafted antibody). Alternatively, it may be desirable to make modifications, such as introducing conservative changes into the antibody molecule. When making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic index of amino acids in conferring interactive biological function on proteins is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydrophilicity characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0254] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as regulated by the hydrophilicity of its neighboring amino acids, correlates with the biological properties 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: aspartic acid (+3.0±1), glutamate (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.3); +0.2) and threonine (-0.4); sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic, non-aromatic 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).

[0255] It is understood that an amino acid can be substituted with another amino acid of similar hydrophilicity and produce a biologically or immunologically altered protein. In such changes, substitution of amino acids with hydrophilicity values ​​within ±2 is preferred, substitution of amino acids with hydrophilicity values ​​within ±1 is particularly preferred, and substitution of amino acids with hydrophilicity values ​​within ±0.5 is even more particularly preferred.

[0256] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account various of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0257] The present disclosure also encompasses isotype modifications. By modifying the Fc region to have a different isotype, different functions can be achieved. For example, changing to IgG 1 It can increase antibody-dependent cellular cytotoxicity, conversion to the A class can improve tissue distribution, and conversion to the M class can improve valency.

[0258] Modified antibodies can be prepared by any technique known to those skilled in the art, including expression via standard molecular biology techniques, or chemical synthesis of polypeptides. Methods for recombinant expression are described elsewhere in this document.

[0259] 2. Fc region modification

[0260] The antibodies disclosed herein may also be engineered to include modifications within the Fc region, typically altering 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 toxicity). In addition, the antibodies disclosed herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, again altering one or more functional properties of the antibody. Each of these embodiments is further described in detail below. The residue numbering in the Fc region is the EU index numbering 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. Pat. No. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702. Such modifications may be used to enhance or inhibit various responses of the immune system, and may have beneficial effects in diagnosis and therapy. Changes in the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fcs. Alterations in the Fc may also alter the half-life of the antibody in therapeutic antibodies, thereby reducing dosing frequency and thus increasing convenience and reducing substance use. This mutation reportedly eliminates heterogeneity of inter-heavy chain disulfide bridges in the hinge region.

[0261] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is increased or decreased. This method is further described in detail in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is changed to, for example, promote light chain and heavy chain assembly or improve or reduce the stability of the antibody. In another embodiment, the antibody is modified to increase its biological half-life. Various methods are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375. Alternatively, as described in U.S. Patent Nos. 5,869,046 and 6,121,022, in order to increase the biological half-life, the antibody can be changed in the CH1 or CL region to contain a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG. In other embodiments, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced by different amino acid residues so that the antibody has an altered affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail in U.S. Patents 5,624,821 and 5,648,260.

[0262] 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 method is further described in PCT Publication WO 94 / 29351. In yet another example, the Fc region is modified to increase or decrease the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase or decrease the affinity of the antibody for Fcγ receptors 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, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 90, 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 further described in PCT Publication WO 00 / 42072. In addition, 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 have been shown to improve binding to FcγRIII. In addition, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A.

[0263] In one embodiment, the Fc region is modified by modifying residues 243 and 264 to reduce the ability of the antibody to mediate effector functions and / or to improve anti-inflammatory properties. 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 by modifying residues 243, 264, 267 and 328 to reduce the ability of the antibody to mediate effector functions and / or to improve anti-inflammatory properties. In another embodiment, the antibody comprises a specific glycosylation pattern. For example, a deglycosylated antibody (i.e., an antibody lacking glycosylation) can be produced. The glycosylation pattern of the antibody can be changed to, for example, increase the affinity or avidity of the antibody for an antigen. Such modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the sites. Such deglycosylation can increase the affinity or avidity of the antibody for an antigen. See, e.g., U.S. Patents 5,714,350 and 6,350,861.

[0264] Antibodies in which the glycosylation pattern includes hypofucosylated or defucosylated glycans, such as hypofucosylated antibodies or defucosylated antibodies having a reduced amount of fucosyl residues on the glycans, can also be prepared. Antibodies can also include glycans having an increased amount of bisected GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of the antibody. Such modifications can be achieved, for example, by expressing the antibody in a host cell in which the glycosylation pathway is genetically engineered to produce a glycoprotein with a specific glycosylation pattern. These cells have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention, thereby producing antibodies 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. Ms704, Ms705 and Ms709 FUT8- / - cell lines were generated by 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 encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit low fucosylation by reducing or eliminating α-1,6 bond-related enzymes. EP 1 176 195 also describes a cell line with low or no enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of an antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 describes a variant CHO cell line Lec13 cells in which the ability to attach fucose to carbohydrates attached to Asn(297) is reduced, also resulting in hypofucosylation of antibodies expressed in the host cells. Antibodies with altered glycosylation profiles can also be produced in eggs, as described in PCT Publication WO 06 / 089231. PCT Publication WO 99 / 54342 describes a cell line engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetamido-glucose transferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibody.

[0265] Alternatively, the fucose residues of the antibody can be cleaved using a fucosidase; for example, fucosidase α-L-fucosidase removes fucosyl residues in antibodies. The antibodies disclosed herein further include antibodies produced in lower eukaryotic host cells, specifically, fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins with mammalian-like or human-like glycosylation patterns. A particular advantage of these genetically modified host cells relative to currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins produced in the cells, allowing the production of compositions of glycoproteins in which a particular N-glycan structure is dominant (see, e.g., U.S. Pat. Nos. 7,029,872 and 7,449,308). These genetically modified host cells have been used to produce antibodies in which a particular N-glycan structure is dominant.

[0266] 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 an enzymatic pathway for producing fucosylated glycoproteins (see, e.g., PCT Publication WO2008112092). In specific embodiments, the antibodies disclosed herein further include those produced in lower eukaryotic host cells and comprising fucosylated and non-fucosylated hybrid and complex N-glycans, including bisecting 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 certain embodiments, the antibody compositions provided herein may comprise antibodies having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2;

[0267] In certain aspects, the hybrid N-glycan is the predominant N-glycan species in the composition. In other aspects, the hybrid N-glycan is a specific N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% hybrid N-glycan in the composition.

[0268] In specific embodiments, the antibody compositions provided herein comprise antibodies having at least one complex N-glycan selected from the group consisting of: GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2;

[0269] and NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; and

[0270] NANA2Gal2GlcNAc2Man3GlcNAc2. In certain aspects, the complex N-glycan is the main N-glycan species in the composition. In other aspects, the complex N-glycan is a specific N-glycan species that contains about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% complex N-glycans in the composition. In certain embodiments, the N-glycan is fucosylated. Generally speaking, fucose is α1,3-linked to GlcNAc at the reducing end of the N-glycan, α1,6-linked to GlcNAc at the reducing end of the N-glycan, α1,2-linked to Gal at the non-reducing end of the N-glycan, α1,3-linked to GlcNac at the non-reducing end of the N-glycan, or α1,4-linked to GlcNac at the non-reducing end of the N-glycan.

[0271] Thus, in a specific aspect of the above glycoprotein composition, the glycoform adopts an α1,3-linked or α1,6-linked fucose form to generate 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); adopts an α1,3-linked or α1,4-linked fucose form to generate a glycoform selected from the group consisting of: GlcNAc(Fuc)Man5GlcNAc2 , GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fuc1-2)Man3GlcNAc2, GalGlcNAc2(Fuc1-2)Man3GlcNAc2, Gal2G lcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc1-2)Man3GlcNAc2 and NANA2Gal2GlcNAc2(Fuc1-2)M an3GlcNAc2; or in the form of α1,2-linked 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.

[0272] In other aspects, the antibodies comprise high mannose N-glycans, including but not limited to Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or N-glycans consisting of Man3GlcNAc2 N-glycan structures. In the above other aspects, complex N-glycans further include fucosylated and non-fucosylated bisected and multiantennary species. As used herein, the terms "N-glycans" and "glycoforms" are used interchangeably and refer to N-linked oligosaccharides, such as asparagine residues attached to a polypeptide via an asparagine-N-acetylglucosamine linkage. N-linked glycoproteins contain N-acetylglucosamine residues attached to the amide nitrogen of an asparagine residue in a protein.

[0273] D. Single-chain antibody

[0274] Single chain variable fragment (scFv) is a fusion of the variable regions of the immunoglobulin heavy chain and light chain connected together via a short (usually serine, glycine) linker. Although the constant region is removed and the linker peptide is introduced, this chimeric molecule retains the specificity of the original immunoglobulin. This modification does not usually change the specificity. Historically, these molecules were produced to help phage display, where it is particularly suitable to express the antigen binding domain in the form of a single peptide. Alternatively, scFv can be directly produced by heavy and light chains derived from subclones of hybridomas. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and therefore lack a common binding site (e.g., protein A / G) for purifying antibodies. These fragments can usually be purified / immobilized using protein L because protein L interacts with the variable region of the kappa light chain.

[0275] Flexible linkers generally contain amino acid residues that contribute to helices and turns, such as alanine, serine, and glycine. However, other residues may also play a role. Tang et al. (1996) used phage display as a method to quickly select custom linkers for single-chain antibodies (scFv) from a protein linker library. A random linker library was constructed in which the genes for the heavy and light chain variable domains were connected by segments encoding 18 amino acid polypeptides of different compositions. The scFv repertoire (approximately 5×10 6 The selected population of variants exhibited a significant increase in binding activity while maintaining considerable sequence diversity. 1054 individual variants were subsequently screened to yield catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed a significant increase in V H A conserved proline in the linker two residues after the C-terminus and a large number of arginines and prolines at other positions served as the only common features of the selected tethers.

[0276] The recombinant antibodies of the present disclosure may also be directed to sequences or portions that allow receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow the formation of multimers together with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chain may be modified with agents such as biotin / antibiotic proteins, thereby allowing two antibody combinations.

[0277] In an independent embodiment, a single-chain antibody can be produced by connecting the receptor light chain and heavy chain using a non-peptide linker or chemical unit. Generally speaking, the light chain and heavy chain will be produced in different cells, purified, and then connected together in an appropriate manner (i.e., the N-terminus of the heavy chain is connected to the C-terminus of the light chain via an appropriate chemical bridge).

[0278] Cross-linking agents are used to form molecular bridges, thereby tying together the functional groups of two different molecules (e.g., stabilizers and coagulants). However, it is expected that dimers or multimers of the same analog or heteromeric complexes containing different analogs may be produced. In order to connect two different compounds in a stepwise manner, heterobifunctional cross-linking agents can be used to eliminate unwanted homopolymer formation.

[0279] Exemplary heterobifunctional cross-linkers contain two reactive groups: one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.) Via the primary amine reactive group, the cross-linker can react with lysine residues of one protein (e.g., a selected antibody or fragment), and via the thiol reactive group, the cross-linker already tethered to the first protein reacts with cysteine ​​residues (free sulfhydryl groups) of another protein (e.g., a selective agent).

[0280] Preferably, a cross-linking agent with reasonable stability in blood will be used. Numerous types of disulfide-containing linkers are known that can be successfully used to conjugate targeting agents and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds can prove to provide greater stability in vivo, thereby preventing the release of the targeting peptide before reaching the site of action. These linkers are therefore a group of linked agents.

[0281] Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "hindered" by adjacent benzene rings and methyl groups. It is believed that steric hindrance of the disulfide bond serves to protect the bond from attack by thiol anions (e.g., glutathione) that may be present in tissues and blood, and thereby helps prevent the conjugate from decoupling prior to delivery of the attached agent to the target.

[0282] Like many other known cross-linking reagents, the SMPT cross-linking reagent is able to cross-link functional groups such as the SH of cysteine ​​or primary amines (e.g., the epsilon amino group of lysine). Another type of possible cross-linking reagent includes heterobifunctional photoreactive phenylazides containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amine groups and the phenylazide (after photolysis) reacts non-selectively with any amino acid residue.

[0283] In addition to sterically hindered crosslinkers, non-sterically hindered linkers may also be employed accordingly. Other useful crosslinkers that are not believed to contain or generate protected disulfide bonds include SATA, SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of such crosslinkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0284] U.S. Patent No. 4,680,338 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and / or proteins, particularly for forming conjugates of antibodies with chelators, drugs, enzymes, detectable labels, and the like. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under a variety of mild conditions. Such linkers are particularly useful in situations where the agent of interest can be directly bonded to the linker and cleavage results in release of the active agent. Specific uses include adding free amino or free sulfhydryl groups to proteins such as antibodies or drugs.

[0285] U.S. Patent No. 5,856,456 provides peptide linkers for linking polypeptide components to form fusion proteins (e.g., single-chain antibodies). The linkers have a length of up to about 50 amino acids, contain at least one occurrence of a charged amino acid (preferably arginine or lysine), followed by a proline, and are characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers suitable for use in a variety of immunodiagnostic and separation techniques.

[0286] E. Purification

[0287] In certain embodiments, the antibodies of the present disclosure may be purified. As used herein, the term "purified" means a composition that can be separated from other components, wherein the protein has been purified to any extent relative to its naturally obtainable state. Therefore, a purified protein also refers to a protein that is separated from the environment in which it can naturally exist. Where the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the major component of the composition, e.g., the protein accounts for about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition.

[0288] Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, the approximate fractionation of the cellular environment into polypeptide and non-polypeptide parts. After separating the polypeptide from other proteins, the polypeptide of interest can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. Other methods for protein purification include precipitation using ammonium sulfate, PEG, antibodies, etc., or precipitation by heat denaturation, followed by centrifugation; gel filtration, reverse phase chromatography, hydroxyapatite chromatography, and affinity chromatography; and combinations of such techniques with other techniques.

[0289] In purifying the antibodies of the present disclosure, it may be necessary to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. Affinity columns that bind to the labeled portion of the polypeptide can be used to purify the polypeptide from other cellular components. As is generally known in the art, it is believed that the order in which the various purification steps are performed can be changed, or certain steps can be omitted, and still be a method suitable for preparing a substantially purified protein or peptide.

[0290] Typically, intact antibodies are fractionated using an agent that binds to the Fc portion of the antibody (i.e., protein A). Alternatively, an antigen can be used to simultaneously purify and select appropriate antibodies. Such methods typically utilize a selection agent that is bound to a carrier (e.g., a column, filter, or bead). The antibody is bound to the carrier, contaminants are removed (e.g., washed off), and the antibody is released by applying conditions (salt, heat, etc.).

