Novel anti-LILRB2 antibody and derivative products

Abstract

The present disclosure provides anti-LILRB2 antibodies or antigen-binding fragments thereof, anti-LILRB2 chimeric antigen receptor proteins, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and uses thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 094,354, filed on 21 October 2020, and U.S. Provisional Patent Application No. 63 / 110,317, filed on 5 November 2021, the disclosures thereof being incorporated herein by reference. Sequence List

[0002] The sequence listing, named "066564-8014WO01_ST25," is 154KB (measured by Microsoft Windows), and is contained in a file created on October 20, 2021, which was filed electronically together with this specification and is incorporated herein by reference. background

[0003] I. Field

[0004] This disclosure generally pertains to the fields of medicine, oncology, and immunology. More specifically, this disclosure pertains to antibodies that bind to LILRB2. [Background technology]

[0002]

[0005] II. Description of related technologies

[0006] Myeloid suppressor cells and tumor-associated macrophages inhibit the anti-cancer immune response both systemically and within the tumor microenvironment, thereby limiting the effectiveness of immune checkpoint blockers. On the other hand, myeloid cell plasticity can enable therapeutic intervention. Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), also known as immunoglobulin-like transcript 4 (ILT4 or ILT-4), leukocyte immunoglobulin-like receptor 2 (LIR2 or LIR-2), and CD85d or CD85D, is a type I membrane protein primarily expressed by myeloid cells (monocytes, macrophages, dendritic cells, and neutrophils) that has emerged as a key immune checkpoint mediating cancer-associated myeloid cell tolerogenic activity. LILRB2 contains a cytoplasmic immune receptor inhibitory tyrosine motif (ITIM) and is involved in the negative regulation of immune cell activation. LILRB2 possesses several ligands (classical and non-classical MHC-I, ANGPTL2 / 5, SEMA4A, complement degradation products [CSP], and CD1c / d) that can play a role in cancer, most of which are known to contribute to immunosuppression in the solid tumor microenvironment. Binding of LILRB2 to its ligands results in inhibitory signals that counteract the stimulation of the immune response. Physiologically, its activity is thought to regulate inflammatory responses and immunocytotoxicity to help focus the immune response and limit autoreactivity. Therefore, LILRB2 is a promising target for modulating the immune response in the treatment of a variety of diseases and conditions, including cancer, chronic viral infections, and autoimmune diseases. Novel anti-LILRB2 antibodies are urgently needed. [Overview of the project]

[0003]

[0007] This disclosure provides anti-LILRB2 antibodies and their antigen-binding fragments, their amino acid and nucleotide sequences, anti-LILRB2 chimeric antigen receptors, and uses thereof.

[0004]

[0008] In one embodiment, the disclosure provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to LILRB2. In certain embodiments, the antibody or antigen-binding fragment modulates the activation of LILRB2 upon binding to LILRB2. In certain embodiments, the antibody or antigen-binding fragment suppresses the activation of LILRB2 upon binding to LILRB2. In certain embodiments, the antibody or antigen-binding fragment specifically blocks the binding of MHC and other ligands (e.g., ANGPTL, SEMA4A, etc.) to LILRB2 upon binding to LILRB2.

[0005]

[0009] In some embodiments, the anti-LILRB2 antibody or its antigen-binding fragment includes a clone-paired heavy chain variable region and a light chain variable region, as shown in Figure 1. In some embodiments, the antibody or its antigen-binding fragment includes (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; or (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32.

[0006]

[0010] In certain embodiments, the antibodies described herein are recombinant fully human antibodies. In certain embodiments, the antibodies described herein are human IgG1, human IgG2, human IgG3, or human IgG4 antibodies. In certain embodiments, the antigen-binding fragments described herein are recombinant scFv (single chain fragment variable) antibodies, Fab fragments, F(ab')2 fragments, or Fv fragments.

[0007]

[0011] In certain embodiments, an isolated recombinant fully human antibody described herein is ligated to one or more conjugate moieties. In some embodiments, the conjugate moieties include antitumor agents, STING (Stimulator of Interferon Genes) agonists, cytokines, clearance modifiers, toxins (e.g., chemotherapeutic agents), immunostimulators (e.g., TLR agonists), detectable labels (e.g., radioisotopes, lantanides, luminescence labels, fluorescent labels, or enzyme-substrate labels), DNA, RNA, or purified moieties.

[0008]

[0012] In another embodiment, isolated nucleic acids encoding isolated recombinant fully human antibodies or antigen-binding fragments thereof are provided herein.

[0013] In another embodiment, a vector comprising an isolated nucleic acid provided herein is provided.

[0009]

[0014] In another embodiment, a host cell containing the vector provided herein is provided. The host cell may be a mammalian cell. The host cell may be a CHO cell.

[0015] In another embodiment, a process for producing antibodies is provided. The method may include the steps of culturing host cells provided herein under conditions suitable for antibody expression, and recovering the antibodies.

[0010]

[0016] In another embodiment, a chimeric antigen receptor (CAR) protein comprising an antigen-binding fragment provided herein is provided.

[0017] In another embodiment, isolated nucleic acids encoding CAR proteins provided herein are provided.

[0011]

[0018] In another embodiment, manipulated cells containing isolated nucleic acids provided herein are provided. In certain embodiments, the cells are T cells, NK cells, or myeloid cells.

[0019] In another embodiment, a method is provided for treating or improving the effects of cancer or chronic viral infection in a subject, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined herein. The method may reduce or eradicate tumor volume in the subject, reduce the number of tumor cells, reduce tumor size, reduce tumor invasion, reduce tumor metastasis, or eradicate the tumor. The cancer may be a solid tumor or a hematological malignancy.

[0012]

[0020] In certain embodiments, cancer is a solid tumor including adrenal cancer, cholangiocarcinoma, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, esophageal cancer, gastroesophageal junction adenocarcinoma (GEA), eye cancer, stomach cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, mesothelioma, melanoma, Merkel cell carcinoma, nasopharyngeal cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pineal gland cancer, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, skin cancer, testicular cancer, thymic carcinoma, thyroid cancer, uterine cancer, and vaginal cancer.

[0013]

[0021] In some embodiments, the cancer is metastatic cancer, recurrent cancer, or drug-resistant cancer.

[0022] In some embodiments, the cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, blast plasmacytoid dendritic cell neoplasms (BPDCN), chronic lymphoblastic leukemia (CLL), chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia (CML), and precursor B-cell acute lymphoblastic leukemia (Pre-B). Hematological malignancies include ALL, diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, HHV8-associated primary exudative lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, heavy chain disease, Hodgkin lymphoma, non-Hodgkin lymphoma, Waldenström macroglobulinemia, multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasms, and polycythemia vera.

[0014]

[0023] Examples of cancers to which the treatment methods described herein apply include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specifically, non-limited examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, stomach cancer, melanoma, and various types of head and neck cancers (including head and neck squamous cell carcinoma).

[0015]

[0024] Antibodies or their antigen-binding fragments may be administered intravenously, intra-arterially, intratumorally, intramuscularly, or subcutaneously.

[0025] In certain embodiments, the method targets chemotherapeutic agents, tumor growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, cancer immunotherapy agents, apoptotic agents, anti-tubulin agents, microtubule inhibitors, anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists, HER1 / EGFR inhibitors, platelet-derived growth factor inhibitors, COX-2 inhibitors, interferons, CTLA4 inhibitors (e.g., ipilimumab (YERVOY®), or tremelimumab, which are anti-CTLA antibodies), PD-1 inhibitors, or PD -L1 inhibitors (e.g., OPDIVO® or nivolumab, KEYTRUDA® or pembrolizumab, TECENTRIQ® or atezolizumab, BAVENCIO® or avelumab, IMFINZI® or durvalumab, LIBTAYO® or semiprimab rwlc, TYVYT® or cintilimab, tislerizumab (BGB-A317), penprimab (AK105), camrelizumab, tripalimab, zimbererimab (GLS-010), retifanlima (e.g., sugemalimab, or CS1003), bispecific antibodies against CTLA-4 and PD-1 or PD-L1 (e.g., anti-PD-1 / CTLA-4 bispecific antibody or AK104), TIM3 inhibitors (e.g., anti-TIM3 antibody), LAG-3 inhibitors (e.g., anti-LAG3 antibody), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, FGFR2b, PDGFR-beta, BlyS, APRIL, BCMA, or VEGF receptors,(or more), TRAIL / A po2, IDH1 inhibitor, ivosidenib, Tibsovo®, IDH2 inhibitor, enasidenib, Idhifa®, smoothed (SMO) inhibitor, glassedegib, arginase inhibitor, IDO inhibitor, epacadostat, BCL-2 inhibitor, venetoclax, Venclexta®, platinum complex derivative, oxaliplatin, kinase inhibitor, tyrosine kinase inhibitor, PI3 kinase inhibitor, BTK inhibitor, ibrutinib, IMBRUVICA®, acalabrutinib, CALQUENCE®,The procedure may further include administering one or more drugs selected from the group consisting of zanubrutinib, ICOS antibody, TIGIT antibody, OX40 antibody, Toll-like receptor (TLR) agonist, STING agonist, TNFR2 antibody, CD40 antibody, 4-1BB antibody, CD47 antibody, SIRP1α antibody or SIRP1a fusion protein, Siglec antibody, antibody against another LILR family member, E-selectin antagonist, antibody that binds to tumor antigen, antibody that binds to markers on the surface of T cells, antibody that binds to markers on the surface of myeloid cells or NK cells, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, plant-derived alkaloids, hormonal therapeutics, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors, engineered T cells, engineered NK cells, or engineered macrophages, and bispecific antibodies.

[0016]

[0026] An isolated fully human recombinant antibody or its antigen-binding fragment may contain an antitumor agent linked thereto. The antitumor agent may be linked to the antibody via a photodissociative linker. The antitumor agent may be linked to the antibody via an acid-sensitive enzyme-cleaving linker or a glutathione-sensitive enzyme-cleaving linker. The antitumor agent may be linked to the antibody via a non-cleaving linker. The antitumor agent may be a toxin, a radioisotope, a cytokine, a STING agonist, or an enzyme.

[0017]

[0027] In another embodiment, a method is provided for detecting cancer cells or cancer stem cells in a sample or subject, comprising the steps of (a) contacting a subject or a sample derived from a subject with an antibody or its antigen-binding fragment as defined herein; and (b) detecting the binding of the antibody to cancer cells or cancer stem cells in the subject or sample. The sample may be a body fluid or biopsy, or blood, bone marrow, sputum, tears, saliva, mucus, serum, ascites, urine, or feces. Detection may include immunohistochemistry, flow cytometry, immunoassay (including ELISA, RIA, etc.), or Western blotting. The method may further include a step at a second time point in which steps (a) and (b) are performed and a change in the detection level compared to the first time point. The isolated recombinant antibody or its antigen-binding fragment may further include labeling such as a peptide tag, enzyme, magnetic particle, chromophore, fluorescent molecule, chemiluminescent molecule, or dye. The isolated recombinant antibody or its antigen-binding fragment may be conjugated into liposomes or nanoparticles.

[0018]

[0028] In further embodiments, a method is provided for treating or improving the effects of a chronic viral infection in a subject, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment specified herein. The antibody or antigen-binding fragment may be administered intravenously, intra-arterially, or subcutaneously. In some embodiments, the chronic viral infection is caused by a virus selected from herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), herpes simplex virus type 3, herpes simplex virus type 4, herpes simplex virus type 5, herpes simplex virus type 6, parvovirus B19, coxsackievirus type A and coxsackievirus type B, hepatitis A virus, hepatitis B virus, hepatitis C virus, cytomegalovirus (CMV), and human immunodeficiency virus (HIV).

[0019]

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

[0020] [Figure 1-1]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-2]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-3]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-4]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-5]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-6]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-7]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-8]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-9]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-10]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-11]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-12]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-13]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-14]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 1-15]

[0030] Figure 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a specific anti-LILRB2 antibody derived from B2-19, which is disclosed in PCT Patent Application No. PCT / US2021 / 015362. [Figure 2]

[0031] Figure 2 shows that the B2-19 antibody mutants, B2-19-12 and B2-19-16, have the same binding affinity to LILRB2 as the B2-19 parent antibody. The binding affinity of B2-19 and selected B2-19-derived mutants to the recombinant LILRB2 extracellular domain (ECD) protein (with a 6×His tag at the C-terminus) was measured by biolayer interferometry (BLI). All measured binding affinity values ​​were nearly identical and within the margin of experimental error, with a KD of approximately 2.0 nM. [Figure 3]

[0032] Figure 3 shows that the B2-19 antibody and its variants have equivalent binding affinity to LILRB2 stably expressed on HEK293 cells. The binding affinity (EC50) of the LILRB2 antibody to HEK293 cells stably expressing LILRB2 was determined by flow cytometry. [Figure 4]

[0033] Figure 4 shows that the B2-19 antibody and its variants have equivalent binding affinity (EC50) to endogenous LILRB2 expressed on primary CD14+CD16- monocytes isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. [Figure 5]

[0034] Figure 5 shows that the B2-19 antibody and its variants specifically bind to LILRB2. The binding specificity of the antibodies to LILRB2 was analyzed by ELISA. [Figure 6]

[0035] Figure 6 shows that the B2-19 antibody and its variants specifically bind to bone marrow cells in human whole blood. The reactivity of the LILRB2 antibody to leukocytes derived from whole blood collected from healthy donors was characterized by flow cytometry. The data shown are the corrected geometric mean fluorescence intensity (MFI) of the samples, i.e., the geometric MFI of anti-LILRB2 stained samples after subtracting the geometric MFI of samples from which the LILRB2 antibody was excluded (fluorescence minus one [FMO] control). Representative data from one donor is shown (N=3 donors). [Figure 7]

[0036] Figure 7 shows that the B2-19 antibody and its variants have equivalent ability to block LILRB2 from binding to HLA-G. The anti-LILRB2 antibody competitively inhibited the binding of HLA-G to HEK293 cells that stably express LILRB2, as determined by flow cytometry efficacy (IC50). [Figure 8A]

[0037] Figures 8A and 8B show that B2-19 and B2-19-16 antibodies, at suboptimal concentrations, had equivalent pro-inflammatory effects on PBMC samples isolated from healthy donors and stimulated with anti-CD3 agonist monoclonal antibodies. Each line represents the correspondence of results from individual donors (human IgG4 isotype control antibody vs. anti-LILRB2 blocking antibody), and data are pooled from six independent experiments. The percentage of donors showing a detectable change in cytokine production / secretion levels corresponding to the enhanced pro-inflammatory effect of the anti-LILRB2 blocking antibody is shown in parentheses, with *p<0.05, **p<0.001, ***p=0.0001, and ****p<0.0001 (paired t-test). Figure 8A shows cytokine levels in PBMCs stimulated with 10 ng / mL anti-CD3 antibody and 15 μg / mL isotype control or B2-19-16. Figure 8B shows cytokine levels in PBMCs stimulated with 10 ng / mL anti-CD3 antibody and isotype controls or B2-19 at 4 μg / mL or 15 μg / mL (experimentally). [Figure 8B]Figures 8A and 8B show that B2-19 and B2-19-16 antibodies, at suboptimal concentrations, had equivalent pro-inflammatory effects on PBMC samples isolated from healthy donors and stimulated with anti-CD3 agonist monoclonal antibodies. Each line represents the correspondence of results from individual donors (human IgG4 isotype control antibody vs. anti-LILRB2 blocking antibody), and data are pooled from six independent experiments. The percentage of donors showing a detectable change in cytokine production / secretion levels corresponding to the enhanced pro-inflammatory effect of the anti-LILRB2 blocking antibody is shown in parentheses, with *p<0.05, **p<0.001, ***p=0.0001, and ****p<0.0001 (paired t-test). Figure 8A shows cytokine levels in PBMCs stimulated with 10 ng / mL anti-CD3 antibody and 15 μg / mL isotype control or B2-19-16. Figure 8B shows cytokine levels in PBMCs stimulated with 10 ng / mL anti-CD3 antibody and isotype controls or B2-19 at 4 μg / mL or 15 μg / mL (experimentally). [Figure 9]

[0038] Figure 9 shows that the B2-19 derived variant exhibits lower polyspecificity than the B2-19 parent antibody, as evidenced by the improved baculovirus particle (BVP) score. The graph shows the OD450nm by ELISA for antibodies coated with 0.5% BVP (v / v relative to stock solution; titer = 5.71 × 10¹² pfu / mL) and conjugated to plates using a 1 / 20,000 dilution of anti-human IgG secondary antibody. The literature has shown that antibodies with a BVP score five times higher than the background tend to exhibit poor pharmacokinetics in humans and non-human primates (Hotzel et al., 2012, mAbs, 753-760). Therefore, B2-19-12 and B2-19-16 are more suitable for therapeutic development than the parent antibody B2-19. [Figure 10]

[0039] Figure 10 shows that the B2-19 mutant does not lose binding activity to a detectable degree after being subjected to heat stress. The data are dose-response curves and calculated potency (EC50) for binding to LILRB2, measured by ELISA. [Figure 11]

[0040] Figure 11 shows that the B2-19 variant does not lose binding activity during freeze-thaw (F / T) cycles. The data are dose-response curves and EC50 values ​​for antibody binding to LILRB2, as measured by ELISA. [Figure 12]

[0041] Figure 12 shows the pharmacokinetics (PK) of B2-19-12 and B2-19-16 in human FcRn transgenic mice. Both B2-19-12 and B2-19-16 exhibit pharmacokinetic parameters within the typical range for human IgG in human FcRn transgenic mice. [Figure 13]

[0042] Figures 13A and 13B show flow cytometry data indicating that B2-19-16 binds to all myeloid cells infiltrating the solid tumor microenvironment, as well as peripheral blood myeloid cells, from solid tumor patients. CD11b is used as a panmyelocyte marker, and CD45 is used as a pantumor-infiltrating leukocyte marker. Figure 13A shows flow cytometry data from tumor tissue samples from three different solid tumor patients. Figure 13B shows flow cytometry data from peripheral blood of solid tumor patient 3. Histograms filled in black: samples incubated with B2-19-16; histograms filled in white: samples incubated with IgG4 isotype controls. [Figure 14]

[0043] Figure 14 shows that the B2-19-16 antibody further enhances the effect of lipopolysaccharide (LPS) on the maturation / activation of monocyte-derived dendritic cells, as indicated by a decrease in the expression level of the tolerogenicity marker, CD209. Each line represents the results from different healthy donor samples, analyzed by flow cytometry of CD209 levels on the cell surface. The percentage of donor samples showing a change in CD209 expression level consistent with the enhanced pro-inflammatory effect is shown in parentheses, with ****p < 0.0001 (paired t-test). [Figure 15]

[0044] Figure 15 shows that when monocyte-derived dendritic cells are matured / activated by LPS stimulation, the B2-19-16 antibody enhances TNF-α production / secretion. Each line represents results from different healthy donors. The percentage of donor samples showing a change in TNF-α concentration level consistent with the enhanced pro-inflammatory effect is shown in parentheses, with *p<0.05 (paired t-test). [Figure 16]

[0045] Figures 16A and 16B show that the B2-19-16 antibody promotes the differentiation of primary monocytes into activated (CD86+) dendritic cells (DCs). The effect of the anti-LILRB2 antibody on monocyte differentiation into DCs in vitro was analyzed by flow cytometry. Figure 16A shows flow cytometry data from two healthy donor samples, and the values ​​shown in each histogram represent the percentage of CD86+ DCs obtained at the end of a 6-day culture under each experimental condition. Figure 16B shows the combination of results from all seven donors analyzed, with **p=0.002 (paired t-test)**. [Figure 17]

[0046] Figure 17 shows flow cytometry data demonstrating that B2-19-16 enhances the expression levels of maturation markers (CD83) and activation markers (CD86, HLA-DR) in immature dendritic cells (DCs) derived from monocytes, while decreasing the expression level of the tolerogenic marker, CD209. The expression level of another immunosuppressive receptor, LILRB4, remains unchanged. Each line represents results from different healthy donor samples, with *p<0.05, **p<0.008, and ns = not significant (paired t-test). [Figure 18]

[0047] Figure 18 shows that B2-19-16 enhances IFN-γ production / secretion in allogeneic CD4+ T cell-macrophage cocultures stimulated by anti-PD-1 blocking antibodies. Each line represents the result from one allogeneic CD4+ T cell-macrophage coculture, with p=0.0032 (repeat measurement, one-way ANOVA). [Figure 19]

