Anti-LILRB2 antibodies and uses thereof

Abstract

The present disclosure provides antibodies, or antigen-binding fragments thereof, that bind to LILRB2 (leukocyte immunoglobulin-like receptor B2), which can block the signaling pathway and up-regulate the immune response to LILRB2-related diseases, disorders, or conditions.

Description

[0001] Cross-references

[0002] This application claims priority to PCT International Application No. PCT / CN2022 / 104494 filed on July 8, 2022 and Chinese Patent Application No. 202310787466.X filed on June 29, 2023. The disclosure of the prior application is deemed to be part of the disclosure of this application and is incorporated into this application in its entirety.

[0003] Reference sequence list

[0004] The sequence listing entitled DCF230536WO-Seqlisting.xml was created on July 6, 2023, is 23,061 bytes in size, and is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to antibodies or antigen-binding fragments thereof that bind to LILRB2 (Leukocyte Immunoglobulin Like Receptor B2; LILRB2) and uses thereof. Background Art

[0006] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0007] Members of the leukocyte immunoglobulin-like receptor (LILR) family are key positive regulators (LILRA) and negative regulators (LILRB) of innate and adaptive immune responses. LILRB family members show different expression patterns on immune cells, among which LILRB2 (leukocyte immunoglobulin-like receptor B2, UniProt: A2IXV5) is mainly restricted to myeloid-derived cells, including monocytes, macrophages, dendritic cells and granulocytes. LILRB2 (also known as monocyte / macrophage immunoglobulin-like receptor 10 (MIR-10), immunoglobulin-like transcript 4 (ILT4), or CD85 antigen-like family member D (CD85D)) is a typical type I transmembrane protein with four extracellular tandem Ig-like domains, a transmembrane region, and a cytoplasmic tail with three immunoreceptor tyrosine inhibitory motifs (ITIMs) (Aiqin Gao et al., ILT4 functions as a potential checkpoint molecule for tumor immunotherapy. Reviews on Cancer. 1869 (2018) 278-285).

[0008] The interaction between LILRB2 and specific ligands is required for molecular processes with unique biological functions. Human LILRB2 was initially shown to bind extensively to MHC-I molecules (also known as human leukocyte antigen (HLA) classical class I molecules (HLA-A, HLA-B, HLA-C) and atypical HLA class I molecules (HLA-E, HLA-F, HLA-G and HLA-H). Subsequent studies have shown that LILRB2 binds to other non-HLA ligands (including angiopoietin-like (ANGPTL) family, myelin-associated glycoprotein (MAG) and oligomeric β-amyloid protein) (William et al., LILRB receptor-mediated regulation of myeloid cell maturation and function. Cancer Immunol Immunother. 2017; 66(8): 1079-1087).

[0009] LILRB2 is a powerful inhibitory molecule with immunosuppressive function. Recent studies have shown that LILRB2 is overexpressed or highly induced in the tumor microenvironment and in malignant tumor cells from hematopoietic tumors and solid tumors. Clinically, the expression level of LILRB2 in tumor cells of NSCLC and breast cancer patients is positively correlated with poor cell differentiation, increased local lymph node metastasis, advanced cancer and low patient survival rate (Aiqin Gao et al., ILT4 functions as a potential checkpoint molecule for tumor immunotherapy. Reviews on Cancer. 1869 (2018) 278-285). Increasing evidence highlights the potential role of LILRB2 as a novel immune checkpoint for tumor immunotherapy.

[0010] Immunotherapy has become a breakthrough strategy for treating human cancer, but there is still a need for more effective cancer treatments using immunotherapy. More specifically, there is still a need for novel anti-LILRB2 antibodies, compositions comprising the same, and methods. Summary of the invention

[0011] For the above purposes, provided herein are novel antibodies or antigen-binding fragments thereof that bind to LILRB2 and uses thereof.

[0012] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising complementary determining regions (CDRs) 1, 2, and 3, wherein VH CDR1 comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 1; VH CDR2 comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 2; VH CDR3 comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 3; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein VL CDR1 comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: NO:4; VL CDR2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:5; VL CDR3 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:6.

[0013] In some embodiments, the VH of the antibody or antigen-binding fragment thereof comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively; and the VL comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively.

[0014] In some embodiments, the VH of the antibody or antigen-binding fragment thereof is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:7, 8, 9, 10, 11 or 12; and the VL is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:13, 14, 15 or 16.

[0015] In some embodiments, the VH of the antibody or antigen-binding fragment thereof differs from the amino acid sequence shown in SEQ ID NO:7, 8, 9, 10, 11 or 12 by no more than 25, 20, 15, 13, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; the VL differs from the amino acid sequence shown in SEQ ID NO:13, 14, 15 or 16 by no more than 20, 15, 13, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid.

[0016] In some embodiments, the VH of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7, 8, 9, 10, 11 or 12; and the VL comprises the amino acid sequence shown in SEQ ID NO:13, 14, 15 or 16.

[0017] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:7 and VL comprises the amino acid sequence of SEQ ID NO:13; VH comprises the amino acid sequence of SEQ ID NO:8 and VL comprises the amino acid sequence of SEQ ID NO:13; VH comprises the amino acid sequence of SEQ ID NO:9 and VL comprises the amino acid sequence of SEQ ID NO:13; VH comprises the amino acid sequence of SEQ ID NO:7 and VL comprises the amino acid sequence of SEQ ID NO:14; VH comprises the amino acid sequence of SEQ ID NO:8 and VL comprises the amino acid sequence of SEQ ID NO:14; VH comprises the amino acid sequence of SEQ ID NO:9 and VL comprises the amino acid sequence of SEQ ID NO:14; VH comprises the amino acid sequence of SEQ ID NO:10 and VL comprises the amino acid sequence of SEQ ID NO:15; or VH comprises the amino acid sequence of SEQ ID NO:11 and VL comprises the amino acid sequence of SEQ ID NO:15; VH comprises the amino acid sequence of SEQ ID NO:12 and VL comprises the amino acid sequence of SEQ ID NO:16.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to LILRB2. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to LILRB2 with an EC of less than 20 nM, less than 10 nM, less than 5 nM, or less than 1 nM. 50 Values ​​specifically bind to LILRB2.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and HLA-A2 (major histocompatibility complex, class I, A2; HLA-A2).

[0020] In some embodiments, the antibody or antigen-binding fragment thereof has an IC of less than 20 nM, less than 10 nM, less than 5 nM, less than 1 nM, or less than 0.6 nM as measured by FACS (fluorescence activated cell sorting; FACS) assay. 50 The value blocks the interaction between human LILRB2 and HLA-A2.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and HLA-G (major histocompatibility complex, class I, G).

[0022] In some embodiments, the antibody or antigen-binding fragment thereof has an IC value of less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, or less than 0.3 nM as measured by a FACS (fluorescence activated cell sorting) assay. 50 The value blocks the interaction between human LILRB2 and HLA-G.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL1 (angiopoietin-like (ANGPTL) 1).

[0024] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL2 (angiopoietin-like protein 2).

[0025] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL4 (angiopoietin-like protein 4).

[0026] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL7 (angiopoietin-like protein 7).

[0027] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human MAG (myelin-associated glycoprotein).

[0028] In some embodiments, the antibody or antigen-binding fragment thereof is capable of reprogramming human monocyte-derived M2 macrophages to an M1 phenotype (pro-inflammatory M1 phenotype).

[0029] In some embodiments, the antibody or antigen-binding fragment thereof is capable of alleviating M2 macrophage-mediated T cell suppression.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof is capable of promoting human dendritic cells to enter a mature state.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof is capable of promoting human monocytes to enter a pro-inflammatory state.

[0032] In some embodiments, the antibody or antigen binding fragment thereof can promote human PBMC (peripheral blood monocular cell (PMBC)) to enter a pro-inflammatory state. In some embodiments, the antibody or antigen binding fragment thereof can enhance the expression of pro-inflammatory cytokines and / or chemokines. In some embodiments, the antibody or antigen binding fragment thereof can upregulate the release of pro-inflammatory cytokines and / or chemokines. In some embodiments, the pro-inflammatory cytokines include IL-2 (interleukin 2 (IL-2)), IFN-γ (interferon γ (interferongamma; IFN-γ)) and / or TNF-α (tumor necrosis factor (tumor necrosis factor; TNF-α)). In some embodiments, the pro-inflammatory cytokine and / or chemokine is selected from the group consisting of IL-2, IFN-γ, TNF-α, granzyme B, PDGF-AA, IL-3, IL-1β, IL-5, TGF-α, IFN-α, CCL4, basic FGF, IL-13, G-CSF, IL-4, PD-L1, CCL20, IL-15, GM-CSF, CCL5, CCL3, PDGF-AB, CCL19, CXCL2, IL-6, VEGF, and combinations thereof.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof is capable of reducing the expression of IL-10 (interleukin 10).

[0034] In some embodiments, the antibody or antigen-binding fragment thereof binds to human LILRB2 with a KD (affinity constant) of less than 15 nM, less than 10 nM, less than 8 nM, or less than 6 nM as measured by a FACS (fluorescence activated cell sorting) assay.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody or an antigen-binding fragment thereof.

[0037] In one aspect, provided herein is an antibody or an antigen-binding fragment thereof that binds to a peptide comprising the amino acid sequence shown in SEQ ID NO:21.

[0038] In one aspect, provided herein are isolated polynucleotides encoding antibodies or antigen-binding fragments thereof that bind to LILRB2.

[0039] In one aspect, provided herein is an isolated vector comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to LILRB2.

[0040] In one aspect, provided herein is a host cell comprising an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to LILRB2 or an isolated vector comprising the above polynucleotide.

[0041] In one aspect, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated vector provided herein, and a pharmaceutically acceptable carrier.

[0042] In one aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof provided above and an anti-PD-1 antibody (eg, Keytruda).

[0043] In one aspect, provided herein is a kit comprising an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated vector provided herein, or a host cell provided herein, or a pharmaceutical composition provided herein.

[0044] In one aspect, provided herein is a use of an antibody or antigen-binding fragment thereof, or an isolated polynucleotide, or an isolated vector, or a host cell, or a pharmaceutical composition, or a kit provided herein in the manufacture of a therapeutic agent for binding to human LILRB2.

[0045] In one aspect, provided herein is the use of an antibody or antigen-binding fragment thereof, or an isolated polynucleotide, or an isolated vector, or a host cell, or a pharmaceutical composition, or a kit that binds to LILRB2 in the manufacture of a therapeutic agent for blocking the interaction between human LILRB2 and HLA-A2, HLA-G, human ANGPTL1, human ANGPTL2, human ANGPTL4, human ANGPTL7, and / or human MAG.

[0046] In one aspect, provided herein is a use of an antibody or antigen-binding fragment thereof, or an isolated polynucleotide, or an isolated vector, or a host cell, or a pharmaceutical composition, or a kit provided herein that binds to LILRB2 in the manufacture of a therapeutic agent for enhancing an immune response.

[0047] In one aspect, provided herein is the use of an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated polynucleotide provided herein, or an isolated vector provided herein, or a host cell provided herein, or a pharmaceutical composition provided herein, or a kit provided herein in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease.