[0291] Those skilled in the art will be aware of various methods for quantifying the degree of purification of a protein or peptide in light of this disclosure. These methods include, for example, determining the specific activity of active fractions, or assessing the polypeptide content in the fractions by SDS / PAGE analysis. Another method for assessing the purity of the fractions is to calculate the specific activity of the fractions, compare it to the specific activity of the initial extract, and calculate the purity therefrom. The actual units used to express the amount of activity will of course depend on the particular analytical technique selected for tracking the purification and whether the expressed protein or peptide exhibits detectable activity.

[0292] It is known that the migration of polypeptides can sometimes vary significantly with different conditions of SDS / PAGE (Capaldi et al., 1977). Therefore, it should be understood that the apparent molecular weight of a purified or partially purified expression product may vary under different electrophoretic conditions.

[0293] V. Cancer Treatment

[0294] A. Formulation and application

[0295] The present disclosure provides pharmaceutical compositions comprising anti-LILRB antibodies and antigens for producing the antibodies. Such compositions comprise a preventively or therapeutically effective amount of the antibody or 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 state government or listed in the U.S. Pharmacopeia or other recognized pharmacopeia for use in animals, and more specifically in humans. The term "carrier" refers to a diluent, excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a specific carrier. Physiological saline solutions, aqueous dextrose solutions, and glycerol solutions can also be used 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, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol and the like.

[0296] If necessary, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, pills, capsules, powders, sustained release formulations, etc. Oral formulations may include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences". Such compositions will contain a preventive or therapeutically effective amount of an antibody or fragment thereof (preferably in a purified form), and an appropriate amount of carrier to provide a form suitable for administration to a patient. The formulation should be suitable for the mode of administration, which may be oral, intravenous, intraarterial, buccal, intranasal, spray, bronchial inhalation, or delivered by mechanical ventilation.

[0297] The antibodies of the present disclosure as described herein can be formulated for parenteral administration, for example, for injection via intradermal, intravenous, intramuscular, subcutaneous, intratumoral or even intraperitoneal routes. Alternatively, the antibodies can be administered directly to the mucosa by topical routes, for example, by nasal drops, inhalation or by a nebulizer. Pharmaceutically acceptable salts include salts and those formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0298] The passive transfer of antibodies, known as artificially acquired passive immunity, generally involves the use of intravenous injection. The form of antibodies can be human or animal plasma or serum, human immunoglobulins collected for intravenous (IVIG) or intramuscular (IG) use, high titer human IVIG or IG from donors who have been immunized or recovered from the disease, and monoclonal antibodies (MAbs). Such immunity generally lasts only for a short period of time, and there is also a potential risk of hypersensitivity reactions and serum sickness (especially hypersensitivity reactions and serum sickness caused by gamma globulins of non-human origin). However, passive immunity provides direct protection. The antibody will be formulated with a carrier suitable for injection (i.e., sterile and injectable).

[0299] In general, the components of the compositions of the present disclosure are supplied separately or mixed together into a unit dosage form (e.g., a dry lyophilized powder or anhydrous concentrate) placed in a hermetically sealed container (e.g., an ampoule or a sachet) indicating the amount of active agent. When the composition is administered by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule with sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0300] The compositions of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and salts formed with cations, such as salts derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0301] B. Cell therapy

[0302] In another aspect, the present disclosure provides immune cells expressing chimeric antigen receptors (CAR). In some embodiments, CAR comprises an antigen binding fragment provided herein. In one embodiment, the CAR protein includes from N-terminus to C-terminus: a leader peptide, an anti-LILRB4 heavy chain variable domain, a linker domain, an anti-LILRB4 light chain variable domain, a human IgG1-CH2-CH3 domain, a spacer, a CD28 transmembrane domain, a 4-1BB intracellular costimulatory signaling domain, and a CD3ζ intracellular T cell signaling domain.

[0303] Also provided are methods of immunotherapy comprising administering an effective amount of immune cells of the present disclosure. In one embodiment, a medical disease or condition is treated by transferring a population of immune cells that elicit an immune response. In certain embodiments of the present disclosure, cancer or infection is treated by transferring a population of immune cells that elicit an immune response. Provided herein are methods of treating cancer or delaying progression in an individual, comprising administering an effective amount of antigen-specific cell therapy to the individual.

[0304] The immune cell can be a T cell (e.g., a regulatory T cell, a CD4+T cell, a CD8+T cell, or a γ-δT cell), a NK cell, an invariant NK cell, a NKT cell, or a macrophage. Also provided herein are methods for producing and engineering immune cells and methods for using and administering the cells for adoptive cell therapy, in which case the cells can be autologous or allogeneic cells. Therefore, the immune cell can be used as an immunotherapy, such as an immunotherapy targeting cancer cells.

[0305] Immune cells can be isolated from subjects, particularly human subjects. Immune cells can be obtained from healthy human subjects, healthy volunteers or healthy donors. Immune cells can be obtained from subjects of interest, such as subjects suspected of having a specific disease or condition, subjects suspected of being susceptible to a specific disease or condition, or subjects undergoing therapy for a specific disease or condition. Immune cells can be collected from any location in the subject where they reside, including but not limited to blood, cord blood, spleen, thymus, lymph nodes and bone marrow. The isolated immune cells can be used directly, or can be stored for a period of time, such as by freezing.

[0306] Immune cells can be enriched / purified from any tissue in which they reside, including, but not limited to, blood (including blood collected by a blood bank or cord blood bank), spleen, bone marrow, tissue removed and / or exposed during surgical procedures, and tissue obtained via a biopsy procedure. Tissues / organs from which immune cells are enriched, separated and / or purified can be separated from surviving subjects and non-surviving subjects, wherein non-surviving subjects are organ donors. In a particular embodiment, immune cells are separated from blood (e.g., peripheral blood or cord blood). In some aspects, immune cells are separated from cord blood with enhanced immunomodulatory ability (e.g., measured according to CD4 positive or CD8 positive T cell suppression). In a particular aspect, immune cells are separated from pooled blood (particularly pooled cord blood) to enhance immunomodulatory ability. Pooled blood can come from 2 or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0307] Immune cell populations can be obtained from subjects who need therapy or suffer from diseases associated with reduced immune cell activity. Therefore, the cells are autologous cells of subjects who need therapy. Alternatively, immune cell populations can be obtained from donors, preferably tissue compatibility matching donors. Immune cell populations can be collected from peripheral blood, cord blood, bone marrow, spleen, or from any other organ / tissue in which immune cells reside in the subject or donor. Immune cells can be separated from a pool of a subject and / or donor (e.g., pooled cord blood).

[0308] When the immune cell population is obtained from a donor different from the subject, the donor is preferably allogeneic, with the proviso that the resulting cells are compatible with the subject so that they can be introduced into the subject. The allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible. To make them compatible with the subject, the allogeneic cells may be treated to reduce immunogenicity.

[0309] Immune cells can be genetically engineered to express antigen receptors, such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, host cells (e.g., autologous or allogeneic T cells) are modified to express T cell receptors (TCRs) with antigen specificity for cancer antigens. In a particular embodiment, NK cells are engineered to express TCRs. NK cells can be further engineered to express CARs. A variety of CARs and / or TCRs (e.g., for different antigens) can be added to a single cell type (e.g., T cells or NK cells).

[0310] Suitable modification methods are known in the art. See, e.g., Sambrook et al., supra; and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and John Wiley & Sons, NY, 1994. For example, cells can be transduced using the transduction techniques described in Heemskerk et al. (2008) and Johnson et al. (2009) to express a T cell receptor (TCR) with antigen specificity for a cancer antigen.

[0311] In some embodiments, the cell comprises one or more nucleic acids encoding one or more antigen receptors introduced via genetic engineering, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is a heterogeneous nucleic acid, i.e., a nucleic acid that is not normally present in the cell or in a sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, such as a nucleic acid that is not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).

[0312] C. Combination therapy

[0313] It may also be desirable to provide combination therapies using the antibodies of the present disclosure in combination with other anti-cancer therapies. These therapies will be provided in a combined amount effective to achieve a reduction in one or more disease parameters. This method may involve contacting the cell / subject with the agent / therapy simultaneously, for example using a single composition or pharmacological formulation comprising both agents, or by contacting the cell / subject with two different compositions or formulations simultaneously, wherein one composition comprises the antibody and the other comprises another agent.

[0314] Alternatively, the antibody can precede or follow another treatment at a time interval ranging from minutes to weeks. It is usually ensured that there is a significant period between the time of each delivery, so that the therapy will still be able to exert a favorable combined effect on the cell / subject. In such cases, it is considered to contact the cells with the two forms within about 12-24 hours of each other, within about 6-12 hours of each other, or via a delay time of only about 12 hours. In some cases, it may be desirable to significantly extend the treatment period; in any case, several 10 days (2, 3, 4, 5, 6 or 7 10 days) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks) will pass between individual administrations.

[0315] It is also conceivable that more than one administration of anti-DC-HIL antibody or other therapy will be required. Various combinations can be used, where the antibody is "A" and another therapy is "B", as exemplified below:

[0316]

[0317] Other combinations are contemplated. In order 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, the target cells or target sites may be contacted with antibodies and at least one other therapy. These therapies will be provided in a combined amount effective to kill or inhibit cancer cell proliferation. This method may involve contacting cells / sites / subjects with agents / therapies simultaneously.

[0318] Specific agents contemplated for combination therapy with the antibodies of the present disclosure include chemotherapy and hematopoietic stem cell transplantation. Chemotherapy may include cytarabine (ara-C) and anthracyclines (most often daunomycin), high-dose cytarabine alone, all-trans retinoic acid (ATRA), and induction chemotherapy, usually anthracyclines, histamine dihydrochloride (Ceplene), and interleukin 2 (Proleukin) after completion of consolidation therapy, gemtuzumab ozogamicin (Mylotarg) for patients older than 60 years with relapsed AML or relapsed acute promyeloid leukemia (APL) who are not candidates for high-dose chemotherapy, clofarabine, and targeted therapy (e.g., kinase inhibitors, farnesyl transferase inhibitors, decitabine, and inhibitors of multidrug resistance protein (MDR1)) or arsenic trioxide.

[0319] In certain embodiments, the agent used in combination therapy is one or more drugs selected from the group consisting of: topoisomerase inhibitors, anthracycline topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolism inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), a combination of daunorubicin and cytarabine, daunorubicin and cytarabine liposomes for injection, Azacytidine, Decitabine, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine dihydrochloride, Interleukin-2, Aldesleukin, Gemtuzumab ozogamicin, FLT-3 inhibitors, midostaurin, Clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors, ivosidenib, IDH2 inhibitors, enasidenib, Smoothened (SMO) inhibitors, glasdegib, arginase inhibitors, IDO inhibitors, epacadostat, BCL-2 inhibitors, venetoclax, Platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, ibrutinib, acalabrutinib, Zanubrutinib, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, CD40 antibody, 4-1BB antibody, CD47 antibody, SIRP1α antibody or fusion protein, E-selectin antagonist, antibody binding to tumor antigen, antibody binding to T cell surface marker, antibody binding to myeloid cell or NK cell surface marker, alkylating agent, nitrosourea agent, antimetabolite, antitumor antibiotic, alkaloid derived from plant, hormone therapy drug, hormone antagonist, aromatase inhibitor and P-glycoprotein inhibitor.

[0320] VI. Antibody Conjugates

[0321] The antibodies disclosed herein can be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of the antibody molecule as a diagnostic or therapeutic agent, at least one desired molecule or moiety is usually linked or covalently bound, or a complex is formed with it. Such molecules or moieties can be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity (e.g., cytotoxic activity). Non-limiting examples of effector molecules that have been linked to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelators, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an analytical method. Non-limiting examples of reporter molecules that 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.

[0322] Antibody-drug conjugates have emerged as a breakthrough approach to cancer therapy. Antibody-drug conjugates (ADCs) consist of a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of a MAb for its antigenic target with a highly potent cytotoxic drug, resulting in an "armed" Mab that delivers its payload (drug) to tumor cells that are enriched for antigenic levels. Targeted delivery of the drug also minimizes its exposure to normal tissues, thereby reducing toxicity and increasing the therapeutic index. The FDA approved two ADC drugs (in 2011) (brentuximab vedotin) and the 2013 The approval of trastuzumab entaxin (trastuzumab bemtansine or T-DM1) validates 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 become more mature, the discovery and development of new ADCs increasingly depends on the identification and validation of new targets suitable for this approach and the generation of target MAbs. Two criteria for ADC targets are upregulated expression / high levels in tumor cells and robust internalization.

[0323] Antibody conjugates are also preferably used as diagnostic agents. Antibody diagnostic agents are generally divided into two categories: those used for in vitro diagnosis, such as those used in a variety of immunoassays; and those used for in vivo diagnostic protocols, generally referred to as "antibody-directed imaging". Many suitable imaging agents are known in the art, as well as methods for attaching them to antibodies (see, e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances, and X-ray imaging agents.

[0324] In the case of paramagnetic ions, mention may be made of, for example, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), calcium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III) ions, with gadolinium being particularly preferred. Ions used in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II) and, in particular, bismuth (III).

[0325] Among the radioisotopes used for therapeutic and / or diagnostic applications, mention may be made of astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 , 152 Eu, Gallium 67 , 3 Hydrogen, iodine 123 ,iodine 125 ,iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, technetium 99m and / or yttrium 90 . 125 I is generally preferred for use in certain embodiments, and 99m and / or indium 111Radiolabeled monoclonal antibodies of the present disclosure may be prepared according to methods well known in the art. For example, monoclonal antibodies may be iodinated by contact with sodium iodide and / or potassium iodide and a chemical oxidant (e.g., sodium hypochlorite) or an enzymatic oxidant (e.g., lactoperoxidase). Monoclonal antibodies according to the present disclosure may be iodinated by ligand exchange (e.g., reduction of pertechnetate with stannous solution, chelation of the reduced technetium to a Sephadex column and application of the antibody to the column), by iodination with technetium. 99m Alternatively, direct labeling techniques can be used, such as by incubation with pertechnetate, a reducing agent (e.g., SNCl 2 ), buffer solution (such as sodium phthalate-potassium solution) and antibody. The intermediate functional group commonly used to bind radioisotopes in the form of metal ions to antibodies is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0326] Among the fluorescent labels contemplated for use as conjugates are 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.