[0048] Figure 19 shows that B2-19-16 enhances the production / secretion of multiple pro-inflammatory cytokines in LPS-stimulated PBMC samples derived from healthy donors, while reducing the production / secretion of the anti-inflammatory cytokine IL-10. Each line represents the correspondence of results from individual donor samples, and the data are pooled from four independent experiments. The percentage of donor samples showing changes in cytokine concentration levels corresponding to the enhanced pro-inflammatory effect is shown in parentheses, with **p<0.001 (paired t-test)**. [Figure 20]

[0049] Figure 20 shows that the B2-19-16 antibody dose-dependently enhances TNF-α production / secretion levels in LPS-stimulated PBMCs. The results represent five donor PBMC samples. [Figure 21]

[0050] Figure 21 shows that the B2-19-16 antibody enhances TNF-α production / secretion by monocyte-derived macrophages stimulated by 2'3'-cGAMP, a STING agonist, in all test donor samples. [Figure 22]

[0051] Figures 22A and 22B show that the B2-19-16 antibody restores the tolerogenic phenotype of PBMC-derived myeloid cells (CD33+) induced by "tumor conditioning." Each line corresponds to the result from a single healthy myeloid cell donor. Figure 22A shows the results of co-culture with SK-MEL-5 melanoma-derived cell lines during immunophenotypic analysis of myeloid cells. Figure 22B shows the results of co-culture with A549 lung adenocarcinoma-derived cell lines during immunophenotypic analysis of myeloid cells. [Figure 23]

[0052] Figure 23 shows that B2-19-16 does not induce LILRB2 internalization in monocyte-derived macrophages from two healthy donors. Anti-CD71 (transferrin receptor) antibody was used as a positive control for receptor:antibody complex internalization. Internalization was monitored for up to 12 hours using the Incucyte Live-Cell Analysis system. [Figure 24]

[0053] Figure 24 shows that B2-19-16 does not induce Fc-mediated depletion of LILRB2+ cells (monocytes derived from PBMCs) in vitro. To assess B2-19-16's ability to induce Fc-mediated monocyte depletion, PBMCs were incubated with up to 40 μg / mL of B2-19-16 (or isotype control) for 20 hours, but no decrease in monocyte viability was observed. In contrast, rituximab, an IgG1 antibody known to deplete B cells, strongly reduced B cell viability in simultaneous incubation with PBMC samples from the same donor. [Modes for carrying out the invention]

[0021]

[0054] The following statements relating to this disclosure are intended solely to illustrate various embodiments of this disclosure. Specific modifications discussed herein shall not be considered limitations to the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, variations, and modifications can be made without departing from the scope of this disclosure, and such equivalent embodiments will be understood to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0022]

[0055] I. Definition

[0056] It should be understood that both the general descriptions set forth above and the following “Modes for Carrying Out the Invention” are merely illustrative and exploratory, and not restrictive to the claimed invention. In this application, unless otherwise explicitly stated, the use of the singular form includes the plural form. In this disclosure, the term “or” is used to mean “and / or” unless it is explicitly stated that only substitutes or substitutes are mutually exclusive. As used herein, “another” may mean at least a second or more. Furthermore, the use of other forms of the term, such as “including” and “included,” in addition to “including,” is not restrictive. Also, terms such as “element” or “component” may include both a single unit containing one or more subunits and an element or component containing one or more elements or components, unless otherwise explicitly stated. Also, the use of the term “part” may include a part or the whole.

[0023]

[0057] Unless the context explicitly indicates otherwise, “a,” “an,” and “that” as used herein include multiple referents.

[0024]

[0058] When referring to measurable values ​​such as quantity or duration, the term “approximately” as used herein is intended to include a variation of no more than ±10% from the specified value. Unless otherwise indicated, all numbers used herein and in the claims to represent properties such as the quantity of components, molecular weight, or reaction conditions shall in all cases be understood to be modified by the term “approximately.” Thus, unless otherwise indicated, the numerical parameters shown herein and in the accompanying claims are approximations that may vary depending on the desired properties to be obtained from the disclosed subject matter. Each numerical parameter shall be understood by applying ordinary rounding techniques, at least in light of the number of significant figures reported, not as an attempt to limit the application of the principle for equivalents to the claims. Numerical ranges and parameters illustrating the broader invention are approximations, but the numerical values ​​shown in specific examples are reported as accurately as possible. However, any numerical value inherently contains a certain degree of error that inevitably arises from the standard deviation found in their respective test measurements.

[0025]

[0059] As used herein, the term “antibody” includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, polyspecific antibody, or bispecific antibody that binds to a specific antigen. Naturally intact antibodies contain two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain contains a variable domain (V H ) and the first steady domain, the second steady domain, and the third steady domain (each, C H1 , C H2 , C H3 It consists of a constant region containing λ or κ; mammalian light chains are classified as λ or κ, but each light chain has a variable domain (V L ) and the steady-state domain (C LA typical IgG antibody has a "Y" shape, and the Y-shaped stem is typically composed of two heavy chains, a second constant domain and a third constant domain, joined together via a disulfide bond. Each arm of the Y-shape contains a single heavy chain variable domain and a first constant domain, which are bound to a single light chain variable domain and a constant domain. The variable domains of the light and heavy chains contribute to binding to the antigen. The variable domains in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, and heavy chain CDRs including HCDR1, HCDR2, and HCDR3). The CDR boundaries for the antibody and antigen-binding fragments disclosed herein follow the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J.Mol.Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. (1985), 186(3):651~63; Chothia, C. and Lesk, AM, J.Mol.Biol. (1987), 196:901; Chothia, C. et al., Nature (1989), 342(6252):877~83; Marie-Paule Lefranc et al., Developmental and Comparative Immunology (2003), 27:55~77; Marie-Paule Lefranc et al., Immunome These can be defined or identified by Research (2005), 1(3); Marie-Paule Lefranc, "Molecular Biology of B cells" (2nd edition), Chapter 26, 481-514, (2015). The three CDRs are more conserved than the CDRs and are interspersed between a series of flankings known as framework regions (FRs), which form the foundation supporting the hypervariable loop. The constant domains of the heavy and light chains are not involved in binding to the antigen and exhibit diverse effector functions. Antibodies are assigned to classes based on the amino acid sequence of their heavy chain constant regions.The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of alpha, delta, epsilon, gamma, and muon heavy chains, respectively. Some of the main antibody classes are further divided into subclasses such as IgG1 (gamma mono-heavy chain), IgG2 (gamma double-heavy chain), IgG3 (gamma triple-heavy chain), IgG4 (gamma quadruple-heavy chain), IgA1 (alpha mono-heavy chain), or IgA2 (alpha double-heavy chain).

[0026]

[0060] The term "antigen" refers to a substance capable of inducing an acquired immune response. More specifically, an antigen is a substance that is specifically bound to an antibody or an antigen receptor on a T lymphocyte. Antigens are typically proteins and polysaccharides, and less frequently, lipids. Appropriate antigens may include, without limitation, parts of bacteria (e.g., coat, capsule, cell wall, flagella, cilia, and toxins), viruses, and other microorganisms. Antigens also include tumor antigens, such as those resulting from mutations within tumors. As used herein, antigens also include immunogens and haptens.

[0027]

[0061] As used herein, the term “antigen-binding fragment” refers to an antibody fragment formed from a portion of an antibody, including one or more CDRs, or any other antibody fragments that bind to an antigen but do not contain an intact native antibody structure. Examples of antigen-binding fragments, to the extent that they are not limited, include diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, polyspecific antibodies, camelid-derived single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments are capable of binding to the same antigen to which the parent antibody binds.

[0028]

[0062] A "Fab fragment" consists of one light chain and one heavy chain of carbon H1 and a variable domain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0029]

[0063] A "Fab' fragment" contains one light chain, the V H domain and the C H 1 domain, and the region between the C H 2 domains, and can form an inter-chain disulfide bond between the two heavy chains of two Fab' fragments so as to form an F(ab')2 molecule. It contains a part of one heavy chain that contains the region between the C H 1 domain and the C H 2 domains.

[0030]

[0064] An "F(ab')2 fragment" contains two light chains and two heavy chains, and forms an inter-chain disulfide bond between the two heavy chains so as to contain a part of the constant region between the C H 1 domain and the C H 2 domains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0031]

[0065] "Fv" with respect to an antibody refers to the smallest antibody fragment that has a complete antigen-binding site. The Fv fragment consists of the variable domain of a single light chain bound to the variable domain of a single heavy chain.​​​​​​​​​​​​​​​The three domains are held together by hydrophobic interactions. The Fc region of the antibody contributes to various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but it does not function in antigen binding.

[0034]

[0068] A "single-chain Fv-Fc antibody" or "scFv-Fc" refers to a modified antibody consisting of scFv attached to the Fc region of the antibody.

[0069] "dsFv" refers to a disulfide-stabilized Fv fragment in which a disulfide bond connects a single light chain variable domain to a single heavy chain variable domain. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" refers to two V molecules linked by three peptide chains: a peptide linker (e.g., a long-chain flexible linker). H The domain and two Vs, each linked via disulfide bridges. L It includes a domain. In some embodiments, dsFv-dsFv' is bispecific, in which each disulfide pair heavy chain and each disulfide pair light chain have different antigen specificities.

[0035]

[0070] A "camelid-derived single-domain antibody," "heavy chain antibody," or "HCAb" is a single-domain antibody derived from a camelid animal. HThis refers to antibodies that contain a domain but do not contain a light chain (Riechmann L. and Muyldermans S.,J., Immunol Methods., December 10, 231(1~2):25~38(1999); Muyldermans S.,J., Biotechnol., June, 74(4):277~302(2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies originally originated from camelid animals (camels, dromedaries, and llamas). Although lacking a light chain, camelid-derived antibodies possess a genuine antigen-binding repertoire (Hamers-Casterman C. et al., Nature (1993), 363:446~8; Nguyen VK. et al., Immunogenetics (2002), 54:39~47; Nguyen VK. et al., Immunology (2003), 109:93~101). The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit brought about by the acquired immune response (Koch-Nolte F. et al., FASEB J. (2007), 21:3490~8).

[0036]

[0071] A "nanobody" refers to an antibody fragment consisting of a VHH domain derived from a heavy chain antibody and two constant domains, the CH2 domain and the CH3 domain.

[0072] A "diabody" or "dAb" comprises a small antibody fragment with two antigen-binding sites, where the fragment is located within the same polypeptide chain. L V connected to the domain H Domain (V H -V L or V L -V H) include (see, for example, Holliger P. et al., Proc Natl Acad Sci US A., July 15, 90(14):6444~8(1993);EP404097;WO93 / 11161). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same antigen (or epitope) or different antigens (or epitopes). In certain embodiments, a “bispecific ds diabody” is a diabody that targets two different antigens (or epitopes).

[0037]

[0073] In certain embodiments, an "scFv dimer" is a divalent (or bivalent) single-chain variable fragment (di-scFv, bi-scFv) that can be manipulated by linking two scFvs. A divalent diabody or bivalent scFv (BsFv, di-scFv, bi-scFv) is a V of one part. H However, the V in other parts L In conjunction with this, another V forms two binding sites, which may target the same antigen (or epitope) or different antigens (or epitopes). H -V L Partial and dimerized V H -V L (linked by a peptide linker). In other embodiments, “scFv dimer” means V H1 and V L1 and work in conjunction, V H2 and V L2 V L1 -V H2 (This is also linked by a peptide linker) and related V H1 -V L2 It is a bispecific diabody containing (linked by a peptide linker).

[0038]

[0074] A "domain antibody" refers to an antibody fragment that contains only the variable domain of the heavy chain or the variable domain of the light chain. In certain cases, two or more Vs are used to create a bivalent domain antibody or a multivalent domain antibody. H The domain is covalently connected to the peptide linker. Two Vs in a bivalent domain antibody H Domains may target the same antigen, or they may target different antigens.

[0039]

[0075] A "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may be located on the same antigen or on two different antigens.

[0040]

[0076] "Binding affinity" generally refers to the strength of the total 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 endogenous binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). D Affinity is expressed by ). Affinity can be measured by common methods known in the art, including the methods described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind rapidly to antigens and maintain binding for extended periods. Various methods for measuring binding affinity are known in the art, and any of these may be used for the purposes of the present invention. Specific and illustrative (illustrative and exemplary) embodiments for measuring binding affinity are described below.

[0041]

[0077] An antibody that "specifically binds to" a particular polypeptide or an epitope on a particular polypeptide, or an antibody "specific to" such a polypeptide, is an antibody that binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. For example, the LILRB2-specific antibody of the present invention is specific to LILRB2. In some embodiments, the antibody that binds to LILRB2 has a molecular weight of ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 The dissociation constant (K) of M D ) has. The dissociation constant used herein is K D This refers to the ratio (k) of the dissociation rate to the association rate, which can be determined by using any common method known in the art, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS, BLI, and flow cytometry. off / k on ) refers to. In a particular embodiment, K D The value can be appropriately determined using flow cytometry.

[0042]

[0078] As used herein, “cancer” means any medical condition characterized by the proliferation or neoplasm, abnormal growth, invasion, or metastasis of malignant cells, and includes both solid tumors and non-solid cancers (hematological malignancies) such as leukemia. As used herein, “solid tumor” means a solid mass of neoplastic and / or malignant cells. Examples of cancer or tumors include hematological malignancies, oral cancers (e.g., lips, tongue, or larynx), digestive tract cancers (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), respiratory tract cancers such as peritoneum, liver and biliary tract, pancreas, pharynx, or lungs (small cell and non-small cell), bone, connective tissue, skin cancers (e.g., melanoma), breast cancer, reproductive tract cancers (fallopian tube, uterus, cervix, testes, ovaries, or prostate), urinary tract cancers (e.g., bladder or kidney), brain cancer, and cancers of endocrine glands such as the thyroid gland. In certain embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, kidney cancer, bladder cancer, hepatocellular carcinoma, and colorectal cancer. In certain embodiments, the cancer is selected from lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and B-cell lymphoma.

[0043]

[0079] As used herein, the term “chimera” means an antibody or antigen-binding fragment having a heavy chain and / or light chain portion derived from one molecular species and a heavy chain and / or light chain residue derived from a different molecular species. In exemplary examples, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal such as a mouse or rabbit. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0044]

[0080] As used herein, the terms “specific binding” or “specifically binding to” refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibody or antigen-binding fragment provided herein binds to LILRB2 ≤ 10 -6 M (for example, ≤ 5 × 10) -7 M, ≤ 2 × 10 -7 M, ≤10 -7 M, ≤ 5 × 10 -8M, ≤ 2 × 10 -8 M, ≤10 -8 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M, or ≤10 -9 Binding affinity (K) of M D ) specifically binds. K used herein D This refers to the ratio (k) of the dissociation rate to the association rate, which can be determined by using any common method known in the art, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry. off / k on ) refers to. In a particular embodiment, K D The value can be appropriately determined using flow cytometry.

[0045]

[0081] As used herein, the ability to “block binding” or the ability to “compete for the same epitope” refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., LILRB2 and an anti-LILRB2 antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 85%, or greater than 90%.

[0046]

[0082] Those skilled in the art will recognize that by confirming whether the former prevents the latter from binding to the LILRB2 antigen polypeptide, it is possible to determine, without unnecessary experimentation, whether a given antibody binds to the same epitope as the antibody of the present disclosure. If the given antibody competes with the antibody of the present disclosure, as demonstrated by the reduced binding of the antibody of the present disclosure to the LILRB2 antigen polypeptide, the two antibodies will bind to the same epitope or a closely related epitope. Alternatively, if the binding of the given antibody to the LILRB2 antigen polypeptide is inhibited by the antibody of the present disclosure, the two antibodies will bind to the same epitope or a closely related epitope.

[0047]

[0083] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to a manipulated receptor capable of grafting desired specificity to an antigen onto immune effector cells such as T cells, NK cells, and macrophages. Typically, a CAR protein comprises an extracellular domain that introduces the desired specificity, a transmembrane domain, and an intracellular domain that transmits a signal to the immune effector cell when the immune effector cell binds to the antigen. In certain embodiments, the extracellular domain comprises a leader peptide, an antigen-recognition region, and a spacer region. In certain embodiments, the antigen-recognition region is derived from an antibody that specifically binds to the antigen. In certain embodiments, the antigen-recognition region is a single-chain variable fragment (scFv) derived from the antibody. In certain embodiments, the single-chain variable fragment (scFv) is derived from a humanized antibody. In certain embodiments, the single-chain variable fragment comprises a heavy-chain variable region fused to a light-chain variable region via a flexible linker.

[0048]

[0084] In reference to amino acid sequences, a "conservative substitution" refers to replacing an amino acid residue with a different amino acid residue that has a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is well known in the art, conservative substitutions typically do not cause significant changes to the conformational structure of a protein, and therefore may preserve the bioactivity of the protein.

[0049]

[0085] As used herein, “effector function” refers to the bioactivity attributable to the binding of the antibody’s Fc region to its effectors, such as the C1 complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the antibody’s Fc region to Fc receptors on effector cells; and phagocytosis.

[0050]

[0086] As used herein, the term “epitope” refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind to the same or closely related epitopes within an antigen if they exhibit competitive binding for that antigen. For example, if an antibody or antigen-binding fragment blocks at least 85%, at least 90%, or at least 95% of the binding of a reference antibody to an antigen, the antibody or antigen-binding fragment can be considered to bind to the same / closely related epitope as the reference antibody.

[0051]

[0087] As used herein, the terms “homologous” and “homologous” are used interchangeably and refer to nucleic acid sequences (or their complementary chains) or amino acid sequences that, when optimally aligned, have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to other sequences.

[0052]

[0088] As used herein, the term “host cell” refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0089] As used herein, the term “humanized” means that the antibody or antigen-binding fragment comprises a CDR derived from a non-human animal, a FR region derived from a human, and, where appropriate, a constant region derived from a human.

[0053]

[0090] An "isolated" substance has been altered from its natural state by human intervention. If an "isolated" composition or "isolated" substance occurs naturally, it is either altered, removed from its original environment, or both. For example, a polynucleotide or polypeptide that exists naturally in a living animal is not "isolated," but is "isolated" if it is sufficiently separated from its natural coexisting material to exist in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, “isolated antibody or antigen-binding fragment” refers to an antibody or antigen-binding fragment having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as determined by electrophoresis (SDS-PAGE, isoelectric focusing, capillary electrophoresis, etc.) or chromatography (ion-exchange chromatography or reverse-phase HPLC, etc.).

[0054]

[0091] A "leader peptide" refers to a peptide approximately 5 to 30 amino acids long that is present at the N-terminus of a newly synthesized protein that forms part of a secretory pathway. Secretory pathway proteins include, but are not limited to, proteins that reside within specific intracellular organelles (endoplasmic reticulum, Golgi apparatus, or endosomes) and are secreted from the cell or inserted into the cell membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of the protein.

[0055]

[0092] Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) is a protein encoded in humans by the LILRB2 gene. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family, found within a gene cluster in the chromosomal region 19q13.4. The encoded protein belongs to class B, a subfamily of LIR receptors, containing two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four intracytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The LILRB2 receptor is expressed on immune cells and transmits negative signals that bind to MHC class I molecules and other ligands on antigen-presenting cells, inhibiting the stimulation of the immune response. The LILRB2 receptor may also play a role in antigen capture and presentation. The LILRB2 receptor is thought to help focus the immune response and limit autoreactivity by regulating inflammatory and cytotoxic responses. Multiple transcript variants encoding different isoforms have been found for this gene. LILRB2 has been shown to interact with PTPN6.

[0056]

[0093] The term "anti-LILRB2 antibody" refers to an antibody that is capable of specifically binding to LILRB2.

[0094] As used herein, “LILRB2-related” disease or condition means any disease or condition caused by, exacerbated by, or otherwise linked to the expression or activity of LILRB2, or to an increase or decrease thereof. In some embodiments, LILRB2-related conditions are immune-related disorders such as cancer, autoimmune diseases, inflammatory diseases, or infectious diseases.

[0057]

[0095] As used herein, the term “association” refers to an association via intramolecular interactions, such as covalent bonds, metallic bonds, and / or ionic bonds, or intermolecular interactions, such as hydrogen bonds or non-covalent bonds.

[0058]

[0096] The term "operatably linked" refers to an arrangement of elements such that the components described as such are configured to perform their usual functions. Thus, a given signal peptide operatably linked to a polypeptide will direct the secretion of the polypeptide from the cell. In the case of a promoter, a promoter operatably linked to a coding sequence will direct the expression of the coding sequence. A promoter, or any other regulatory element, does not need to be contiguous with the coding sequence insofar as it functions to direct its expression. For example, there may be an intervening sequence between a promoter sequence and a coding sequence that is not translated but is transcribed, and the promoter sequence can still be considered "operatably linked" to the coding sequence.