[0048] In one aspect, provided herein is a method for enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated polynucleotide provided herein, or an isolated vector provided herein, or a host cell provided herein, or a pharmaceutical composition provided herein, or a kit provided herein.

[0049] In one aspect, provided herein is a method for diagnosing or preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated polynucleotide provided herein, or an isolated vector provided herein, or a host cell provided herein, or a pharmaceutical composition provided herein, or a kit provided herein.

[0050] In some embodiments, the disease, disorder or condition comprises a tumor, and optionally, at least one tumor cell expresses LILRB2.

[0051] In some embodiments, the subject is a mammal, including a human.

[0052] In one aspect, provided herein is a binding epitope on LILRB2, wherein the binding epitope comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, LILRB2 is human LILRB2. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The following is a brief description of the drawings, which are presented for the purpose of illustrating exemplary embodiments disclosed herein and not for the purpose of limiting the same.

[0054] Figure 1 Binding of 043c, 1E1 and J-19 to LILRB2 ECD recombinant protein measured by ELISA is shown. The average OD450 values ​​obtained with the indicated antibodies are listed at the top of each column. OD450 is an abbreviation for optical density at 450 nm. hIgG4 iso represents human IgG4 isotype control.

[0055] Figure 2 The binding of 043c to EL4 (EL4 murine tumor cell line)-LILRB2 cells measured by FACS is shown. MFI is the abbreviation for mean fluorescence intensity and Ab conc. is the abbreviation for the concentration of the antibody (μg / ml). hIgG4 iso stands for human IgG4 isotype control.

[0056] Figure 3 A-3B shows the difference between 043c and 1E1 from healthy donor #1045 ( Figure 3 A) and #6004( Figure 3 B) Binding of isolated human primary monocytes. MFI is the abbreviation for mean fluorescence intensity and Ab conc. is the abbreviation for the concentration of the antibody (μg / ml). hIgG4 iso stands for human IgG4 isotype control.

[0057] Figure 4 A-4I shows the expression of 043c, 1E1 and J-19 with LILRA1 ( Figure 4 A), LILRA2( Figure 4 B) LILRA3 Figure 4 C) LILRA4 Figure 4 D) LILRA5( Figure 4 E) LILRB1 Figure 4 F), LILRB3( Figure 4 G)、LILRB4( Figure 4 H)、LILRB5( Figure 4 I) Binding of ECD recombinant proteins. The average OD450 values ​​obtained with the specified antibodies are listed at the top of each column. OD450 is an abbreviation for optical density at 450 nm. hIgG4 iso represents human IgG4 isotype control, and PC represents positive control.

[0058] Figure 5The activity of 043c, 1E1 and J-19 in blocking the interaction between LILRB2 and HLA-A2, HLA-G, ANGPTL1, ANGPTL2, ANGPTL4, ANGPTL7 or MAG as measured by FACS-based competition assay is shown. The inhibition rate obtained with the indicated antibodies is listed at the top of each bar. hIgG4 iso represents human IgG4 isotype control.

[0059] Figure 6 Shown are the activities of 043c, 1E1, and J-19 in blocking the interaction between LILRB2 and HLA-G as measured by a FACS-based competition assay. IC values ​​obtained with the indicated antibodies 50 The values ​​are listed to the right of each graph legend. Ab conc. is the abbreviation for the concentration of the antibody (μg / ml).

[0060] Figure 7 The activity of 043c in blocking the interaction between LILRB2 and HLA-A2 as measured by a FACS-based competition assay is shown. The IC obtained with 043c 50 The values ​​are listed to the right of the graph legend. Ab conc. is the abbreviation for the concentration of the antibody (μg / ml).

[0061] Figure 8 A-8B shows the effects of 043c, 1E1 and J-19 in promoting human PBMC to enter a pro-inflammatory state. TNFα ( Figure 8 A) and IL10( Figure 8 B) Secretion. Cytokine level values ​​are listed at the top of each bar. hIgG4 iso represents human IgG4 isotype control and vehicle represents negative control without any antibody treatment.

[0062] Fig. 9 A-9B shows the effects of 043c, 1E1 and J-19 in promoting human monocytes to enter a pro-inflammatory state. TNFα ( Fig. 9 A) and IL10( Fig. 9 B) Secretion. Cytokine level values ​​are listed at the top of each bar. hIgG4iso represents human IgG4 isotype control and vehicle represents negative control without any antibody treatment.

[0063] Fig.10A-10B shows the effects of 043c, 1E1 and J-19 on reprogramming human monocyte-derived M2 macrophages to pro-inflammatory M1 phenotype. M1 macrophage population conversion (CD86 高 CD163 低 , Fig.10 A) and TNFα secretion ( Fig.10 B) The values ​​of percentage of M1 macrophage population and TNFα level are listed at the top of each bar. hIgG4 iso represents human IgG4 isotype control, vehicle represents negative control without any antibody treatment, and M1 control represents positive control using monocyte-derived M1 macrophages.

[0064] Fig.11 A-11C shows the effects of 043c, 1E1 and J-19 in alleviating T cell suppression mediated by M2 macrophages. HTRF (fluorescence resonance energy transfer) was used to detect IL-2 ( Fig.11 A) IFNγ( Fig.11 B) and TNFα( Fig.11 C). The cytokine level values ​​are listed at the top of each bar. M2:T=1:0.5, M2:T=1:1, M2:T=1:2, M2:T=1:4 and M2:T=1:8 represent M2 macrophages mixed with autologous T cells in different ratios. hIgG4 iso represents human IgG4 isotype control, and vehicle represents negative control without any antibody treatment.

[0065] Fig.12 The effects of 043c, 1E1 and J-19 on promoting the maturation of human monocyte-derived dendritic cells were shown. CD86 was detected by FACS under the treatment of the indicated antibodies 高 CD163 低 Dendritic cell population conversion. Population percentage values ​​are listed at the top of each bar. hIgG4 iso represents human IgG4 isotype control and vehicle represents negative control without any antibody treatment.

[0066] Fig.13 A-13B shows the 043c ( Fig.13 A) and 1E1( Fig.13 B) Binding epitope of the peptide. HDX% deuteration is the abbreviation for percentage of hydrogen-deuterium exchange.

[0067] Fig.14Binding of 043c and 043c-derived humanized variants (043c, hu043.01, hu043.02, hu043.03, hu043.04, hu043.05 and hu043.06) to EL4 (EL4 murine tumor cell line)-LILRB2 cells is shown. ECs obtained with the indicated antibodies 50 The values ​​are listed to the right of each graph legend. MFI is the abbreviation for mean fluorescence intensity, and Ab conc. is the abbreviation for the concentration of the antibody (μg / ml). hIgG4 iso stands for human IgG4 isotype control.

[0068] Fig.15 A-15B shows the expression of 043c, hu043.01, hu043.02, hu043.03, hu043.04, hu043.05, hu043.06, J-19 ( Fig.15 A) and hu043.07, hu043.08( Fig.15 B) Activity of blocking the interaction between LILRB2 and HLA-G. IC obtained with the indicated antibodies 50 The values ​​are listed to the right of each graph legend. Ab conc. is the abbreviation for the concentration of the antibody (μg / ml). hIgG4 iso stands for human IgG4 isotype control.

[0069] Fig.16 The effects of hu043.07, hu043.08, 043c, 1E1 and J-19 in promoting human monocytes into a pro-inflammatory state are shown. TNFα secretion from LPS (lipopolysaccharide)-induced human primary monocytes treated with the indicated antibodies was detected by HTRF (fluorescence resonance energy transfer). The cytokine level values ​​are listed at the top of each column. hIgG4 iso represents human IgG4 isotype control and vehicle represents a negative control without any antibody treatment.

[0070] Fig.17 A-17B shows the effects of hu043.07, hu043.08, 043c, 1E1 and J-19 on reprogramming human monocyte-derived M2 macrophages to a pro-inflammatory M1 phenotype. The M1 macrophage population conversion (CD86 高 CD163 低 , Fig.17 A) and TNFα secretion ( Fig.17B) The values ​​of percentage of M1 macrophage population and TNFα level are listed at the top of each bar. hIgG4iso represents human IgG4 isotype control, vehicle represents negative control without any antibody treatment, and M1 control represents positive control using monocyte-derived M1 macrophages.

[0071] Figures 18A-18C The effect of hu043.08 in monotherapy and in combination with Keytruda on alleviating M2 macrophage-mediated T cell suppression was shown. IL-2 ( Fig.18A )、IFNγ( Fig.18B ) and TNFα( Fig. 18C ) secretion. The cytokine level values ​​are listed at the top of each bar. hIgG4 iso represents human IgG4 isotype control and vehicle represents negative control without any antibody treatment. DETAILED DESCRIPTION

[0072] The disclosure will be explained in more detail below. This specification is not intended to be a list of all the different ways in which the invention can be implemented or all the features that can be added to the invention. For example, features shown in relation to one embodiment may be incorporated into other embodiments, and features shown in relation to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the disclosure without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used in the testing practice of the present disclosure, preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terms will be used.

[0074] LILRB2 (leukocyte immunoglobulin-like receptor B2, also known as MIR-10, ILT4 or CD85D) is a cell surface receptor that negatively regulates immune responses by recruiting tyrosine phosphatases such as SHP1 or SHP2. LILRB2 is primarily expressed in monocytes, macrophages and bone marrow cells, and by endothelial cells, placental trophoblasts and decidual macrophages, suggesting that LILRB2 may play an important role in a variety of physiological functions (Jian He et al., Overexpression of ANGPTL2 and LILRB2 as predictive and therapeutic biomarkers for metastasis and prognosis in colorectal cancer. Int J Clin Exp Pathol. 2018; 11(5): 2281-2294).

[0075] The function of LILRB2 is to inhibit myeloid cell activation, antigen presentation and immune response. ILT4 is also highly expressed in various tumor cells and stromal cells (such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM)) in the TME (tumor microenvironment), directly regulating tumor growth, progression and metastasis (Aiqin Gao et al., Tumor-derived ILT4 induces T cell senescence and suppresses tumor immunity. J Immunother Cancer. 2021; 9(3): e001536).

[0076] LILRB2 is a potential checkpoint molecule for tumor immunotherapy. LILRB2 blockade can promote anti-tumor immunity.

[0077] The present disclosure provides examples of antibodies and antigen-binding fragments thereof that bind to LILRB2, and uses of the antibodies and antigen-binding fragments thereof in the manufacture of therapeutic agents (e.g., drugs) for binding to LILRB2.

[0078] definition

[0079] As used herein, the term "antibody" is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as these structures express the desired antigen-binding activity.

[0080] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, an antigen binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain).

[0081] As used herein, the term "humanized antibody" refers to a non-human antibody that contains minimal sequence derived from non-human (eg, mouse) immunoglobulin and contains sequence derived from human immunoglobulin.

[0082] As used herein, the term "subject" refers to humans and non-human animals. Non-human animals include all mammals such as mice, rats, rabbits, cats, dogs, pigs, monkeys, chimpanzees, gorillas, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

[0083] As used herein, the term "polynucleotide" refers to a nucleotide polymer of any length of at least two nucleotides, and includes, but is not limited to, DNA, RNA, DNA / RNA hybrids, and modified forms thereof.