[0327] Another class of antibody conjugates encompassed by the present disclosure are those conjugates intended to be used primarily in vitro, in which the antibody is attached to a secondary binding ligand and / or an enzyme (enzyme tag) that, upon contact with a chromogenic substrate, produces a colored product. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydroperoxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and antibiotic protein and streptavidin compounds. The use of such labels is well known to those skilled in the art and is 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.

[0328] Another known method of site-directed attachment of molecules to antibodies involves reacting the antibody with a hapten-based affinity tag. Essentially, hapten-based affinity tags react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigenic reactions. However, this is disadvantageous because it results in reduced binding of the antibody conjugate to the antigen.

[0329] Molecules containing an azide group can also be used to form covalent bonds with proteins via reactive imino intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). Specifically, 2-azido analogs and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens and Haley, 1987; Atherton et al., 1985). 2-Azido and 8-azido nucleotides have also been used to localize nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binders.

[0330] Several methods are known in the art for linking or conjugating antibodies to their conjugated moieties. Some methods of linking involve the use of metal chelator complexes, using, for example, organic chelators such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycoluril-3 to link to antibodies (U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moiety is bound to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl) propionate.

[0331] In other embodiments, it is contemplated to selectively introduce sulfhydryl groups into the Fc region of the immunoglobulin by using reaction conditions that do not change the antibody combining site for derivatization of the immunoglobulin. The disclosed antibody conjugates produced according to this method exhibit improved durability, specificity, and sensitivity (U.S. Pat. No. 5,196,066, which is incorporated herein by reference). The literature has also disclosed site-directed attachment of effector or reporter molecules, wherein the reporter or effector molecules are conjugated to carbohydrate residues in the Fc region (O'Shannessy et al., 1987). It is reported that this method produces diagnostically and therapeutically promising antibodies that are currently under clinical evaluation.

[0332] VII. Immunoassay Methods

[0333] In yet other embodiments, the present disclosure relates to immunoassay methods for binding, purifying, removing, quantifying and otherwise generally detecting LILRB-associated cancers. While such methods may be applied in a conventional sense, another use is in the quality control and monitoring of vaccines and other viral stocks, where antibodies according to the present disclosure may be used to assess the content or integrity (i.e., long-term stability) of the H1 antigen in the virus. Alternatively, the methods may be used to screen various antibodies for appropriate / desired response characteristics.

[0334] Some immunoassay methods include, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluorescent immunoassay, chemiluminescent assay, bioluminescent assay, and western blot. In particular, competitive assays for detecting and quantifying LILRB are also provided. The procedures for various suitable immunoassay methods have been described in the scientific literature, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), DeJager et al. (1993), and Nakamura et al. (1987). In general, the immunocombination method includes obtaining a sample suspected of containing a LILRB-associated cancer and, as the case may be, contacting the sample with a first antibody according to the present disclosure under conditions effective to allow the formation of immune complexes.

[0335] These methods include methods for detecting or purifying LILRB or LILRB-associated cancer cells from a sample. The antibody is preferably attached to a solid support (e.g., in the form of a column matrix), and a sample suspected of containing LILRB-associated cancer cells is applied to the immobilized antibody. Unwanted components are washed away from the column, leaving the LILRB-expressing cells immunocomplexed with the immobilized antibody, which are then collected by removing the organism or antigen from the column.

[0336] Immunobinding methods also include methods for detecting and quantifying the amount of LILRB-associated cancer cells or associated components in a sample and detecting and quantifying any immune complexes formed during the binding process. Here, a sample suspected of containing LILRB-associated cancer cells is obtained and the sample is contacted with an antibody that binds LILRB or a component thereof, followed by detecting and quantifying the amount of immune complexes formed under specific conditions. For antigen detection, the biological sample analyzed can be any sample suspected of containing LILRB-associated cancer, 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.

[0337] Contacting the selected biological sample with the antibody under effective conditions for a period of time sufficient to allow the formation of an immune complex (primary immune complex) is generally a matter of adding the antibody composition to the sample and incubating the mixture for a period of time sufficient to allow the antibody to form an immune complex with the LILRB (i.e., bind to the LILRB). Thereafter, the sample-antibody composition, e.g., tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove any non-specifically bound antibody species so that only those antibodies that are specifically bound within the primary immune complex are detected.

[0338] In general, detection of immune complex formation is well known in the art and can be achieved by applying numerous methods. These methods are generally based on the detection of labels or markers (such as any of those radioactive labels, fluorescent labels, biological labels and enzyme labels). Patents on 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, as known in the art, additional advantages can be found by using secondary binding ligands (such as second antibodies and / or biotin / antibiotic protein ligand binding configurations).

[0339] The antibody used for detection can itself be linked to a detectable label, wherein only this label is then detected, thereby determining the amount of the primary immune complex in the composition. Alternatively, the first antibody bound within the primary immune complex can be detected by means of a second binding ligand having binding affinity for the antibody. In these cases, the second binding ligand can be linked to a detectable label. The second binding ligand itself is usually an antibody, which can therefore be referred to as a "secondary" antibody. The primary immune complex is contacted with a labeled secondary binding ligand or antibody under effective conditions for a period of time sufficient to allow the formation of a secondary immune complex. The secondary immune complex is then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the residual label in the secondary immune complex is then detected.

[0340] Other methods include detecting the primary immune complex by a two-step method. A second binding ligand, such as an antibody with binding affinity to the antibody, is used to form a secondary immune complex as described above. After washing, the secondary immune complex is contacted with a third binding ligand or antibody with binding affinity to the second antibody again under effective conditions for a period of time sufficient to allow the immune complex (tertiary immune complex) to form. The third ligand or antibody is connected to a detectable label, thereby allowing detection of the tertiary immune complex thus formed. If necessary, this system can provide signal amplification.

[0341] An immunodetection method uses two different antibodies. The target antigen is detected using a first biotinylated antibody, and then the biotin attached to the complex biotin is detected using a second antibody. In the 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 antibodies bind to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubating in a continuous solution with streptavidin (or antibiotic protein), biotinylated DNA and / or complementary biotinylated DNA, wherein each step adds additional biotin sites to the antibody / antigen complex. The amplification step is repeated until a suitable amplification degree is reached, at which point the sample is incubated in a solution containing a second step antibody for biotin. This second step antibody is, for example, labeled with an enzyme that can detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. Under appropriate amplification conditions, a conjugate visible on a macroscopic scale can be produced.

[0342] Another known immunoassay method utilizes an immuno-PCR (polymerase chain reaction) method. The PCR method is similar to the Cantor method in that it is incubated with biotinylated DNA, but rather than using multiple rounds of streptavidin and biotinylated DNA incubations, the DNA / biotin / streptavidin / antibody complex is washed with a low pH or high salt buffer to release the antibody. The resulting wash solution is then used to perform a PCR reaction in the presence of suitable primers and appropriate controls. At least in theory, the huge amplification capacity and specificity of PCR can be used to detect single antigen molecules.

[0343] 1.ELISA

[0344] Immunoassays are binding assays in their simplest and most direct sense. Certain preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) known in the art. Immunohistochemical assays using tissue sections are also particularly useful. However, it is readily appreciated that assays are not limited to such techniques, and Western blots, dot blots, FACS analyses, etc. may also be used.

[0345] In an exemplary ELISA, an antibody of the present disclosure is immobilized to a selected surface that exhibits protein affinity, such as a well of a polystyrene microtiter plate. Subsequently, a test composition suspected of containing LILRB-associated cancer cells is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding another anti-LILRB antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection can also be achieved by adding a second anti-LILRB4 antibody, followed by a third antibody that has binding affinity for the second antibody, wherein the third antibody is linked to a detectable label.

[0346] In another exemplary ELISA, a sample suspected of containing LILRB4-associated cancer cells is immobilized on a well surface and subsequently contacted with an anti-LILRB4 antibody of the present disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-LILRB4 antibody is detected. In the case where the initial anti-LILRB4 antibody is linked to a detectable label, the immune complex can be detected directly. Again, the immune complex can be detected using a second antibody that has binding affinity for the first anti-LILRB4 antibody, wherein the second antibody is linked to a detectable label.

[0347] Regardless of the format used, ELISA has certain common features, such as coating, incubation and binding, washing to remove non-specifically bound substances, and detection of bound immune complexes. These formats are described below.

[0348] When coating a plate with an antigen or antibody, the wells of the plate are generally incubated with the antigen or antibody solution overnight or for a specified period of hours. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surface of the wells is then "coated" with a nonspecific protein that is antigenically neutral to the test antiserum. These proteins include bovine serum albumin (BSA), casein, or milk powder solutions. The coating allows blocking of nonspecific adsorption sites on the fixed surface and thus reduces the background caused by nonspecific binding of antisera on the surface.

[0349] In ELISA, it may be more common to use a secondary or tertiary detection format rather than a direct procedure. Thus, after the protein or antibody is bound to the wells, coated with a non-reactive material to reduce background, and washed to remove unbound material, the fixed surface is contacted with the biological sample to be tested under conditions effective to allow the formation of immune complexes (antigen / antibody). Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a combination of the secondary binding ligand or antibody with a labeled tertiary antibody or third binding ligand.

[0350] "Under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions preferably include diluting the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents also tend to help reduce nonspecific background.

[0351] "Suitable" conditions also mean that the incubation is carried out at a temperature or time period sufficient to allow effective binding. The incubation step is generally carried out at a temperature of preferably about 25°C to 27°C for about 1 to 2 to about 4 hours, or can be incubated overnight at about 4°C.

[0352] After all incubation steps in ELISA, the contacted surface is washed to remove uncomplexed material. Preferred washing procedures include washing with solutions such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the initially bound material and subsequent washing, the presence of even trace amounts of immune complexes can be determined.

[0353] In order to provide a detection means, the second or third antibody will have a relevant label to allow detection. Preferably, this label will be an enzyme that develops color when incubated with an appropriate chromogenic substrate. Thus, for example, it is necessary to contact or incubate the first and second immune complexes with urease, glucose oxidase, alkaline phosphatase or hydroperoxidase conjugated antibodies for a period of time (e.g., incubating for 2 hours at room temperature in a solution containing PBS (e.g., PBS-Tween)) under conditions that promote the formation of other immune complexes.

[0354] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified, for example by reacting with a chromogenic substrate (e.g., urea, or bromocresol purple, or 2,2'-azo-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) or H 2 O 2 (In the case of peroxidase as enzyme marker)) and incubated together. Subsequently, quantification is achieved by measuring the colorimetry of the color produced, for example using a visible spectrum spectrophotometer.

[0355] 2. Western Blot

[0356] Western blotting (or, protein immunoblotting) is an analytical technique used to detect specific proteins in a given tissue homogenate or extract sample. It uses gel electrophoresis to separate native or denatured proteins based on the length of the polypeptide (denaturing conditions) or based on the three-dimensional structure of the protein (native / non-denaturing conditions). The proteins are then transferred to a membrane (usually nitrocellulose or PVDF), where they are probed (detected) using antibodies specific for the target protein.

[0357] Samples can be obtained from intact tissues or cell cultures. In most cases, solid tissues are first mechanically broken down using a blender (for larger sample volumes), a homogenizer (for smaller volumes), or by sonication. Cells can also be ruptured by one of the above mechanical methods. However, it should be noted that bacteria, viruses, or environmental samples can be sources of protein and therefore protein imprinting is not limited to cell studies. Selected detergents, salts, and buffers can be used to promote cell lysis and solubilization of proteins. Protease and phosphatase inhibitors are usually added to prevent the sample from being digested by its own enzymes. Tissue preparation is usually carried out at low temperatures to avoid protein denaturation.

[0358] Gel electrophoresis is used to separate proteins in a sample. Separation of proteins can be based on isoelectric point (pi), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the handling of the sample and the nature of the gel. This is a very useful way to measure proteins. Two-dimensional (2-D) gels can also be used, which diffuse proteins from a single sample in two dimensions. Proteins are separated in the first dimension based on their isoelectric point (pH that gives them a neutral net charge) and in the second dimension based on their molecular weight.

[0359] In order to make protein available for antibody detection, it is moved from the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter paper is placed on top of it. The entire stack is placed in a buffer solution, which moves the protein forward on the filter paper with it under capillary action. Another method for transferring protein is called electroblotting and uses electric current to pull protein from the gel to PVDF or nitrocellulose membrane. Protein moves from the gel to the membrane while maintaining the organization it has in the gel. The result of this imprinting method is that the protein is exposed to a thin surface layer for detection (see below). The two types of membranes are selected for their non-specific protein binding properties (i.e., all proteins are equally combined). Protein binding is based on hydrophobic interactions and charged interactions between membrane and protein. Compared to PVDF, nitrocellulose membranes are cheaper, but much more fragile and cannot withstand repeated detection. The uniformity and overall efficiency of protein transfer from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, the proteins are detected using either a labeled primary antibody or using an unlabeled primary antibody followed by indirect detection using labeled protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0360] 3. Immunohistochemistry

[0361] The antibodies disclosed herein can also be used with fresh frozen and / or formalin fixed, paraffin embedded tissue blocks prepared for immunohistochemistry (IHC) studies. Methods for preparing tissue blocks from these particle samples have been successfully used in previous IHC studies for various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0362] Briefly, cryosections can be prepared as follows: rehydrate 50 ng of frozen "crushed" tissue in phosphate buffered saline (PBS) in a small plastic capsule at room temperature; pellet the particles by centrifugation; resuspend them in a viscous embedding medium (OCT); invert the capsule and / or pellet them again by centrifugation; snap freeze in -70°C isopentane; cut the plastic capsule and / or remove the frozen tissue cylinder; mount the tissue cylinder on a cryo-microtome chuck; and / or cut 25-50 serial sections from the capsule. Alternatively, serial section cutting can be performed using intact frozen tissue samples.

[0363] Permanent sections can be prepared by a similar method involving rehydrating 50 mg of sample in a plastic microcentrifuge tube; aggregation; resuspending in 10% formalin for 4 hours; washing / aggregation; resuspending in warm 2.5% agar; aggregation; cooling in ice water to harden agar; removing tissue / agar block from tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 permanent serial sections. Again, whole tissue samples can be substituted.

[0364] 4. Immunoassay kit

[0365] In yet other embodiments, the present disclosure relates to an immunoassay kit for use in conjunction with the above-described immunoassay method. Since the antibody can be used to detect LILRB-associated cancer cells, the antibody can be included in the kit. Thus, the immunoassay kit will contain a first antibody that binds to LILRB, and optionally an immunoassay reagent, in a suitable container member.