[0059]

[0097] The "sequence identity percentage (%)" for an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in the candidate sequence that are identical to those in the reference sequence, after the sequences have been aligned to achieve the maximum number of identical amino acids (or nucleic acids) and gaps have been introduced where necessary. Conservative substitutions of amino acid residues may or may not be considered identical. Alignment aimed at determining the identity percentage of amino acid (or nucleic acid) sequences can be achieved using publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J.Mol.Biol. (1990), 215:403~410; Stephen F et al., Nucleic Acids Res. (1997), 25:3389~3402), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology (1996), 266:383~402; Larkin MA et al., Bioinformatics (2007), 23:2947~8), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or they may customize the parameters to suit the alignment, for example, by selecting an appropriate algorithm.

[0060]

[0098] In the claims, the term “or” is used to mean “and / or” unless it is expressly indicated that it refers to substitutes only or substitutes that are mutually exclusive; however, this disclosure supports both the definition referring to substitutes only and the definition referring to “and / or.” As used herein, “another” may mean at least a second or more.

[0061]

[0099] The terms “polynucleotide” or “nucleic acid” include both single-stranded and double-stranded nucleotide polymers. The nucleotides in polynucleotides may be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine derivatives and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide ligation modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladetes, and phosphoramidates.

[0062] [000100] The terms “polypeptide” or “protein” mean a sequence of at least two amino acids linked to one another by peptide bonds. Polypeptides and proteins may also include additional amino acid segments (e.g., they may be glycosylated) and / or otherwise processed or modified. Those skilled in the art will notice that a “polypeptide” or “protein” may be a complete polypeptide chain produced by a cell (with or without a signal sequence) or a functional segment thereof. Those skilled in the art will further notice that a polypeptide or protein may include more than one polypeptide chain linked, for example, by one or more disulfide bonds or otherwise associated. The terms “polypeptide” or “protein” also include amino acid polymers, in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers.

[0063] [000101] Common pharmaceutically acceptable carriers useful in the present invention are typical. Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, PA, 15th edition (1975) describes compositions and formulations suitable for pharmaceutically delivering the fusion proteins disclosed herein. Generally, the properties of the carrier will depend on the specific administration method employed. For example, parenteral formulations typically include injectable solutions containing pharmaceutically and physiologically acceptable fluids as a medium, such as water, physiological saline, equilibrium salt solution, dextrose aqueous solution, or glycerol. For solid compositions (e.g., in powder, pill, tablet, or capsule form), common non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may also contain small amounts of non-toxic auxiliary substances, such as humectants or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0064] [000102] As used herein, the term “subject” means a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, meaning a human being who visits a healthcare provider for the diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” The subject may be susceptible to or prone to a disease or disorder, and may or may not present symptoms of the disease or disorder.

[0065] [000103] As used herein, the terms “therapeutic effective dose” or “effective dose” refer to a dose or concentration of a drug that is effective in treating a disease or condition. For example, with respect to the use of a monoclonal antibody or its antigen-binding fragment disclosed herein for treating cancer, the therapeutic effective dose is a dose or concentration of a monoclonal antibody or its antigen-binding fragment that can cause regression of tumor volume, eradicate all or part of a tumor, inhibit or slow the growth of tumor or the invasion of cancer cells into other organs, inhibit the growth or proliferation of cells mediating a cancerous condition, inhibit or slow the metastasis of tumor cells, improve any symptoms or markers associated with a tumor or cancerous condition, prevent or delay the onset of a tumor or cancerous condition, or a combination of some of these.

[0066] [000104] As used herein, “treating” a condition or “treatment” a condition includes preventing or mitigating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the onset of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, resulting in complete or partial regression of a condition, or curing a combination of a condition or parts thereof.

[0067] [000105] As used herein, the term “vector” refers to a medium into which a polynucleotide encoding a protein has been operably inserted so as to result in the expression of that protein. A vector may be used to transform, transduce into, or transfect a host cell so as to result in the expression of the genetic elements it contains within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector may contain various elements for controlling expression, including a promoter sequence, a transcription start sequence, an enhancer sequence, a selectable element, and a reporter gene. In addition, a vector may contain an origin of replication. A vector may also contain material that aids in its entry into cells, including but not limited to viral particles, liposomes, or protein coatings. A vector may be an expression vector or a cloning vector. This disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding an antibody or an antigen-binding fragment thereof, and at least one promoter (e.g., SV40, CMV, EF-1α) and at least one selectable marker operably linked to the nucleic acid sequence.Examples of vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages, and M13 phages, plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, and pGEX. This includes, but is not limited to, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0068] [000106] II. Anti-LILRB2 antibody and antigen-binding fragment [000107] In one embodiment, the present disclosure provides an anti-LILRB2 antibody and its antigen-binding fragment. In some embodiments, the anti-LILRB2 antibody and its antigen-binding fragment are derived from or modified from a particular anti-LILRB2 antibody (the disclosure of which is incorporated herein by reference) disclosed in PCT Patent Application PCT / US2021 / 015362. In some embodiments, the anti-LILRB2 antibody disclosed in PCT Patent Application PCT / US2021 / 015362 was prepared using a phage display method. Briefly, the phage display method involves a large library of human scFvs that are panned against and phage-displayed against a target protein, i.e., LILRB2. Human scFvs selected to specifically bind to the target protein can be sequenced and then subcloned into a human IgG expression vector to produce a desired fully human antibody.

[0069] [000108] In some embodiments, the anti-LILRB2 antibody and its antigen-binding fragment disclosed herein retain the same potent ligand-blocking activity as the anti-LILRB2 antibody disclosed in PCT Patent Application No. PCT / US2021 / 015362, as well as the ability to bind to LILRB2 with high affinity and specificity, but with improved properties in terms of the development of therapeutic or diagnostic products. In some embodiments, such antibodies and their antigen-binding fragments have significantly different physicochemical properties compared to the anti-LILRB2 antibody disclosed in PCT Patent Application No. PCT / US2021 / 015362, resulting in improved manufacturability, stability, and pharmacokinetic properties. In certain embodiments, such antibodies and their antigen-binding fragments have lower immunogenicity in humans.

[0070] [000109]Specific anti-LILRB2 antibody [000110] In certain embodiments, the anti-LILRB2 antibody disclosed herein is derived from B2-19, which is an antibody having the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 2. In certain embodiments, the anti-LILRB2 antibody disclosed herein has enhanced manufacturability, stability and / or pharmacokinetic profile compared to B2-19, while substantially retaining the ability to bind to LILRB2 with the same level of specificity and affinity. In addition, the anti-LILRB2 antibody disclosed herein retains the same ligand-blocking activity.

[0071] [000111] In certain embodiments, the LILRB2 antibody disclosed herein has a clone-paired heavy chain variable region (VH) amino acid sequence and a light chain variable region (VL) amino acid sequence, as shown in Figure 1.

[0072] [000112] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32.

[0073] [000113] In one embodiment, the anti-LILRB2 antibody and antigen-binding fragment provided herein are single-domain antibodies comprising all or part of the heavy chain variable domain provided herein. Further information on such single-domain antibodies is available in the Art (see, for example, U.S. Patent No. 6,248,516).

[0074] [000114] In certain embodiments, the anti-LILRB2 antibody and fragments provided herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region and / or a light chain constant region. The heavy chain constant region comprises a CH1 region, a hinge region, and / or a CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises Cκ or Cλ.

[0075] [000115] The antibodies or antigen-binding fragments provided herein may be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, bivalent antibodies, or anti-idiotype antibodies. Recombinant antibodies are antibodies prepared in vitro using recombinant methods, rather than in animals.

[0076] [000116] Antibody variant [000117]The antibodies and their antigen-binding fragments provided herein also encompass a variety of their variants. In certain embodiments, the antibodies and their antigen-binding fragments encompass a variety of variants of the exemplary antibodies provided herein.

[0077] [000118] In certain embodiments, an antibody variant comprises one or more modifications or substitutions within one or more variable region sequences and / or constant regions (e.g., Fc regions) provided herein. Such variants retain the specific binding affinity and ligand-blocking ability to LILRB2 of their parent antibody, but possess one or more desired properties conferred by the modification(s) or substitution(s). For example, an antibody variant may have improved antigen-binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamidation or deamination, improved or increased effector function(s), reduced or depleted effector function(s), improved binding to the FcRn receptor, extended pharmacokinetic half-life, increased pH sensitivity, or reduced immunogenicity and / or conjugation compatibility (e.g., introduction of one or more cysteine ​​residues).

[0078] [000119] The parent antibody sequence may be screened using methods known in the art, e.g., "alanine scanning mutagenesis," to identify suitable or preferred residues for modification or substitution (see, e.g., Cunningham and Wells (1989), Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) may be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and the modified antibody may be prepared and screened for the desired properties. If a substitution at a particular amino acid position supports the desired functional change, this position may be identified as a potential residue for modification or substitution. Potential residues may be further evaluated by substitution with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0079] [000120] Affinity variant [000121] Affinity variants may contain modifications or substitutions within the heavy chain variable region sequence or light chain variable region sequence provided herein. Affinity variants retain or improve upon the LILRB2-specific binding affinity of the parent antibody to LILRB2.

[0080] [000122] Various methods known in the art can be used to achieve this objective. For example, phage display technology can be used to create and express libraries of antibody variants (such as Fab variants or scFv variants) and then screen them for their binding affinity to LILRB2. As another example, computer software can be used to virtually simulate the binding of an antibody to LILRB2 and to identify amino acid residues on the antibody that form the binding interface. Such residues may be avoided in substitution to prevent a reduction in binding affinity, or they may be targeted for substitution to result in stronger binding.

[0081] [000123] In certain embodiments, the antibody or antigen-binding fragments provided herein include substitutions of one or more amino acid residues within one or more CDR sequences and / or within one or more FR sequences. In certain embodiments, affinity variants include a total of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer substitutions within the CDR sequences and / or FR sequences.

[0082] [000124] In certain embodiments, the anti-LILRB2 antibody and its antigen-binding fragment include one or more variable region sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the anti-LILRB2 antibody and its antigen-binding fragment provided herein, while maintaining a binding affinity to LILRB2 at a level equivalent to or exceeding that of the parent antibody. In some embodiments, substitutions, insertions, or deletions occur within the CDR region. In some embodiments, substitutions, insertions, or deletions occur within a region outside the CDR (e.g., within the FR).

[0083] [000125] Glycosylated mutant [000126] The anti-LILRB2 antibodies and antigen-binding fragments provided herein also include glycosylation variants, which may be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.

[0084] [000127] In some embodiments, the anti-LILRB2 antibodies and antigen-binding fragments provided herein include specific glycosylation patterns. For example, an aglycosylated antibody may be produced (i.e., the antibody lacks glycosylation). The glycosylation pattern of an antibody may be modified, for example, to increase the antibody's affinity or binding affinity to an antigen. Such modifications may be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions may be made, resulting in the removal of one or more glycosylation sites in the variable region framework, thereby eliminating glycosylation at that site. Such aglycosylation may increase the antibody's affinity or binding affinity to an antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0085] [000128] Antibodies such as low-fucosylated antibodies or afucosylated antibodies can also be produced, in which the glycosylation pattern includes low-fucosylated or afucosylated glycans, and the amount of fucosyl residues on the glycans is reduced. Antibodies may also include glycans in which the amount of branched GlcNac structures is increased. Such modifications to the glycosylation pattern have been shown to increase the ADCC activity of the antibody. Such modifications can be achieved, for example, by expressing an antibody in a host cell in which the glycosylation pathway has been 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), so that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The FUT8- / - cell lines, Ms704, Ms705, and Ms709, were created by targeting the disruption of the FUT8 gene in CHO / DG44 cells using two substitution vectors (see U.S. Patent Publication No. 20040110704). As another example, EP1176195 describes a cell line in which the FUT8 gene, which encodes a fucosyltransferase, is functionally disrupted, such that antibodies expressed within such cell lines exhibit hypofucosylation by reducing or eliminating the α-1,6 binding-related enzyme. EP1176195 also describes a cell line with low or no enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL1662).PCT Publication WO2003 / 035835 describes Lec13 cells, a mutant CHO cell line in which the ability to conjugate fucose to Asn(297)-linked carbohydrates is reduced, which also results in lower fucosylation of antibodies expressed in these host cells. As described in PCT Publication WO06 / 089231, antibodies with modified glycosylation profiles can also be produced in chicken eggs. Alternatively, antibodies with modified glycosylation profiles can also be produced in plant cells such as those of the genus Lemna (U.S. Patent No. 7,632,983). Methods for producing antibodies in plant systems are disclosed in U.S. Patents No. 6,998,267 and No. 7,388,081. PCT Publication WO1999 / 054342 describes cell lines engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed within the engineered cell lines exhibit an increased branched GlcNac structure, resulting in increased ADCC activity of the antibodies. When fucosylation on the antibody is minimal, low fucosylation is also called non-fucosylation.

[0086] [000129] Alternatively, the fucose residues of antibodies may be cleaved using a fucosidase enzyme; for example, α-L-fucosidase removes fucosyl residues from antibodies. The antibodies disclosed herein further include antibodies produced in lower eukaryotic host cells, in particular fungal host cells such as yeast, which have been genetically engineered to produce glycoproteins having a mammalian glycosylation pattern or a human-like glycosylation pattern. A particular advantage of these genetically modified host cells over currently used mammalian cell lines is their ability to control the glycosylation profile of the glycoproteins produced in the cells so that a glycoprotein composition in which a particular N-glycan structure is dominant is obtained (see, for example, U.S. Patents 7,029,872 and 7,449,308). These genetically modified host cells have been used to produce antibodies that predominantly have a particular N-glycan structure.

[0087] [000130] In addition, fungi such as yeast or filamentous fungi lack the ability to produce fucosylated glycoproteins, so antibodies produced in such cells will lack fucose unless the cells are further modified to include an enzymatic pathway for producing fucosylated glycoproteins (see, for example, PCT Publication WO2008112092). In certain embodiments, the antibodies disclosed herein further include antibodies comprising N-glycans that are produced in the host cells of lower eukaryotes, are hybrid N-glycans of fucosylated N-glycans and non-fucosylated N-glycans, and are also complex N-glycans comprising bibranched and multibranched molecular 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 include antibodies having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2;GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In certain embodiments, the hybrid N-glycan is the dominant N-glycan molecular species in the composition. In further embodiments, the hybrid N-glycan is a specific N-glycan molecular species comprising about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the hybrid N-glycan in the composition.

[0088] [000131] In certain embodiments, the antibody compositions provided herein include an antibody having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2;GalGlcNAcMan3GlcNAc2;NANAGalGlcNAcMan3GlcNAc2;GlcNAc2Man3GlcNAc2;GalGlcNAc2Man3GlcNAc2;Gal2GlcNAc2Man3GlcNAc2;NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In certain embodiments, the complex N-glycan is the major N-glycan molecular species in the composition. In further embodiments, the complex N-glycan is a specific N-glycan molecular species comprising about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycan in the composition. In certain embodiments, the N-glycan is a fucosylated N-glycan. Generally, fucose is an α1,3-linked fucose with GlcNAc at the reducing end of GlcNAcN-glycan, an α1,6-linked fucose with GlcNAc at the reducing end of GlcNAcN-glycan, an α1,2-linked fucose with Gal at the non-reducing end of GalN-glycan, an α1,3-linked fucose with GlcNac at the non-reducing end of GlcNacN-glycan, or an α1,4-linked fucose with GlcNAc at the non-reducing end of GlcNAcN-glycan.

[0089] [000132]Therefore, in a particular embodiment of the glycoprotein composition described above, the sugar forms are Man5GlcNAc2(Fuc), GlcNAcMan5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2GlcNAc2Man To produce sugar forms selected from the group consisting of 3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc), sugar forms of α1,3-linked fucose or α1,6-linked fucose; GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fu To produce a sugar form selected from the group consisting of c1-2)Man3GlcNAc2, GalGlcNAc2(Fuc1-2)Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc1-2)Man3GlcNAc2, and NANA2Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, use α1,3-linked fucose or α The sugar form is an α1,2-linked fucose sugar form, which is selected from the group consisting of Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2, NANAGal2(Fuc1-2)GlcNAc2Man3GlcNAc2, and NANA2Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2.

[0090] [000133] In further embodiments, the antibody comprises a high-mannose N-glycan, including but not limited to Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or an N-glycan consisting of the Man3GlcNAc2 structure of the N-glycan. In further embodiments of the above, the complex N-glycan further comprises fucosylated bibranched molecular species and fucosylated multibranched molecular species, as well as non-fucosylated (or afucosylated) bibranched molecular species and non-fucosylated (or afucosylated) multibranched molecular species. As used herein, the terms “N-glycan” and “sugar type” are used interchangeably and refer to N-linked oligosaccharides, for example, N-linked oligosaccharides that are linked to an asparagine residue of a polypeptide by an asparagine-N-acetylglucosamine linkage. N-linked glycoproteins contain N-acetylglucosamine residues linked to the amide nitrogen of asparagine residues within the protein.

[0091] [000134] Cysteine-manipulated mutant [000135]The anti-LILRB2 antibodies and antigen-binding fragments provided herein also include cysteine-manipulated variants, which involve the introduction of one or more free cysteine ​​amino acid residues.

[0092] [000136] Free cysteine ​​residues are free cysteine ​​residues that are not part of a disulfide crosslink. Cysteine-manipulated mutants are useful for conjugation of cytotoxic compounds and / or imaging compounds, labels, or radioisotopes at the manipulated cysteine ​​site, for example, via maleimide or haloacetyl. Methods for manipulating antibody or antigen-binding fragments to introduce free cysteine ​​residues are known in the art; see, for example, WO2006 / 034488.

[0093] [000137]Fc variant [000138] The anti-LILRB2 antibodies and antigen-binding fragments disclosed herein may also be manipulated to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or effector function (e.g., antigen-dependent cell-mediated cytotoxicity). Furthermore, the antibodies disclosed herein may be chemically modified (e.g., one or more chemical moieties may be conjugated to the antibody), modified to alter its glycosylation, or modified to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues within the Fc region is the numbering of the EU index by Kabat. The antibodies disclosed herein also include antibodies in which the Fc region is modified (or blocked) to result in altered effector function. See, for example, U.S. Patent No. 5,624,821; WO2003 / 086310; US2004 / 0002587; US2005 / 0152894; US2005 / 0249723; WO2006 / 019447. Such modifications can be used to enhance or suppress a variety of immune system responses, which may have beneficial effects in diagnosis and therapy. Modifications to the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fc. Changes to Fc also alter the antibody half-life within therapeutic antibodies, which can lead to reduced administration frequency, thus increasing convenience and reducing material usage. This mutation has been reported to invalidate the heterogeneity of inter-heavy-chain disulfide crosslinks within the hinge region.

[0094] [000139] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is increased or decreased. This method is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is changed, for example, to facilitate the assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another embodiment, the antibody is modified to extend its biological half-life. A variety of methods are possible. For example, one or more of the following mutations: T252L, T254S, T256F may be introduced, as described in U.S. Patent No. 6,277,375. Alternatively, to extend the biological half-life, the antibody may be modified within the CH1 or CL region to contain a salvage receptor that binds to an epitope extracted from two loops of the CH2 domain in the Fc region of IgG, as described in U.S. Patents No. 5,869,046 and 6,121,022. In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from the amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be replaced with a different amino acid residue so that the antibody's affinity for the effector ligand is altered, while retaining the parental antibody's ability to bind to the antigen. The effector ligand whose affinity is altered may be, for example, the Fc receptor or the C1 component of complement. This method is described in more detail in U.S. Patents No. 5,624,821 and No. 5,648,260.

[0095] [000140] In another example, one or more amino acid residues within the range of amino acid positions 231-239 are modified, thereby altering the antibody's ability to bind to complement. This technique is further described in PCT Publication WO1994 / 029351. In yet another example, the Fc region is located at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 33 By modifying one or more amino acids at sites 3, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, antibodies are modified to increase or decrease their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or increase or decrease their affinity for Fcγ receptors. This technique is further described in PCT Publication WO2000 / 042072. Furthermore, binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 are mapped, and variants with improved binding are also described. Specific mutations at positions 256, 290, 298, 333, 334, and 339 were shown to improve binding to FcγRIII. In addition, the following combination mutants: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A were shown to improve binding to FcγRIII.

[0096] [000141] In one embodiment, the Fc region is modified by modifying residues 243 and 264 so that the antibody has reduced its ability to mediate effector function and / or increased its 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 so that the antibody has reduced its ability to mediate effector function and / or increased its anti-inflammatory properties.