[0084] As used herein, the term "vector" refers to a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers) that regulate expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase).

[0085] As used herein, the term "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate cells.

[0086] As used herein, the term "IL-2" is interleukin 2, and the terms "IL-2" and "IL2" are used interchangeably herein. The term "IL-3" is interleukin 3, and the terms "IL-3" and "IL3" are used interchangeably herein. The term "IL-4" is interleukin 4, and the terms "IL-4" and "IL4" are used interchangeably herein. The term "IL-5" is interleukin 5, and "IL-5" and "IL5" are used interchangeably herein. The term "IL-6" is interleukin 6, and the terms "IL-6" and "IL6" are used interchangeably herein. The term "IL-8" is interleukin 8, and the terms "IL-8" and "IL8" are used interchangeably herein. The term "IL-10" is interleukin 10, and the terms "IL-10" and "IL10" are used interchangeably herein. The term "IL-13" is interleukin 13, and the terms "IL-13" and "IL13" are used interchangeably herein. The term "IL-15" is interleukin 15, and the terms "IL-15" and "IL15" are used interchangeably herein. The term "IL-1β" or the term "IL-1F2" is interleukin 1β, and the terms "IL-1β (IL-1beta)", "IL-1β", "IL-1F2", "IL1β (IL1beta)", "IL1β" and "IL1F2" are used interchangeably herein. The term "IL-1α" or the term "IL-1Fa" is interleukin 1α, and the terms "IL-1α (IL-1alpha)", "IL-1Fa", "IL-1α", "IL1α (IL1alpha)", "IL1Fa" and "IL1α" are used interchangeably herein.

[0087] As used herein, the term "IFNγ" is interferon gamma. The term "IFN-α" is interferon α1, and the terms "IFN-α (IFN-alpha)", "IFN-α", "IFNα" and "IFNα" are used interchangeably herein. The term "IFN-β" is interferon β, and the terms "IFN-β (IFN-beta)", "IFN-β", "IFNβ", "IFNβ" are used interchangeably herein.

[0088] As used herein, the term "TNFα" is tumor necrosis factor, and the terms "TNFα" and "TNF-α" are used interchangeably herein.

[0089] As used herein, the term "PDGF-AA" is platelet-derived growth factor subunit Aα, the term "PDGF-AB" is platelet-derived growth factor subunit Aβ, and the term "PDGF-BB" is platelet-derived growth factor subunit Bβ.

[0090] As used herein, the term "TGF-α" is transforming growth factor alpha, and the terms "TGFα" and "TGF-α" are used interchangeably herein.

[0091] As used herein, the term "CCL2" or the term "MIP-1" or the term "JE" is a CC motif chemokine ligand 2, the term "CCL3" or the term "MIP-1α" is a CC motif chemokine ligand 3, the term "CCL4" or the term "MIP-1β" is a CC motif chemokine ligand 4, the term "CCL5" is a CC motif chemokine ligand 5, the term "CCL19" or the term "MIP-3β" is a CC motif Chemokine ligand 19, the term "CCL20" or the term "MIP-3α" is the CC motif chemokine ligand 20, the term "MIP-3" is the CC motif chemokine ligand 23, the term "RANTES" is the CC motif chemokine ligand 5, the term "CCL22" or the term "MDC" is the CC motif chemokine ligand 22, and the term "CCL23" or the term "MPIF-1" is the CC motif chemokine ligand 23.

[0092] As used herein, the term "basic FGF" is fibroblast growth factor 2.

[0093] As used herein, the term "G-CSF" is colony stimulating factor 3 and the term "GM-CSF" is colony stimulating factor 2.

[0094] As used herein, the term "B7-H1" or the term "PD-L1" is programmed cell death 1 ligand 1.

[0095] As used herein, the term "CXCL2" or the term "GRO[beta]" is CXC motif chemokine ligand 2, and the term "CXCL8" is CXC motif chemokine ligand 8.

[0096] As used herein, the term "VEGF" is vascular endothelial growth factor.

[0097] Antibodies and their antigen-binding fragments

[0098] Provided herein are antibodies or antigen-binding fragments thereof that bind to LILRB2 or anti-LILRB2 antibodies or antigen-binding fragments thereof.

[0099] Typically, antibodies are composed of two types of polypeptide chains, light chains and heavy chains. An antibody may contain two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, each containing a variable region (or variable domain, VH) and multiple constant regions (or constant domains), are bound to each other via disulfide bonds within their constant domains. The light chains, each containing a variable region (or variable domain, VL) and a constant region (or constant domain), are each bound to a heavy chain via a disulfide bond. The variable regions of both the light and heavy chains contain three highly variable regions sandwiched between more conservative framework regions (FR).

[0100] These highly variable regions, called complementarity determining regions (CDRs), form the loops that comprise the primary antigen-binding surface of an antibody. CDRs are important for recognizing antigenic epitopes. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by an antibody's antigen-binding domain.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0102] a heavy chain variable region (VH), comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 1; the VH CDR2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 2; and the VH CDR3 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 3; and

[0103] A light chain variable region (VL), comprising CDRs 1, 2 and 3, wherein the VL CDR1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:4; VL CDR2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:5; and VL CDR3 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:6.

[0104] The Kabat definition is used to define the CDRs. The Kabat definition is well known to those skilled in the art, see, for example, Kabat, EA et al. Sequences of Proteins of Immunological Interest, 1991.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof may have a VH comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 are shown in SEQ ID NOs: 1, 2, and 3, respectively.

[0106] Furthermore, the antibody or antigen-binding fragment thereof may have a VL comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 are shown in SEQ ID NOs: 4, 5, and 6, respectively.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof has a VH comprising CDRH1 (heavy chain CDR1) of SEQ ID NO: 1, CDRH2 (heavy chain CDR2) of SEQ ID NO: 2, and CDRH3 (heavy chain CDR3) of SEQ ID NO: 3.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof has a VL comprising CDRL1 (light chain CDR1) of SEQ ID NO:4, CDRL2 (light chain CDR2) of SEQ ID NO:5, and CDRL3 (light chain CDR3) of SEQ ID NO:6.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof can have a VH, wherein the VH is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:7, 8, 9, 10, 11 or 12.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof can have a VL, wherein the VL is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:13, 14, 15 or 16.

[0111] Methods for determining the percent identity between two amino acid sequences are known in the art, including but not limited to BLAST (basic local alignment search tool; BLAST) on NCBI.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof can have a VH, wherein the VH differs from the amino acid sequence shown in SEQ ID NO:7, 8, 9, 10, 11 or 12 by no more than 25, 20, 15, 13, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids.

[0113] In addition, in some embodiments, the antibody or its antigen-binding fragment may have a VL, wherein the VL differs from the amino acid sequence shown in SEQ ID NO:13, 14, 15 or 16 by no more than 20, 15, 13, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH selected from the group of SEQ ID NOs: 7, 8, 9, 10 and 11; and / or a VL selected from the group of SEQ ID NOs: 13, 14 and 15.

[0115] Any of the VH sequences provided in the present disclosure (SEQ ID NOs: 7-11) can be paired with any of the VL sequences provided in the present disclosure (SEQ ID NOs: 13-15).

[0116] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may have a VH of the following sequence: SEQ ID NO: 7 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions; SEQ ID NO: 8 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions; SEQ ID NO: 9 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions; SEQ ID NO: 10 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions; or SEQ ID NO: 11 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions.

[0117] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may have a VL with the following sequence: SEQ ID NO: 13 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions; SEQ ID NO: 14 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions; or SEQ ID NO: 15 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions.

[0118] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof, wherein VH comprises the amino acid sequence of SEQ ID NO: 7 and VL comprises the amino acid sequence of SEQ ID NO: 13,

[0119] VH comprises the amino acid sequence of SEQ ID NO: 8 and VL comprises the amino acid sequence of SEQ ID NO: 13,

[0120] VH comprises the amino acid sequence of SEQ ID NO:9 and VL comprises the amino acid sequence of SEQ ID NO:13,

[0121] VH comprises the amino acid sequence of SEQ ID NO:7 and VL comprises the amino acid sequence of SEQ ID NO:14,

[0122] VH comprises the amino acid sequence of SEQ ID NO: 8 and VL comprises the amino acid sequence of SEQ ID NO: 14,

[0123] VH comprises the amino acid sequence of SEQ ID NO:9 and VL comprises the amino acid sequence of SEQ ID NO:14,

[0124] VH comprises the amino acid sequence of SEQ ID NO: 10 and VL comprises the amino acid sequence of SEQ ID NO: 15,

[0125] VH comprises the amino acid sequence of SEQ ID NO: 11 and VL comprises the amino acid sequence of SEQ ID NO: 15; or

[0126] VH comprises the amino acid sequence of SEQ ID NO:12 and VL comprises the amino acid sequence of SEQ ID NO:16.

[0127] The antibody or antigen-binding fragment thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody or an antigen-binding fragment thereof.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

[0130] In some embodiments, antigen binding fragments include, for example, Fab, Fab', F(ab')2, Fv fragments and scFv fragments.

[0131] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

[0132] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof.

[0133] In a non-limiting example, a humanized antibody is a human antibody in which a hypervariable region (e.g., CDR) residue is replaced by a hypervariable region (e.g., CDR) residue from a non-human antibody (e.g., mouse, rat, or rabbit antibody). In some embodiments, a humanized antibody is a human antibody in which a framework region residue is replaced by a corresponding non-human (e.g., mouse) framework region residue.

[0134] In some embodiments, a humanized antibody may also contain at least a portion of a human immunoglobulin constant region (Fc). Humanized antibodies can be produced using molecular biology methods known in the art.

[0135] In some embodiments, the antibody or antigen-binding fragment thereof is a polyclonal, monoclonal, or multispecific antibody or antigen-binding fragment thereof.

[0136] In some embodiments, the antibodies or antigen-binding fragments thereof provided by the present disclosure specifically bind to LILRB2, exemplary LILRB2s include human LILRB2.

[0137] In some embodiments, the anti-LILRB2 antibody or antigen-binding fragment thereof can block signaling pathways and upregulate immune responses. In some embodiments, the antibody or antigen-binding fragment thereof described herein is a LILRB2 agonist. In some embodiments, the antibody or antigen-binding fragment thereof described herein is a LILRB2 antagonist.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof has an EC less than 20 nM, less than 10 nM, less than 5 nM, or less than 1 nM as measured by standard techniques in the art, such as the Octet rule, Biacore, or FACS. 50 (50% effective concentration) values ​​specifically bind to LILRB2.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof binds to human LILRB2 with a KD (affinity constant) of less than 15 nM, less than 10 nM, less than 8 nM, or less than 6 nM as measured by standard techniques in the art, such as the Octet Rule, Biacore, or FACS.

[0140] Typically, the KD value or affinity constant is the equilibrium dissociation constant or Kd / Ka ratio between an antibody and its antigen. Kd refers to the dissociation constant and Ka refers to the association constant.