[0366] In certain embodiments, the antibody may be pre-bound to a solid support, such as a column matrix and / or a microtiter plate well. The immunodetection reagents in the kit may be in any of a variety of forms, including those detectable labels that are bound or linked to a given antibody. Detectable labels that are bound or linked to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0367] Other immunodetection reagents suitable for use in the kits of the present invention include two-component reagents comprising a secondary antibody having binding affinity for the first antibody, and a third antibody having binding affinity for the second antibody, the third antibody being linked to a detectable label. As described above, a variety of exemplary labels are known in the art and all such labels can be used in conjunction with the present disclosure.

[0368] The kit may further comprise an appropriate aliquot of a LILRB composition, whether labeled or unlabeled, which can be used to prepare a standard curve for a detection assay. The kit may contain the antibody-label conjugate in fully conjugated form, in intermediate form, or the individual portions conjugated by the user of the kit. The components of the kit may be packaged in an aqueous medium or in lyophilized form.

[0369] The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody or preferably an appropriately aliquoted antibody may be placed. The kit of the present disclosure will also typically include means for containing the antibody, antigen, and any other reagent containers in a tightly enclosed form for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials are retained.

[0370] 5. Flow Cytometry and FACS

[0371] The antibodies of the present invention can also be used in flow cytometry or FACS. Flow cytometry is a laser or impedance-based technology that is used in many detection analyses, including cell counting, cell sorting, biomarker detection, and protein engineering. The technology suspends cells in a fluid stream and passes them through an electronic detection device that allows multi-parameter analysis of the physical and chemical characteristics of up to thousands of particles per second at the same time. Flow cytometry is commonly used to diagnose diseases, especially blood cancers, but has many other applications in basic research, clinical practice, and clinical trials.

[0372] Fluorescence activated cell sorting (FACS) is a specific type of cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell. In general, the technique involves entraining a cell suspension in the center of a narrow, fast-flowing liquid stream. The stream is configured so that there is a large spacing between cells (relative to their diameter). A vibration mechanism causes the cell stream to split into individual droplets. Just before the stream splits into droplets, the stream passes through a fluorescence measurement station where the fluorescence of each cell is measured. A charging ring is placed exactly where the stream splits into droplets. The ring is charged, the fluorescence intensity is then measured, and when the stream splits, the opposite charge is trapped on the droplets. The charged droplets then fall via an electrostatic deflection system that transfers the droplets to containers based on their charge.

[0373] In certain embodiments, for use in flow cytometry or FACS, antibodies of the present disclosure are labeled with a fluorophore and then allowed to bind to cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine.

[0374] VIII. Examples

[0375] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention, and therefore may be considered to constitute preferred embodiments thereof. However, in light of the present disclosure, it will be appreciated by those skilled in the art that many changes may be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0376] Example 1

[0377] Mice. C57 BL / 6J and NOD-SCID IL2Rγ null (NSG) mice were purchased from the Animal Core Facility at the University of Texas Southwestern Medical Center (UTSW) and maintained there. Apoe knockout (apoe-KO, Apoe tm1Unc ) Mouse 31Purchased from Jackson Laboratory. Animal studies have been approved and conducted under the supervision of the Institutional Animal Care and Use Committee (IACUC) of UT Southwestern. Each experiment used mice of the same sex and age match (4-8 weeks) and randomly assigned to each group; and for tumor size measurement and intravital imaging experiments, the mice were blinded to the treatment conditions. The minimum number of mice in each group was calculated based on the results of previous related studies. For the subcutaneous tumor model, according to (width × width × length) cm 3 Calculation of tumor size. The maximum tumor measurement allowed by the UTSW IACUC is a tumor diameter of 2 cm.

[0378] Cell culture. 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO. 2 and normal content O 2 Culture at 37°C in 5% CO 2 and normal content of O 2 Human umbilical vein / vascular endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial cell growth medium plus growth factors, cytokines and supplements (EGM-BulletKit, Lonza). The cells were incubated at 37°C in 5% CO 2 and normal content of O 2 Human monocytic AML cells THP-1 (ATCC, TIB-202), MV4-11 (ATCC, CRL-9591) and U937 (ATCC, CRL-1593.2) and mouse AML cells WEHI-3 (ATCC, TIB-68) were cultured in RPMI 1640 supplemented with 10% FBS. The cells were incubated at 37°C in 5% CO 2 and normal content of O 2 Mouse AML cells C1498 (ATCC, TIB-49) were cultured in DMEM supplemented with 10% FBS. All cell lines were routinely tested using the Mycoplasma contamination kit (R&D systems).

[0379] Primary human leukemic cells. Primary human AML and B-ALL samples were obtained from the tissue bank of UTSW. Informed consent was obtained according to a protocol reviewed and approved by the Institutional Review Board of UTSW. The UTSW group included 105 AML patients with AML subtypes represented by the French-American-British (FAB) classification, minimally mature acute myeloblastic leukemia (M1, n=9), mature acute myeloblastic leukemia (M2, n=34), acute promyelocytic leukemia (M3, n=10), acute myelomonocytic leukemia (M4, n=34), acute monocytic leukemia (M5, n=25), acute erythroid leukemia (M6, n=2), and acute megakaryoblastic leukemia (M7, n=1), as well as patients with undifferentiated leukemia (AUL; n=1) and transient myeloproliferative disorders (TAM; n=2). Samples were frozen in FBS containing 10% DMSO and stored in liquid nitrogen.

[0380] Normal human monocytes and macrophages. Normal human monocytes (CD14 + Cells) were isolated from the mononuclear cell fraction of normal peripheral blood by AutoMACSPro separation system (Miltenyi Biotech, Auburn, CA). Briefly, the buffy coat was purchased from Interstate Blood Bank (Memphis, TN) and the mononuclear cell layer was separated by Ficoll Hypaque (17144003, GE Lifesciences) density gradient separation. Monocytes were treated with red blood cell lysis buffer to remove red blood cells and then incubated with CD14 microbead conjugated antibody (130-050-201, Miltenyi Biotech, Auburn, CA) at 4°C for 15 minutes. CD14 positive cells were then isolated using a positive selection procedure according to the manufacturer's protocol. One million CD14 + The cells were seeded in each well of a 6-well plate in macrophage culture medium (Iscove's modified Dulbecco's medium, IMDM (12440053, Thermo fisher) supplemented with 10% human AB serum (MT35060CI, Fisher Scientific), 1% NEAA (11-140-050, Fisher), 2 μM L-alanine-L-glutamine (SH3003402, Fisher)) and cultured for 7 days. After incubation, most of the cells adhered to the plastic surface and stained positive for CD14 and other markers specific for macrophages.

[0381] TCGA analysis. Data were obtained from the TCGA acute myeloid leukemia database (version: August 16, 2016). Patients were classified into AML subtypes (FAB classification) M0 (undifferentiated acute myeloid leukemia) (n=16), M1 (n=42), M2 (n=39), M3 (n=16), M4 (n=35), M5 (n=18), M6 (n=2), M7 (n=3); two cases were not classified according to subtype. The mRNA levels of the indicated genes were determined by RNA-seq (polyA+IlluminaHiSeq). RESM normalized counts were reported and the data were analyzed and visualized using UCSC Xena (xena.ucsc.edu). To analyze overall survival, 160 patients with available survival data were divided into three groups based on whether they had high, intermediate, or low gene expression, and then analyzed by Xena Kaplan Meier plots (http: / / xena.ucsc.edu / survival-plots / ).

[0382] Flow cytometry. Primary antibodies used included: anti-human CD45-PE (BD Pharmingen, HI30, 1:100), CD45-FITC (BD Pharmingen, HI30, 1:100), CD45-APC (BD Pharmingen, HI30, 1:100), anti-human CD34-FITC (BD Pharmingen, 55582, 1:100), anti-human CD19-PE (eBioscience, HIB19, 1:100), anti-human CD20-PE (BD Pharmingen, 555623, 1:100), anti-human CD11b-APC (eBioscience, ICRF44, 1:100), anti-human LILRB4-APC (eBioscience, ZM4.1, 1:100), anti-human LILRB4-PE (Biolegend, ZM4.1, 1:100), anti-human CD14-APC (eBioscience, 61D3, 1:100), anti-human CD33-APC (Biolegend, P67.6, 1:100), anti-human CD4-APC (eBioscience, RPA-T4, 1:100), anti-human CD3-FITC (BioLegend, HIT3a, 1:100), anti-human CD3-Pacific Blue (BD Pharmingen, SP34-2, 1:100), anti-human CD8-PE (BD Pharmingen, 555367, 1:100), anti-human CD28-APC (eBioscience, CD28.2, 1:100), anti-human CD40L-APC (eBioscience, 24-31, 1:100), anti-human PD1-APC (Biolegend, EH12.2H7, 1:100), anti-human TIM3-APC (eBioscience, F38-2E2, 1:100), anti-human TIGIT-APC (eBioscience, Bioscience, MBSA43, 1:100), anti-human LAG3-APC (eBioscience, 3DS223H, 1:100), anti-human FasL-PE (eBioscience, 24-31, 1:100), anti-uPAR-APC (Biolegend, VIM5, 1:100), anti-mouse CD3-APC (BioLegend, 17A2, 1:200), anti-mouse CD8a-PE (BioLegend, 53-6.7, 1:200), anti-mouse CD45-PE (BD Pharmingen, 30-F11, 1:200), anti-mouse CD49b-APC (eBioscience, DX5, 1:200), anti-mouse CD49f-PE (eBioscience, GoH3, 1:200), anti-mouse CD11b-APC (BioLegend, M1 / 71, 1:200), anti-mouse CD11b-PE (BioLegend, M1 / 71, 1:200), anti-mouse CD11c-APC (eBioscience, N418, 1:200), anti-mouse F4 / 80-APC (BioLegend, BM8, 1:200), anti-His tag-APC (R&D systems, AD1.1.10, 1:400) and IgG isotype control APC (eBioscience, P3.6.2.8.1, 1:400) antibodies. Cells were analyzed on Calibur or analyzed and sorted on FACSAria. Flow data were analyzed by Flowjo software. For analysis of human hematopoietic engraftment in NSG mice, previously published protocols were followed. PI staining was used to exclude dead cells in analysis and sorting. For intracellular staining, the inventors followed the fixation / methanol two-step protocol from eBioscience. Briefly, antibodies against LILRB4 (anti-LILRB4-Alexa Fluor 647, Biolegend, ZM4.Human primary AML cells were stained for surface expression of 5-mercaptoethanol (1:100) and CD33 (anti-human CD33-FITC, Biolegend, HIM3-4, 1:100) and the fixable cell viability dye eFluor 450 (Bioscience, catalog number 65-0863-14, 1:100) followed by fixation (IC fixation buffer, eBioscience, catalog number 00-8222) and methanol treatment. Subsequently, the cells were stained with anti-p-SHP-2 (Y580)-PE (Cell signaling, catalog number 13328S, 1:100), anti-pIKKα / β (S176 / 180) (16A6) (Cell signaling, catalog number 2697, 1:100), anti-NFκB (S529)-PE (eBioscience, B33B4WP, 1:100), anti-uPAR-PE (Biolegend, VIM5, 1:100), anti-arginase-1 (D4E3M) (Cell signaling, catalog number 93668, 1:100), rabbit IgG isotype control-PE (Cell signaling, catalog number 5742, 1:100), mouse IgG isotype control-PE (eBioscience, m2a-15F8, 1:100), and anti-rabbit IgG-PE (Jackson Immunoresearch The cells were stained for intracellular antigens using ELISA (Lab, catalog number 111-116-144, 1:400) for flow cytometry analysis.

[0383] Virus construction and infection. For retroviral packaging, plasmid constructs XZ201-IRES-GFP and XZ201-human lilrb4 (hlilrb4)-IRES-GFP were mixed with PCL-ECO (2:1) and then transfected into 293T cells using Lipofectamine 2000 (Invitrogen). For lentiviral packaging, CRISPER / Cas-9-based gRNA (guide RNA) constructs and other constructs for gene overexpression (including pLentiLox3.7-luciferase-IRES-GFP, ZsGreen-hlilrb4 and ZsGreen-hlilrb4-intΔ, pLVX-plaur-IRES-tdTomato, pLVX-arg1-IRES-tdTomato) were mixed with psPAX2 and pMD2.G (Addgene) at a ratio of 4:3:1 and transfected into 293T cells using Lipofectamine 2000 (Invitrogen). Virus-containing supernatants were collected 48-72 hours after transfection and used for infection as previously described.

[0384] CRISPR / Cas9-based gene knockout in AML cells. Human AML cells were infected with a lentivirus expressing doxycycline-inducible Cas9 (pCW-Cas9, Addgene 50661). After selection with 1 μg / ml puromycin, surviving cells were infected with a lentivirus expressing sgRNA, which was generated by replacing puro-mcherry with GFP selected from a plasmid modified with pSLQ1651 (Addgene 51024). Scrambled control sgRNA (sgRNA 5'-GAACGACTAGTTAGGCGTGTA-3' (SEQ ID NO: 211)) and sgRNA targeting lilrb4 (sgRNA1 5'-TGTTACTATCGCAGCCCTGT-3' (SEQ ID NO: 212) designed by an online tool (http: / / crispr.mit.edu);

[0385] sgRNA2 5'-GTAGGTCCCCCCGTGCACTG-3' (SEQ ID NO: 213); sgRNA3 5'-CCTGTGACCTCAGTGCACGG-3' (SEQ ID NO: 214)), apoe-targeting sgRNA (sgRNA1 5'-CTTTTGGGATTACCTGCGC-3' (SEQ ID NO: 215); sgRNA2 5'-AACTGGCACTGGGTCGCTTT-3'(SEQ ID NO:216)), sgRNA targeting shp-1 (sgRNA1 5'-TAAGACCTACATCGCCAGCC-3'(SEQ ID NO:217); sgRNA2 5'-GAAGAACTTGCACCAGCGTC-3'(SEQ ID NO:218)), sgRNA targeting shp-2 (sgRNA1 5'-GAGACTTCACACTTTCCGTT-3'(SEQ ID NO:219); sgRNA2 5'-TACAGTACTACAACTCAAGC-3'(SEQ ID NO:220)), sgRNA targeting ship (sgRNA1 5'-CACGCAGAGCGCGTATGCCC-3'(SEQ ID NO:221); sgRNA2 5'-TGGCAACATCACCCGCTCCA-3'(SEQID NO:222)) were cloned into sgRNA plasmids respectively. After being treated with 1 μg / ml doxycycline (Sigma, catalog number PHR1789) for 1 week, these cells were stained with anti-LILRB4 antibody and LILRB4-negative cells were sorted as lilrb4 knockout cells. For apoe-, shp-1-, shp-2- and ship-knockout cells, GFP was expressed. + Cells were sorted into 96-well plates with single cells per well. After cell amplification, knockout cells were verified by Western blotting. To induce CRISPR / Cas9 in vivo to achieve gene knockout, the inventors fed mice with doxycycline as described. Briefly, 7 days after THP-1 cells transfected with Cas9 / lilrb4-sgRNA were implanted, mice were treated with 2 mg doxycycline per mouse for 5 days via daily gavage to achieve Cas9 expression in transplanted leukemia cells. Gene knockout was verified by flow cytometry.