[0097] [000142] In one embodiment, the Fc region is modified by modifying residues 234, 235, and 329 to alanine or glycine (L234A-L235A-P329G) so that the antibody loses its ability to mediate effector function.

[0098] [000143] In certain embodiments, the anti-LILRB2 antibody or antigen-binding fragment comprises one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants may have an extended pharmacokinetic half-life because they bind to FcRn at an acidic pH, allowing FcRn to avoid degradation within lysosomes, and subsequently be translocated and released from the cell. In this field, methods for improving the binding affinity to FcRn by manipulating antibodies and their antigen-binding fragments are well known. For example, see Vaughn, D. et al., Structure, 6(1):63~73, 1998; Kontermann, R. et al., "Antibody Engineering", Vol. 1, Chapter 27: "Engineering of the Fc region for improved PK", Springer, 2010; Yeung, Y. et al., Cancer Research (2010), 70:3269~3277; and Hinton, P. et al., J. Immunology (2006), 176:346~356.

[0099] [000144] In certain embodiments, the anti-LILRB2 antibody or antigen-binding fragment comprises one or more amino acid substitutions that modify antibody-dependent cytotoxicity (ADCC). Certain amino acid residues in the CH2 domain of the Fc region may be substituted to result in enhanced ADCC activity. Alternatively, or in addition, the carbohydrate structure on the antibody may be altered to enhance ADCC activity. In this technical field, methods for modifying ADCC activity by manipulating antibodies have been described, for example, Shields RL. et al., J Biol Chem. (2001), 276(9):6591~604; Idusogie EE. et al., J Immunol. (2000), 164(8):4178~84; Steurer W. et al., J Immunol. (1995), 155(3):1165~74; Idusogie EE. et al., J Immunol. (2001), 166(4):2571~5; Lazar GA. et al., PNAS (2006), 103(11):4005~4010; Ryan MC. et al., Mol. Cancer Ther. (2007), 6:3009~3018; Richards JO. et al., Mol Cancer See Ther. (2008), 7(8):2517~27; Shields RL et al., J. Biol. Chem, 2002, 277:26733~26740; Shinkawa T. et al., J. Biol. Chem (2003), 278:3466~3473.

[0100] [000145] In certain embodiments, the anti-LILRB2 antibody or antigen-binding fragment comprises one or more amino acid substitutions that modify complement-dependent cytotoxicity (CDC) by, for example, improving or reducing binding to C1q and / or CDC (see, e.g., WO99 / 51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO1994 / 029351 for other examples of Fc region variants).

[0101] [000146] In certain embodiments, the anti-LILRB2 antibody or antigen-binding fragment includes one or more amino acid substitutions within the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include the introduction of a protrusion into a first Fc polypeptide and the introduction of a void into a second Fc polypeptide, in which case the protrusion may be positioned within the void to facilitate interaction between the first Fc polypeptide and the second Fc polypeptide to form a heterodimer or complex. Methods for obtaining antibodies with these modifications are known in the Art, for example, as described in U.S. Patent No. 5,731,168.

[0102] [000147]Antigen-binding fragment [000148] In the art, a variety of antigen-binding fragments are known and, for example, their variable sequences can be developed based on the exemplary antibodies provided herein and the anti-LILRB2 antibodies provided herein, including different variants thereof (affinity variants, glycosylated variants, Fc variants, cysteine-modified variants, etc.).

[0103] [000149] In certain embodiments, the anti-LILRB2 antigen-binding fragments provided herein are camelid-derived single-domain antibodies, diabodies, single-chain Fv fragments (scFv), scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragments, Fab, Fab', F(ab')2, ds-diabodies, nanobodies, domain antibodies, single-domain antibodies, or bivalent domain antibodies.

[0104] [000150] Single-chain variable fragments (scFv) are fusions of the heavy-chain and light-chain variable regions of an immunoglobulin, linked together by a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker peptide. This modification typically preserves the specificity. Historically, these molecules were created to facilitate phage display, where expressing the antigen-binding domain as a single peptide is extremely convenient. Alternatively, scFv can be created directly from heavy and light chains subcloned from hybridomas. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules and therefore lack the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments are often purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0105] [000151] Flexible linkers are generally composed of helix-promoting amino acid residues and turn-promoting amino acid residues, such as alanine, serine, and glycine. However, other residues can also function. Tang et al. (1996) used phage display as a means to rapidly select custom linkers for single-chain antibodies (scFv) from a protein linker library. A random linker library was constructed in which genes for heavy chain variable domains and genes for light chain variable domains were linked by segments encoding 18-amino acid polypeptides with variable composition. scFv repertoire (approximately 5 × 10⁻¹⁶) 6Different members of the tether were presented on filamentous phages and subjected to affinity selection using haptens. The selected population of mutants exhibited a significant increase in binding activity while retaining considerable sequence diversity. Subsequently, screening of 1054 individual mutants yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed that the general features of the selected tethers were conserved proline two residues after the C-terminus of VH within the linker, and a proliferation of arginine and proline at other positions.

[0106] [000152] The recombinant antibodies of this disclosure may also be accompanied by sequences or portions that enable dimerization or multimerization of the receptor. Such sequences include sequences derived from IgA that enable multimerization together with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chain may be modified with a drug such as biotin / avidin that enables a combination of two antibodies.

[0107] [000153] In a separate embodiment, a single-chain antibody may be created by linking the light chain and heavy chain of a receptor using a non-peptide linker or chemical unit. Generally, the light chain and heavy chain would be produced and purified in distinctly different cells and then linked together in an appropriate manner (i.e., the N-terminus of the heavy chain joined to the C-terminus of the light chain via appropriate chemical crosslinking).

[0108] [000154] Crosslinking reagents, such as stabilizers and flocculants, are used to form molecular crosslinks that link the functional groups of two different molecules. However, it is conceivable that dimers or polymers of the same analog or heteromeric complex may be created, composed of different analogs. To link two different compounds stepwise, heterobifunctional crosslinking agents may be used that eliminate undesirable homopolymer formation.

[0109] [000155] An exemplary heterobifunctional crosslinking agent contains two reactants, one of which reacts with a primary amine group (e.g., N-hydroxysuccinimide) and the other which reacts with a thiol group (e.g., pyridyl disulfide, maleimide, halogen, etc.). Via the primary amine reactant, the crosslinking agent can react with lysine residues (or more) of one protein (e.g., a selected antibody or fragment), and via the thiol reactant, the already linked crosslinking agent reacts with cysteine ​​residues (free sulfhydryl groups) of another protein (e.g., a selective agent).

[0110] [000156] It is preferable to use a crosslinking agent that has reasonable stability in the blood. Several types of disulfide bond-containing linkers are known that can be successfully used to conjugate a targeting agent with a therapeutic / preventive agent. Linkers containing sterically hindered disulfide bonds have been found to provide greater stability in vivo and may prevent the release of the targeted peptide before it reaches the site of action. Therefore, these linkers constitute one group of linkers.

[0111] [000157] Another crosslinking agent is SMPT, a bifunctional crosslinking agent containing a disulfide bond that is "sterically hindranced" by adjacent benzene rings and methyl groups. The steric hindrance by the disulfide bond is thought to protect the bond from attack by thiolate anions such as glutathione that may be present in tissues and blood, thereby helping to prevent decoupling of the conjugate before delivery of the conjugated drug to the target site.

[0112] [000158]SMPT crosslinking reagents, like many other known crosslinking reagents, provide the ability to crosslink functional groups such as the SH of cysteine ​​or primary amines (e.g., the epsilon-amino group of lysine). Another possible type of crosslinking agent includes heterobifunctional photoreactive phenyl azides containing cleaved disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamide)ethyl-1,3'-dithiopropionate. The N-hydroxysuccinimidyl group reacts with primary amino groups, and the phenyl azide (upon photodegradation) reacts non-selectively with any amino acid residue.

[0113] [000159] In addition to obstructing crosslinkers, non-obstructing linkers may also be incorporated herein. Other useful crosslinkers that do not contain or result in protective disulfides 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 a flexible linker.

[0114] [000160] U.S. Patent No. 4,680,338 describes a bifunctional linker useful for producing ligand conjugates with amine-containing polymers and / or amine-containing proteins for forming antibody conjugates with chelating agents, drugs, enzymes, detectable labels, etc. U.S. Patents No. 5,141,648 and No. 5,563,250 disclose cleavage-type conjugates containing unstable bonds that can be cleaved under a variety of mild conditions. This linker is particularly useful in that the drug of interest can be directly bound to the linker, resulting in the release of the active drug as a result of cleavage. Specific uses include the addition of free amino or free sulfhydryl groups to proteins such as antibodies or drugs.

[0115] [000161] U.S. Patent No. 5,856,456 provides a peptide linker for use in linking polypeptide components to produce fusion proteins, such as single-chain antibodies. The linker is up to about 50 amino acids long, contains proline following the presence of at least one charged amino acid (preferably arginine or lysine), and is characterized by increased stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses an aminooxy-containing linker useful in various immunodiagnostic and separation methods.

[0116] [000162] A variety of techniques can be used to produce such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods (1992), 24:107-117; and Brennan et al., Science (1985), 229:81), recombinant expression by host cells such as E. coli (e.g., antibody fragments, Fab, Fv, and ScFv), screening from phage display libraries discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form an F(ab')2 fragment (Carter et al., Bio / Technology (1992), 10:163-167). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0117] [000163] In certain embodiments, the antigen-binding fragment is an scFv. The creation of scFvs is described, for example, in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. The scFv can be fused to an effector protein at the amino or carboxyl terminus to result in a fusion protein (see, for example, "Antibody Engineering," edited by Borrebaeck).

[0118] [000164] Multispecific antibodies [000165] In certain embodiments, the anti-LILRB2 antibody and its antigen-binding fragment provided herein are polyspecific. As used herein, the term “polyspecific” encompasses molecules having more than one specificity, e.g., bispecificity, trispecificity, tetraspecificity. In certain embodiments, the polyspecific antibody and its antigen-binding fragment provided herein are capable of specifically binding to a first epitope and a second epitope of LILRB2, but the first epitope and the second epitope of LILRB2 are significantly different from or do not overlap with each other. In certain embodiments, the polyspecific antibody and its antigen-binding fragment provided herein are capable of specifically binding to LILRB2 and a second antigen different from LILRB2.

[0119] [000166] In certain embodiments, the second antigen is an immune-associated target. As used herein, immune-associated targets include biomolecules involved in stimulating, inhibiting, or modulating an immune response, optionally, a cellular immune response. Examples of immune-associated targets are immunomodulatory molecules expressed by cancer cells, stromal cells (such as fibroblasts and vascular cells), or immune cells. In some embodiments, immunomodulatory molecules may mediate co-stimulatory signals to enhance an immune response, or they may mediate co-inhibitory signals to suppress an immune response. Thus, in some embodiments, the second antigen is an immunomodulatory molecule.

[0120] [000167] In some embodiments, the immunomodulatory molecules are PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, Fc receptor, FCRL(1~6), A2AR, CD160, 2B4, TGF-β, TGF-βR, VISTA, BTLA, TIGIT, LAIR1, LILRB1, LILRB3, LILRB4, LILRB5, LILRA(1~6), OX40, CD2, CD27, CD28, CD30, CD40, CD47, SIRPA, CLEC-1, clever-1 / s tabilin-1, ADGRE, TREM1, TREM2, CD122, ICAM-1, IDO, NKG2D / C, SLAMF7, MS4A4A, SIGLEC(7~15), NKp80, NKG2A, CD160, CD161, CD300, CD 163, B7-H3, B7-H4, LFA-1, ICOS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, TNFR2, TLR(1-9), IL-2, IL-7, IL-15, IL-21, CD16, and CD83.

[0121] [000168] In certain embodiments, the second antigen includes a tumor antigen. As used herein, “tumor antigen” means tumor-specific antigens (e.g., those specific to tumor cells and not found on normal non-tumor cells) and tumor-associated antigens (e.g., those found on both tumor and non-tumor cells but differentially expressed within tumor cells or found within the tumor microenvironment). Tumor-specific antigens may also include tumor neoantigens (e.g., those expressed in cancer cells due to somatic mutations that alter protein sequences or create fusion proteins between two unrelated sequences).

[0122] [000169] Examples of tumor antigens, without limitation, include prostate-specific antigen (PSA), CA-125, ganglioside G (D2), ganglioside G (M2) and ganglioside G (D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-like peptide, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, FGFR2b, CD44v6, cancer-related mucin, VEGF, VEGFR (e.g., VEGFR3), estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-kit receptor, transferrin receptor, Claudin Contains 18.2, GPC-3, Nectin-4, ROR1, Metseline, BCMA, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, BCR-ABL, E2APRL, H4-RET, IGH-IGK, MYL-RAR, IL-2R, CO17-1A, TROP2, Ephrin A, or LIV-1.

[0123] [000170] The multispecific antibodies and their antigen-binding fragments provided herein may be in appropriate formats known in the art. For example, exemplary bispecific formats include bispecific diabody, scFv-based bispecific formats, IgG-scFv fusion, bivariable domain (DVD)-Ig, Quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole), BiTE, CrossMab, CrossFab, Duobody, SEEDbody, leucine zipper, dual-acting Fab (DAF)-IgG, and Mab 2 It can be a bispecific format (see, for example, Brinkmann et al., 2017, Mabs, 9(2):182-212). Bispecific molecules can have either a symmetric or asymmetric architecture.

[0124] [000171] The polyspecific antibodies and antigen-binding fragments provided herein can be prepared by any suitable method known in the art. In one embodiment, two immunoglobulin heavy-light chain pairs having different antigen specificities are co-expressed in host cells in a recombinant manner (see, for example, Milstein and Cuello, Nature, 305:537 (1983)) and then purified by affinity chromatography to produce a bispecific antibody.

[0125] [000172] Conjugate [000173] In some embodiments, the anti-LILRB2 antibody and its antigen-binding fragment further comprises a conjugate moiety. The conjugate moiety can be linked to the antibody and its antigen-binding fragment. The conjugate moiety is a proteinaceous or nonproteinaceous portion that can be linked to the antibody or its antigen-binding fragment. Various conjugate moieties are envisioned to be linked to antibodies or antigen-binding fragments provided herein (see, for example, "Conjugate Vaccines," Contributions to Microbiology and Immunology, JMCruse and RE Lewis, Jr. (eds.), Carger Press, New York (1989)). These conjugate moieties can be linked to antibodies or antigen-binding fragments by means of covalent linkage, affinity linkage, insertion, coordination linkage, complexation, association, blending, or addition.

[0126] [000174] In certain embodiments, the antibodies and antigen-binding fragments disclosed herein may be manipulated to include specific sites outside the epitope-binding moiety that can be utilized for binding to one or more conjugate moieties. For example, such sites may include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent linkage to the conjugate moiety.

[0127] [000175] In certain embodiments, the antibody may be indirectly conjugated to a conjugate moiety, or conjugated via another conjugate moiety. For example, an antibody or antigen-binding fragment may be conjugated to biotin, and then indirectly conjugated to a second conjugate which is conjugated to avidin.

[0128] [000176] Examples of conjugate portions include, but are not limited to, immunomodulators, antitumor drugs, STING (Stimulator of Interferon Genes) agonists, cytokines, clearance modifiers, toxins (e.g., chemotherapeutic agents), immune cell stimulants (e.g., TLR agonists), detectable labels (e.g., radioisotopes, lantanides, luminescence labels, fluorescent labels, or enzyme-substrate labels), DNA, RNA, or purified portions.

[0129] [000177] Examples of immunomodulators include, but are not limited to, the immunomodulatory molecules disclosed herein (e.g., PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, Fc receptor, FCRL(1-6), A2AR, CD160, 2B4, TGF-β, TGF-βR, VISTA, BTLA, TIGIT, LAIR1, LILRB1, LILRB3, LILRB4, LILRB5, LILRA(1-6), OX40, CD2, CD27, CD28, CD30, CD40, CD47, SIRPA, CLEC-1, clever-1 / stabilin-1, This includes ADGRE, TREM1, TREM2, CD122, ICAM-1, IDO, NKG2D / C, SLAMF7, MS4A4A, SIGLEC(7-15), NKp80, NKG2A, CD160, CD161, CD300, CD163, B7-H3, B7-H4, LFA-1, ICOS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, TNFR2, TLR(1-9), IL-2, IL-7, IL-15, IL-21, CD16, and CD83), or functional fragments thereof, their ligands, and their ligand-binding proteins.

[0130] [000178] Examples of antitumor drugs, without limitation, include chemotherapeutic agents, growth inhibitors, cytotoxic agents, drugs used in radiotherapy, anti-angiogenic agents, cancer immunotherapy agents, apoptotic agents, antitubulin agents, anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists, HER1 / EGFR inhibitors, platelet-derived growth factor inhibitors, COX-2 inhibitors, interferons, CTLA4 inhibitors (e.g., ipilimumab (YERVOY®) or tremelimumab, which are anti-CTLA antibodies), PD-1 inhibitors or PD-L1 Inhibitors (e.g., OPDIVO® or nivolumab, KEYTRUDA® or pembrolizumab, TECENTRIQ® or atezolizumab, BAVENCIO® or avelumab, IMFINZI® or durvalumab, LIBTAYO® or semiprimab rwlc, TYVYT® or cintilimab, tislerizumab (BGB-A317), penprimab (AK105), camrelizumab, tripalimab, zimbererimab (GLS-010), retifanlimab, Sugemalimab (or CS1003), bispecific antibodies against CTLA-4 and PD-1 or PD-L1 (e.g., anti-PD-1 / CTLA-4 bispecific antibody or AK104), TIM3 inhibitors (e.g., anti-TIM3 antibody), LAG-3 inhibitors (e.g., anti-LAG3 antibody), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, FGFR2b, PDGFR-beta, BlyS, APRIL, BCMA, or VEGF receptors (multiple targets), TRAIL / Ap o2, IDH1 inhibitor, ivosidenib, Tibsovo®, IDH2 inhibitor, enasidenib, Idhifa®, smoothed (SMO) inhibitor, glassedegib, arginase inhibitor, IDO inhibitor, epacadostat, BCL-2 inhibitor, venetoclax, Venclexta®, platinum complex derivative, oxaliplatin, kinase inhibitor, tyrosine kinase inhibitor, PI3 kinase inhibitor, BTK inhibitor, ibrutinib, IMBRUVICA®, acalabrutinib, CALQUENCE®,This includes zanubrutinib, TLR agonists, STING agonists, ICOS antibodies, TIGIT antibodies, CD40 antibodies, 4-1BB antibodies, CD47 antibodies, OX40 antibodies, TNFR2 antibodies, antibodies against other LILR family members, Siglec antibodies, SIRP1a antibodies or SIRP1a fusion proteins, E-selectin antagonists, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to myeloid or NK cell surface markers, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, plant-derived alkaloids, hormonal therapeutics, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors, and engineered T cells, engineered NK cells, or engineered macrophages.

[0131] [000179] A “toxin” can be any drug that is harmful to cells, may damage cells, or may kill them. Examples of toxins, without limitation, include taxol, deruxtecan, citcaresin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), meltansine, emtansine, DM1, mytansinoid DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and their analogues, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine), The formulation includes cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisplatin, cis-dichlorodiamine platinum(II) (DDP), anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin binders.

[0132] [000180] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharidooxidase, or β-D-galactosidase), and radioisotopes (e.g., 123 I, 124 I,125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P, other lanthanides), a luminescent label, a chromophore moiety, digoxigenin, biotin / avidin, a DNA molecule, or gold for detection.

[0133] [000181] In certain embodiments, the conjugate moiety can be a clearance modifier that helps to extend the half-life of the antibody. Exemplary examples include water-soluble polymers such as PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol / propylene glycol, and the like. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers conjugated to the antibody can vary and, when more than one polymer is conjugated, can be the same molecule or different molecules.

[0134] [000182] In certain embodiments, the conjugate moiety can be a purification moiety such as magnetic beads. [000183] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are used as a basis for conjugates.