[0141] In addition, the antibodies or antigen-binding fragments thereof provided herein block the interaction between human LILRB2 and a typical MHC (major histocompatibility complex (MHC)) class I molecule. In some embodiments, the antibodies or antigen-binding fragments thereof block the interaction between human LILRB2 and HLA-A2. In some embodiments, the antibodies or antigen-binding fragments thereof block the interaction between human LILRB2 and HLA-G.

[0142] In some embodiments, the antibody or antigen-binding fragment thereof has an IC value of less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, or less than 0.3 nM as measured by standard techniques in the art, such as FACS (fluorescence activated cell sorting) assay. 50 The value blocks the interaction between human LILRB2 and HLA-G.

[0143] Typically, IC 50 The value represents the concentration at which the substance exerts half of its maximal inhibitory effect. This value is typically used to characterize antagonists of biological processes.

[0144] In some embodiments, the antibody or antigen-binding fragment thereof has an IC of less than 20 nM, less than 10 nM, less than 5 nM, less than 1 nM, or less than 0.6 nM as measured by standard techniques in the art, such as FACS (fluorescence activated cell sorting) assay. 50 The value blocks the interaction between human LILRB2 and HLA-A2.

[0145] The antibody or antigen-binding fragment thereof blocks the interaction between LILRB2 (eg, human LILRB2) and an ANGPTL (angiopoietin-like protein) family molecule (eg, human ANGPTL).

[0146] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL1 (angiopoietin-like protein 1).

[0147] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL2 (angiopoietin-like protein 2).

[0148] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL3 (angiopoietin-like protein 3).

[0149] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL4 (angiopoietin-like protein 4).

[0150] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL6 (angiopoietin-like protein 6).

[0151] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL7 (angiopoietin-like protein 7).

[0152] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human MAG (myelin-associated glycoprotein).

[0153] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRA1 (leukocyte immunoglobulin-like receptor A1).

[0154] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRA2 (leukocyte immunoglobulin-like receptor A2).

[0155] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRA3 (leukocyte immunoglobulin-like receptor A3).

[0156] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRA4 (leukocyte immunoglobulin-like receptor A4).

[0157] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRA5 (leukocyte immunoglobulin-like receptor A5).

[0158] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRB1 (leukocyte immunoglobulin-like receptor B1).

[0159] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRB3 (leukocyte immunoglobulin-like receptor B3).

[0160] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRB4 (leukocyte immunoglobulin-like receptor B4).

[0161] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are unable, barely able, or unlikely to bind to LILRB5 (leukocyte immunoglobulin-like receptor B5).

[0162] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB2 can upregulate the release of pro-inflammatory cytokines and / or chemokines (for example, but not limited to, IL-2, IFN-γ and / or TNF-α) or enhance the expression of pro-inflammatory cytokines and / or chemokines.

[0163] In some embodiments, the pro-inflammatory cytokine and / or chemokine is selected from the group consisting of IL-2, IFN-γ, TNF-α, granzyme B, PDGF-AA, IL-3, IL-1β, IL-5, TGF-α, IFN-α, CCL4, basic FGF, IL-13, G-CSF, IL-4, PD-L1, CCL20, IL-15, GM-CSF, CCL5, CCL3, PDGF-AB, CCL19, CXCL2, IL-6, VEGF, and combinations thereof.

[0164] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein upregulate an immune response by reducing the expression of anti-inflammatory interleukins (eg, IL-10).

[0165] In some embodiments, the antibodies or antigen binding fragments thereof provided herein can promote human PBMC to enter a pro-inflammatory state. In some embodiments, the state of human PBMC can be measured by detecting the release of anti-inflammatory interleukins (e.g., IL-10) / or pro-inflammatory cytokines (e.g., TNF-α).

[0166] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can promote human monocytes to enter a pro-inflammatory state. In some embodiments, the state of human PBMC can be measured by detecting the release of anti-inflammatory interleukins (e.g., IL-10) and / or pro-inflammatory cytokines (e.g., TNF-α).

[0167] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of reprogramming human monocyte-derived M2 macrophages to a pro-inflammatory M1 phenotype. In some embodiments, the effect of the above-mentioned antibodies or antigen-binding fragments thereof on macrophage phenotype conversion is measured by quantifying CD86 高 CD163 低 CD86 and CD163 are known markers expressed on M1 and M2 macrophages, and those skilled in the art of the present disclosure know that M1 and M2 macrophages can be distinguished by detecting the expression levels of CD86 and CD163.

[0168] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can reduce the inhibitory function of M2 macrophages. In some embodiments, the release of IL-2, IFNγ, granzyme B, TNFα, PDGF-AA, IL-3, IL-1β, IL-1F2, IL-5, TGF-α, IFN-α, CCL4, MIP-1β, basic FGF, IL-13, G-CSF, IL-4, B7-H1, PD-L1, CCL20, MIP-3α, IL-15, GM-CSF, CCL5, RANTES, CCL3, MIP-1α, PDGF-AB, PDGF-BB, CCL19, MIP-3β, CXCL2, GROβ, IL-6 and / or VEGF is measured to reduce the inhibitory function of M2 macrophages. In some embodiments, the release of IL-2, IFNγ and TNFα is measured to reduce the inhibitory function of M2 macrophages.

[0169] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can promote immature dendritic cells (DCs) to enter a mature state. 高 CD163 低 The effect of the above-mentioned antibody or its antigen-binding fragment on the maturation of dendritic cells is determined by measuring the percentage of the population (M1%) and the release of pro-inflammatory cytokines and chemokines, wherein the pro-inflammatory cytokines and chemokines are selected from the following groups: TNFα, IL-6, IL-8, CXCL8, IFN-β, IL-10, CCL2, JE, MCP-1, VEGF, CCL20, MIP-3α, CCL3, MIP-1α, CCL22, MDC, IL-1α, IL-1F1, CCL23, MPIF-1 and combinations thereof.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof upregulates an immune response to a LILRB2-associated disease, disorder, or condition (eg, a tumor).

[0171] In some embodiments, the antibody or antigen-binding fragment thereof upregulates the immune response by reprogramming human monocyte-derived M2 macrophages to an M1 phenotype. In some embodiments, the antibody or antigen-binding fragment thereof upregulates the immune response by reprogramming tumor-associated macrophages from an M2 phenotype to an M1 phenotype.

[0172] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein reduce the suppressive function of M2 macrophages. In some embodiments, the antibodies or antigen-binding fragments thereof upregulate the immune response by reducing T cell suppression mediated by the M2 phenotype of tumor-associated macrophages.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof upregulates an immune response by promoting dendritic cells (eg, human dendritic cells) to enter a mature state.

[0174] In some embodiments, the antibody or antigen-binding fragment thereof upregulates an immune response by promoting monocytes (eg, human monocytes) to enter a pro-inflammatory state.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof upregulates an immune response by promoting PBMCs (eg, human PBMCs) into a pro-inflammatory state.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof blocks binding of LILRB2 to tumor-associated macrophages. In some embodiments, LILRB2 blockade reprograms tumor-associated macrophages (TAMs) to a pro-inflammatory phenotype. In some embodiments, LILRB2 blockade inhibits regulatory T cell (Treg) infiltration, e.g., in the tumor microenvironment.

[0177] In one aspect, the present disclosure also provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof that binds to LILRB and an anti-PD-1 antibody. An exemplary anti-PD-1 antibody is Keytruda.

[0178] The above antibodies or antigen-binding fragments thereof combined with anti-PD-1 antibodies can induce a greater release of pro-inflammatory cytokines / chemokines.

[0179] The combination of the above antibodies or antigen-binding fragments thereof with anti-PD-1 antibodies can alleviate the inhibitory function of M2 macrophages.

[0180] The above antibodies or antigen-binding fragments thereof synergize with anti-PD-1 antibodies to revitalize T cell function.

[0181] The combination of the above antibodies or antigen-binding fragments thereof with anti-PD-1 antibodies can improve the immune response.

[0182] In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds to a peptide comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the peptide of SEQ ID NO: 21 may be a binding epitope on LILRB2. In some embodiments, the peptide of SEQ ID NO: 21 may be a partial binding epitope on LILRB2.

[0183] In one aspect, provided herein is a binding epitope on LILRB2, wherein the binding epitope comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, LILRB2 is human LILRB2.

[0184] In this disclosure, the term "binding epitope" refers to a molecular region on the surface of an antigen (or therapeutic target) that is capable of eliciting an immune response and combining with specific antibodies generated by such a response.

[0185] Polynucleotides, vectors and host cells

[0186] The present disclosure provides isolated polynucleotides encoding antibodies or antigen-binding fragments thereof that bind to LILRB2 or anti-LILRB2 antibodies or antigen-binding fragments thereof.

[0187] A polynucleotide is a polymer of DNA, RNA, a DNA / RNA hybrid, or a modified form thereof. In some embodiments, a polynucleotide is a polymer of DNA. In some embodiments, a polynucleotide is a polymer of RNA.

[0188] DNA or RNA encoding an antibody or antigen-binding fragment thereof that binds to LILRB2 can be readily isolated and sequenced using known procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody). Encoding DNA or RNA can also be obtained synthetically.

[0189] Using recombinant techniques known in the art, an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to LILRB2 can be inserted into a vector for further cloning (amplification of the DNA) or expression. Many vectors are available. Vector components 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.

[0190] In one aspect, provided herein is an isolated vector comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to LILRB2 or an anti-LILRB2 antibody or antigen-binding fragment thereof.

[0191] The present disclosure provides a vector comprising a polynucleotide of separation provided herein. In some embodiments, the vector provided herein encodes an antibody or its antigen-binding fragment, at least one promoter (e.g., SV40, CMV, EF-1α) operably connected to a nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, parvoviruses (e.g., SV40), lambda phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0192] The vector comprising the polynucleotide sequence encoding the above-mentioned antibody or its antigen-binding fragment can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA or RNA in the vector herein are the above-mentioned prokaryotes, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella, as well as Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces.

[0193] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for anti-LILRB2 antibody encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as 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. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger.

[0194] In one aspect, provided herein is a host cell comprising the above-described isolated polynucleotide or the above-described isolated vector.

[0195] Suitable host cells for expressing antibodies (eg, glycosylated antibodies) or antigenic fragments thereof provided herein are derived from multicellular organisms. However, vertebrate cells are of greatest interest, and propagation of vertebrate cells in culture (tissue culture) has become a routine method. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture [293 or 293 cells subcloned for growth in suspension culture], Graham et al., J. Gen Virol. [Journal of General Virology] 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. [Reproduction Biology] 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293 and its derivatives.

[0196] Host cells are transformed with the above expression or cloning vectors for producing antibodies or antigen-binding fragments thereof that bind to LILRB2 or anti-LILRB2 antibodies or antigen-binding fragments thereof and cultured in a known nutrient medium modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, antibodies can be produced by homologous recombination as is known in the art. In certain embodiments, host cells are capable of producing antibodies or antigen-binding fragments thereof provided herein.

[0197] In one aspect, the present disclosure also provides a method for expressing an antibody or antigen-binding fragment thereof provided herein, the method comprising culturing a host cell provided herein under conditions of expressing a vector of the present disclosure. The host cell used to produce an antibody or antigen-binding fragment thereof provided herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), minimal essential medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's 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. 102:255 (1980); U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Re. 30,985 can be used as a culture medium for the host cells. Any of these media may be supplemented as necessary 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 (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at appropriate concentrations. The culture conditions (such as temperature, pH, etc.) are those previously used with the host cell selected for expression, and these culture conditions will be apparent to those skilled in the art.