[0386] Leukemia cell and T cell co-culture analysis. In the co-culture analysis, human T cells (5×10 4 / well) and irradiated (28Gy) designated human leukemia cells were mixed in a U-shaped bottom 96-well plate. For non-contact co-culture of T cells and leukemia cells, leukemia cells were cultured in the upper chamber of a U-shaped bottom 96-well plate with a transwell plug-in (pore size, 3 μM, #09-761-80, Thermo Fisher). T cells isolated from healthy donors were placed in the lower chamber of a 96-well transwell plate. Irradiated designated leukemia cells (if not indicated, E: T ratio = 2: 1) were added to the upper chamber and treated with designated antibodies, proteins and reagents. After culturing for 5 to 7 days with anti-CD3 / CD28 coated beads (11161D, Thermo Fisher) and 50U / ml rhIL-2, representative cells were photographed using an inverted microscope, and T cells were stained with anti-CD3 antibodies and analyzed by flow cytometry.

[0387] For primary AML or B-ALL samples, the CD33 + and CD19 + These leukemic cells were combined with autologous CD3 + T cells or allogeneic T cells from healthy donors were cultured together (E:T ratio = 2: 1). After 14 days of culture with anti-CD3 / CD28 coated beads (11161D, Thermo Fisher) and 50U / ml rhIL-2, representative cells were photographed using an inverted microscope, and T cells were stained with anti-CD3, anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry.

[0388] For cytotoxicity assays, human CD8 T cells isolated from PBMCs of healthy donors were stimulated with anti-CD3 / CD28 / CD137 coated beads (11163D, Thermo Fisher) in 96-well plates. + T cells (5×10 4 Then, add the specified 5×10 3 Leukemic cells and 50 to 500 μg / ml anti-LILRB4 antibody or control IgG. Cell number was determined in triplicate wells on day 7. Or the specified leukemic cells were cultured with T cells at the specified E:T ratio for 4 to 6 hours in triplicate wells. Anti-CD3 and anti-CD8 were used to detect human CTL cells; the specified live THP-1 was positive for GFP and negative for PI. Cell supernatants from co-cultures of stimulated CTL cells and THP-1 cells treated with anti-LILRB4 or IgG were used to detect cytokine production using a human cytokine array (AAH-CYT-6, RayBiotech).

[0389] For mouse leukemia / T cell co-cultures, splenocytes from wild-type C57bl / 6 were cocultured with 2.5 × 10 4 Irradiated (28Gy) mouse leukemia C1498 cells were co-cultured in U-bottom 96-well plates for 60 hours. Anti-CD3 / CD28 coated beads (11452D, Thermo Fisher), 50U / ml recombinant human IL-2, and 5% serum from wild-type C57bl / 6 mice or from apoe-KO mice were added to the culture medium. In some experiments, 50μg / ml lipid-bound APOE protein (APOE-POPC) was added to the culture medium. Lipidation of APOE recombinant protein was performed as described.

[0390] Transendothelial migration assay. To measure the ability of AML cells to migrate through endothelial cells, 3 × 10 5 After 3 days, 1×10 5 The designated leukemia cells were seeded in the upper chamber. In the designated experiments, the leukemia cells were treated with antibodies or proteins in the upper chamber. After 18 hours, the cells in the lower chamber were counted.

[0391] Short-term infiltration analysis of leukemic cells and homing analysis of hematopoietic stem / progenitor cells (HSPC). Cells (5×10 6 cells / mouse) into NSG mice. Animals were treated with 10 mg / kg of anti-LILRB4 antibody or control IgG immediately after injection of leukemic cells. After 20 hours, mice were sacrificed. Peripheral blood, bone marrow, liver and spleen were collected and single cell suspensions were detected by flow cytometry. In the specified experiments, CFSE, GFP or specified markers such as anti-human CD45 and anti-human CD33 were used to detect leukemic target cells. The number of leukemic cells in the recipient liver, spleen and bone marrow is reported as a ratio relative to the number of cells in the peripheral blood.

[0392] To test the infiltration ability of mouse leukemia cells, 5 × 10 6 C1498-GFP-hLILRB4 cells or C1498-GFP were added to wild-type C57BL / 6J or APOE-null mice. After 20 hours, the mice were sacrificed. GFP was used to detect leukemic cells by flow cytometry. The number of leukemic cells in the recipient liver, spleen and bone marrow was normalized to the number in peripheral blood and reported as a ratio.

[0393] To test the homing ability of HSPCs, 1×10 7Human umbilical cord blood mononuclear cells were injected into NSG mice. Mice were treated with 10 mg / kg of anti-LILRB4 antibody or control IgG immediately after injection of monocytes and sacrificed 20 hours later. Anti-human CD45 and anti-human CD34 were used to detect human HSPCs by flow cytometry. Similarly, to test the infiltration capacity of normal human monocytes, 5×10 PBMCs from healthy donors were injected into NSG mice. 6 CD14 positive selected monocytes were labeled by CFSE and injected intravenously into NSG mice. Mice were treated with 10 mg / kg of anti-LILRB4 antibody or control IgG immediately after monocyte injection and sacrificed 20 hours later. CFSE positive cells were analyzed by flow cytometry.

[0394] Depletion of innate immune cells. NK cell depletion was performed by intraperitoneal injection of 50 μl of anti-asialo GM1 antibody (CL8955, Cedarlane) 3 days before leukemia cell implantation, which resulted in >90% depletion of CD45+CD49b+ NK cells in the circulation of NSG mice. Macrophages were depleted by treating NSG mice with clodronate (dichloromethylene bisphosphonate) liposomes (SKU8909, Clodrosome) (200 μl stock solution 3 days before leukemia cell implantation), which resulted in >70% depletion of CD45+CD11b+F4 / 80+ macrophages in the circulation of NSG mice. NSG mice were neutropenic by intraperitoneal injection of 200 μg of anti-Ly-6G mAb (BP0075-1, Bioxcell) on days -3, -2, -1, and 0 after leukemia cell implantation, which resulted in >70% depletion of CD45+CD11b+F4 / 80+ macrophages in the circulation of NSG mice. + CD11b + CD11c - Neutrophil depletion >80%.

[0395] Human AML xenografts. Xenografts were performed essentially as described. 2,3 ,6 ,7 Briefly, 6-8 week old NSG mice were used for transplantation. For each mouse injected intravenously, 1×10 6 Individual human leukemic cells were resuspended in 200 μl PBS. Mice were immediately given 10 mg / kg of anti-LILRB4 antibody or control IgG intravenously. Three to four weeks after transplantation, peripheral blood, bone marrow, spleen, and liver were evaluated for engraftment. Bioluminescence imaging (maximum, 3×10 8 p / sec / cm 2 / sr; minimum value, 5×10 6 p / sec / cm 2 / sr) to monitor leukemic growth over time. For survival curve experiments, the death of mice was recorded when moribund animals were euthanized. For patient-derived primary xenografts (PDXs), each NSG mouse was given 5 to 10 × 10 6 Individual human primary peripheral blood or bone marrow mononuclear cells, which contain leukemic cells and other normal compartments, such as normal hematopoietic stem cell progenitors and autologous T cells. Mice were immediately given 10 mg / kg anti-LILRB4 antibody or control IgG intravenously and treated twice a week until euthanasia. For AML#11, mice were given 10 mg / kg anti-LILRB4 antibody or control IgG intravenously 7 days after leukemic cell implantation and treated twice a week until euthanasia. Leukemia growth was monitored over time by performing flow cytometry on human cells in peripheral blood. More than 1% of human leukemic cells in mouse tissues were considered successful engraftment of primary AML cells. Engraftment of peripheral blood, bone marrow, spleen, and liver was evaluated one to four months after transplantation.

[0396] For the hPBMC humanized model, 1 × 10 7 1×10 human PBMCs were implanted subcutaneously into each NSG mouse. Three weeks after implantation, the mice had 30 to 50% human T cell engraftment. 6 Individual human AMLTHP-1 cells, including wild-type, lilrb4-KO THP-1 cells, or THP-1 cells stably expressing luciferase (THP-1-Luc-GFP cells). Mice were immediately given 10 mg / kg anti-LILRB4 antibody or control IgG intravenously and treated twice a week until euthanasia. Luminescence imaging (maximum, 1×10 8 p / sec / cm 2 / sr; minimum value, 5×10 6 p / sec / cm 2 / sr) to monitor tumor growth over time. Tumor size was determined by caliper measurement (width × width × length). For the inducible lilrb4 knockdown experiment, 1×10 6 Each NSG mouse was intravenously injected with 10 Cas9 / lilrb4-sgRNA-transfected THP-1 cells, followed by an intravenous injection of 0.5 × 10 6 7 days after THP-1 and T cell implantation, mice were treated for 5 days by gavage with 2 mg doxycycline per mouse per day to achieve Cas9 expression in transplanted THP-1 cells. 3 weeks after implantation, engraftment of peripheral blood, bone marrow, spleen and liver was evaluated.

[0397] For the human umbilical cord blood (hCB)-xenograft model, 2 × 10 4 Human CD34 + hCB cells were implanted into each NSG mouse. Six weeks after implantation, the mice had 10 to 50% human cell engraftment. 1×10 6 THP-1 cells stably expressing luciferase (THP-1-Luc-GFP cells) were added to the mice. Mice were immediately given 10 mg / kg of anti-LILRB4 antibody or control IgG intravenously. Luminescence imaging (maximum, 1×10 8 p / sec / cm 2 / sr; minimum value, 5×10 6 p / sec / cm 2 / sr), monitoring tumor growth over time. Analysis of human normal blood cell lineages by flow cytometry.

[0398] Mouse AML allografts. The procedure for mouse AML allografts is similar to that for human AML xenografts. Briefly, 6-8 week old wild-type C57bl / 6 mice were used for transplantation. For each mouse implanted intravenously or subcutaneously, 1×10 6 Mouse leukemia cells expressing human LILRB4 were resuspended in 200 μl PBS. Mice were given 10 mg / kg anti-LILRB4-N297A antibody or control IgG intravenously on day 7 after leukemia cell implantation and treated twice a week until euthanasia. Three weeks after transplantation, engraftment of peripheral blood, bone marrow, spleen and liver was evaluated. For mice implanted subcutaneously, tumor size was determined by caliper measurement (width×width×length). For survival curve experiments, the death of mice was recorded when moribund animals were euthanized. For CD8+T depletion, 10 mg / kg anti-CD8 antibody (YTS169.4.2, Bioxcell) was injected intravenously 3 days after leukemia cell implantation and treated twice additionally every 3 days. To determine whether anti-LILRB4 antibody treatment produces tumor-specific memory T cells against tumors or against LILRB4, the inventors injected spleen cells (5×10 6 Four out of five transplanted mice rejected control C1498-GFP mouse leukemia cells, and these mice were not susceptible to 3-fold higher numbers (3 × 10 6 None of the five mice adoptively transferred splenocytes from untreated mice rejected the control C1498-GFP mouse leukemia cells.

[0399] Chimeric receptor reporter assay. A stable chimeric receptor reporter cell system was constructed as described to test the ability of ligands to bind to the ECD of individual LILRB, PirB, gp49B1, and LILRB4 site mutations and trigger activation or inhibition of chimeric fusion intracellular domains of paired immunoglobulin-like receptor β, which signal through the adapter DAP-12 to activate the NFAT promoter. If an agonist or antagonist binds to the ECD and activates or inhibits the chimeric signaling domain, an increase or decrease in GFP expression is observed, respectively. Competition assays were used to screen for LILRB4 blocking antibodies. Briefly, APOE protein (CI02, Novoprotein; 10 μg / ml) or human AB serum (10%, diluted in PBS) was pre-coated on 96-well plates for 3 h at 37°C. After washing twice with PBS, 2×10 4 LILRB4 reporter cells were seeded in each well; at the same time, the indicated anti-LILRB4 antibodies were added to the culture medium. After 16 hours, GFP expression was analyzed by flow cytometry. + The percentage of cells is reported. The threshold for activation is 2-fold that of negative control treatment.