[0135] [000184] Polynucleotides and Recombinant Methods [000185] This disclosure provides isolated polynucleotides encoding anti-LILRB2 antibodies and their antigen-binding fragments. In certain embodiments, the isolated polynucleotide comprises one or more nucleotide sequences encoding the variable region of the exemplary antibody provided herein. The DNA encoding the monoclonal antibody is readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[0136] [000186] Isolated polynucleotides encoding the anti-LILRB2 antibody and antigen-binding fragments can be inserted into a vector for further cloning (DNA amplification) or expression using recombination methods known in the art. Many vectors are available. The components of a vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0137] [000187] The Disclosure provides vectors (e.g., expression vectors) comprising a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages, and M13 phages, plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, This includes, but is not limited to, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0138] [000188] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment can be introduced into a host cell for cloning or gene expression. Host cells suitable for cloning or expression of DNA in the vectors herein are prokaryotic cells, yeast cells, or higher eukaryotic cells as described above. Prokaryotes suitable for this purpose include Gram-negative or Gram-positive bacteria, such as the Enterobacteriaceae family, including genera Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and Shigella, as well as eubacteria such as Bacillus, B. subtilis and B. licheniformis, Pseudomonas, P. aeruginosa, and Streptomyces.

[0139] [000189] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding anti-LILRB2 antibodies. Saccharomyces cerevisiae or common baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, Schizosaccharomyces pombe; for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotlerans, and K. marxianus are Kluyveromyces host species; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma freesia (EP 244,234); Neurospora crassa; Schwanniomyces Numerous other genera, species, and strains of filamentous fungi, such as Schwanniomyces occidentalis, as well as filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus host species like A. nidulans and A. niger, are also commercially available and useful herein.

[0140] [000190] The host cells suitable for the expression of glycosylated antibodies or antigen fragments provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and mutants, as well as corresponding, permissible insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori, have been identified. A variety of virus strains for transfection, such as the L-1 mutant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are commercially available and such viruses can be used as the viruses herein, in particular for transfection of Spodoptera frugiperda cells, according to the present invention. Plant cell cultures of cotton, maize, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

[0141] [000191]However, the primary objective is vertebrate cells, and the propagation of vertebrate cells in cultures (tissue cultures) has become a standard procedure. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7; ATCC:CRL1651); and the human fetal kidney cell line (293 cells or 293 cells subcloned for propagation in suspension cultures; Graham et al., J. Gen). Virol. (1977), 36:59); Baby hamster kidney cells (BHK; ATCC:CCL10); Chinese hamster ovary cells (CHO), CHO cells lacking non-hydrofolate reductase (DHFR) activity, CHO-DHFR (Urlaub et al., Proc. Natl. Acad. Sci. USA (1980), 77:4216); Mouse Sertoli cells (TM4; Mather, Biol. Reprod. (1980), 23:243~251); Monkey kidney cells (CV1; ATCC:CCL70); African green monkey kidney cells (VERO-76; ATCC:CRL-1587); Human cervical cancer cells (HELA; ATCC:CCL2); Canine kidney cells (MDCK; ATCC:CCL34); Buffalo rat hepatocytes (BRL These include 3A;ATCC:CRL1442); human lung cells (W138;ATCC:CCL75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562;ATCC:CCL51); TRI cells (Mather et al., Annals NYAcad.Sci. (1982), 383:44~68); MRC 5 cells; FS4 cells and human hepatoma cell lineage (Hep G2). In some preferred embodiments, the host cell is 293F cells.

[0142] [000192] Host cells are transfected with the expression vector or cloning vector described above for the production of anti-LILRB2 antibodies and cultured in a standard nutrient medium modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. In another embodiment, the antibody may be produced by homologous recombination known in the art.

[0143] [000193] The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercial media such as Ham F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the culture media described in Ham et al., Meth. Enz., 58:44 (1979), Barnes et al., Anal. Biochem., (1980) 102:255, U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655 or 5,122,469; WO90 / 03430; WO87 / 00195; or U.S. Patent Reissue No. 30,985 may also be used as culture media for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN® drugs), trace elements (typically defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be incorporated at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH, along with the host cells selected for expression, are already established culture conditions and will be apparent to those skilled in the art.

[0144] [000194]When using recombinant DNA methods, antibodies may be produced intracellularly, in the pericellular lumen, or secreted directly into the culture medium. If antibodies are produced intracellularly, the first step is to remove host cells or lysed fragments, such as particulate lysates, by centrifugation or ultrafiltration. Carter et al., Bio / Technology (1992), 10:163-167, describe a procedure for isolating antibodies secreted into the pericellular lumen of E. coli cells. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell lysates can be removed by centrifugation. If antibodies are secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon ultrafiltration unit or a Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be incorporated into one of the aforementioned steps to inhibit protein degradation, and antibiotics may be incorporated to prevent the growth of accidental contaminants.

[0145] [000195] Anti-LILRB2 antibodies and their antigen-binding fragments prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, but affinity chromatography is the preferred purification method.

[0146] [000196] In certain embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma single-chain, human gamma double-chain, or human gamma quadruple-chain (Lindmark et al., J.Immunol.Meth. (1983), 62:1~13). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. (1986), 5:1567~75). The matrix to which the affinity ligand is conjugated is very often agarose, but other matrices are also available. Mechanically stable matrices, such as controlled-pore glass or poly(styrenedivinyl)benzene, allow for higher flow rates and shorter processing times than those achievable with agarose. If the antibody contains a CH3 domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification are also available, depending on the antibody being recovered, including fractionation on ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE® chromatography on anion-exchange or cation-exchange resins (such as polyaspartate columns), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0147] [000197] Following any preliminary purification step(s), the mixture containing the antibody of interest and the impurities may be subjected to low-pH hydrophobic interaction chromatography, preferably carried out using an elution buffer with a pH between approximately 2.5 and 4.5, and preferably at a low salt concentration (e.g., approximately 0 to 0.25 M salt).

[0148] [000198] Purification [000199] In certain embodiments, the antibodies for this disclosure may be purified. As used herein, the term “purified” is intended to mean a composition that can be isolated from other components, in which case the protein is purified to any degree compared to its naturally occurring state. Thus, purified protein also means a protein that has been released from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation would mean a composition that forms the majority component of the composition, such as the protein or peptide constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein in the composition.

[0149] [000200] Protein purification methods are well known to those skilled in the art. These techniques involve, at one level, crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. Once the polypeptide is separated from other proteins, the polypeptide of interest may be further purified using chromatography and electrophoresis to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Other methods for protein purification include centrifugation followed by precipitation with ammonium sulfate, PEG, antibodies, etc., or thermal denaturation; gel filtration chromatography, reverse-phase chromatography, hydroxyl apatite chromatography, and affinity chromatography; and combinations of such techniques and other techniques.

[0150] [000201] In the purification of antibodies according to this disclosure, it may be desirable to express polypeptides in a prokaryotic or eukaryotic expression system and extract the proteins using denaturing conditions. Polypeptides may be purified from other cellular components using affinity columns that bind to the tagging portion of the polypeptide. As is generally known in the art, the order in which various purification steps are performed may vary, and certain steps may be omitted, but a method suitable for preparing substantially purified proteins or peptides may still result.

[0151] [000202] Generally, complete antibodies are fractionated by utilizing a drug (i.e., protein A) that binds to the Fc portion of the antibody. Alternatively, antigens can be used to purify and simultaneously select the appropriate antibody. Such methods often utilize a selection agent bound to a support such as a column, filter, or beads. The antibody is bound to the support, impurities are removed (e.g., by washing), and the antibody is released by applying conditions (e.g., salt, heat).

[0152] [000203] In light of this disclosure, a variety of methods are known to those skilled in the art for quantifying the purity of a protein or peptide. These methods include, for example, a step of determining the specific activity of an active fraction, or a step of evaluating the number of polypeptides in the fraction by SDS / PAGE analysis. Another method for evaluating the purity of a fraction is to calculate the specific activity of the fraction and compare this to the specific activity of an initial extract, thereby calculating the purity. The actual units used to express the amount of activity will, of course, depend on the specific assay method chosen to track the purification and track whether the expressed protein or peptide exhibits detectable activity.

[0153] [000204] It is known that polypeptide electrophoresis can vary significantly depending on the SDS-PAGE conditions (Capaldi et al., 1977). Therefore, it can be observed that the apparent molecular weight of purified or partially purified expression products may vary under different electrophoretic conditions.

[0154] [000205] III. Pharmaceutical Compositions [000206] The present disclosure further provides a pharmaceutical composition comprising an anti-LILRB2 antibody or an antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.

[0155] [000207] Acceptable pharmaceutical carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid carriers, gel carriers, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents or sequestering agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or a variety of combinations thereof.

[0156] [000208] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, fragrances, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. Incorporation of one or more antioxidants, such as methionine, into a composition comprising the antibody or antigen-binding fragment and conjugate provided herein reduces the oxidation of the antibody or antigen-binding fragment. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving the stability of the antibody and maximizing its shelf life. Accordingly, in certain embodiments, compositions are provided comprising one or more antibodies or antigen-binding fragments disclosed herein and one or more antioxidants, such as methionine. Furthermore, methods are provided for preventing oxidation of the antibodies or antigen-binding fragments provided herein, extending their shelf life, and / or improving their efficacy, by mixing the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0157] [000209] For further example, acceptable pharmaceutical carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose, and lactated Ringer's injection; non-aqueous media such as plant-derived fixative oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungiostatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate buffer or citrate buffer; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone; emulsifiers such as Polysorbate 80 (TWEEN-80); chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers, including phenol or cresol, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl esters of p-hydroxybenzoic acid, thimerosal, benzalkonium chloride, and benzethonium chloride, may be added to the pharmaceutical composition in a multi-dose container. Suitable excipients may include, for example, water, physiological saline, dextrose, glycerol, or ethanol. Suitable non-toxic adjuncts may include, for example, humectants or emulsifiers, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0158] [000210] The pharmaceutical composition may be a solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation, or powder. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, and magnesium carbonate.

[0159] [000211] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as a solution, suspension, emulsion, or a solid form suitable for yielding a solution, suspension, or emulsion. Preparations for injection may include a sterile solution and / or pyrogen-free solution ready for injection, a sterile-dried soluble product such as a lyophilized powder, including a subcutaneous tablet ready for combination with a solvent immediately before use, a sterile suspension ready for injection, a sterile-dried insoluble product ready for combination with a medium immediately before use, and a sterile emulsion and / or pyrogen-free emulsion. The solution may be an aqueous solution or a non-aqueous solution.

[0160] [000212] In certain embodiments, a unit dose of parenteral preparation is packaged in an ampoule, a vial, or a syringe with a needle. All preparations for parenteral administration are sterile and pyrogen-free preparations, as is known and practiced in the Art.

[0161] [000213] In certain embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients or other pharmacological components that improve the stability of the powder or a reconstituted solution prepared from the powder. Possible excipients include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain buffers such as citrate buffer, sodium phosphate buffer, or potassium phosphate buffer, or, in one embodiment, other such buffers known to those skilled in the art, with a pH of approximately neutral. A subsequent lyophilization process under standard conditions known to those skilled in the art, following sterile filtration of the solution, yields the desired formulation. In one embodiment, the resulting solution will be dispensed into vials for lyophilization. Each vial may contain a single dose or a multi-dose dose of the anti-LILRB2 antibody or its antigen-binding fragment or a composition thereof. Overfilling the vial with a small amount exceeding the amount required for the dose or set of doses (e.g., about 10%) is acceptable to facilitate accurate sampling and accurate administration. The lyophilized powder can be stored under appropriate conditions, such as about 4°C to room temperature.

[0162] [000214] Reconstitution of lyophilized powder for injection with water results in a formulation for use in parenteral administration. In one embodiment, sterile water and / or pyrogen-free water, or other suitable liquid carrier, is added to the lyophilized powder for reconstitution. The exact amount may be determined empirically, depending on the selected treatment being administered.

[0163] [000215] In certain embodiments, a pharmaceutical composition comprising an anti-LILRB2 antibody or its antigen-binding fragment as described herein further comprises one or more additional therapeutic agents co-administered with the anti-LILRB2 antibody or its antigen-binding fragment. Candidate substances for the additional therapeutic agents are disclosed in Section IV below. It can be understood that the additional therapeutic agents may be co-formulated with the anti-LILRB2 antibody or its antigen-binding fragment, or may be mixed with the anti-LILRB2 antibody or its antigen-binding fragment immediately before administration, such as in an intravenous bag as described in Section IV.

[0164] [000216] IV. Method of use of anti-LILRB2 antibody [000217] LILRB2 has been identified as a key regulator of the myeloid phenotype. Activation of LILRB2 suppresses the pro-inflammatory activity of myeloid cells. While myeloid cells with an inhibitory / anti-inflammatory phenotype can downmodulate T cell activation, proliferation, and cytotoxic activity, modulation of LILRB2 has potential therapeutic use in conditions and disorders including cancer, infectious diseases (e.g., chronic viral infections), autoimmune diseases, and inflammatory diseases.

[0165] [000218] Accordingly, the Disclosure also provides therapeutic methods using anti-LILRB2 antibodies or antigen-binding fragments provided herein. In some embodiments, the method includes the step of administering a therapeutically effective amount of the antibody or antigen-binding fragment provided herein to a subject in need thereof, thereby treating or preventing a LILRB2-related condition or LILRB2-related disorder. In some embodiments, the LILRB2-related condition or LILRB2-related disorder is cancer, infectious disease, autoimmune disease, and inflammatory disease.

[0166] [000219] Examples of cancer are generally classified into solid tumors and hematological malignancies. Solid tumors include non-small cell lung cancer (squamous cell lung cancer / non-squamous cell lung cancer), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal cell-like breast cancer, ductal carcinoma, and lobular breast cancer), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, multiple myeloma, mycosis fungoides, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synoviomas / synovial sarcomas, mesothelioma, and eucytoma. This includes, but is not limited to, ng's sarcoma, leiomyosarcoma, rhabdomyosarcoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, seminomas, mast cell-derived tumors, EBV-positive and EBV-negative PTLD, nasopharyngeal carcinoma, spinal cord tumors, brainstem glioma, astrocytoma, medulloblastoma, craniopharyngioma, epithelioma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendrone glioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0167] [000220]Solid tumors are characterized by multiple biological features, including persistent proliferative signaling, evasion of growth suppression, resistance to cell death, conferral of immortality through replication, induction of angiogenesis, activation of invasion and metastasis, protumor-promoting inflammation, evasion of immune destruction, genomic instability and mutation, and disregulated cellular energetic states. Treatment approaches have evolved from cytotoxic chemotherapy targeting rapidly dividing cells to small molecules inhibiting selective signaling pathways, and to monoclonal antibodies targeting cell surface proteins. More recently, the concepts of cancer immunotherapy mediated by the reactivation of endogenous antitumor immunity, or cancer immunotherapy mediated by cell therapies utilizing synthetic immunity, have shown promise. Despite these advances, the majority of patients with advanced solid tumors still do not survive long-term. The use of immune checkpoint inhibitors, such as anti-CTLA-4 or anti-PD-1 / PD-L1, has resulted in long-term progression-free survival and overall survival, but in a small number of patients.

[0168] [000221] Novel immunotherapies targeting different aspects of immunobiology, and different tumor-infiltrating cells, such as tumor-infiltrating cells that target LILRB2 as an inhibitory receptor expressed on myeloid cells, including myeloid suppressor cells (MDSCs), tolerogenic DCs, and tumor-associated macrophages (TAMs), are needed to improve outcomes. These myeloid cells are functionally described as immunosuppressive cells because their immunosuppressive / anti-inflammatory phenotype inhibits the activation, proliferation, and cytotoxic activity of tumor antigen-specific T cells.

[0169] [000222] In some embodiments, depletion of immunosuppressive cells can restore the suppressive effect on tumor antigen-specific T cells in order to treat solid tumors. [000223] In some embodiments, blocking LILRB2 on bone marrow cells may also relieve its inhibitory effect on antigen-presenting cells (APCs), including DCs, monocytes, macrophages, neutrophils, or myeloid leukemia cells, which express LILRB2. Increased antigen-presenting activity may result in T cell activation, cytotoxicity, and cytokine production by T cells.

[0170] [000224] In some embodiments, antibodies targeting LILRB2 reprogram immunosuppressive myeloid cells in the tumor microenvironment and / or tumor margins into pro-inflammatory myeloid cells, resulting in T cell recruitment and activation.

[0171] [000225] In some embodiments, the antibody targeting LILRB2 blocks the binding of one or more ligands involved in the immunosuppressive tumor microenvironment (TME), such as HLA-G, ANGPTL2, CD1c / d, CSP, and SEMA4A.

[0172] [000226] In some embodiments, antibodies that target LILRB2 promote the activation of primary myeloid cells and primary lymphocytes. [000227] In some embodiments, antibodies targeting LILRB2 enhance the differentiation, maturation, and activation of dendritic cells (DCs).

[0173] [000228] In some embodiments, antibodies targeting LILRB2 polarize bone marrow cells derived from solid tumor cancer patients into a pro-inflammatory phenotype. [000229] In some embodiments, antibodies targeting LILRB2 mitigate the inhibitory effect of patient-derived monocytic MDSCs (M-MDSCs) on autologous T cell proliferation and cytokine release.

[0174] [000230] In some embodiments, antibodies targeting LILRB2 restore and / or circumvent the “tumor conditioning” effect of cancer cells on bone marrow cells. [000231] In some embodiments, antibodies that target LILRB2 enhance the effects of pro-inflammatory stimuli, such as anti-CD3 agonist antibodies, STING agonists, TLR agonists, and anti-PD-1 blocking antibodies.

[0175] [000232] In some embodiments, antibodies targeting LILRB2 inhibit tumor growth in animal models, either as monotherapy or in combination with anti-PD-1, anti-PD-L1, anti-CTLA-4, or other T cell or myeloid checkpoint inhibitors.

[0176] [000233] Hematological malignancies include acute lymphoblastic / lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, blast plasmacytoid dendritic cell neoplasm (BPDCN), chronic lymphoblastic leukemia (CLL), chronic lymphocytic leukemia (CML), chronic myelomonocytic leukemia (CMML), classical Hodgkin lymphoma (CHL), diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, heavy chain disease, HHV8-associated primary exudative lymphoma, lymphoid malignancies, multiple myeloma (MM), myelodysplasia, myelodysplastic syndrome (MDS), non-Hodgkin lymphoma, plasmablastic lymphoma, and precursor B-cell acute lymphoblastic leukemia (Pre-B). This includes, but is not limited to, ALL, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, myeloproliferative neoplasms, and Waldenström macroglobulinemia.

[0177] [000234] A chronic viral infection is a disease in which a virus is not eliminated from the specific cells of an infected individual and remains there. Chronic viral infections can be caused by a variety of viruses, including, but are not limited to, herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), herpes simplex virus type 3, herpes simplex virus type 4, herpes simplex virus type 5, herpes simplex virus type 6, parvovirus B19, coxsackievirus type A and coxsackievirus type B, hepatitis A virus, hepatitis B virus, hepatitis C virus, cytomegalovirus (CMV), and human immunodeficiency virus (HIV).

[0178] [000235] Autoimmune diseases and inflammatory diseases include acquired immunodeficiency syndrome (AIDS, a viral disease with autoimmune components), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac disease - herpetiform dermatitis, chronic fatigue syndrome, immunodeficiency syndrome (CFIDS), chronic Inflammatory demyelinating polyneuropathy (CIPD), pemphigoid scarring, cold agglutinin disease, Crest syndrome, Crohn's disease, Degos disease, dermatomyositis (juvenile), discoid lupus erythematosus, essential mixed cold globulinemia, fibromyalgia (fibromyositis), Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis Myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, autoimmune syndrome of polyglandular disease, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, systemic scleroderma, progressive systemic sclerosis (PSS), systemic sclerosis (SS), Sjögren's syndrome, Stiffman syndrome, systemic lupus erythematosus (SLE), Takayasu's arteritis, temporal arteritis / gigantitis This includes, but is not limited to, cellular arteritis, inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, inflammation of the intestinal mucosa, colitis and associated wasting diseases, uveitis, vitiligo, and Wegener's granulomatosis, Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, ventilator-induced lung injury, viral infections, and autoimmune diabetes. Inflammatory disorders include, for example, chronic inflammatory disorders and acute inflammatory disorders.

[0179] [000236] The therapeutically effective dose of the antibody or antigen-binding fragment provided herein will depend on a variety of factors known in the art, such as body weight, age, medical history, current medication status, the health status of the subject, and the potential for cross-reactivity, allergies, hypersensitivity, and adverse side effects, as well as the route of administration and the severity of disease occurrence. The dose may be reduced or increased by a person skilled in the art (e.g., a physician or veterinarian) depending on these circumstances or requirements, and as indicated by other circumstances or requirements.