[0198] Pharmaceutical composition

[0199] In one aspect, the present disclosure further provides a pharmaceutical composition comprising the above-mentioned antibody or its antigen-binding fragment, and one or more pharmaceutically acceptable carriers. In some embodiments, the present disclosure provides a pharmaceutical composition comprising the above-mentioned antibody or its antigen-binding fragment and an anti-PD-1 antibody (e.g., Keytruda).

[0200] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to LILRB2, and one or more pharmaceutically acceptable carriers.

[0201] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising an isolated vector containing a polynucleotide encoding an antibody or an antigen-binding fragment thereof that binds to LILRB2 or an anti-LILRB2 antibody or an antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers.

[0202] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising a host cell containing a polynucleotide encoding an antibody or an antigen-binding fragment thereof that binds to LILRB2 (anti-LILRB2 antibody or antigen-binding fragment thereof), or a vector comprising the above polynucleotide.

[0203] The pharmaceutical compositions provided herein can be formulated in any manner known in the art, such as pharmaceutical compositions provided herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage).

[0204] The pharmaceutical composition is formulated to be compatible with its intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0205] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0206] Suitable components may include, for example, antioxidants, fillers, adhesives, disintegrating agents, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers (such as sugars and cyclodextrins). Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA (ehtylenediaminetetraacetic acid; EDTA), sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylhydroxymethoxybenzene, butylhydroxytoluene and / or propyl gallate. As disclosed herein, one or more antioxidants (such as methionine) are included in the composition comprising the antibody or its antigen-binding fragment and conjugate provided herein to reduce the oxidation of the antibody or its antigen-binding fragment. This reduction in oxidation can prevent or reduce the loss of binding affinity, thereby improving antibody stability and maximizing the shelf life. Therefore, in certain embodiments, a pharmaceutical composition comprising one or more antibodies or their antigen-binding fragments as disclosed herein and one or more antioxidants (such as methionine) is provided.

[0207] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection), non-aqueous vehicles (e.g., fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., sodium bisulfate), local anesthetics (e.g., procaine hydrochloride), suspending and dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinyl pyrrolidone), emulsifiers (e.g., polysorbate 80 (Tween-80)), sequestrants or chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethyleneglycol tetraacetic acid)). The antimicrobial agent used as a carrier can be added to the pharmaceutical composition in a multi-dose container, which contains phenol or cresol, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or agents (such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin).

[0208] In some embodiments, the pharmaceutical composition may be a liquid solution, suspension, or emulsion.

[0209] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any known form, such as liquid solutions, suspensions, emulsions, or solid forms suitable for producing liquid solutions, suspensions, or emulsions. Injectable preparations can include sterile and / or pyrogen-free solutions for injection, sterile dry soluble products (such as lyophilized powders) (including subcutaneous tablets) prepared for combination with solvents before use, sterile suspensions for injection, sterile dry insoluble products prepared for combination with carriers before use, and sterile and / or pyrogen-free emulsions. The solution can be aqueous or non-aqueous.

[0210] In some embodiments, the unit dose of parenteral formulations is packaged in an ampoule, a vial, or a syringe with a needle.All preparations for parenteral administration should be sterile and pyrogen-free, as known and practiced in the art.

[0211] In some 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 that improve the stability of the powder or a reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may contain a buffer (such as citrate, sodium phosphate, or potassium phosphate) at about neutral pH in one embodiment, or other such buffers known to those skilled in the art. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution will be dispensed into vials for freeze-drying. Each vial may contain a single dose or multiple doses of an antibody or antigen-binding fragment thereof that binds to LILRB2, or a combination thereof. It is acceptable to overfill vials with a small amount (e.g., about 5%, 10%, or 15%) higher than that required for one dose or a group of doses to facilitate accurate sample extraction and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0212] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, sterile and / or pyrogen-free water or other liquid suitable carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the given selected therapy and can be determined empirically.

[0213] Reagent test kit

[0214] In one aspect, the present disclosure provides a kit comprising the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned isolated polynucleotide, the above-mentioned isolated vector and / or the pharmaceutical composition provided herein.

[0215] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of: a chemotherapeutic agent, an anticancer drug, a radiotherapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy, a cell therapy, a gene therapy, a hormone therapy, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, and a cytokine.

[0216] If desired, such a kit may further include one or more of a variety of known pharmaceutical kit components, such as containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to those skilled in the art. Instructions such as inserts or labels indicating the amounts of the components to be administered, directions for administration, and / or directions for mixing the components may also be included in the kit.

[0217] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein (optionally conjugated with a detectable moiety), which can be used to detect a LILRB2-related disease, disorder or condition. The kit may further comprise instructions for use.

[0218] Use in the manufacture of therapeutic agents

[0219] The present disclosure provides use of the above-mentioned antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition, or kit in the manufacture of a therapeutic agent for binding to human LILRB2.

[0220] In some embodiments, provided herein is a use of an antibody or an antigen-binding fragment thereof in the manufacture of a medicament for blocking the interaction between human LILRB2 and HLA-A2, HLA-G, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL6, ANGPTL7 and / or MAG.

[0221] In some embodiments, the therapeutic agent includes, but is not limited to, a drug, an antibody, a fusion protein, a vaccine, a CAR-T, and an antibody-drug conjugate. In some embodiments, the therapeutic agent is a biologically active substance that can treat or alleviate a LILRB2-related disease, disorder, or condition.

[0222] In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof, an isolated polynucleotide, an isolated vector, a host cell, a pharmaceutical composition, or a kit for the manufacture of a therapeutic agent for upregulating or enhancing an immune response to a disease, disorder or condition (e.g., a tumor).

[0223] In some embodiments, provided herein is a use of an antibody or antigen-binding fragment thereof, an isolated polynucleotide, an isolated vector, a host cell, a pharmaceutical composition, or a kit for the manufacture of a therapeutic agent for upregulating or enhancing an immune response to a LILRB2-related disease, disorder or condition (e.g., a tumor).

[0224] In some embodiments, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition, or kit in the manufacture of a therapeutic agent for enhancing the immune response to a tumor.

[0225] In some embodiments, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof in the manufacture of a therapeutic agent for enhancing immune response by reprogramming human monocyte-derived M2 macrophages to an M1 phenotype.

[0226] In some embodiments, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof in the manufacture of a therapeutic agent for alleviating the inhibitory function of M2 macrophages.

[0227] In some embodiments, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof in the manufacture of a therapeutic agent for enhancing immune response and alleviating T cell suppression mediated by the M2 phenotype of tumor-associated macrophages by reprogramming tumor-associated macrophages from M2 phenotype to M1 phenotype macrophages.

[0228] In some embodiments, the present invention provides the use of the above-mentioned antibodies or antigen-binding fragments thereof in the manufacture of therapeutic agents for promoting PBMC to enter a pro-inflammatory state. In addition, in some embodiments, the present invention provides the use of the above-mentioned antibodies or antigen-binding fragments thereof in the manufacture of therapeutic agents for promoting human PBMC to enter a pro-inflammatory state.

[0229] In some embodiments, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof in the manufacture of a therapeutic agent for promoting dendritic cells (eg, human dendritic cells) to enter a mature state.

[0230] In some embodiments, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof in the manufacture of a therapeutic agent for promoting monocytes (eg, human monocytes) to enter a pro-inflammatory state.

[0231] In some embodiments, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof in the manufacture of a therapeutic agent for promoting PBMC (eg, human PBMC) to enter a pro-inflammatory state.

[0232] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein enhance the immune response by upregulating the expression of proinflammatory cytokines (including but not limited to IL-2, IFN-γ, TNF-α, granzyme B, PDGF-AA, IL-3, IL-1β, IL-5, TGF-α, IFN-α, CCL4, basic FGF, IL-13, G-CSF, IL-4, PD-L1, CCL20, IL-15, GM-CSF, CCL5, CCL3, PDGF-AB, CCL19, CXCL2, IL-6 and VEGF).

[0233] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein enhance immune responses by downregulating the expression of anti-inflammatory interleukins (eg, IL-10).

[0234] The present disclosure provides use of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned isolated polynucleotide, the above-mentioned isolated vector, or the above-mentioned pharmaceutical composition, or the above-mentioned kit in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

[0235] In some embodiments, the disease, disorder or condition is a LILRB2-associated disease.

[0236] In some embodiments, the disease comprises a tumor, which may be, without limitation, a blood tumor or a solid tumor. In some embodiments, the tumor may be malignant or benign. In some embodiments, the disease is cancer (e.g., lymphoma).

[0237] Treatment

[0238] The present disclosure provides a method for diagnosing, preventing or treating a LILRB2-related disease, disorder or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, an isolated polynucleotide provided herein, an isolated vector provided herein, a host cell provided herein, a pharmaceutical composition provided herein and / or a kit provided herein.

[0239] In some embodiments, the LILRB2-associated disease, disorder, or condition is characterized by expression or overexpression of LILRB2.

[0240] In some embodiments, the LILRB2-associated disease, disorder or condition comprises cancer. In some embodiments, the cancer is a LILRB2-expressing cancer. The term "LILRB2-expressing cancer" refers to a cancer characterized by expression of LILRB2 protein in cancer cells, tumor-infiltrating immune cells, or immunosuppressive cells, or expression of LILRB2 in cancer cells, tumor-infiltrating immune cells, or immunosuppressive cells at levels significantly higher than expected in normal cells.

[0241] In some embodiments, the subject has been identified as having cancer cells or tumor-infiltrating immune cells or immunosuppressive cells that express LILRB2, optionally at levels significantly higher than those normally found on non-cancerous cells or non-immunosuppressive cells.

[0242] The present disclosure further provides a method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated polynucleotide provided herein, or an isolated vector provided herein, or a host cell provided herein, or a pharmaceutical composition provided herein, or a kit provided herein.

[0243] In some embodiments, an antibody or antigen-binding fragment thereof that binds to LILRB2, or an isolated polynucleotide provided herein, or an isolated vector provided herein, or a host cell provided herein, or a pharmaceutical composition provided herein, or a kit provided herein upregulates the expression of pro-inflammatory cytokines and / or downregulates the expression of anti-inflammatory interleukins.

[0244] Without limitation, in some embodiments, the expression level of IL-2, IFN-γ and / or TNF-α is increased. In some embodiments, the expression level of IL-10 is decreased.

[0245] In some embodiments, a subject may benefit from modulation of LILRB2 activity.

[0246] In some embodiments, the subject is a mammal. In some embodiments, the subject comprises a human. In some embodiments, the subject is a human (or human patient) and may be an adult or a juvenile (e.g., a person under the age of 18). In some embodiments, the subject is a non-human animal, including but not limited to non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, rabbits), swine (e.g., pigs), equines, canines, felines, bovines, and other domestic, farm, and zoo animals.

[0247] The therapeutically effective amount of the antibodies or antigen-binding fragments provided herein will depend on various factors known in the art, such as body weight, age, past medical history, current medication, the health status of the subject and the possibility of cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease development. According to these and other circumstances or required indications, a person skilled in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage.