[0400] Fast protein liquid chromatography (FPLC) and mass spectrometry. 10% human AB serum in PBS was loaded onto a 16 / 60 Superdex 200 gel filtration column and eluted with PBS and 2 mM EDTA. Eighty fractions (40 ml) were collected and each fraction (0.5 ml) was analyzed by chimeric receptor reporter assay. Active fractions (#26-30) were loaded onto a PAGE gel and processed for LC-MS / MS analysis (Orbitrap Elite) for protein identification at the UTSW Proteome Research Center. The recombinant or purified proteins used for validation were ZA2G (MBS145455, MyBioSource), AMBP (13141-H08H1, Sino Biological Inc), TTHY (12091-H08H, Sino Biological Inc), PEDF (11104-H08H, Sino Biological Inc), A2MG (MBS173010, MyBioSource), HEMO (MBS143111, MyBioSource), ANGT (MBS173525, MyBioSource), A1AT (MBS173006, MyBioSource), S100A9 (pro-814, Prospecbio), HORN (EBP08267, Biotrend USA), VTDB (CSB-EP009306HU, Biotrend USA), LRG1 (pro-141, Prospecbio), A1BG (RPE570Hu01, Cloud-Clone Corp), CRSP3 (RD172262100, BioVendor), APOA1 (16-16-120101-LEL, Athens Research & Technology), APOA2 (16-16-120102, Athens Research & Technology), APOA4 (16-16-120104, Athens Research & Technology), APOB (16-16-120200, Athens Research & Technology), APOC1 (16-16-120301, Athens Research & Technology), APOC2 (16-16-120302, Athens Research & Technology), APOC3 (16-16-120303, Athens Research&Technology), hAPOE(16-16-120500,Athens Research & Technology), mAPOE (CJ05, Novoprotein), APOE2 (350-12, Peprotech), APOE3 (350-02, Peprotech), APOE4 (350-04, Peprotech), PODXL2 (1524-EG-050, R&D systems), CD44 (12211-H08H, Beijing Sino Biological Technology Co., Ltd.), HCK (PV6128, Thermo Fisher), VEGFR3 (10806-H08H, Beijing Sino Biological Technology Co., Ltd.), NRG3 (16071-H08H, Beijing Sino Biological Technology Co., Ltd.), PI16 (H00221476-P01, Novusbio), hMAG (8940-MG-050, R&D systems), mMAG (8580-MG-100, R&D systems), CNTF(303-CR-050,R&D systems), ANGPTL-7(914-AN-025 / CF,R&D systems), Integrin-α1β1(7064-AB-025,R&D systems), Integrin-α2β1(5698-AB-050,R&D systems), Integrin-α2β3(7148-AB-025,R&D systems), Integrin-α3β1(2840-A3-050,R&Dsystems), Integrin-α4β1(5668-A4-050,R&D systems), Integrin-α4β7(5397-A3-050,R&Dsystems), Integrin-α5β1(3230-A5-050,R&D systems systems), integrin-α5β3(3050-AV-050,R&Dsystems), integrin-α5β5(2528-AV-050,R&D systems), integrin-α5β6(CT039-H2508H,Beijing Sino Biological Science & Technology Co., Ltd.), integrin-α5β6(CT051-M2508H,Beijing Sino Biological Science & Technology Co., Ltd.), integrin-α5β8(4135-AV-050,R&D systems), integrin-α6β4(5497-A6-050,R&Dsystems), integrin-α8β1(CT016-H2508H,Beijing Sino Biological Science & Technology Co., Ltd.), integrin-α9β1(5438-A9-050,R&D systems), integrin-α10β1(5895-AB-050,R&D systems), integrin-α11β1(6357-AB-050, R&D systems), integrin-αEβ7(5850-A3-050, R&D systems), integrin-αXβ2(CT017-H2508H, Beijing Sino Biological Technology Co., Ltd.) and normal mouse serum(NS03L, Milliporesigma).

[0401] Biolayer interferometry. Binding interaction analysis between LILRB4-Fc and APOE2, APOE3, and APOE4 was performed on an Octet RED96 (ForteBio, Pall Corporation). All interaction studies were performed with Protein A dip-and-read biosensors (ForteBio). All binding experiments were performed at 30°C using Octet Red and kinetic buffer. LILRB4-Fc-coated biosensors (25 μg / ml LILRB4-Fc loading for 420 s) were washed in kinetic buffer before monitoring the association (300 s) and dissociation (600 s) of APOE. Background wavelength shift was measured with a reference sensor loaded with LILRB4-Fc alone.

[0402] Surface plasmon resonance (SPR). Biacore 2000 and CM5 chips were used to analyze the binding of recombinant APOE to the extracellular domain of LILRB4 fused to hFc as previously described. 2 Recombinant protein A (Pierce) was pre-immobilized in two flow cells using an amine coupling kit from GE. LILRB4-hFc was injected into one of the flow cells to be captured by protein A. For the protein A-coupled cell control, each binding sensing profile of the sample flow cell containing captured LILRB4-hFc was calibrated. After each injection of the antigen solution to induce the binding reaction and the dissociation period of the infusion of the operating buffer, the binding reaction was induced by injecting a solution containing 10 mM Na 3 PO 4 The Protein A surface was regenerated with a regeneration solution of 500 mM NaCl (pH 2.5). All captured LILRB4-hFc was completely removed in the presence and absence of APOE binding and another cycle was started. All measurements were performed at 25°C with a flow rate of 30 μL / min.

[0403] Microscale thermophoresis (MST). MST experiments were performed on a Monolith NT.115 system (NanoTemper Technologies) using 80% LED and 20% IR laser power. The laser on and off times were set to 30 seconds and 5 seconds, respectively. Recombinant LILRB4-ECD protein (SinoBio) was labeled with 4488-NHS (NanoTemper Technologies) and applied at a final concentration of 5.9 nM. A double dilution series was prepared for unlabeled His-APOE (CI06, Novoprotein) in PBS, and each dilution point was similarly transferred to the LILRB4-ECD solution. The final concentration of His-APOE ranged from 0.36 nM to 12 μM. Samples were filled into standard-treated capillaries (NanoTemper Technologies) for measurement.

[0404] Western blotting and co-immunoprecipitation. Whole cells were lysed in Laemmli sample buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Roche Diagnostics). Samples were separated on SDS-PAGE gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad) for protein detection.The primary antibodies used included: anti-SHP-1 (Cell signaling, 3759, 1:1000), anti-phospho-SHP-1Tyr564 ​​(Cell signaling, 8849, 1:500), anti-phospho-SHP-1Tyr564 ​​(Invitrogen, PA537708, 1:500), anti-SHP-2 (Cell signaling, 3397, 1:1000), anti-phospho-SHP-2Tyr580 (Cell signaling, 3703, 1:500), anti-SHIP1 (Cell signaling, 2727, 1:1000), anti-phospho-SHIP1 Tyr1020 (Cell signaling, 3941, 1:500), anti-NFκB p65 (Cell signaling, 8242, 1:1000), anti-IKKα (Cell signaling, 1 Cell signaling, 11930, 1:1000), anti-IKKβ (Cell signaling, 8943, 1:1000), anti-phospho-IKKα / βSer176 / 180 (Cell signaling, 2697, 1:500), anti-IκBα (Cell signaling, 4814, 1:1000), anti-phospho-IκBαSer32 (Cell signaling, 2859, 1:500), anti-lamin-B2 (Cell signaling, 12255, 1:1000) and anti-arginase-1 (Cell signaling, 9819, 1:1000), anti-uPAR (Invitrogen, MON R-4-02, 1:500), anti-LILRB4 (Santa cruz, sc-366213, 1:200), anti-APOE (Creative diagnostics, DCABH-2367, 1:250), anti-β-actin (Sigma-Aldrich, A2066, 1:1000) and anti-α-tubulin (Sigma-Aldrich, MABT205, 1:1000), as well as horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell signaling, 7074, 1:1,000 and 7076, 1:1,000) and chemiluminescent substrate (Invitrogen). Specific cell compartments were isolated using the NE-PER nuclear / cytoplasmic extraction kit (Thermo fisher, 78833) or the plasma membrane protein extraction kit (Abcam, ab65400).Proteins from the plasma membrane fraction were further incubated with anti-LILRB4 antibody and dyno beads protein A (Thermofisher, 10001D) for further immunoprecipitation and western blotting.

[0405] Immunohistochemistry. Paraffin sections of tumors were stained with hematoxylin and immunostained. Antibodies used were against LILRB4 (laboratory generated, 1:100), CD3 (Abcam, ab16669, 1:100), PD-1 (Thermo Fisher, J116, 14-9989-82, 1:100) and arginase-1 (Cell signaling, 9819S, 1:100). Images were observed using Hamamatsu NanoZoomer 2.0-HT (Meyer instruments Inc., Houston, TX) and viewed in NPDview 2 software (Hamamatsu, Japan).

[0406] Cytokine antibody array and arginase activity analysis. To detect secreted proteins from leukemia cells, conditioned media were applied to a human cytokine antibody array (AAH-CYT-1000, RayBio) for semi-quantitative detection of 120 human proteins. Image J (NIH) was used for quantification. Arginase activity was measured in conditioned media from designated leukemia cells using the QuantiChrom Arginase Assay Kit (DARG-100, BioAssay Systems).

[0407] RNA-seq analysis. RNA was purified from sorted cells using the Qiagen RNeasy Mini kit and then reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. RNA-seq was performed at the UTSW Genomics and Microarray Core Facility. cDNA was sonicated using a Covaris S2 ultrasonic processor, and libraries were prepared using a KAPA High Throughput Library Preparation Kit. Samples were end-repaired, and 3' ends were adenylated and barcoded with multiple adapters. PCR-amplified libraries were purified with AmpureXP beads and validated on an Agilent 2100 bioanalyzer. Before normalization and pooling, samples were quantified by Qubit (Invitrogen) and then run on an Illumina Hiseq 2500 instrument using PE100SBS v3 reagents to generate 51-bp single-end reads. Prior to mapping, reads were trimmed to remove low-quality regions in the ends. Trimmed reads were mapped relative to the human genome (HM19) using TopHat v2.0.1227 using the UCSC iGenomes GTF file from Illumina.

[0408] The method of data normalization and analysis is based on the use of "internal standards" that characterize some aspect of the system behavior, such as technical variability, as described elsewhere. 2 Genes with (fold change)>2, P<0.01 and RPKM>0.1 were considered to be significantly differently expressed between the two conditions and were used for pathway analysis and upstream transcription factor analysis. Pathway analysis was performed using DAVID (david.ncifcrf.gov / tools.jsp). Upstream transcription factor analysis was performed using QIAGEN's ingenuity tool (world wide web at ingenuity.com / ).

[0409] Molecular docking of LILRB4 and APOE. The docking of LILRB4 and APOE was performed on the ZDOCKpro module packaged in Insight II. The general protocol for running ZDOCK includes two consecutive computational steps described as geometric search and energy search, which are run in the programs ZDOCK and RDOCK, respectively. The LILRB4 crystal structure (3P2T) and the APOE3 structure (2L7B) were obtained from the PDB database. The top 50 ZDOCK poses were provided to RDOCK for improvement. The poses with higher scores in both ZDOCK and RDOCK were selected as candidate complexes for LILRB4 / APOE interaction analysis.

[0410] Statistical analysis. Representative data from four independent experiments or independent samples as indicated are presented as dot plots (mean ± SD) or box plots (median (line), 25th-75th percentiles (box outlines), and minimum and maximum values ​​(whiskers)). Statistical significance of two-sample comparisons was calculated by two-tailed Student's t-test. Statistical significance of survival was calculated by log-rank test. Multivariate analysis of TCGA data was analyzed by Cox regression. Differences were considered statistically significant if p < 0.05. ns, not significant; p values ​​are expressed as exact values. Pearson's correlation analyses were performed with RStudio software (R Foundation).

[0411] Preparation of LILRB4 and h193 complex. DNA encoding the extracellular domain of LILRB4 (residues 1-196 of the mature protein) was cloned into the vector pET21a (Novagen) with NdeI and XhoI restriction sites and expressed in E. coli strain BL21 (DE3) (Novagen). Bacteria were cultured in LB medium containing the corresponding antibiotic (100 μg / ml ampicillin) in a shaking incubator at 37°C. When the culture reached an OD of 0.8-1.0, the cells were cultured for 1 h. 600 When the expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), the cells were cultured for 4-6 hours before harvesting. The centrifuged cells were suspended in PBS buffer (20 mM Na 3 PO 4, 280 mM NaCl, 6 mM KCl, pH 7.4) and disrupted using a homogenizer (JNBIO, China). Inclusion bodies of recombinant proteins were purified and refolded as described with some modifications. Briefly, aliquots of inclusion bodies were diluted dropwise in stirring refolding buffer (100 mM Tris-HCl, 2 mM EDTA, 400 mM L-arginine, 0.5 mM oxidized glutathione and 5 mM reduced glutathione) at 4°C for 8 hours. The refolded protein was concentrated and buffer exchanged with a solution containing 20 mM Tris-HCl and 50 mM NaCl (pH 8.0) using an Amicon 8400 concentrator. Subsequently, in the same exchange buffer as above, 16 / 60 The protein was further purified by gel filtration chromatography on a 75PG column (GE Healthcare). Qualified peak isolated proteins were concentrated for further studies or crystallization. The h193-scFv was constructed as VL-(GGGGS)4-VH and cloned into the vector pET21a (Novagen) with NdeI and XhoI restriction sites. The scFv was overexpressed in E. coli as inclusion bodies and then refolded and purified as LILRB4 protein as described above. LILRB4 and h193-scFv were then mixed together at a molar ratio of 1:2 and incubated on ice for 1 hour. The LILRB4 and h193-scFv complex was purified by 16 / 60 Further purification was performed by 75PG (GE Healthcare) chromatography to purify the LILRB4 / Hu193-scFv complex from any excess LILRB4.

[0412] Crystallization of LILRB4 / h193 complex. LILRB4 / h193-scFv complex crystals were grown in a sedimentation drop form by vapor diffusion. A total of 1 μl of 5 mg / ml or 10 mg / ml complex protein solution was mixed with an equal volume of reservoir solution. Crystals were grown at 18°C ​​in 1% w / v tryptic protein, 0.05M HEPES sodium (pH 7.0), 20% w / v polyethylene glycol 3,350. Crystals were frozen in liquid nitrogen in a reservoir solution supplemented with 17% glycerol (volume / volume) as a cryoprotectant. X-ray diffraction data were collected at 100K and indexed, integrated and scaled with HKL2000. The complex structure was solved by the molecular replacement method using Phaser from the CCP4 program suite, with the structures of LILRB4 (PDB: 3P2T) and antibody (PDB: 4OQT) as search models, respectively. Initial constrained rigid body refinement and manual modeling were performed using REFMAC5 and COOT, respectively, and further refined using Phenix.

[0413] Example 2

[0414] LILRB4 expressed on leukemic cells inhibits T cell proliferation. To identify novel mechanisms of AML development and immune regulation, the present inventors analyzed the relationship between gene expression of known co-stimulatory and co-inhibitory receptors and overall survival of AML patients, as recorded in the TCGA database. Protein expression and expression of mRNA encoding leukocyte immunoglobulin-like receptor B4 (LILRB4), an immunosuppressive receptor restrictedly expressed on monocytic cells (Kang et al., 2016; Hirayasu and Arase, 2015; Trowsdale et al., 2015) and monocytic AML cells (FAB M4 and M5 AML subtypes) (Dobrowolska et al., 2013) ranked at the top of the list of negative correlations with AML patient survival ( Figure 2A and Fig. 6A Importantly, the level of LILRB4 protein on the surface of neoplastic monocytes was higher than that on the surface of normal monocytes ( Figure 2B ).