[0180] [000237] In certain embodiments, the antibodies or antigen-binding fragments provided herein may be administered in therapeutically effective doses ranging from about 0.0001 mg / kg to about 100 mg / kg. In certain embodiments of these embodiments, the antibodies or antigen-binding fragments are administered in doses of about 50 mg / kg or less, and in certain embodiments of these embodiments, the doses are 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In some embodiments, the antibodies or antigen-binding fragments are administered in doses of about 4000 mg or less, and in certain embodiments of these embodiments, the doses are 800 mg or less, 400 mg or less, 240 mg or less, 80 mg or less, 40 mg or less, or 0.8 mg or less. In certain embodiments, the dosage may vary throughout the course of treatment. For example, in certain embodiments, the initial dosage may be higher than subsequent dosages. In certain embodiments, the dosage may fluctuate throughout the course of treatment depending on the patient's response.

[0181] [000238] The dosage regimen may be adjusted to produce the desired optimal response (e.g., therapeutic response). For example, a single dose may be administered, or multiple divided doses may be administered over time.

[0182] [000239] The antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as parenteral routes (e.g., intravenous routes including subcutaneous, intraperitoneal, intravenous infusion, intramuscular, or intradermal injection) or routes other than parenteral routes (e.g., oral, intranasal, intraocular, sublingual, intrarectal, or local).

[0183] [000240] In some embodiments, the antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more further therapeutic means or therapeutic agents. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with radiotherapy and / or in combination with another therapeutic agent, such as another immunoactivator, anti-angiogenic agent, chemotherapeutic agent, or anticancer drug.

[0184] [000241] In certain embodiments of these embodiments, the antibodies or antigen-binding fragments disclosed herein, which are administered in combination with one or more further therapeutic agents, may be administered simultaneously with one or more further therapeutic agents, but in certain embodiments of these embodiments, the antibodies or antigen-binding fragments and the further therapeutic agents may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered “in combination” with another therapeutic agent does not have to be administered simultaneously with this agent, nor does it have to be administered as a drug in the same composition. In this specification, the term “in combination” is used even when the antibody or antigen-binding fragment and the second agent are administered via different routes, so an antibody or antigen-binding fragment administered before or after another agent is considered to be administered “in combination” with this agent. Where possible, further therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein shall be administered according to the schedule listed in the drug information sheet for the further therapeutic agent, or according to the Prescriber's Digital Reference (available online only at pdr.net), or according to protocols well known in the art.

[0185] [000242] In certain embodiments, the agent for combination therapy is an antineoplastic composition. As used herein, “antineoplastic composition” means a composition useful in the treatment of cancer and comprising at least one active therapeutic agent. Examples of therapeutic agents include, for example, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, cancer immunotherapy agents, apoptotic agents, antitubulin agents, as well as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva®)), platelet-derived growth factor inhibitors (e.g., Gleevec® (imatinib mesylate)). ), COX-2 inhibitors (e.g., celecoxib), interferon, CTLA4 inhibitors (e.g., the anti-CTLA antibody ipilimumab (YERVOY®) or tremelimumab), PD-1 or PD-L1 inhibitors (e.g., OPDIVO® or nivolumab, KEYTRUDA® or pembrolizumab, TECENTRIQ® or atezolizumab, BAVENCIO® or avelumab, IMFINZI® or duopropyl alcohol), Ruvalumab, LIBTAYO® or semiprimab rwlc, TYVYT® or cintilimab, tislerizumab (BGB-A317), penprimab (AK105), camrelizumab, tripalimab, zimbererimab (GLS-010), retifanlimab, sugemalimab, or CS1003), bitargeted antibodies against CTLA-4 and PD-1 or PD-L1 (e.g., anti-PD-1 / CTLA-4 bispecific antibody or AK104), TIM3 inhibitors (e.g., (Anti-TIM3 antibodies), LAG-3 inhibitors (e.g., anti-LAG3 antibodies), cytokines, TLR agonists, STING agonists, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, FGFR2b, PDGFR-beta, BlyS, APRIL, BCMA, or VEGF receptors (multiple targets), TRAIL / Apo2, IDH1 inhibitors, ivosidenib, Tibsovo®, IDH2 inhibitors, enasidenib,Idhifa®, smoothed (SMO) inhibitors, Glasdegib, arginase inhibitors, IDO inhibitors, epacadostat, BCL-2 inhibitors, venetoclax, Venclexta®, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, ibrutinib, IMBRUVICA®, acalabrutinib, CALQUENCE®, zanubrutinib, ICOS antibody, TIGIT antibody, CD40 antibody, 4-1BB antibody, Siglec antibody, OX40 antibody, TNFR2 antibody, other LILR family members Other agents for treating cancer, including but not limited to antibodies against -, CD47 antibodies, SIRP1a antibodies or SIRP1a fusion proteins, E-selectin antagonists, antibodies that bind to tumor antigens, antibodies that bind to markers on the surface of T cells, antibodies that bind to markers on the surface of bone marrow cells or NK cells, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, plant-derived alkaloids, hormonal therapeutics, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, and other bioactive and organic chemical agents, engineered T cells, engineered NK cells, or engineered macrophages, and bispecific antibodies.

[0186] [000243] In certain embodiments, the agent for combination therapy is a chemotherapeutic agent. As used herein, “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents that can be administered by the methods described herein include alkylating agents such as CYTOXAN®, which is thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocon, metsuredopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethiolmamine; acetogenins (particularly bractacin and bractacinone); camptothecin (including its synthetic analog, topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (particularly cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictiin; spongstatin; chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembiquin, phenesterine, prednimustine, trophosphamide, uracil mustard, and other nitrogenous mustards; carmustine Nitrosoureas such as chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; engine antibiotics (e.g., calichemycin, in particular calichemycin gamma II and calichemycin omega II (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33:183-186 (1994)); ginemicins, including ginemicin A; bisphosphonates such as clodronate; esperamicin and others;Adriamycin® (registered trademark) (morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolidodoxorubicin, and deoxyd Antibiotics such as sorbicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and solubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); and leaf ointments such as denopterin, methotrexate, pteropterin, and trimethrexate. Acid analogs; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal cortical antibodies such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; acegraton; aldofsphamide glycoside Side; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatrexate; defofamin; demecolsin; diazion; eflorumitin; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; meitansinoids such as meitansin and anthamitosin; mitogluzone; mitoxantrone; mopidamol; intracrin; pentostatin; fenmetrazine; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; lyzoxin; schizofuran; spirogermanium; tenuazonic acid; triadiquan; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, vercalin A, loridine A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel, Taxol® (Bristol-Myers Squibb Oncology, Princeton, NJ), and the Cremophor-free albumin-modified nanoparticle formulation of paclitaxel, Abraxane® (American Pharmaceuticals) Partners, Schaumberg, Illinois), and docetaxel Taxotere® (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine Gemzar®; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine Navelbine®; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens with irinotecan accompanied by 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; Difluoromethylomitin (DMFO); Retinoids such as retinoic acid; Capecitabine; Combretastatin; Leucovorin (LV); Oxaliplatin, including oxaliplatin treatment regimens (FOLFOX);This includes, but is not limited to, PKC-alpha, Raf, H-Ras, EGFR inhibitors (e.g., erlotinib (Tarceva®)), and VEGF-A, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0187] [000244] Further non-limiting exemplary chemotherapeutic agents that may be administered in the manner described herein include, for example, anti-estrogen agents and selective estrogen receptor modulators (SERMs), such as tamoxifen (including Nolvadex®, which is tamoxifen), raloxifen, droloxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and Fareston®, which is toremifene. Antihormone agents that act to modulate or inhibit the effects on cancer; for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate (Megase®), exemestane (Aromasin®), formestan, fadrozol, borozole (Rivisor®), letrozole (Femara®), and anastrozole (Arimidex®), which inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland. Aromatase inhibitors; as well as antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; troxacitabine (a cytosine analog, 1,3-dioxolane nucleoside); antisense oligonucleotides, in particular antisense oligonucleotides that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Ralf, and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., the ribozyme Angiozyme®) and HER2 expression inhibitors; gene therapy vaccines, such as the vaccines Allovectin®, Leuvectin®, and Vaxid®; rIL-2, which is Proleukin®; topoisomerase 1 inhibitor, which is Lurtotecan®; rmRH, which is Abarelix®; and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0188] [000245] In certain embodiments, the agent for combination therapy is an anti-angiogenic agent. As used herein, “anti-angiogenic agent” means a low molecular weight substance, polynucleotide (e.g., including inhibitory RNA (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or a conjugate or fusion protein thereof that directly or indirectly inhibits angiogenesis, angiogenesis, or unwanted vascular permeability. It should be understood that anti-angiogenic agents include agents that bind to angiogenic factors or their receptors and block their angiogenesis. For example, anti-angiogenic agents that can be administered by the methods described herein may include antibodies against angiogenic agents, or other antagonists, such as antibodies against VEGF-A (e.g., bevacizumab (Avastin®)), or antibodies against VEGF-A receptors (e.g., KDR receptor or Flt-l receptor), anti-PDGFR inhibitors such as Gleevec® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, Sutent® / SUl1248 (sunitinib malate), AMG706, or small molecules described in international patent application WO2004 / 113304). Anti-angiogenic agents also include natural angiogenic inhibitors, such as angiostatins and endostatins. For example, see Klagsbrun and D'Amore (1991), Annu. Rev. Physiol., 53:217~39; Streit and Detmar (2003), Oncogene, 22:3172~3179; Ferrara and Alitalo (1999), Nature Medicine, 5(12):1359~1364; Tonini et al. (2003), Oncogene, 22:6549~6556; and Sato (2003), Int. J. Clin. Oncol., 8:200~206.

[0189] [000246] In certain embodiments, the agent for combination therapy is a growth inhibitor. As used herein, “growth inhibitor” means a compound or composition that inhibits the growth of cells (such as cells expressing VEGF) in vitro or in vivo. Thus, growth inhibitors that can be administered in the methods herein may be growth inhibitors that significantly reduce the percentage of cells in the S phase (such as cells expressing VEGF). Examples of growth inhibitors include, but are not limited to, agents that block the progression of the cell cycle (in phases other than the S phase), such as agents that induce G1 phase arrest and M phase arrest. Classical M phase blockers include vinca alkaloids (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Drugs that halt the G1 phase, such as DNA alkylating agents like tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also have an effect on halting the S phase. Further information can be found, for example, on page 13 of Chapter 1, "Cell cycle regulation, oncogenes, and antineoplastic drugs," by Murakami et al., in "The Molecular Basis of Cancer," edited by Mendelsohn and Israel (WBSaunders, Philadelphia, 1995). Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semi-synthetic analog of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel inhibit intracellular mitosis by stabilizing microtubules through promoting microtubule assembly from tubulin dimers and preventing depolymerization.

[0190] [000247] The dosage of drugs for combination therapy may be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of specific drugs. Typically, the attending physician will determine the dosage of drugs for combination therapy to treat each individual patient by taking into account various factors such as age, weight, overall health, diet, sex, drugs being administered, route of administration, and severity of the condition being treated. For illustrative purposes only and not intended to limit the disclosure, the dosage for combination therapy may be approximately 0.0001 to 1 g per kg of body weight per day, approximately 0.0001 to 0.001 g per kg of body weight per day, or approximately 0.01 mg to 1 g per kg of body weight per day. The dosage unit also refers to the quantity in milligrams per square meter of body surface area, mg / m 2 It can also be expressed in units.

[0191] [000248] In the combination therapies described herein, each therapeutic agent may be administered simultaneously (e.g., in the same drug or at the same time), or synchronously (i.e., by separate drugs, in any order, successively, or sequentially). Sequential administration may be useful when the therapeutic agents in a combination therapy, for example, a chemotherapeutic agent administered at least daily and a biotherapeutic agent administered less frequently, such as once a week, once every two weeks, or once every three weeks, are in different dosage forms (one drug is a tablet or capsule and the other is a sterile liquid) and / or are administered on different dosing schedules.

[0192] [000249]In certain embodiments, the LILRB2 antibody of the present disclosure and the second drug are combined or co-formulated in a single dosage form. In certain embodiments, the LILRB2 antibody of the present disclosure and the second drug are administered separately. Concurrent administration of the LILRB2 antibody of the present disclosure and the second drug may be maintained throughout the treatment period, but anti-cancer activity can also be achieved by subsequent administration of a single separate compound (e.g., the LILRB2 antibody following an initial combination treatment, or alternatively, the second drug following an initial combination treatment). In some embodiments, the LILRB2 antibody is administered prior to administration of the second drug, while in other embodiments, the LILRB2 antibody is administered after administration of the second drug. In some embodiments, at least one of the therapeutic agents in the combination therapy is administered using the same dosing regimen (dose, frequency, and duration of treatment) typically employed when the agent is used as a monotherapy for treating the same cancer. In other embodiments, the patient is administered a lower total amount, e.g., a lower dose, lower dosing frequency, and / or shorter treatment duration total amount, for at least one of the therapeutic agents in the combination therapy than when the agent is used as a monotherapy.

[0193] [000250]The combination therapy of the present invention may be used before surgery to remove a tumor, after surgery, before radiation therapy, during radiation therapy, or after radiation therapy. The combination therapy of the present invention can be used to treat tumors large enough to be found by palpation or by imaging methods well known in the art such as MRI, ultrasound, or CAT scan. In some embodiments, the combination therapy of the present invention is used to treat advanced tumors having a diameter of at least about 200 mm 3 , 300 mm 3 , 400 mm 3 , 500 mm 3 , 750 mm 3 , or up to, 1000 mm 3 in diameter.

[0194] [000251] The present disclosure further provides a method for detecting the presence or amount of LILRB2 in a sample using an anti-LILRB2 antibody or an antigen-binding fragment thereof, comprising the steps of contacting the sample with the antibody or an antigen-binding fragment thereof and determining the presence or amount of LILRB2 in the sample. Methods for detecting LILRB2 using an anti-LILRB2 antibody include, but are not limited to, ELISA, RIA, Western blotting, flow cytometry, and immunohistochemistry.

[0195] [000252] In some embodiments, the present disclosure provides a method for diagnosing a LILRB2-related disease or condition in a subject, comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of LILRB2 in the sample; and c) correlating the presence of LILRB2 with a LILRB2-related disease or condition in the subject.

[0196] [000253] In some embodiments, the Disclosure provides a kit comprising an antibody or its antigen-binding fragment provided herein, conjugated with an optionally detectable portion. The kit may be useful in the detection of LILRB2 or in the diagnosis of LILRB2-related diseases.

[0197] [000254] In some embodiments, the disclosure also provides the use of antibodies or antigen-binding fragments provided herein in the manufacture of pharmaceuticals for treating LILRB2-related diseases or conditions in a subject, and in the manufacture of diagnostic reagents for diagnosing LILRB2-related diseases or conditions.

[0198] [000255] V. Chimeric antigen receptor [000256] In another embodiment, the present disclosure provides chimeric antigen receptor (CAR) proteins that bind to LILRB2 (anti-LILRB2 CAR proteins). In certain embodiments, the CAR protein comprises an antigen-recognition region, i.e., an antibody or antigen-binding fragment that recognizes LILRB2 as described herein, and other intramembrane and intracellular components. In some embodiments, the anti-LILRB2 CAR protein comprises a LILRB2 antigen-recognition region, a transmembrane domain, and an intracellular costimulatory signaling domain. In certain embodiments, the single-chain anti-LILRB2 CAR protein also comprises a leader peptide, a spacer region, and an intracellular T cell signaling domain.

[0199] [000257] In a particular embodiment, the antigen recognition region comprises a plurality of polypeptide chains. [000258] In some embodiments, the CAR protein comprises a first polypeptide containing the heavy chain variable domain of the antibody and a polypeptide containing the light chain variable domain of the antibody, wherein the first or second polypeptide further comprises a transmembrane domain, and the heavy chain variable domain of the antibody and the light chain variable domain of the antibody together form an antigen recognition region.

[0200] [000259] In some embodiments, the CAR protein comprises a first polypeptide comprising an antibody heavy chain variable domain and a second polypeptide comprising an antibody light chain variable domain and an antibody light chain constant domain, the first polypeptide further comprising a transmembrane domain, and the antibody heavy chain variable domain, the antibody light chain variable domain and the antibody light chain constant domain together form an antigen recognition region. In some embodiments, the first portion further comprises an intracellular costimulatory signaling domain and a CD3ζ intracellular T cell signaling domain.

[0201] [000260] In some embodiments, the CAR protein comprises a first polypeptide comprising an antibody heavy chain variable domain and an antibody heavy chain constant domain, and a second polypeptide comprising an antibody light chain variable domain, the first polypeptide further comprising a transmembrane domain, and the antibody heavy chain variable domain, the antibody heavy chain constant domain, and the antibody light chain variable domain together form an antigen recognition region. In some embodiments, the first portion further comprises an intracellular costimulatory signaling domain and a CD3ζ intracellular T cell signaling domain.

[0202] [000261] In some embodiments, the CAR protein comprises a first polypeptide containing an antibody heavy chain variable domain and a second polypeptide containing an antibody light chain variable domain, the second polypeptide further comprising a transmembrane domain, and the antibody heavy chain variable domain, the antibody light chain variable domain and the antibody light chain constant domain together form an antigen recognition region. In some embodiments, the second portion further comprises an intracellular costimulatory signaling domain and a CD3ζ intracellular T cell signaling domain.

[0203] [000262] In some embodiments, the CAR protein comprises a first polypeptide comprising an antibody heavy chain variable domain and an antibody heavy chain constant domain, and a second polypeptide comprising an antibody light chain variable domain, the second polypeptide further comprising a transmembrane domain, and the antibody heavy chain variable domain, the antibody heavy chain constant domain, and the antibody light chain variable domain together form an antigen recognition region. In some embodiments, the second portion further comprises an intracellular costimulatory signaling domain and a CD3ζ intracellular T cell signaling domain.

[0204] [000263] In certain embodiments, the CAR protein is a single-chain polypeptide comprising an anti-LILRB2 scFv, i.e., an anti-LILRB2 heavy chain variable domain and an anti-LILRB2 light chain variable domain linked by a linker domain, as described herein. In one embodiment, the CAR protein comprises, from the N-terminus to the C-terminus: a leader peptide, an anti-LILRB2 heavy chain variable domain, a linker domain, an anti-LILRB2 light chain variable domain, a hinge region, a transmembrane domain, and an intracellular costimulatory signaling domain. In some embodiments, the CAR protein further comprises a CD3ζ intracellular T cell signaling domain.

[0205] [000264] In certain embodiments, the linker domain generally consists of helix-promoting amino acid residues and turn-promoting amino acid residues, such as alanine, serine, and glycine. However, other residues may also function. In some embodiments, the linker domain is inserted between VH and VL of scFv. In some embodiments, the linker domain is located between the transmembrane domain and the intracellular costimulatory signaling domain. In some embodiments, the linker domain is located between the intracellular T cell signaling domain and the intracellular costimulatory signaling domain. In some embodiments, the linker domain contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 128).

[0206] [000265] In some embodiments, the transmembrane domain is a CD8α transmembrane domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity compared to a naturally occurring CD8α transmembrane domain polypeptide (SEQ ID NO: 129). In some embodiments, the CD8α transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 130.

[0207] [000266] In some embodiments, the transmembrane domain is a CD28 transmembrane domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity compared to a naturally occurring CD28 transmembrane domain polypeptide (SEQ ID NO: 131). In some embodiments, the CD28 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 132.

[0208] [000267] The intracellular co-stimulatory signaling domain comprises an amino acid sequence capable of producing a co-stimulatory signal in response to the binding of an antigen to a CAR. In some embodiments, the signaling of the co-stimulatory signaling domain results in the production of cytokines and the proliferation of T cells or NK cells expressing the cytokines. In some embodiments, the intracellular co-stimulatory signaling domain is the CD28 intracellular co-stimulatory signaling domain, the 4-1BB intracellular co-stimulatory signaling domain, the ICOS intracellular co-stimulatory signaling domain, the OX-40 intracellular co-stimulatory signaling domain, or any combination thereof. In some embodiments, the CD28 co-stimulatory domain has the polypeptide sequence of SEQ ID NO: 133. In some embodiments, the CD28 intracellular co-stimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 134. In some embodiments, the 4-1BB intracellular co-stimulatory signaling domain has the polypeptide sequence of SEQ ID NO: 135. In some embodiments, the 4-1BB intracellular co-stimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 136.