[0248] In certain embodiments, the antibodies or antigen binding fragments provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose can be changed during the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequent administered dose. In certain embodiments, the administered dose can be changed during the course of treatment, depending on the subject's response.

[0249] Dosage regimens may be adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0250] The antibodies or antigen-binding fragments thereof provided herein can be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

[0251] Example

[0252] Example 1. Generation of reagents

[0253] 1.1 Reference Antibodies

[0254] Reference antibodies 1E1 and J-19 were produced according to patents US2018 / 0298096 A1 (SEQ ID NOs: 69 and 77) and WO2019 / 126514A2 (SEQ ID NOs: 13 and 14), respectively. The variable region sequences of 1E1 and J-19 are shown in Table 1. They are both human IgG4 monoclonal antibodies with S228P mutation in the constant region.

[0255] Table 1.1 Variable region sequences of E1 and J-19

[0256]

[0257] 1.2 Stable cell lines

[0258] Cell lines 293-F / LILRB2 and EL4 / LILRB2 stably expressing human LILRB2 (SEQ ID NO: 23) were generated for fusion tumor screening and in vitro assays. HEK293-F cells were transfected with a GFP-tagged human LILRB2 expression plasmid (Sino Biological) and selectively cultured in medium containing 200 μg / mL hygromycin for 2 weeks to generate 293-F / LILRB2. Single cell clones were then isolated by limiting dilution and screened by FACS to obtain monoclonal cell lines stably expressing human LILRB2. EL4 cells were transfected with an untagged human LILRB2 expression plasmid (Genery) and selectively cultured in medium containing 500 μg / mL hygromycin to generate a stable pool of EL4 / LILRB2.

[0259] 1.3 Recombinant proteins

[0260] Human LILRB2 extracellular domain (ECD) recombinant protein with human Fc tag (AAH36827.1, Gly24-His458) or 6xHis-tag (Q8N423.4, Gln22-His458) was purchased from R&D systems for immunization and fusion tumor screening.

[0261] A series of recombinant proteins of LILRA / LILRB family members and potential ligands of LILRB2 were also purchased for in vitro assays. Human LILRA1 (NP_006854.1, Met1-Asn461), LILRA3 (AAH28208.1, Met1-Glu439), LILRA4 (P59901.2, Met1-Asn446), LILRA5 (NP_067073.1, Met1-Arg268), LILRB1 (ADJ55949.1, Met1-His458), LILRA2 (NP_006854.1, Met1-Asn461), LILRA3 (AAH28208.1, Met1-Glu439), LILRA4 (P59901.2, Met1-Asn446), LILRA5 (NP_067073.1, Met1-Arg268), LILRA2 (NP_006854.1, Met1- RB2 (AAH36827.1, Met1-Val461), LILRB3 (AAI04994.1, Met1-Glu443), LILRB4 (AAI04994.1, Met1-Glu443), LILRB5 (NP_006831.1, Met1-Gly458), and MAG (P20916-1, Met1-Pro516) ECD recombinant proteins were purchased from Sino Biological Technology Co., Ltd. Recombinant proteins of human LILRA2 ECD (NP_001124389, Gly24-Asn449), ANGPTL2 (Q9UKU9-1, Thr260-His493), ANGPTL4 (Q9BY76, Gly26-Ser406, Lys163Ala, Arg164Ala), ANGPTL7 with His-tag (O43827, Met1-Pro346) and ANGPTL1 with Flag-tag (O95841, Gly24-Asp491) were purchased from R&D Systems. Recombinant proteins of HLA-A2 complex (MHC-HM431) and HLA-G complex tetramer (HLG-HM41CT) were purchased from Kactus.

[0262] Example 2. Fusion tumor development and screening

[0263] 2.1 Immunity and Fusion

[0264] Four mice from different strains (CD-1, NZB / w, C57BL / 6, SJL) were immunized with Fc-tagged human LILRB2 ECD recombinant protein using a rapid immunization strategy. The serum titers of immunized mice were detected by ELISA analysis using 6xHis-tagged human LILRB2 ECD recombinant protein as antigen. When the serum titers reached a high level, a final boost was performed. Three days after the final boost, the combined spleen cells and lymph node cells were harvested and fused with SP2 / 0 mouse myeloma cells. The fused cells were then inoculated into 384-well plates for screening.

[0265] 2.2 Primary screening and secondary screening

[0266] 10-12 days after fusion, the supernatant harvested from each well of the primary screening was analyzed by ELISA using 6xHis-tagged human LILRB2 ECD recombinant protein as antigen. The fusion tumor cells in the positive wells were expanded and confirmed by rescreening using the same assay as the primary screening. The fusion tumor cells that secreted antibodies with the highest human LILRB2 binding activity were then subcloned.

[0267] 2.3 Fusion tumor subcloning and screening

[0268] The selected fusion tumor cells were limitedly diluted into a 96-well plate at a density of 1 cell / well to obtain monoclonal fusion tumor cells. The supernatants harvested from these monoclonal cells were screened by the same ELISA analysis as in Example 2.2 and FACS determination using 293-F / LILRB2. The antibodies secreted by the positive strains were quantified by biolayer interferometry, and then the human LILRB2 binding affinity and the activity of blocking the interaction between LILRB2 and its ligand were determined (refer to the method described in Example 3.2.4). As shown in Table 2, the monoclonal antibodies secreted by strain 8G2F10 showed nanomolar human LILRB2 binding affinity and good blocking activity.

[0269] Table 2.8G2F10 Characterization Summary

[0270]

[0271] The heavy chain variable region (VH) and light chain variable region (VL) of the monoclonal antibody secreted by strain 8G2F10 were sequenced. Their sequences are shown as SEQ ID NO: 12 (VH) and SEQ ID NO: 16 (VL).

[0272] Example 3. Chimeric Antibody Production and Characterization

[0273] 3.1 Chimeric Antibody Production

[0274] Based on the sequencing results of the monoclonal antibody secreted by strain 8G2F10 (VH, SEQ ID NO: 12; VL, SEQ ID NO: 16), a human IgG4 chimeric antibody with an S228P mutation in the Fc region was generated and named 043c, where the suffix "c" represents chimeric.

[0275] 3.2 Chimeric Antibody Characterization

[0276] 3.2.1 Binding activity

[0277] Using 6xHis-tagged human LILRB2 ECD recombinant protein as the antigen, the human LILRB2 binding activity of 043c and two benchmark antibodies (1E1 and J-19) was detected by ELISA analysis. Briefly, 0.5 μg / mL anti-LILRB2 antibody was incubated in an ELISA plate coated with human LILRB2 ECD recombinant protein at 37°C for 1 hour. After washing, horseradish peroxidase (HRP)-labeled detection antibody was added and incubated at 37°C for 1 hour. Color development was performed by adding 100 μl / well of TMB (3,3',5,5'-Tetramethylbenzidine (TMB)) solution. After incubation at room temperature (RT) for 10-15 minutes, the reaction was stopped by adding 100 μl 1N HCl. The optical density at 450 nm was then immediately read using a plate reader. Figure 1 As shown, 043c, 1E1 and J-19 strongly bound to the human LILRB2 ECD recombinant protein.

[0278] The binding of 043c to cell membrane human LILRB2 was detected by FACS using EL4 / LILRB2 and human primary monocytes. Figure 2 ) and isolated from healthy human donors (donors #1045 and #6004, Figure 3 Table 3 summarizes the EC values ​​analyzed using a four-parameter nonlinear fit in GraphPad Prism 9.0. 50 and the highest MFI value.

[0279] Table 3. Binding of 043c and 1E1 to membrane LILRB2

[0280]

[0281] 3.2.2 Affinity detection

[0282] The binding affinity of 043c, 1E1 and J-19 to human LILRB2 ECD recombinant protein was determined using biolayer interferometry (Octet). The association and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​of each antibody were calculated and summarized in Table 4. 043c (KD = 0.74 nM) showed higher antigen binding affinity than 1E1 (KD = 8.07 nM) and J-19 (KD = 2.14 nM) in the test.

[0283] Table 4. Human LILRB2 binding affinity of 043c, 1E1 and J-19

[0284] Ab Code ka(1 / Ms) kd(1 / s) KD(nM) 043c 7.56E+05 5.57E-04 0.74 1E1 2.53E+05 2.04E-03 8.07 J-19 8.30E+05 1.78E-03 2.14

[0285] 3.2.3 Binding specificity

[0286] Using LILRA1( Figure 4 A), LILRA2( Figure 4 B) LILRA3 Figure 4 C) LILRA4 Figure 4 D) LILRA5( Figure 4 E) LILRB1 Figure 4 F), LILRB3( Figure 4 G)、LILRB4( Figure 4 H)、LILRB5( Figure 4 I) The ECD recombinant protein was used as an antigen to detect the binding of 043c, 1E1 and J-19 to LILRA / LILRB family members by ELISA analysis (refer to the method described in Example 3.2.1). 043c, 1E1 and J-19 did not bind to other LILRA / LILRB family members except LILRB2, showing good binding specificity.

[0287] 3.2.4 Blocking activity

[0288] The activity of 043c, 1E1, and J-19 in blocking the interaction between LILRB2 and its ligands was evaluated by competitive FACS. Briefly, EL4 / LILRB2 cells were pre-incubated with 10 μg / mL 043c, 1E1, J-19, or IgG4 isotype for 30 minutes. Recombinant proteins of HLA-A2 complex, HLA-G complex tetramer, ANGPTL1, ANGPTL2, ANGPTL4, ANGPTL7, or MAGECD were then added and incubated for another 30 minutes. Blocking activity was determined by quantifying the blockade of ligand binding to EL4 / LILRB2 cells. Figure 5 As shown in the results, 043c, 1E1 and J-19 can almost completely block the interaction between LILRB2 and HLA-A2, HLA-G, ANGPTL2 or ANGPTL7, and can partially block the interaction between LILRB2 and ANGPTL1 or ANGPTL4. 043c and 1E1 also showed strong LILRB2 / MAG interaction blocking activity, while J-19 could not block this interaction well.

[0289] In addition, titration tests were performed to further evaluate the activity of 043c, 1E1, and J-19 in blocking the interaction between LILRB2 and MHC class I ligands ( Figure 6 and Figure 7 Table 5 summarizes the IC values ​​analyzed using a four-parameter nonlinear fit using GraphPad Prism 9.0.50 value and maximum blocking rate.

[0290] Table 5. Blocking activity of 043c, 1E1 and J-19

[0291]

[0292] 3.2.5 Human PBMC activation assay

[0293] The effects of 043c, 1E1 and J-19 on PBMC activation were studied. Briefly, peripheral blood mononuclear cells (PBMCs) collected from healthy human donors (donor #LP200714, LP200717, LP200818, LP200824) were pre-incubated with 6.67 μg / ml anti-LILRB2 antibody or hIgG4 isotype control at 37°C overnight and then primed with LPS (2 ng / ml) for 24 hours. The TNFα ( Figure 8 A) and IL-10( Figure 8 B) Quantification. 043c, 1E1 and J-19 up-regulated the release of the pro-inflammatory cytokine TNFα, while down-regulated the release of the anti-inflammatory cytokine IL-10, indicating that blocking LILRB2 with 043c promotes LPS-primed human PBMCs to enter a pro-inflammatory state.