[0415] The previous study reported that the extracellular domain of LILRB4 inhibits T cell activity (Vlad et al., 2010). To test whether LILRB4 expressed on AML cells has T cell inhibitory function, the present inventors co-cultured LILRB4-positive leukemia cells, LILRB4-negative leukemia cells or normal hematopoietic cells with autologous T cells or T cells from healthy donors. Only LILRB4-positive monocytic AML cells significantly inhibited T cell proliferation ( Figure 2C and Figures 7A to 7F ). The inventors subsequently deleted lilrb4 from human monocytic AML THP-1 and MV4-11 cells and found that after lilrb4 gene knockout (lilrb4-KO), the T cell suppressive ability of AML cells was significantly reduced and restored by forced expression of wild-type lilrb4 (such as lilrb4-KO-wt), but not by mutant lilrb4 with a deleted intracellular domain (such as lilrb4-KO-intΔ) ( Figure 2D and Figures 8A to 8E In addition, when wild-type THP-1 cells and human T cells were cultured in separate transwells, LILRB4-mediated T cell suppression was also observed and could be rescued by anti-LILRB4 blocking antibodies ( Figures 8F to 8L Blockade of LILRB4 resulted in an increase in cytotoxic T cells, a decrease in AML cells, and an increase in T cell cytokine release ( Figures 8M to 8OThese in vitro data suggest that AML cells require intracellular signaling of LILRB4, but not the extracellular domain, for suppressing T cell activity (Vlad et al., 2010).

[0416] Subsequently, the present inventors used humanized mouse xenograft models and immune competent mouse models to study LILRB4 function during immune checkpoint blockade. Subcutaneous implantation of THP-1 cells, but not lilrb4-KO THP-1 cells, led to exacerbation of AML in human T cell reconstituted mice, which was blocked by anti-LILRB4 treatment (Mosier et al., 1988) ( Figures 9A to 9G In a disseminated leukemia model established in humanized mice, doxycycline-induced LILRB4 deletion also attenuated leukemia progression and restored T cells ( Figures 2E to 2F and Figures 9H to 9J ). In addition, the inventors subcutaneously implanted mouse C1498 AML cells expressing human LILRB4 (hlilrb4-C1498) into C57BL / 6 mice to establish a syngeneic immune competent mouse model. In order to exclude the anti-tumor effect from the Fc effector function, the inventors treated tumor-bearing mice with anti-LILRB4 with Fc glycosylation site N297A mutation (Ha et al., 2011). LILRB4 blockade effectively reduced tumor burden and prolonged survival; elimination of CD8 + T cells abolish the anti-tumor effects of anti-LILRB4 antibodies ( Figures 2G to 2H These results suggest that the tumor-supporting effects of LILRB4 depend on host T cell suppression. Anti-LILRB4 antibody treatment generates tumor-specific memory T cells ( Fig.2I Similar results were obtained in a disseminated hlilrb4-C1498 isogenic mouse model ( Figures 10A to 10D Finally, blockade of LILRB4 significantly reduced leukemia development in primary human monocytic AML-derived xenografts ( Figures 2J to 2K ) and increased the number of transplantable autologous human T cells. Overall, the in vitro and in vivo results indicate that LILRB4 signaling in monocytic AML cells inhibits T cell-mediated anti-tumor immunity.

[0417] LILRB4 promotes AML cell migration and infiltration. One of the characteristic features of monocytic AML is the enhanced extramedullary infiltration of tumor cells (Straus et al., 1980). The present inventors observed that antibody blockade of LILRB4 resulted in a significant reduction in leukemia infiltration into the viscera (including bone marrow, liver and brain). Although anti-LILRB4 antibody treatment did not reduce CD8 + Size of subcutaneous C1498 tumors in T cell-depleted C57BL / 6 mice ( Figure 2G), but treatment with anti-LILRB4 antibody did result in a decrease in the infiltration of leukemic cells into the liver ( Fig. 10A ). The inventors hypothesized that in addition to T cell suppression, LILRB4 promotes leukemic infiltration. To test this hypothesis, the inventors performed transendothelial migration and homing assays and monitored leukemic infiltration associated with LILRB4 expression on leukemic cells. LILRB4-depleted human AML THP-1 cells had lower transendothelial migration in vitro than cells expressing LILRB4. Loss of lilrb4 reduced homing and engraftment of AML cells to hematopoietic organs, resulting in prolonged survival and delayed weight loss in xenografted mice. In contrast, forced expression of human LILRB4 in mouse AML C1498 or WEHI-3 cells had the opposite effect ( Figures 12A to 12D Antibody-mediated LILRB4 blockade showed the same effect as lilrb4 knockout in AML cells expressing LILRB4 ( Figures 12E to 12G This effect depends on LILRB4 expression in leukemia cells and its intracellular signaling, rather than the Fc effector function of the antibody ( Figure 3A and Figures 12H to 12J Furthermore, LILRB4 blockade reduced the infiltration capacity of primary monocytic AML cells ( Figures 3B to 3D and Figures 13A to 13C ). The results consistent with the previous study showed that LILRB4 + The circulating frequency of AML blasts was significantly lower than that of LILRB4 - The circulating frequency of AML blasts (Dobrowolska et al., 2013) and LILRB4 + Chronic lymphocytic leukemia cells are associated with lymphoid tissue damage (Colovai et al., 2007). + The bone marrow, liver, and brain, to which AML cells tend to migrate, have certain immune privileges (Crispe et al., 2006; Carson et al., 2006; Fujisaki et al., 2001). Therefore, LILRB4-mediated migration that supports enhanced extramedullary infiltration of monocytic AML cells may also contribute to immune evasion.

[0418] APOE is an extracellular binding protein of LILRB4. Anti-LILRB4 antibodies blocked the immunosuppression and migration of AML cells, indicating that the functions of LILRB4 are ligand-dependent. v β 3, which was previously identified as a ligand for gp49B1, the mouse LILRB4 ortholog (Castells et al., 2001). However, the different integrin-αβ complexes did not activate the human LILRB4 reporter cells. Surprisingly, human serum and mouse serum were able to activate the LILRB4 reporter but not the other LILRB reporters ( Figure 4A Through protein liquid chromatography separation followed by reporter analysis and mass spectrometry, the inventors identified APOE as a reporter that specifically activates LILRB4 and mouse PirB ( Figure 4B Serum from wild-type but not APOE-null mice activated the LILRB4 reporter ( Figure 4C In addition, both liposome-reconstituted APOE protein (APOE-POPC) and lipid-free APOE activated LILRB4 reporter cells ( Figure 4D ). The binding of APOE to THP-1 cells was significantly reduced by lilrb4-KO ( Figure 4E ). Microscale thermophoresis (MST), surface plasmon resonance (SPR), and biolayer interferometry (Octet) were used to confirm the specific binding of recombinant APOE to LILRB4. The dissociation constant determined by MST was 210 nM ( Figure 4F Mutagenesis studies have shown that P35 and W106 in the N-terminal domain of APOE and the first Ig domain of LILRB4 and Y121 in the linker region between the two Ig domains are essential for APOE-mediated LILRB4 activation ( Figures 4G to 4H ).

[0419] The finding that APOE activates the immunosuppressive receptor LILRB4 is consistent with the well-documented immunosuppressive function of APOE (Grainger et al., 2004; Ali et al., 2005). To determine whether the T cell suppressive activity of LILRB4 is dependent on APOE, the inventors examined the proliferation of T cells co-cultured with control or apoe-knockout human AML cells. AML cells deficient in APOE restored T cell proliferation and inhibited the migration of leukemic cells ( Fig. 4I Moreover, when C1498 cells expressing LILRB4 ectopically were treated with wild-type mouse serum, the percentage of T cells in the co-culture was significantly reduced compared to those cells treated with apoe-knockout mouse serum ( Figures 4J to 4K Addition of liposomal reconstituted APOE to co-cultures of mouse splenocytes and AML cells expressing LILRB4 reduced the percentage of T cells ( Figure 4L In addition, LILRB4 expression significantly increased C1498 cells infiltrating the bone marrow and liver in wild-type mice, but not in APOE-null recipients ( Figure 4M ). These data indicate that APOE activates LILRB4 on human monocytic AML cells to inhibit T cell proliferation and support AML cell migration.

[0420] LILRB4-mediated intracellular signaling controls AML cell migration and T cell inhibition. The present inventors sought to identify downstream signaling of LILRB4 required for T cell inhibition and leukemic infiltration. The phosphatases SHP-1, SHP-2, and SHIP can be recruited to the intracellular domain of LILRB (Kang et al., 2016). Phosphorylation levels of SHP-2, but not SHP-1 or SHIP, were lower in lilrb4-KO AML cells than in wild-type cells ( Figure 5A and Fig.15A ) Deletion of SHP-2, but not SHP-1 or SHIP, reversed THP-1 cell suppression of T cells ( Figure 5B and Fig. 15B ) and reduced the short-term and long-term infiltration of THP-1 cells ( Figures 5C to 5D ). The results indicate that SHP-2 is a mediator of LILRB4 signaling.

[0421] Ingenious pathway analysis revealed that the activities of key transcription factors NFkB1 and RELA in the NF-κB pathway (DiDonato et al., 2012), which is positively regulated by SHP-2 (You et al., 2001), were most significantly inhibited by the loss of lilrb4 ( Figure 5E ). Consistently, phosphorylation of IKKα / β and nuclear NF-κB levels were reduced in lilrb4-KO AML cells ( Figures 5F to 5G and Fig.15A Inhibition of NF-κB signaling restored T cell suppression and reduced AML cell infiltration in a LILRB4-dependent manner ( Figures 5H to 5I and Figures 15C to 15D Thus, the effects of LILRB4 activation are mediated via the NF-κB pathway, which is particularly robust in monocytic AML among AML subtypes (Baumgartner et al., 2002).

[0422] This is consistent with the results that AML cells inhibit T cell proliferation in transwells ( Figure 2D ), the conditioned medium of wild-type THP-1 cells inhibited T cell activity, but the conditioned medium of lilrb4-KO cells did not ( Figure 5J The protein was present at a higher level in the conditioned medium of WT THP-1 cells than in the lilrb4-KO counterparts ( Fig.16A), uPAR is highly expressed by monocytic AML cells (Bene et al., 2004). uPAR (NF-κB target) is known to promote cancer invasion, metastasis, survival and angiogenesis (Su et al., 2016; Wang et al., 2000). The addition of recombinant uPAR reduced the proliferation of T cells co-cultured with lilrb4-KOTHP-1 cells in a dose-dependent manner ( Figure 5K This activity of uPAR may be mediated through downstream effectors in AML cells, as uPAR does not directly and effectively reduce T cell proliferation ( Fig. 16B ).

[0423] Arginase-1 (ARG1), like uPAR, is expressed significantly less in lilrb4-KO AML cells than in wild-type cells ( Figures 16C to 16D ). ARG1 is upregulated and inhibits T cell proliferation through uPAR-mediated signaling (Hu et al., 2014; Ilkovitch and Lopez, 2009), and can enhance immunosuppressive function through APOE (Baitsch et al., 2011) and NF-κB (Hagemann et al., 2008). The present inventors hypothesize that ARG1 is a key downstream effector of LILRB4-NF-kB-uPAR signaling. ARG1 can be secreted by AML cells to inhibit T cell activity 27 Recombinant ARG1 reduces T cell proliferation in co-culture with lilrb4-KO, apoe-KO, and shp-2-KO AML or primary AML cells ( Figure 5L and Figures 16E to 16G In addition, addition and overexpression of uPAR or ARG1 rescued the migration ability of lilrb4-KO AML cells in vitro and in vivo, respectively ( Fig.16H and Figure 5M ). Overall, the results indicate that LILRB4 / SHP-2 / NF-κB / uPAR / ARG1 is a signaling pathway that suppresses immune activity and supports leukemic migration in monocytic AML cells (Figures 17 to 18).

[0424] Targeting LILRB4 not only resulted in a specific attack on monocytic AML cells, but also reactivated multiple immune cell types including T cells and possibly monocytes / macrophages (Kang et al., 2016). Importantly, because LILRB4 is restrictedly expressed on normal monocytic cells (Kang et al., 2016), LILRB4 signaling may be different from that of leukemic cells (Figure 19) and LILRB4 blockade did not significantly interfere with normal hematopoiesis ( Figures 20A to 20B), so LILRB4 targeting may have minimal toxicity. Finally, LILRB4 is also expressed on certain other types of cancer and on myeloid-derived suppressor cells, tolerogenic dendritic cells, and tumor-associated macrophages (Kang et al., 2016; de Goeje et al., 2015; Chang et al., 2002; Suciu-Foca et al., 2007). Targeting LILRB4 may therefore enable the combination of immunotherapy and targeted therapy in cancer treatment.

[0425] The present inventors also found that LILRB4 is expressed on bone marrow-derived suppressor cells (MDSCs) isolated from solid cancer patients ( FIG. 21A ). The expression of LILRB4 is positively correlated with the autologous T cell suppressive ability of MDSCs ( FIG. 21B ). The present inventors performed anti-LILRB4 treatment in MDSC / T cell co-culture and found that anti-LILRB4 treatment increased IFNγ secretion of co-cultured T cells ( Fig. 21C ).

[0426] Example 3

[0427] The inventors subsequently generated a batch of rabbit monoclonal antibodies (mAbs) against the extracellular domain (ECD) of human LILRB4. The amino acid and nucleic acid sequences of the heavy and light chain variable regions of exemplary LILRB4 antibodies are shown in Figures 28A to 28C , Figures 29A to 29C , Figures 30A to 30C and Figures 31A to 31C The ELISA binding results of eight exemplary LILRB4 mAbs binding to LILRB4 ECD are shown in Fig. 22 and Table 6. Kinetic binding measurements (sensograms) of exemplary LILRB4 antibodies using the Octet assay are shown in Fig.26 And Table 7.

[0428] like Fig.23A As described in , LILRB4 ECD has two Ig domains, D1 and D2. The two residues (W106 and Y121) whose mutations significantly reduce the activation of LILRB4 by APOE are located in the first Ig domain D1 and in the linker or hinge region between the two Ig domains, respectively. Fig. 23B As shown in , among the eight exemplary mAbs that bind to the LILRB4 ECD, seven mAbs except B4-193 bind to the D1 domain of LILRB4.