[0209] [000268] As provided herein, a “hinge region” is a polypeptide that connects an antigen-binding region to a transmembrane domain. In some embodiments, the hinge region connects a heavy chain variable region to the transmembrane domain. In some embodiments, the hinge region connects a heavy chain constant region to the transmembrane domain. In some embodiments, the hinge region connects a light chain variable region to the transmembrane domain. In some embodiments, the hinge region connects a light chain constant region to the transmembrane domain. In some embodiments, the binding affinity of the antigen-binding region to the antigen is increased compared to the absence of the hinge region. In some embodiments, steric hindrance between the antigen-binding region and the antigen is reduced in the presence of the hinge region. In some embodiments, the hinge region is a CD8α hinge region. In some embodiments, the hinge region is a CD28 hinge region.

[0210] [000269] In some embodiments, the intracellular T cell signaling domain includes the signaling domain of the zeta (ζ) chain of the human CD3 complex, i.e., the CD3ζ intracellular T cell signaling domain. In some embodiments, the intracellular T cell signaling domain is CD3zIso1, a protein with the amino acid sequence of SEQ ID NO: 137. In some embodiments, the intracellular T cell signaling domain is CD3zIso3, a protein with the amino acid sequence of SEQ ID NO: 138, encoded by the nucleic acid sequence of SEQ ID NO: 139.

[0211] [000270] For example, a CAR protein is a single-chain polypeptide comprising, from the N-terminus to the C-terminus: a CD8α leader peptide, an anti-LILRB2 scFv, a CD8α hinge region, a CD8α transmembrane domain (or CD28 transmembrane domain), a 4-1BB intracellular costimulatory signaling domain (or a CD28 intracellular costimulatory signaling domain, or a CD28 intracellular costimulatory signaling domain followed by a 4-1BB intracellular costimulatory signaling domain), and a CD3ζ intracellular T cell signaling domain located in one of two isoforms (CD3zIso1 or CD3zIso3).

[0212] [000271] In certain embodiments, the anti-LILRB2 CAR protein provided herein exhibits high binding affinity to LILRB2. In certain embodiments, the CAR protein provided herein exhibits binding affinity to LILRB2 (EC2 measured by ELISA) of 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, or less than 0.05 nM. 50 ) has. For the purposes of this application, EC by ELISA 50The values ​​may be determined as follows: Recombinant LILRB2 ECD-6×His-tagged protein was coated onto a high-binding 96-well clear plate (Corning-Costar, Fisher Scientific) at a concentration of 1 μg / ml (100 μl per well) at 4°C for 14-16 hours. The coated plate was briefly washed with PBS, pH 7.4, and blocked in PBS with 5% skim milk powder, 200 μl per well, at 37°C for 2 hours. For binding, the assay plate was covered and incubated at 37°C for 45 minutes. A series of dilutions were added to the 96-well plate, examining monoclonal antibodies (IgG or scFv fragments) starting at 10 μg / ml and decreasing the titer by one-third in 12 steps. Next, wash the plate three times with PBS containing Tween 20 (0.05% concentration), and then once with PBS. Add a secondary antibody with an anti-human antibody, anti-rabbit antibody, or other species of IgG-specific antibody accompanied by an HRP conjugate (Jackson ImmunoResearch) according to the dilution ratio suggested by the manufacturer, for incubation at room temperature for 1 hour. Detection is performed over 10 minutes by adding HRP substrate, tetramethylbenzidine (TMB, ThermoFisher), and stopped by adding 50 μl of 2N H2SO4 per well. Read the absorbance of the plate at 450 nm using a plate reader (SpectraMax M4, Molecular Device). Collect the data and perform EC 50 For the calculation, a 4-parameter fitted curve is used and graphed with GrapPad Prism 7 software.

[0213] [000272] In another embodiment, the disclosure provides polynucleotide molecules encoding the CAR proteins described herein. In some embodiments, the polynucleotide molecule further comprises a promoter active in eukaryotic cells. In some embodiments, the promoter is a JeT promoter. The JeT promoter is a recombinant promoter with transcriptional activity equivalent to that of many potent mammalian promoters. The JeT promoter consists of five key elements: (1) a TATA box; (2) a transcription start site (Inr); (3) a CAT consensus sequence; (4) a CArG element; and finally, (5) four Spl transcription-binding sites (GGGCGG) (US2002 / 0098547A1) located within two tandem sequences. In some embodiments, the polynucleotide molecule is an expression vector. In some embodiments, the vector is made based on Clontech's pLVX-EF1alpha-IRES-ZsGreen, or pSIN-EF1alpha-IRES-Puromycin or pSIN-EF1alpha. In one example, the polynucleotide molecule of this disclosure comprises, in order, the following elements: (1) the JeT promoter; (2) a sequence encoding the CD8-alpha reader; (3) a sequence encoding the heavy chain variable region; (4) a sequence encoding the linker; (5) a sequence encoding the light chain variable region; (6) a sequence encoding the CD8 hinge domain and TM domain; (7) a sequence encoding the 4-1BB costimulatory domain; and (8) a sequence encoding the CD3-zeta activation domain. In one example, the elements described above are flanked by a 5' homologous arm and a 3' homologous arm that facilitate the insertion of the polynucleotide molecule into a target locus, for example, the T cell receptor alpha constant (TRAC) locus.

[0214] [000273]VI. Manipulated cells expressing anti-LILRB2 CAR protein [000274] In another aspect, the present disclosure provides engineered immune cells that express the CAR proteins described herein. The immune cells are T cells (e.g., regulatory T cells, CD4 + T cells, CD8 +It can be a T cell, or a gamma-delta T cell), a natural killer (NK) cell, an invariant NK cell, an NKT cell, or a macrophage. In the present specification, in addition to methods for producing and manipulating immune cells, methods for using and administering cells for adoptive cell therapy are also provided, and in these cases, the cells may be autologous cells or allogeneic cells. Therefore, the engineered immune cells are LILRB2 + It can be used as an immunotherapy such as an immunotherapy targeting cancer cells.

[0215] [000275]The expression of the CAR protein enables the engineered immune cells to bind to target cells such as cancer cells by recognizing an antigen present on the target cells. When bound to the target cells, the engineered immune cells are activated, then proliferate, become cytotoxic, and ultimately proceed to destroy the target cells. CAR-T cell immunotherapy has been proven successful in clinical trials for treating refractory acute B-cell lymphoblastic leukemia and B-cell non-Hodgkin lymphoma and has been approved by the US FDA (Hartmann J et al., EMBO Mol Med (2017), 9:1183-97). In recent years, in addition to CAR-T cells, CAR NK cells and CAR macrophages have also been developed as immunotherapy options (Kloess S et al., Transfusion Medicine and Hemotherapy (2019), 46:4-13; Klichinsky M et al., AACR Annual Meeting, 2017, Abstract, 4575). Therefore, in certain embodiments of the present disclosure, the immune cells expressing the CAR protein described herein are T cells, NK cells, or macrophages.

[0216] [000276]Immune cells can be isolated from a subject, particularly a human subject. Immune cells can be obtained from a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, a subject undergoing treatment for a particular disease or condition, a healthy volunteer or a healthy donor, or from a blood bank. Immune cells can be recovered from any location where they are resident in the subject, including but not limited to blood, cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells may be used directly or may be stored for a period of time, such as by freezing.

[0217] [000277]Immune cells can be enriched / purified from any tissue in which they are resident, including but not limited to blood (including blood collected by a blood bank or cord blood bank), spleen, bone marrow, tissue excised and / or exposed during a surgical procedure, and tissue obtained via a biopsy procedure. The tissue / organs from which the immune cells are enriched, isolated, and / or purified can be isolated from either a living subject or a non-living subject, where in this case the non-living subject is an organ donor. In certain embodiments, the immune cells are isolated from blood such as peripheral blood or cord blood. In some aspects, immune cells isolated from cord blood have enhanced immunomodulatory ability as measured by suppression of CD4-positive T cells or CD8-positive T cells. In specific aspects, the immune cells are isolated from pooled blood, particularly pooled cord blood, for enhancement of immunomodulatory ability. The pooled blood can be derived from two or more sources, such as three, four, five, six, seven, eight, nine, ten or more donors (e.g., donor subjects).

[0218] [000278] A population of immune cells may be obtained from a subject in need of treatment, or from a subject suffering from a disease associated with reduced immune cell activity. Therefore, the cells may be autologous to the subject in need of treatment. Alternatively, a population of immune cells may be obtained from a donor, preferably a histocompatibility-matched donor. The immune cell population may be collected from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are normally present in the subject or donor. Immune cells may be isolated from a pool of subjects and / or donors, such as pooled umbilical cord blood.

[0219] [000279] When a population of immune cells is obtained from a donor that is significantly different from the target, the donor is preferably an allogeneic donor, provided that the cells obtained are suitable for introduction into the target. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible. To ensure suitability for the target, allogeneic cells may be treated or genetically engineered, for example, by deleting T cell receptors or inhibiting T cell signaling pathways to minimize graft-versus-host disease (see Kim and Cho, "Recent Advances in Allogeneic CAR-T Cells," Biomolecules (2020), 10:263).

[0220] [000280] Immune cells can be genetically engineered to express CARs using appropriate modification methods known in the art. See, for example, Sambrook and Ausubel, "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY," Greene Publishing Associates and John Wiley & Sons, NY, 1994. In some embodiments, the immune cells include one or more nucleic acids, which are introduced via genetic engineering, and which encode one or more CAR proteins. In certain embodiments, the nucleic acids encoding the CAR proteins are inserted into the genome of the immune cells using gene editing techniques, such as CRISPR / Cas technology. In one example, the nucleic acids encoding the CAR proteins are inserted into the T cell receptor alpha constant (TRAC) locus (see, for example, Eyquem J et al., Nature (2017), 543:113-117).

[0221] [000281] Immunotherapy is also provided which includes the step of administering an effective amount of the immune cells of this disclosure. In some embodiments, a medical disease or medical disorder is treated by the transfer of a population of immune cells described herein that induces an immune response. In certain embodiments, the medical disease or medical disorder is cancer. In certain embodiments, the medical disease or medical disorder is an autoimmune disease or an inflammatory disease.

[0222] [000282] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but only to illustrate specific embodiments that fall within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive ability and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein, while still remaining within the boundaries of the invention. It is the inventors' intention that such variations also fall within the scope of the invention.

[0223] Example 1 [000283]This example illustrates the design of an anti-LILRB2 antibody variant. [000284] The inventors previously identified an anti-LILRB2 antibody, named B2-19, which has high binding affinity to LILRB2 (see PCT Patent Application No. PCT / US2021 / 015362, the disclosure of which is incorporated herein in its entirety). Analysis of the variable domain sequence of the phage display-derived parental B2-19 antibody has been reported to be harmful in other clinical-stage antibodies (Lu et al., mAbs, 2019, 11:1, 45-57; and Yang et al., mAbs, 2017, 9:4, 646-653), leading to the identification of isomerization motifs (DG, DD, DD, DS), deamidation motifs (NN, NG), and oxidation motifs (W). Analysis of the variable domain of B2-19 using IgBLAST (Ye et al., Nucleic Acids Res, 2013, W34-W40) also identified mutations within the framework region that could pose an immunogenic risk. Thus, point mutations in B2-19 were designed to eliminate toxicity within the CDR: isomerization motifs (B2-19-1 to B2-19-6), deamidation motifs (B2-19-7 to B2-19-8), and oxidation sites (B2-19-9 and B2-19-10). To minimize the risk of unwanted immunogenicity, framework residues within the VH and VL variable domains were germline-treated (B2-19-11 or germline-treated / germ antibody). Antibody variants were cloned into pcDNA3.4 vectors, expressed in a mammalian Expi293F system, and purified using RoboColumn Eshmuno A 0.6 mL. Binding affinity was measured by BLI using a Gator system (R&D systems model number: 8429-T4) with an anti-human IgG Fc (HFC) probe, an antibody loaded at 5 μg / mL, and a recombinant LILRB2 ECD-6X His-tagged protein. Data were analyzed by global fit. B2-19-12 was designed by combining mutations that reduce the risk of immunogenicity and eliminate all isomerization motifs without significant loss of binding affinity.

[0224] [000285] Further mutants were designed to further optimize B2-19. First, mutants 14-18 were created by stacking additional mutations on mutant 12 to remove the deamidation motif. Mutants 20-32 and 41-63 were designed to reduce surface-exposed hydrophobic residues as well as Arg and Lys residues, which correlated with high polyspecificity and poor pharmacokinetics (Sharma et al., PNAS, 2014, 111, 52, 18601-18606; and Shehata et al., Cell Reports, 2019, 28, 3300-3308). To potentially improve thermal stability, mutants 34 and 35 were manipulated by grafting the B2-19 lambda light chain CDR onto the human kappa light chain 1-39 framework and the 3-20 framework using a previously described method (Lehmann et al., mAbs, 7:6, 1058-1071). Similarly, variants 38-40 were designed by grafting CDRs onto the VH3-23 and VL2-23 frameworks, which have been reported to possess favorable biophysical properties in clinical-stage antibodies (Jain et al., PNAS, 2017, 114, 5, 944-949). Variants 36 and 37 were designed to improve the Fv charge symmetry parameter (FvCSP) to potentially reduce viscosity in high-concentration formulations (Sharma et al., PNAS, 2014, 111, 52, 18601-18606). All of these antibody variants were cloned into custom antibody vectors based on vectors described in the literature (Tiller et al., J Immunol Methods, 2008, 329, 112-124) and expressed using the Expi293 expression system (approximately 6 mL of culture, ThermoFisher model number: A14635). As shown in Table 1, the off-rate was measured from the supernatant using biolayer interferometry (BLI) with an HFC probe, loaded with an antibody variant, and measuring binding to the recombinant LILRB2 ECD-6X His-tagged protein using a Gator system (R&D systems model number: 8429-T4).

[0225] [000286]

[0226] [Table 1]

[0227] Example 2 [000287]This example illustrates the binding affinity, specificity, and biological properties of the B2-19 antibody variant.

[0228] [000288] The binding affinity of B2-19 and selected mutants to recombinant LILRB2 ECD-6X His-tagged protein was measured by BLI. As shown in Figure 2, the B2-19 antibody mutant has the same binding affinity to LILRB2 as the B2-19 parent antibody. All measured binding affinity values ​​were similar and within the range of experimental error. D It was approximately 2.0 nM.

[0229] The binding ability of the [000289]B2-19 antibody variant to HEK293 cells stably expressing LILRB2 was analyzed by flow cytometry. 50,000 cells were incubated with 4-fold series dilutions (10–0.00015 μg / mL) of the test anti-LILRB2 antibody in 100 μL final volume at 4°C for 30 minutes. After washing, the bound antibody was detected using a goat anti-human Fc-specific secondary antibody conjugated to Alexa Fluor 647. The results were analyzed using FlowJo software, and dose-response curves were plotted using GraphPad Prism. As shown in Figure 3, the B2-19 antibody and its variants exhibit equivalent binding ability to LILRB2 stably expressed on HEK293 cells. The data shown are geometric mean MFIs for samples examined in a double plate, analyzed by flow cytometry (BD FACS Celesta).

[0230] [000290] To measure the binding ability of the B2-19 antibody variant on monocytes, CD14 + CD16 -50,000 monocytes were isolated from PBMCs of healthy donors and pre-incubated with 400 μg / mL human IgG derived from human serum at 4°C for 10 minutes to block the Fc gamma receptor. Then, they were rapidly incubated at 4°C for 30 minutes with a 4-fold series dilution (10–0.000038 μg / mL) of the test anti-LILRB2 antibody directly conjugated to Alexa Fluor 647 in a final volume of 100 μL. The data shown are geometric mean MFIs from dipset samples obtained from one donor in a flow cytometer (BD FACS Celesta). Results were analyzed using FlowJo software, and dose-response curves were plotted using GraphPad Prism. As shown in Figure 4, the B2-19 antibody and its variants were found to be primary CD14 + CD16 - It has equivalent binding ability to endogenous LILRB2 on monocytes.

[0231] [000291] The binding specificity of the B2-19 antibody variant to LILRB2 was measured by ELISA. ELISA plates were coated with 5 μg / mL recombinant LILR / LAIR1 ECD fused with a 6×His tag protein at the C-terminus, and incubated with an antibody at a concentration of 10 nM (10 times higher than the concentration that resulted in saturation of B2-19 antibody binding to LILRB2, as determined in pilot experiments). Incubation was performed at room temperature for 2 hours. The bound antibody was detected using an HRP-conjugated goat anti-human Fc-specific secondary antibody and a TMB substrate. Optical density at 450 nm was measured using a SpectraMax M5 Spectrophotometer (Molecular Device), and the data were analyzed using SoftMax Pro. As shown in Figure 5, the B2-19 antibody and its variants specifically bind to LILRB2.

[0232] [000292] To measure the binding specificity of the B2-19 antibody variant to bone marrow cells, the reactivity of the antibody on leukocytes derived from whole blood collected from healthy donors was characterized by flow cytometry. The B2-19 antibody and its variants were directly conjugated with Alexa Fluor 647. Following the protocol available in the literature (Hensley et al., J Vis Exp, 2012, 67:4302), 100 microliters of whole blood were incubated with antibodies for cell surface markers and anti-LILRB2 antibody. Samples were analyzed by flow cytometry (BD FACS Celesta), and the results were analyzed using FlowJo software. As shown in Figure 6, the B2-19 antibody and its variants specifically bind to bone marrow cells in whole blood. The data shown are the corrected geometric MFI of the samples, i.e., the geometric MFI of anti-LILRB2 stained samples with the geometric MFI of the sample excluding LILRB2 antibody (FMO control) deducted. Representative data from one donor is shown (N=3 donors).

[0233] [000293] To measure the activity of the B2-19 antibody mutant in blocking the binding of LILRB2 to HLA-G, 50,000 HEK293 cells stably expressing LILRB2 were subjected to a 4-fold series dilution of anti-LILRB2 antibody (10-0.00015 μg / mL) in the presence of 10 μg / mL (EC2) for binding, as determined in a pilot experiment. 80 The samples were incubated with His-tagged soluble HLA-G at concentrations corresponding to the approximate number of HLA-Gs. After washing, the bound HLA-Gs were detected using an anti-His antibody directly conjugated to allophycocyanin (APC). All incubations were performed at 4°C for 30 minutes. Samples were analyzed by flow cytometry (BD FACS Celesta). Results were analyzed using FlowJo software, and dose-response curves were plotted using GraphPad Prism. As shown in Figure 7, the B2-19 antibody and its variants exhibit equivalent blocking activity against HLA-G binding.

[0234] [000294]To measure the pro-inflammatory effect of the B2-19 antibody variant, PBMCs were isolated from healthy donors using Ficoll-Paque Plus (GE Healthcare) and incubated for 3 days in a 96-well round-bottom plate in the presence of 10 ng / mL anti-CD3 mAb (HIT3a) in the presence of anti-LILRB2 antibody or isotype control. The levels of cytokine concentrations were measured in the culture supernatant at the end of the 3-day incubation using the Human Cytokine Premixed Magnetic Luminex Performance Assay (R&D Systems). As shown in FIGS. 8A and 8B, the B2-19 antibody and the B2-19-16 antibody have equivalent pro-inflammatory effects on PBMCs isolated from healthy donors and stimulated with suboptimal anti-CD3 mAb concentrations.

[0235] Example 3 [000295]This example illustrates the reduction of multispecificity of the anti-LILRB2 antibody variant. [000296]The high BVP multispecificity ELISA scores of the antibodies showed a correlation with rapid clearance in cynomolgus monkeys and humans (Hotzel et al., mAb, 2012, 4, 6, 753-760), as well as a shortened half-life in humans (Shehata et al., Cell Reports, 2019, 28, 3300-3308). Therefore, a BVP ELISA was performed to evaluate the multispecificity of B2-19 and selected variants to determine which antibodies reduce the risk of exhibiting poor pharmacokinetics (PK) in humans. The baculovirus particles were produced by LakePharma. Briefly, 2 L of Sf9 cells were cultured with the baculovirus, isolated, resuspended in PBS pH 7.4, and stored at -80°C. Using the Bradford method, the total protein of the BVP preparation was measured to be 2.3 mg / mL, and the titer was 5.71×10 12The concentration was pfu / mL. BVP ELISA was performed as previously described (Hotzel et al., mAb, 2012, 4, 6, 753-760). Briefly, MaxiSorp plates were coated with 0.5% BVP in pH 9.6 carbonate buffer overnight at 4°C, washed once with 300 μL of PBS per well, and blocked with 200 μL of PBS + 0.5% BSA per well for 1 hour. After washing, the plates, which had been blocked three times with 150 μg / mL antibody in 300 μL of PBS and PBS + 0.5% BSA per well, were incubated for 1 hour, followed by six washes with 300 μL of PBS per well. The conjugated antibodies were detected for 1 hour after adding 100 μL of goat anti-human Fc-HRP conjugate per well, diluted to 1 / 20,000 or 1 / 40,000 in PBS + 0.5% BSA. The plates were washed three times with 300 μL of PBS per well, developed with TMB, and stopped with hydrochloric acid after 10 minutes. The BVP score was used to determine the OD of the sample. 450nm This was calculated by dividing by the background signal for the secondary antibody alone on BVP. At a secondary dilution of 1 / 20,000, a BVP score of wild-type B2-19 above the 5-fold cutoff indicates a risk of PK malformation in humans and non-human primates. As shown in Figure 9 and Table 2, the BVP scores of B2-19-12 and B2-19-16 both below the 5-fold cutoff indicate a reduced risk of PK malformation. A commercially available positive control (MEDNA type: H1308) and other therapeutic antibodies (e.g., rituxan, ixekizumab, 4E10) were used in the assay as references correlated with literature values ​​(Jain et al., PNAS, 2017, 114, 5, 944-949; and Shehata et al., Cell Reports, 2019, 28, 3300-3308).