[0294] 3.2.6 Human monocyte activation assay

[0295] The effects of 043c, 1E1 and J-19 on monocyte activation were also studied. Briefly, human monocytes isolated from PBMCs of healthy donors (donor #LP200714, LP200717, LP200818, LP200824) were pre-incubated with 6.67 μg / ml anti-LILRB2 antibody or hIgG4 isotype control at 37°C overnight and then primed with LPS (2 ng / ml) for 24 hours. The released TNFα ( Fig. 9 A) and IL-10( Fig. 9 Similar to the results of the PBMC activation assay, 043c, 1E1, and J-19 upregulated the release of the pro-inflammatory cytokine TNFα, while downregulated the release of the anti-inflammatory cytokine IL-10, indicating that blocking LILRB2 with 043c promotes LPS-primed human monocytes to enter a pro-inflammatory state.

[0296] 3.2.7 Macrophage polarization assay

[0297] The effects of 043c, 1E1, and J-19 on macrophage differentiation were investigated using a macrophage polarization assay. Briefly, human monocytes isolated from PBMCs of healthy donors were treated with M-CSF (colony stimulating factor 1) for 5 days in the presence of anti-LILRB2 antibody or hIgG4 isotype control, and then polarized for an additional 2 days with IL-4 and IL13, which are commonly used for anti-inflammatory M2 macrophage polarization. At the end of the study, CD86 高 CD163 低 The effect of the test antibodies on macrophage phenotype switching was determined by measuring the percentage of the M1 population (M1%) and the release of TNFα. Human monocytes were also directly polarized into pro-inflammatory M1 macrophages using GM-CSF and IFNγ as system controls (M1 controls). Similar to the M1 controls, 043c, 1E1, and J-19 significantly upregulated the M1 macrophage population ( Fig.10 A) and the release of the pro-inflammatory cytokine TNFα ( Fig.10 B), showing that blockade of LILRB2 with 043c reprograms human monocyte-derived M2 macrophages to a pro-inflammatory M1 phenotype.

[0298] 3.2.8 M2-T co-culture assay

[0299] The effects of 043c, 1E1, and J-19 in alleviating M2 macrophage-mediated T cell suppression were investigated using an M2 macrophage-T cell (M2-T) co-culture assay. Briefly, human monocytes isolated from PBMCs of healthy donors were treated with M-CSF for 5 days in the presence of 5 μg / ml anti-LILRB2 antibody or hIgG4 isotype control, and then polarized to an anti-inflammatory M2 phenotype with IL-4 and IL13 for an additional 2 days. Monocyte-derived macrophages differentiated with or without anti-LILRB2 antibody were mixed with autologous T cells at different ratios of 1:0.5, 1:1, 1:2, 1:4, and 1:8 in the presence of 10 ng / ml OKT3 (a mouse monoclonal antibody of the immunoglobulin IgG2a isotype). Released IL-2 in supernatants harvested after 48 hours of co-culture, and IFNγ and TNFα in supernatants harvested after 120 hours of co-culture were quantified using the HTRF kit (Cisbio).

[0300] At all M2-T mixing ratios, 043c, 1E1, and J-19 significantly upregulated IL-2 ( Fig.11 A) IFNγ( Fig.11 B) and TNFα( Fig.11C). The cytokine / chemokine release curve of the supernatant harvested after 120 hours of equal ratio co-culture (M2-T mixing ratio = 1:1) was also measured by Luminex. As shown in Table 6, more pro-inflammatory cytokines and chemokines (such as granzyme B, IL-3, IL-5 and CCL4) were found to be upregulated under the treatment of 043c. The results show that blocking LILRB2 with 043c alleviates the inhibitory function of M2 macrophages.

[0301] Table 6. Cytokine / chemokine release in M2-T co-culture assay

[0302]

[0303]

[0304] 3.2.9 DC differentiation assay

[0305] The effects of 043c, 1E1 and J-19 on dendritic cell (DC) maturation were investigated using a DC differentiation assay. Briefly, human monocytes isolated from PBMCs of healthy donors were treated with GM-CSF and IL-4 for six days to differentiate into immature DC cells. Monocyte-derived immature DC cells were then cultured with disk-coated or soluble anti-LILRB2 antibodies (5 μg / ml) for another two days, followed by priming with LPS (10 ng / ml) for 6 hours. At the end of the study, CD86 was quantified by FACS. 高 CD163 低 The effect of the test antibodies on dendritic cell maturation was determined by measuring the percentage of the population and by quantifying the release of pro-inflammatory cytokines and chemokines by Luminex. Fig.12 As shown in Table 7, 043c, 1E1 and J-19 significantly upregulated CD86 高 CD163 低 DC populations, as well as the release of pro-inflammatory cytokines and chemokines (e.g., TNFα, IL-6, IFN-β, and CCL3), indicating that blocking LILRB2 with 043c promotes the entry of immature DCs into a mature state.

[0306] Table 7. Cytokine / chemokine release in DC differentiation assay

[0307]

[0308]

[0309] 3.2.10 Epitope analysis

[0310] Epitope identification of 043c, 1E1 and J19 was performed by Octet. Briefly, the HIS1K sensor was hydrolyzed for 10 minutes, followed by capture of 6xHis-tagged human LILRB2 ECD recombinant protein to 0.3 nm. The LILRB2-captured sensor was then exposed to Ab1 and Ab2 in series or directly to Ab2. The binding signals of Ab2 and LILRB2 under different conditions were detected respectively. Finally, the relative binding signal of the series Ab2 was calculated by standardization. A high relative binding signal indicates low competition between Ab2 and Ab1 for binding to the antigen. Similarly, a low relative association signal indicates high competition between Ab2 and Ab1. As shown in Table 8, J-19 and 1E1 competed for binding to LILRB2, while 043c did not compete with J-19 and 1E1.

[0311] Table 8. Epitope identification of 043c, 1E1 and J-19

[0312]

[0313] The binding epitopes of 043c and 1E1 on LILRB2 were further mapped using hydrogen deuterium exchange mass spectrometry (HDX-MS). Binding of 043c resulted in a decrease in hydrogen deuterium exchange in the peptide "NGQFHIPSITWE" (binding site 1, SEQ ID NO: 21), indicating that this region on LILRB2 is critical for the binding of 043c ( Fig.13 A). Interestingly, binding of 1E1 resulted in reduced hydrogen-deuterium exchange in a different peptide "LYREKKSASWITRIRPEL" (binding site 2, SEQ ID NO: 22) ( Fig.13 B).

[0314] Combining the results obtained using Octet and HDX-MS, it can be concluded that 043c has a unique binding epitope different from 1E1 and J-19.

[0315] Example 4. Antibody humanization

[0316] 4.1 Humanized Design

[0317] 043c was humanized using the complementary determining region (CDR) transplantation method. Briefly, IGHV1-46*01 (IMGT allele name) and IGKV1-33*01 (IMGT allele name) were first selected as humanized templates for heavy and light chains based on their homology to the original mouse antibody sequence. CDRs were defined using the Kabat definition, except for the heavy chain CDR1, which was defined using a combination of the Kabat and Chothia systems. For transplantation, different combinations of CDRs and typical residues from 043c were transplanted onto the template. The resulting variants (human IgG4 antibodies with S228P mutations in the constant region) were generated and named hu043.01 to hu043.08, where the prefix "hu" means "humanized" and the numbers in the suffix represent the serial number. All variants were tested in multiple in vitro assays to select the best variants that retain the properties of the parent antibody.

[0318] 4.2 Characterization of humanized variants

[0319] 4.2.1 Binding activity

[0320] The binding of 043c and 043c-derived humanized variants to cell membrane human LILRB2 was detected by FACS assay using EL4 / LILRB2 cells. Fig.14 As shown, hu043.01, hu043.02, hu043.03, hu043.04, hu043.05 and hu043.06 retained the same human LILRB2 binding activity as the parent antibody 043c. Table 9 summarizes the EC values ​​analyzed using a four-parameter nonlinear fit in GraphPadPrism 9.0. 50 and the highest MFI value.

[0321] Table 9. Binding of 043c and 043c-derived humanized variants to EL4 / LILRB2

[0322] Ab Code 043c hu043.01 hu043.02 hu043.03 hu043.04 hu043.05 hu043.06 <![CDATA[EC 50 (nM)]]> 0.45 0.42 0.35 0.41 0.43 0.42 0.40 Highest MFI 45273 45828 45663 46324 45027 45096 45722

[0323] 4.2.2 Affinity detection

[0324] The binding affinity of the humanized variants derived from 043c to human LILRB2 was determined using biolayer interferometry (Octet) or surface plasmon resonance (Biacore). The association and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​for each antibody were calculated and summarized in Table 10. hu043.01, hu043.02, hu043.03, hu043.04, hu043.05, hu043.06, hu043.07 and hu043.08 retained the same human LILRB2 binding affinity as the parent antibody 043c.

[0325] Table 10. Human LILRB2 binding affinity of 043c-derived humanized variants

[0326]

[0327]

[0328] 4.2.3 Blocking activity

[0329] The activity of 043c and 043-derived humanized variants in blocking the interaction between LILRB2 and HLA-G was evaluated by competitive FACS ( Fig.15 A and Fig.15 B, refer to the method described in Example 3.2.4). Table 11 summarizes the IC values ​​analyzed using a four-parameter nonlinear fitting analysis using GraphPadPrism 9.0. 50 The values ​​and the highest blocking rates were as follows. hu043.01, hu043.02, hu043.03, hu043.04, hu043.05, hu043.06, hu043.07 and hu043.08 retained the same LILRB2 / HLA-G interaction blocking activity as the parent antibody 043c.

[0330] Table 11. Blocking activity of 043c and humanized variants derived from 043c

[0331]

[0332] 4.2.4 Functional Verification

[0333] The functions of hu043.07 and hu043.08 were verified using the monocyte activation assay (method described in reference to Example 3.2.6) and the macrophage polarization assay (method described in reference to Example 3.2.7). hu043.07 and hu043.08 upregulated the release of the pro-inflammatory cytokine TNFα in the monocyte activation assay ( Fig.16 ). They also significantly upregulated the M1 macrophage population ( Fig.17 A) and the release of the anti-inflammatory cytokine TNFα ( Fig.17 B) In both studies, hu043.07 and hu043.08 showed similar potency to 1E1, J-19, and the parental antibody 043c.

[0334] The function of hu043.08 or hu043.08 in combination with Keytruda (anti-PD-1) was further explored using the M2-T co-culture assay. Briefly, human monocytes isolated from PBMCs of healthy donors were treated with M-CSF for 5 days in the presence of 50nM hu043.08 or hIgG4 isotype control, and then polarized to the anti-inflammatory M2 phenotype with IL-4 and IL13 for 2 days. In the presence of 10ng / ml OKT3, monocyte-derived macrophages differentiated with or without hu043.08 were mixed with autologous T cells in a 1:1 ratio, with or without the addition of 5nM Keytruda. The IL-2 released in the supernatant harvested after 24 hours of co-culture, and the IFNγ and TNFα in the supernatant harvested after 96 hours of co-culture were quantified by the HTRF kit (Cisbio). In the test, Hu043.08 significantly upregulated IL-2 ( Fig.18A )、IFNγ( Fig.18B ) and TNFα( Fig. 18C ) release. However, Keytruda alone only slightly upregulated the release of pro-inflammatory cytokines (Figure 18). Interestingly, in the test, the combination of hu043.08 and Keytruda induced more release of these pro-inflammatory cytokines (Figure 18). The results showed that hu043.08 alleviated the inhibitory function of M2 macrophages, and hu043.08 synergized with anti-PD-1 antibodies to revitalize T cell function.