[0429] Table 6. EC values ​​of LILRB4 antibodies binding to human LILRB4 determined by ELISA 50

[0430] Antibody EC50(nM) B4-15-1 0.99 B4-116-1 2.58 B4-116-2 1.7 B4-49 1.01 B4-72-2 0.29 B4-86 1.02 B4-87 2.54 B4-193 1.81

[0431] Table 7. Kinetic binding constants of LILRB4 antibodies determined using Octet biosensor chip

[0432] Antibody Kd Kon Koff <![CDATA[R 2 ]]> B4-116-1 8.20E-10 2.31E+05 1.89E-04 0.9972 B4-116-2 4.95E-10 2.05E+05 1.01E-04 0.9983 B4-49 2.33E-11 1.11E+05 2.58E-06 0.834 B4-87 2.38E-10 7.04E+04 1.68E-05 0.9991 B4-86 3.44E-10 7.96E+04 2.74E-05 0.9992 B4-72-2 3.73E-09 4.26E+04 1.59E-04 0.6988 B4-193 1.33E-10 1.31E+05 1.74E-05 0.9997

[0433] The present inventors then used the LILRB4 D1 domain (D1), D2 domain (D2), stalk region (SR), D1+D2, D2+SR and full-length ECD (D1+D2+SR) of human LILRB4 recombinant protein to determine the binding domain of B4-193. Fig. 24B As shown in , in this particular ELISA assay, B4-193 only binds to the full-length ECD of human LILRB4.

[0434] The inventors then used wild-type and Y121A mutant human LILRB4 ECD recombinant proteins to determine the contribution of amino acid Y121 on LILRB4 to the binding of B4-193. Fig.25 As shown in , the Y121A mutation of LILRB4 significantly reduced the binding of B4-193 to LILRB4.

[0435] The present inventors used APOE competition assay ( Fig.32 ), LILRB4-reporter cell / K562-co-culture analysis ( Fig.33 ) and LILRB4 reporter cell analysis ( Fig.34 and Fig.35 ) measured the effect of exemplary LILRB4 mAbs in modulating LILRB4 activity.

[0436] The inventors also tested the cross-reactivity of exemplary anti-LILRB4 antibodies with LILRB family members, LILRA family members, and cynomolgus monkey LILRB4 (cynoB4) ( Fig.36 and Fig.37 ).

[0437] like Fig.38 As shown in , exemplary anti-LILRB4 antibody B4-193 rescues THP-1 cells from suppressing T cells.

[0438] like Figures 39A to 39C As shown in , exemplary anti-LILRB4 antibodies 128-3 and B4-193 inhibited leukemia progression in THP-1 xenograft mice without significant adverse effects on mouse body weight.

[0439] Example 4

[0440] The inventors further generated a group of humanized anti-LILRB4 antibodies based on B4-193. The amino acid sequences of the heavy and light chain variable regions and their CDRs of the humanized B4-193 antibody (h193) are shown in Figures 28B to 28C , Figures 30B to 30C , Table 4 and Table 5. The present inventors used flow cytometry, ELISA and Octet analysis to measure the binding of h193 antibody to human LILRB4, and the results are shown in Fig.40 And Table 8.

[0441] Table 8. h193 antibody binding to human LILRB4

[0442]

[0443]

[0444] The present inventors also tested the cross-reactivity of h193 antibody with LILRB family members, LILRA family members and cynomolgus monkey LILRB4 (cynoB4) ( Figures 41A to 41M and Fig.42 ). The results show that the h193 antibody does not cross-react with LILRA family members or LILRB family members, except LILRB4. The H193 antibody can also bind to the cynomolgus monkey LILRB4 protein to varying degrees.

[0445] The present inventors further measured the antagonistic and agonistic effects of the h193 antibody using ApoE competition assay and K562 co-culture assay. Fig.43A (ApoE competition assay) and Fig.43B As shown in (K562 cell co-culture assay), most of the h193 antibodies inhibited ApoE-induced LILRB4 activity.

[0446] The inventors also used cytokine array analysis to measure the effect of h193 antibody in regulating cytokine release (Figures 44 to 47). Briefly, human PBMC (1.5×10 6 10 cells / ml) were treated with h193 antibody (20 μg / ml) for 48 hours and then subjected to cytokine array to measure the levels of 120 cytokines. In another analysis, human PBMCs were mixed with THP-1 cells (0.3×10 6 Human IgG was used as a negative control. Figures 44A to 44D As shown in Figure 2, h193 antibody treatment reduced the secretion of certain immunomodulatory and inflammatory cytokines only in PBMCs. Figures 45A to 45D As shown in , h193 antibody treatment increased the secretion of certain immunomodulatory and inflammatory cytokines in PBMCs co-cultured with THP-1 cells.

[0447] In a xenograft mouse model, h193 antibody inhibited leukemia progression ( Fig.48 ).

[0448] Example 5

[0449] This example illustrates the molecular basis of the interaction between LILRB4 and the h193 antibody through crystallization of the LILRB4 / h193 complex. The D1 and D2 domains of LILRB4 and the single-chain Fv fragment (scFv) of the h193 antibody were expressed as inclusion bodies in E. coli and soluble proteins were obtained by in vitro refolding methods. The LILRB4-D1D2 / h193 scFv complex was then prepared for crystal screening and the complex structure was determined by molecular replacement with The resolution is determined by

[0450] The overall structure revealed that the h193 antibody mainly binds to the connecting region between D1 and D2 of LILRB4 ( Fig.49A ). Antibodies utilize heavy chains (V H ) and light chain (V L ) Both interact with LILRB4 and involve h193 antibody V H and V L All three CDR loops in the V L The exception is the CDR2 loop in ( Fig.49A and Table 9). Specifically, the h193 antibody binds to the D1D2 hinge loop and the BC and C'E loops of the D2 domain of the LILRB4 molecule. Residues K100, G96, and S99 in the hinge loop of the D1 and D2 domains of LILRB4 form three hydrogen bonds with residues in both the HCDR1 (S32) and HCDR2 (S59 and D53) regions ( Fig.49B ), while residue R124 in the BC loop of the D2 domain not only forms two hydrogen bonds with V H Residue H101 of HCDR3 interacts with V L Residue I93 (LCDR3) in Fig.49C In addition, H153 in the BC loop of LILRB4-D2 contacts R94 of the HCDR3 loop by forming two hydrogen bonds ( Fig.49C Residues Q122 in the BC loop and Q154 in the C'E loop of LILRB4-D2 form three hydrogen bonds with residues W32 and N30 of the LCDR1 loop ( Fig.49C ). In summary, the h193 antibody targets three loops of LILRB4 through continuous contacts including multiple hydrogen bond interactions. In particular, residues Q122 and H153 that contribute to important contacts with h193 antibody binding are unique in LILRB4 among all LILR molecules. This may contribute to the specific binding of h193 antibody to LILRB4.

[0451] Table 9. Interactions between h193 and LILRB4

[0452]

[0453] The numbers indicate the number of atom-to-atom contacts between antibody residues and LILRB4 residues, which were analyzed by the contact program in the CCP4 suite (distance cutoff = ).

[0454] The numbers in parentheses indicate the number of hydrogen bonds between the antibody residues and LILRB4 residues.

[0455] All methods disclosed and claimed herein can be performed and executed without undue experimentation according to the present invention. Although the compositions and methods of the present invention have been described according to preferred embodiments, it should be clear to those skilled in the art that changes can be applied to the methods described herein and the steps or step sequences of the methods without departing from the concept, spirit and scope of the present invention. More specifically, it is apparent that certain agents that are chemically and physiologically related can replace the agents described herein while obtaining the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0456] References

[0457] The following references are specifically incorporated herein by reference, to the extent they pr...

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof specifically binds to LILRB4 and comprises: (a) A heavy-chain variable region comprising: The amino acid sequence of heavy-chain complementarity-determining region 1 as shown in SEQ ID NO: 100, The amino acid sequence of heavy-chain complementarity-determining region 2 as shown in SEQ ID NO: 101, and The amino acid sequence of heavy-chain complementarity-determining region 3 as shown in SEQ ID NO: 102, 224 or 227; and (b) A light-chain variable region comprising: The amino acid sequence of light-chain complementarity-determining region 1 as shown in SEQ ID NO: 104, The sequence KAS or the amino acid sequence of light-chain complementarity-determining region 2 as shown in SEQ ID NO: 233, and The amino acid sequence of light-chain complementarity-determining region 3 as shown in SEQ ID NO: 105 or 234.

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

3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated monoclonal antibody is a murine antibody.

4. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is a recombinant ScFv antibody, a Fab fragment, an F(ab')2 fragment or an Fv fragment.

5. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated monoclonal antibody is a rabbit antibody or a chimeric antibody.

6. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein (a) the heavy-chain variable region has the amino acid sequence shown in SEQ ID NO: 99; and (b) the light-chain variable region has the amino acid sequence shown in SEQ ID NO:

103.

7. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated monoclonal antibody is a humanized antibody.

8. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein (a) the heavy-chain variable region has the amino acid sequence shown in SEQ ID NO: 223, 225, 226, 228, 229, 230 or 231; and (b) the light-chain variable region has the amino acid sequence shown in SEQ ID NO: 232, 235, 236 or 237.

9. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

10. An isolated nucleic acid encoding the isolated monoclonal antibody according to any one of claims 1 to 8.

11. A vector comprising the isolated nucleic acid according to claim 10.

12. A host cell comprising the vector according to claim 11.

13. The host cell according to claim 12, wherein the host cell is a mammalian cell.

14. The host cell according to claim 12, wherein the host cell is a CHO cell.

15. A method for producing an antibody, comprising culturing the host cell according to claim 12 under conditions suitable for expressing the antibody and recovering the antibody.

16. A chimeric antigen receptor protein, comprising the antigen-binding fragment according to any one of claims 1 to 8.

17. An isolated nucleic acid encoding the chimeric antigen receptor protein according to claim 16.

18. A vector comprising the isolated nucleic acid according to claim 17.

19. An engineered cell comprising the isolated nucleic acid according to claim 17.

20. The engineered cell according to claim 19, wherein the cell is a T cell, an NK cell or a macrophage.

21. Use of the antibody or its antigen-binding fragment according to any one of claims 1 to 8 or the engineered cell according to claim 19 or 20 in the preparation of a medicament for treating or ameliorating the effects of cancer in a subject, the treatment or amelioration comprising administering to the subject a therapeutically effective amount of the antibody or its antigen-binding fragment or the engineered cell, the cancer being leukemia.

22. The use according to claim 21, wherein the treatment or amelioration reduces or eradicates the tumor burden in the subject.

23. The use according to claim 21, wherein the treatment or amelioration reduces the number of tumor cells.

24. The use according to claim 21, wherein the treatment or amelioration eradicates the tumor of the subject.

25. The use according to claim 21, wherein the leukemia is selected from the group consisting of chronic myelomonocytic leukemia, chronic myeloid leukemia, or acute myeloid leukemia, acute promyelocytic leukemia or M3 acute myeloid leukemia, acute myelomonocytic leukemia or M4 acute myeloid leukemia, acute monocytic leukemia or M5 acute myeloid leukemia, and acute lymphoblastic leukemia.

26. The use according to claim 21, wherein the antibody or its antigen-binding fragment is administered intravenously, intraarterially, intratumorally or subcutaneously.

27. The use according to claim 21, further comprising administering to the subject one or more drugs selected from the group consisting of: topoisomerase inhibitors, anthracycline topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolism inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine, combinations of daunorubicin and cytarabine, daunorubicin and cytarabine liposomes for injection, azacitidine, decitabine, all-trans retinoic acid, arsenic, arsenic trioxide, histamine dihydrochloride, interleukin-2, aldesleukin, gemtuzumab ozogamicin, FLT-3 inhibitors, midostaurin, clofarabine, farnesyl transferase inhibitors, IDH1 inhibitors, ivosidenib, IDH2 inhibitors, enasidenib, smoothened inhibitors, glasdegib, arginase inhibitors, IDO inhibitors, ikaroxostat, BCL-2 inhibitors, venetoclax, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, ibrutinib, acalabrutinib, zanubrutinib, PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, CD40 antibodies, 4-1BB antibodies, CD47 antibodies, SIRP1α antibodies or fusion proteins, E-selectin antagonists, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to myeloid cell or NK cell surface markers, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, plant-derived alkaloids, hormone therapy drugs, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors.

28. The use according to any one of claims 21 to 27, wherein the isolated monoclonal antibody or its antigen-binding fragment further comprises an anti-tumor drug linked thereto.

29. The use according to claim 28, wherein the anti-tumor drug is linked to the antibody via a photo-labile linker.

30. The use according to claim 28, wherein the anti-tumor drug is linked to the antibody via an enzyme-cleavable linker.

31. The use according to claim 28, wherein the anti-tumor drug is a toxin, a radioisotope, a cytokine or an enzyme.

32. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 for the preparation of a medicament for detecting leukemia cells in a sample or a subject, said detection comprising: (a) contacting the subject or a sample from the subject with the antibody or antigen-binding fragment thereof; and (b) detecting the binding of the antibody to leukemia cells in the subject or sample.

33. Use according to claim 32, wherein the sample is a body fluid or a biological specimen.

34. Use according to claim 32, wherein the sample is blood, bone marrow, sputum, tears, saliva, mucus, serum, urine or feces.

35. Use according to claim 32, wherein the detection comprises immunohistochemistry, flow cytometry, ELISA, RIA or Western blotting.

36. Use according to claim 32, wherein the detection comprises FACS.

37. Use according to claim 32, which further comprises performing steps (a) and (b) a second time and determining the change in the detection level compared to the first time.

38. Use according to claim 32, wherein the isolated monoclonal antibody or antigen-binding fragment thereof further comprises a label.

39. Use according to claim 38, wherein the label is a peptide tag, an enzyme, a magnetic particle or a chromophore.

40. Use according to claim 38, wherein the label is a fluorescent molecule.

41. Use according to claim 38, wherein the label is a chemiluminescent molecule.

42. Use according to claim 38, wherein the label is a dye.

43. Use according to any one of claims 21 to 42, wherein the isolated monoclonal antibody or antigen-binding fragment thereof is conjugated to a liposome or a nanoparticle.

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