[0236] [000297]

[0237] [Table 2]

[0238] Example 4 [000298]This example illustrates the thermal stability of the anti-LILRB2 antibody variant. [000299] To evaluate the thermal stability of B2-19-12 and B2-19-16, antibodies were incubated at 40°C in PBS pH 7.5 or formulation buffer (FB: 20 ​​mM histidine-HCl, 7% sucrose, 0.02% w / v PS80, pH 5.5) at approximately 2 mg / mL for 4 weeks. Initial samples (T0), as well as aliquots collected after 2 weeks (2W) and 4 weeks (4W), were analyzed for aggregation and fragmentation by size exclusion chromatography (SEC). As shown in Table 3, an increase in the percentage of high molecular weight (%HMW) in PBS and FB was observed for both molecules, but remained below 6%. A slight increase in the percentage of low molecular weight (%LMW) was also observed, but this remained below 0.6%. Antibody binding activity was also evaluated by ELISA in PBS and FB before and after incubation at 40°C. As shown in Figure 10, coupled EC 50 A slight decrease (approximately 2-3 times) was observed after a 4-week incubation period. Overall, these results suggest that none of the molecules significantly aggregate, fragment, or lose binding activity over time under thermal stress.

[0239] [000300]

[0240] [Table 3]

[0241] Example 5 [000301]This example illustrates the stability of an anti-LILRB2 antibody variant under freeze-thaw stress.

[0242] [000302] To evaluate the stability of B2-19-12 and B2-19-16 against freeze-thaw (F / T) stress, antibodies were exposed to freezing at -70°C and thawing at room temperature (approximately 20°C) for one or three cycles (1C) in the formulation buffer FB (20 mM histidine-HCl, 7% sucrose, 0.02% w / v PS80, pH 5.5) at a concentration of 20 mg / mL. Dynamic light scattering (DLS) was performed by WuXi Biologics, but did not reveal the aggregation tendency as assessed by Z-mean (nm) / PDI (polydispersion index) (see Table 4). Antibody activity before and after the freeze-thaw cycle was evaluated by ELISA. As shown in Figure 11, no significant change in binding activity was observed for either B2-19-12 or B2-19-16 after freeze-thaw cycles.

[0243] [000303]

[0244] [Table 4]

[0245] Example 6 [000304]This example illustrates the pharmacokinetics of B2-19 antibody variants in human FcRn transgenic mice. Since this model demonstrated the ability to predict antibody PK in humans, human FcRn transgenic mice were used to evaluate the pharmacokinetics (PK) of B2-19-12 and B2-19-16 (Avery et al., mAbs, 2016, 8, 1064). Specifically, 6-8 week old female Fcgrt m1DcrTwelve homozygous female Tg(FCGRT)32Dcr mice (JAX strain number: 014565) were obtained from Jackson Laboratory. Following the schedule shown in Table 5, the animals were administered 5 mg / kg by a single intravenous injection, and 60 μL of blood was collected. Serum concentrations of B2-19-12 and B2-19-16 were measured using a sandwich ELISA format. Briefly, the assay utilized recombinant LILRB2 ECD-6X His-tagged protein, coated onto a 96-bottomed microtitration plate, as the capture reagent. Diluted samples and B2-19-12 or B2-19-16 standards were added to the coated plate. HRP-conjugated goat anti-human IgG, along with B2-19-12 or B2-19-16, was used as the detection reagent, resulting in an immunocomplex. Serum concentration-time plots were created for B2-19-12 and B2-19-16 (Figure 12). Pharmacokinetic parameters (Table 6) indicate similar exposures for B2-19-12 and B2-19-16, and terminal half-lives and clearances are within the typical range for human IgG in FcRn transgenic mice.

[0246] [000305]

[0247] [Table 5]

[0248] [000306]

[0249] [Table 6]

[0250] Example 7 [000307]This example illustrates the characterization of the bioactivity of B2-19-16, a B2-19 antibody variant, on multiple primary immune cell lines.

[0251] [000308] Using mechanical methods and PBS-10 mM EDTA, tissue samples derived from solid tumors were dissociated into single cells. In some cases, peripheral blood samples were also obtained from the same donor as tumor tissue samples. The resulting cells were stained with antibodies for B2-19-16 and human bone marrow cell markers at 4°C using standard methods, and the stained samples were analyzed by flow cytometry. As shown in Figures 13A and 13B, B2-19-16 binds to all bone marrow cells infiltrating the solid tumor microenvironment from solid tumor patients, as well as peripheral blood bone marrow cells. CD11b was used as a panmyelocyte marker, and CD45 was used as a pantumor-infiltrating leukocyte marker.

[0252] [000309] Classical monocytes were isolated from healthy donor PBMCs and differentiated into immature dendritic cells with GM-CSF and IL-4 over 6 days. Subsequently, monocyte-derived immature dendritic cells were incubated with an antibody (100 nM) in the presence of 100 ng / mL of LPS to induce dendritic cell maturation. After 2 days, TNF-α levels were measured in the culture medium supernatant, and cells were analyzed by flow cytometry. As shown in Figure 14, treatment with the B2-19-16 antibody resulted in a decrease in the expression level of the tolerogenicity marker CD209. In addition, as shown in Figure 15, B2-19-16 further enhanced LPS-induced TNF-α production compared to isotype-treated conditions. These results indicate that B2-19-16 enhances the pro-inflammatory effect of LPS on monocyte-derived immature DCs. This data suggests that blocking LILRB2 by B2-19-16 mitigates the inhibitory signal of LILRB2 to DC activation pathways such as TLRs.

[0253] [000310] Classical monocytes were isolated from freshly prepared PBMCs and placed in 3 mL of complete DC medium (StemXVivo Dendritic Cell Base Media, 50 μg / mL gentamicin, 50 ng / mL GM-CSF, 35 ng / mL IL-4) using a 6-well plate, with 1 × 10 cells per mL.6 During a 6-day culture at individual densities, cells were treated with 15 μg / mL of B2-19-16 or an isotype control. GM-CSF (50 ng / mL) and IL-4 (35 ng / mL) were added again on day 3. On day 6, the resulting DCs were analyzed by flow cytometry. As shown in Figures 16A and 16B, treatment of primary monocytes with B2-19-16 was pro-inflammatory (CD86). + This promoted their differentiation into DCs. This finding is key because diverse tumor microenvironment-associated myeloid cell populations, including tolerogenic DCs, originate from circulating monocytes and are desirable for reprogramming these cells into pro-inflammatory cells.

[0254] [000311] Classical monocytes were isolated from healthy donor PBMCs and differentiated into immature dendritic cells (DCs) with GM-CSF and IL-4 over 6 days. Subsequently, the monocyte-derived immature DCs were incubated with an antibody (100 nM) in the absence of any other stimuli, and their phenotypes were analyzed by flow cytometry after 2 days. As shown in Figure 17, B2-19-16 enhanced the expression levels of the maturation marker (CD83) and activation marker (CD86, HLA-DR) in immature DCs, while decreasing the expression of the tolerogenicity marker, CD209. In contrast, the cell surface expression level of another immunosuppressive receptor, LILRB4, remained unchanged. Therefore, B2-19-16 promotes the differentiation of immature DCs into DCs that exhibit enhanced ability to induce adaptive immunity.

[0255] [000312] Macrophages were differentiated from classical monocytes isolated from PBMCs of healthy donors using 100 ng / mL M-CSF over a period of 6 days. On day 6, CD4 +T cells were isolated from healthy, unrelated donor PBMCs and suspended in fresh medium containing 100 ng / mL of M-CSF and antibody (100 nM each). This mixture was then added to differentiated macrophages. At the 6-day endpoint, INF-γ levels in the medium supernatant were measured by ELISA. The anti-PD-1 antibody used was the clone EH12.2H7. As shown in Figure 18, B2-19-16 combined with the anti-PD-1 blocking antibody was allogeneic CD4 + This study enhances IFN-γ production in T cell-macrophage co-cultures compared to each antibody alone.

[0256] Using Ficoll-Paque (GE Healthcare), PBMCs were isolated from healthy donors and incubated for 3 days in 96-well round-bottom plates with 50 ng / mL of LPS in the presence of a 3-fold dilution series of the anti-LILRB2 antibody B2-19-16 or isotype control. Cytokine concentration levels were measured in the culture medium supernatant at the end of the 3-day incubation using the Human Cytokine Premixed Magnetic Luminex Performance Assay. As shown in Figure 19, the B2-19-16 antibody enhanced the concentration levels of various pro-inflammatory cytokines produced in response to LPS stimulation. In addition, as shown for TNF-α in Figure 20, the B2-19-16 antibody dose-dependently enhanced cytokine production by LPS-stimulated PBMCs.

[0257] [000314] Classical monocytes were isolated from freshly prepared PBMCs using a classical monocyte isolation kit. Classical monocytes were placed in a flat-bottomed 96-well plate in complete macrophage medium (X-VIVO 10, 4 mM L-glutamine, 0.5 mg / mL penicillin / streptomycin, and 100 ng / mL M-CSF) at a rate of 1 × 10 cells per 1 mL. 6Cells were cultured at a density of 100 μL per well. The cells were differentiated into macrophages over 7 days, with the medium changed on day 4. On day 7, the medium was replaced with complete macrophage medium containing 5 μg / mL of 2'3'-cGAMP, a STING agonist, and 15 μg / mL of B2-19-16 or an isotype control, and incubation was continued for 2 days. During this 2-day incubation, the levels of TNF-α accumulated in the medium were measured using the human cytokine Luminex assay. As shown in Figure 21, B2-19-16 enhances the stimulating effect of the cGAS-STING pathway in monocyte-derived macrophages from all test donors, as indicated by the increased TNF-α concentration levels.

[0258] [000315] The cancer cell lines SK-MEL-5 and A549 were placed in 200 μL of culture medium (DMEM, 10% heat-inactivated FBS) in a flat-bottomed 96-well plate, with 1 × 10 per mL. 4 The cells were seeded at a density of 10¹⁴ cells. The following day, bone marrow cells were isolated from freshly prepared PBMC samples obtained from healthy donors using CD33 MicroBeads. The culture medium of the cancer cell culture was used to isolate the bone marrow cells at a density of 1 × 10¹⁴ cells. 5 The cells were replaced with 200 μl of co-culture medium (X-VIVO 10, 5% FBS, 50 ng / ml GM-CSF) containing 15 μg / mL of B2-19-16 or an isotype control. In the wells, bone marrow cells without cancer cells were seeded (1 × 10⁶ per well). 5 (7-AAD) cells were used as a control for tumor conditioning effects. The cultures were maintained for 5 days. On day 5, cells were detached using 10 mM EDTA in PBS and analyzed by flow cytometry. Based on forward scatter (FSC) and side scatter (SSC) signals, as well as CD11b expression, myeloid cells were classified as viable cells (7-AAD). -Gated from ). The phenotype of myeloid cells was assessed by measuring changes in the expression levels of cell surface markers associated with anti-inflammatory (CD163 and CD209) or pro-inflammatory activity (CD64). As shown in Figures 22A and 22B, "tumor conditioning" of myeloid cells resulted in upregulation of the expression of the inhibitory receptor CD209 and the scavenger receptor CD163. In contrast, the decrease in the expression of the Fcγ receptor CD64 by tumor conditioning indicates that the resulting macrophages exhibit reduced antibody-mediated phagocytic activity. The presence of B2-19-16 in these cancer cell-myeloid cell co-cultures reversed these changes, suggesting that treatment with B2-19-16 may preserve the potential for pro-inflammatory phagocytosis in myeloid cells within cancer. Importantly, the observation of B2-19-16 activity in the presence of cancer cell lines with significantly different histological origins suggests that B2-19-16 may offer broad therapeutic benefits.

[0259] [000316] Classical monocytes were purified from freshly prepared PBMCs using a classical monocyte isolation kit. Classical monocytes were placed in a flat-bottomed 96-well plate in complete macrophage medium (X-VIVO 10, 4 mM L-glutamine, 0.5 mg / mL penicillin / streptomycin, 100 ng / mL M-CSF) at a rate of 1 × 10 cells per 1 mL. 6Cells were cultured at a density of 100 μL per well. The medium was changed on day 4, and the cells were differentiated into macrophages over 7 days. On day 7, B2-19-16 was labeled with Fabfluor-pH Red Antibody Labeling Dye. The fluorescence emission level of this dye increases at low pH. Anti-CD71 (transferrin receptor) antibody was also simultaneously labeled and used as a positive control for the internalization of the receptor:antibody complex. After adding the antibody:FabFluor complex (final concentration: 4 μg / ml) to the cells in a triple-well array, the assay plate was placed in an Incucyte S3 live-cell analysis system (Essen Bioscience), and the phase image and red fluorescence image were rapidly scanned at 10x magnification, and then scanned every 20 minutes for 12 hours. The images were analyzed using Incucyte software for total integrated intensity to determine the level of FabFluor-labeled internalization antibody. As shown in Figure 23, the anti-CD71 antibody is efficiently internalized (detectable within cells, with increasing fluorescence over time), whereas B2-19-16 is not efficiently internalized.

[0260] [000317] Using a U-shaped bottom 94-well plate, PBMC (1 × 10 cells per well) 6PBMCs were seeded in 200 μL of X-VIVO 10 medium supplemented with 50 ng / ml of IL-2, and in a 3-fold dilution series (40-0.002 μg / mL) of B2-19-16, IgG4 (isotype control), or rituximab (positive control). For each donor, the procedure was as described above, but PBMCs seeded in the absence of antibodies were used as their respective untreated controls. The cells were incubated at 37°C for 20 hours. The cells were washed with PBS and incubated at room temperature for 10 minutes with 400 μg / mL of human IgG diluted in FACS buffer [PBS, 0.5% (wt / vol) BSA] (50 μL per well) to block the Fcγ receptor. Immediately afterward, an antibody cocktail diluted in FACS buffer (clone M5E2, an anti-CD14 antibody, and clone HIB19, an anti-CD19 antibody) was added (50 μL per well), mixed with the cell suspension by pipetting, and the cells were incubated on ice for 30 minutes. The cells were washed with FACS buffer and resuspended in 100 μL of PBS containing 7-AAD diluted to 1:20 (vol / vol). The cells were collected in a BD FACSCelesta flow cytometer fitted with a high-throughput sample acquisition module, and 1 × 10⁶ cells were collected. 5 Individual data were recorded. The data were analyzed using FlowJo 10.5.3. Live cells were identified by excluding 7-AAD. Within the surviving PBMC population, monocytes were CD14 + While it was identified as a cell, B cells were identified as CD19 + Cells were identified. For each cell type, cell viability at a given antibody concentration was calculated as a percentage of the untreated control value obtained for the same PMBC donor. Antibody concentration-cell viability curves were created by plotting the data using GraphPad Prism 9.1.0. As shown in Figure 24, B2-19-16 does not induce monocyte, Fc-dependent depletion. In contrast, B cell depletion is observed in simultaneous incubation of the same donor sample in the presence of rituximab.

[0261] [000318] In light of this disclosure, all compositions and methods disclosed and claimed herein can be prepared and performed without unnecessary experimentation. Although the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and steps or sequences of steps described herein, without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related, while achieving the same or similar results, may be substituted with the agents described herein. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are deemed to be within the spirit, scope, and concept of the present invention as defined by the accompanying claims.

Claims

1. An anti-LILRB2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, (a) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 25, and the light chain variable region has the amino acid sequence of SEQ ID NO: 26; or (b) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 31, and the light chain variable region has the amino acid sequence of SEQ ID NO:

32. Anti-LILRB2 antibody or its antigen-binding fragment.

2. The antibody or antigen-binding fragment according to claim 1, further comprising an immunoglobulin constant region, optionally an IgG constant region, optionally a human IgG constant region, optionally an IgG4 constant region, or optionally a hinge-stabilized IgG4 constant region.

3. Diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv) ２ dsFv - dsFv', Fv fragment, Fab, Fab', F(ab') ２ The antibody or its antigen-binding fragment according to claim 1, which is a bispecific antibody, a DS diabody, or a bivalent antibody.

4. An antibody or antigen-binding fragment according to claim 1, which blocks the binding of LILRB2 to one or more ligands, wherein the ligand is selected from the group consisting of HLA-G, classical MHC-I, ANGPTL, CD1c / d, CSP, and SEMA4A.

5. An antibody or antigen-binding fragment thereof according to claim 1, which modulates the activation of LILRB2, and which suppresses the activation of LILRB2 or antagonistizes LILRB2 signaling.

6. A polyspecific antibody or antigen-binding fragment according to claim 1, comprising PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, Fc receptor, FCRL (1-6), A2AR, CD160, 2B4, TGF-β, TGF-βR, VISTA, BTLA, TIGIT, LAIR1, LILRB1, LILRB3, LILRB4, LILRB5, LILRA (1-6), OX40, CD2, CD27, CD28, CD30, CD40, CD47, SIRPA, CLEC-1, clever-1 / stabilin-1, AD An antibody or its antigen-binding fragment that specifically binds to a second antigen selected from GRE, TRIME1, TRIME2, CD122, ICAM-1, IDO, NKG2D / C, SLAMF7, MS4A4A, SIGLEC (7-15), NKp80, NKG2A, CD160, CD161, CD300, CD163, B7-H3, B7-H4, LFA-1, ICOS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, TNFR2, TLR (1-9), IL-2, IL-7, IL-15, IL-21, CD16, and CD83.

7. An antibody or antigen-binding fragment according to claim 1, which is linked to one or more conjugate portions, wherein the conjugate portions include an immunomodulator, an antitumor agent, a clearance modifier, a toxin, a detectable label, DNA, RNA, a cytokine, or a purified portion.

8. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

9. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

10. A vector comprising an isolated polynucleotide as described in claim 9.

11. A host cell comprising the vector according to claim 10.

12. A method for expressing an antibody or an antigen-binding fragment thereof, comprising the step of culturing the host cells described in claim 11 under conditions in which the antibody or the antigen-binding fragment thereof is expressed.

13. A chimeric antigen receptor (CAR) protein comprising an antigen-binding fragment according to any one of claims 1 to 7.

14. An isolated nucleic acid encoding the CAR protein according to claim 13.

15. A vector comprising the isolated nucleic acid described in claim 14.

16. A manipulated cell comprising the isolated nucleic acid described in claim 14, wherein the manipulated cell is a T cell, an NK cell, or a macrophage.

17. A pharmaceutical composition according to claim 8 for use in a method for treating or improving the effects of cancer in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the antibody or an antigen-binding fragment thereof.

18. A composition comprising the antibody or antigen-binding fragment thereof according to claim 1 for use in a method for detecting cancer cells or cancer stem cells in a sample or subject, wherein the method is: (a) The step of bringing a sample of a subject or a sample derived from a subject into contact with the antibody or an antigen-binding fragment thereof; (b) the step of detecting the binding of the antibody in the subject or sample to cancer cells or cancer stem cells, A composition in which the sample is body fluid, biopsy, blood, bone marrow, sputum, tears, saliva, mucus, serum, ascites, urine, or feces.

19. The composition according to claim 18, wherein the method further comprises (c) a step of performing steps (a) and (b) at a second time point and determining the change in the detection level compared to the first time point.

20. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 for use in a method for enhancing T cell activation or enhancing dendritic cell maturation and activation in a subject; modulating the phenotype of anti-inflammatory macrophages and tolerogenic DCs; polarizing bone marrow cells derived from cancer patients of solid tumors to a pro-inflammatory phenotype; or mitigating the inhibitory effect of patient-derived monocytic MDSCs (M-MDSCs) on autologous T cell proliferation and cytokine release, wherein the method comprises the step of administering the antibody or antigen-binding fragment thereof to a subject.

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