[0335] Exemplary heavy chain variable regions and light chain variable regions of 043c-derived humanized antibodies (i.e., hu043.01, hu043.02, hu043.03, hu043.04, hu043.05, hu043.06, hu043.07, and hu043.08) are shown in Table 12.

[0336] Table 12. VL and VH of humanized antibodies derived from 043c

[0337] Code Relink Code Light chain code hu043.01 hu043VH6 hu043VL4 hu043.02 hu043VH7 hu043VL4 hu043.03 hu043VH8 hu043VL4 hu043.04 hu043VH6 hu043VL2 hu043.05 hu043VH7 hu043VL2 hu043.06 hu043VH8 hu043VL2 hu043.07 hu043VH9 hu043VL1 hu043.08 hu043VH10 hu043VL1

[0338] *The prefix "hu" means "humanized", "VH" means "heavy chain variable region", "VL" means "light chain variable region", and the numbers in the suffix indicate the serial number.

[0339] The amino acid sequences in the present disclosure are listed in Table 13. The CDRs of VH and VL are underlined and the CDRs are defined using the Kabat definition.

[0340] Table 13. Amino acid sequence

[0341]

[0342]

[0343] *The prefix "hu" means "humanized", "VH" means "heavy chain variable region", "VL" means "light chain variable region", "H" means "heavy chain", "L" means "light chain", and "CDR" means "complementarity determining region". The numbers in the suffix indicate the serial number.

[0344] Amino acids are represented by standard single-letter codes according to the standard IUPAC (International Union of Pureans Applied Chemistry; IUPAC) amino acid abbreviations.

[0345] Although particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents may occur that are not or may not currently be foreseen by the applicant or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that binds to LILRB2 (leukocyte immunoglobulin-like receptor B2), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH), comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 1; the VH CDR2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 2; and the VH CDR3 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO: 3; and A light chain variable region (VL), comprising CDRs 1, 2 and 3, wherein the VL CDR1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:4; the VL CDR2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:5; and the VL CDR3 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, And the VL comprises CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the VH is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 11 or 12; and The VL is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:13, 14, 15 or 16.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the VH differs from the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 11 or 12 by no more than 25, 20, 15, 13, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; The VL differs from the amino acid sequence shown in SEQ ID NO:13, 14, 15 or 16 by no more than 20, 15, 13, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11 or 12; and The VL comprises the amino acid sequence shown in SEQ ID NO:13, 14, 15 or 16.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO: 13, The VH comprises the amino acid sequence of SEQ ID NO: 8 and the VL comprises the amino acid sequence of SEQ ID NO: 13, The VH comprises the amino acid sequence of SEQ ID NO:9 and the VL comprises the amino acid sequence of SEQ ID NO:13, The VH comprises the amino acid sequence of SEQ ID NO:7 and the VL comprises the amino acid sequence of SEQ ID NO:14, The VH comprises the amino acid sequence of SEQ ID NO: 8 and the VL comprises the amino acid sequence of SEQ ID NO: 14, The VH comprises the amino acid sequence of SEQ ID NO:9 and the VL comprises the amino acid sequence of SEQ ID NO:14, the VH comprises the amino acid sequence of SEQ ID NO: 10 and the VL comprises the amino acid sequence of SEQ ID NO: 15; The VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO: 15; or The VH comprises the amino acid sequence of SEQ ID NO:12 and the VL comprises the amino acid sequence of SEQ ID NO:

16.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof specifically binds to LILRB2, or The antibody or antigen-binding fragment thereof has an EC of less than 20 nM, less than 10 nM, less than 5 nM or less than 1 nM. 50 Values ​​specifically bind to LILRB2.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and HLA-A2.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof has an IC of less than 20 nM, less than 10 nM, less than 5 nM, less than 1 nM or less than 0.6 nM. 50 The value blocks the interaction between human LILRB2 and HLA-A2.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and HLA-G.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the antibody or antigen-binding fragment thereof has an IC of less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM or less than 0.3 nM. 50 The value blocks the interaction between human LILRB2 and HLA-G.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL1.

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL2.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL4.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human ANGPTL7.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human LILRB2 and human MAG. 17 . The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 , wherein the antibody or antigen-binding fragment thereof is capable of reprogramming human monocyte-derived M2 macrophages to an M1 phenotype.

18. The antibody or antigen-binding fragment thereof of any one of claims 1-17, wherein the antibody or antigen-binding fragment thereof reduces M2 macrophage-mediated T cell suppression.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof promotes human dendritic cells to enter a mature state.

20. The antibody or antigen-binding fragment thereof of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof promotes human monocytes to enter a pro-inflammatory state.

21. The antibody or antigen-binding fragment thereof of any one of claims 1-20, wherein the antibody or antigen-binding fragment thereof promotes human PBMCs to enter a pro-inflammatory state.

22. The antibody or antigen-binding fragment thereof of any one of claims 1-21, wherein the antibody or antigen-binding fragment thereof upregulates the release of pro-inflammatory cytokines and / or chemokines.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein the pro-inflammatory cytokine and / or chemokine is selected from the group consisting of IL-2, IFN-γ, TNF-α, granzyme B, PDGF-AA, IL-3, IL-1β, IL-5, TGF-α, IFN-α, CCL4, basic FGF, IL-13, G-CSF, IL-4, PD-L1, CCL20, IL-15, GM-CSF, CCL5, CCL3, PDGF-AB, CCL19, CXCL2, IL-6, VEGF, and combinations thereof.

24. The antibody or antigen-binding fragment thereof of any one of claims 1-23, wherein the antibody or antigen-binding fragment thereof reduces the expression of IL-10.

25. The antibody or antigen-binding fragment thereof of any one of claims 1-24, wherein the antibody or antigen-binding fragment thereof binds to human LILRB2 with a KD (affinity constant) of less than 15 nM, less than 10 nM, less than 8 nM or less than 6 nM.

26. The antibody or antigen-binding fragment thereof of any one of claims 1-25, wherein the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, wherein the antibody or antigen-binding fragment thereof is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody or an antigen-binding fragment thereof.

28. An antibody or an antigen-binding fragment thereof, which binds to a peptide comprising the amino acid sequence shown in SEQ ID NO:

21.

29. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 28.

30. An isolated vector comprising the isolated polynucleotide of claim 29.

31. A host cell comprising the isolated polynucleotide of claim 29 or the isolated vector of claim 30.

32. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 28, or the isolated polynucleotide as described in claim 29, or the isolated vector as described in claim 30, or the host cell as described in claim 31, and a pharmaceutically acceptable carrier.

33. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, or the isolated polynucleotide according to claim 29, or the isolated vector according to claim 30, or the host cell according to claim 31, or the pharmaceutical composition according to claim 32.

34. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 28 and an anti-PD-1 antibody.

35. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, or the isolated polynucleotide according to claim 29, or the isolated vector according to claim 30, or the host cell according to claim 31, or the pharmaceutical composition according to claim 32, or the kit according to claim 33, or the pharmaceutical composition according to claim 34 in the manufacture of a therapeutic agent for binding to human LILRB2.

36. The use of claim 35, wherein the antibody or antigen-binding fragment thereof of any one of claims 1 to 28, or the isolated polynucleotide of claim 29, or the isolated vector of claim 30, or the host cell of claim 31, or the pharmaceutical composition of claim 32, or the kit of claim 33, or the pharmaceutical composition of claim 34 that binds to LILRB2 is used in the manufacture of a therapeutic agent for blocking the interaction between human LILRB2 and HLA-A2, HLA-G, human ANGPTL1, human ANGPTL2, human ANGPTL4, human ANGPTL7 and / or human MAG.

37. Use of an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 28, or an isolated polynucleotide as described in claim 29, or an isolated vector as described in claim 30, or a host cell as described in claim 31, or a pharmaceutical composition as described in claim 32, or a kit as described in claim 33, or a pharmaceutical composition as described in claim 34 in the manufacture of a therapeutic agent for enhancing an immune response.

38. Use of an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 28, or an isolated polynucleotide as described in claim 29, or an isolated vector as described in claim 30, or a host cell as described in claim 31, or a pharmaceutical composition as described in claim 32, or a kit as described in claim 33, or a pharmaceutical composition as described in claim 34 in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease.

39. The use of claim 38, wherein the disease comprises a tumor, and optionally at least one tumor cell expresses LILRB2.

40. A method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-28, or an isolated polynucleotide as described in claim 29, or an isolated vector as described in claim 30, or a host cell as described in claim 31, or a pharmaceutical composition as described in claim 32, or a kit as described in claim 33, or a pharmaceutical composition as described in claim 34.

41. The method of claim 40, wherein the antibody or antigen-binding fragment thereof reprograms human monocyte-derived M2 macrophages to an M1 phenotype, The antibody or antigen-binding fragment thereof reduces T cell suppression mediated by the M2 phenotype of tumor-associated macrophages, The antibody or antigen-binding fragment thereof promotes human dendritic cells to enter a mature state. The antibody or antigen-binding fragment thereof promotes human monocytes to enter a pro-inflammatory state, and / or The antibody or antigen-binding fragment thereof promotes human PBMC to enter a pro-inflammatory state.

42. The method of claim 40 or 41, wherein the antibody or antigen-binding fragment thereof upregulates the release of pro-inflammatory cytokines and / or chemokines selected from the group consisting of IL-2, IFN-γ, TNF-α, granzyme B, PDGF-AA, IL-3, IL-1β, IL-5, TGF-α, IFN-α, CCL4, basic FGF, IL-13, G-CSF, IL-4, PD-L1, CCL20, IL-15, GM-CSF, CCL5, CCL3, PDGF-AB, CCL19, CXCL2, IL-6, VEGF, and combinations thereof.

43. The method of any one of claims 40-42, wherein the antibody or antigen-binding fragment thereof decreases expression of IL-10.

44. A method of diagnosing or preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-28, or an isolated polynucleotide as described in claim 29, or an isolated vector as described in claim 30, or a host cell as described in claim 31, or a pharmaceutical composition as described in claim 32, or a kit as described in claim 33, or a pharmaceutical composition as described in claim 34.

45. The method of any one of claims 40-44, wherein the disease, disorder or condition comprises a tumor, optionally at least one tumor cell expresses LILRB2.

46. ​​The method of any one of claims 40-45, wherein the subject is a mammal, including a human.

47. A binding epitope on LILRB2, wherein the binding epitope comprises the amino acid sequence shown in SEQ ID NO:

21.

48. The binding epitope on LILRB2 of claim 47, wherein the LILRB2 is human LILRB2.

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