Anti-tmem176b antibody, pharmaceutical composition and use

Abstract

Provided are an anti-Tmem176b antibody, a pharmaceutical composition and a use. Specifically, the anti-Tmem176b antibody is an anti-Tmem176b monoclonal antibody. Specifically, provided is an anti-Tmem176b antibody or an antigen-binding fragment thereof; the anti-Tmem176b antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1-HCDR3; and the light chain variable region comprises LCDR1-LCDR3, wherein the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 5, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 6, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 7; and the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 8, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO: 9, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO: 10. The anti-Tmem176b antibody has a good anti-tumor effect.

Description

Anti-Tmem176b antibodies, pharmaceutical compositions and uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202311595484.4 and application date November 24, 2023, and claims its priority. The entire content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present invention belongs to the field of biomedicine and relates to an anti-Tmem176b antibody, a pharmaceutical composition and uses thereof. Specifically, the anti-Tmem176b antibody is a monoclonal antibody against Tmem176b. Background Art

[0004] Tmem176b is a member of the MS4A family of four-transmembrane structural proteins, which is located on the membranes of various organelles in cells and has the function of regulating intracellular Ca2 + Tmem176b is expressed in lymphocytes and is closely related to monocytes, macrophages, dendritic cells and RORγT. + Cells. Studies have shown that reducing TMEM176B expression in dendritic cells can activate the Caspase-1 / IL-1β signaling pathway and enhance dendritic cell anti-tumor immunity, thereby increasing the body's sensitivity to immune checkpoint blockade (ICB) therapy. Other reports have shown that TMEM176B is highly expressed in human colorectal tumor tissue, has a significant negative correlation with cancer patient prognosis, and has a clear correlation with clinical sensitivity to ICB therapy.

[0005] SHP-1 (also referred to herein as Shp1) is a Src homology domain 2 (SH2) protein tyrosine phosphatase-1 (SH2-containing protein tyrosine phosphatase, non-receptor type 6 (PTPN6)). It is primarily expressed in the cytoplasm of hematopoietic cells and is a key regulator of intracellular phosphorylation levels. This family of proteins comprises two proteins, SHP-1 and SHP-2. The gene encoding SHP-1 is located at 12p13 and possesses two N-terminal SH2 domains, a phosphorylation domain, and a C-terminal tyrosine phosphorylation site. In T lymphocytes, SHP-1 dephosphorylates TCR-proximal activation signals such as PLCγ1 and SLP76, downregulating TCR signaling and thereby inhibiting T cell activation, proliferation, and maturation. Studies have shown that peripheral T cells deficient in SHP-1 expression also exhibit enhanced TCR-induced apoptosis.

[0006] There is still a need to develop new anti-tumor methods. Summary of the Invention

[0007] After in-depth research and creative work, the inventors discovered the interaction between Tmem176b and Shp1. After tumor-derived Tmem176b is delivered to T cells, it inhibits TCR proximal signaling molecules by recruiting Shp1, thereby inhibiting the activation, proliferation and anti-tumor function of T cells. This interaction plays an important regulatory role in the process of tumor immune escape. In view of the unique mechanism of Tmem176b regulating T cell activation, the designed blocking monoclonal antibody can inhibit Shp1 from approaching the TCR activation signal complex by binding to the extracellular segment of Tmem176b on the surface of T cells, thereby relieving the inhibitory effect of Shp1 on TCR signals. The experimental results showed a significant tumor inhibitory effect, and the combination with PD-1 monoclonal antibody can further enhance the anti-tumor effect, and has good anti-tumor prospects. The following invention is thus provided:

[0008] One aspect of the present invention relates to an anti-Tmem176b antibody or an antigen-binding fragment thereof, wherein the anti-Tmem176b antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0009] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 7, and

[0010] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 10.

[0011] In some embodiments of the present invention, the anti-Tmem176b antibody or antigen-binding fragment thereof, wherein,

[0012] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2.

[0013] In some embodiments of the present invention, the anti-Tmem176b antibody or antigen-binding fragment thereof, wherein,

[0014] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0015] In some embodiments of the present invention, the anti-Tmem176b antibody or antigen-binding fragment thereof, wherein the heavy chain constant region of the antibody is Ig gamma-1chain C region or Ig gamma-4chain C region; and the light chain constant region is Ig kappa chain C region.

[0016] In some embodiments of the present invention, the anti-Tmem176b antibody or its antigen-binding fragment, wherein the anti-Tmem176b antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

[0017] In some embodiments of the present invention, the anti-Tmem176b antibody or antigen-binding fragment thereof, wherein,

[0018] The antibody includes non-CDR regions, and the non-CDR regions are derived from a species other than murine, such as a human antibody or a rabbit antibody.

[0019] In some embodiments of the present invention, the anti-Tmem176b antibody or antigen-binding fragment thereof, wherein:

[0020] The anti-Tmem176b antibody binds to the EC of Tmem176b 50 is less than or equal to 0.003 μg / mL, less than or equal to 0.002 μg / mL, or less than or equal to 0.001 μg / mL;

[0021] Preferably, the EC 50 It was measured by Capture ELISA method.

[0022] In some embodiments of the present invention, the anti-Tmem176b antibody or antigen-binding fragment thereof, wherein:

[0023] The anti-Tmem176b antibody binds to Tmem176b with a KD of less than or equal to 5E-8, less than or equal to 1E-8, or less than or equal to 1E-9;

[0024] Preferably, the EC 50 It was determined by Biacore detection method.

[0025] In some embodiments of the present invention, the anti-Tmem176b antibody or antigen-binding fragment thereof, wherein,

[0026] The amino acid sequence of the heavy chain of the anti-Tmem176b antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain is shown in SEQ ID NO: 4.

[0027] The anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of the present invention is used for treating or preventing tumors;

[0028] Preferably, the tumor is a tumor in which TMEM176B-positive exosomes are present in the patient's blood;

[0029] Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematologic malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0030] In the present invention, unless otherwise specified, the Tmem176b (also referred to as TMEM176B) is mouse Tmem176b or human Tmem176b.

[0031] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of the present invention.

[0032] Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.

[0033] Another aspect of the present invention relates to a host cell comprising the isolated nucleic acid molecule of the present invention or the recombinant vector of the present invention.

[0034] Another aspect of the present invention relates to an antibody-drug conjugate, which comprises an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is an anti-Tmem176b antibody or antigen-binding fragment thereof described in any one of the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug.

[0035] In some embodiments of the present invention, in the antibody-drug conjugate, the antibody or antigen-binding fragment thereof is connected to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond;

[0036] Preferably, the molar ratio of the antibody or antigen-binding fragment thereof to the small molecule drug is 1:(2-4), such as 1:2, 1:3 or 1:4.

[0037] The antibody-drug conjugate according to any one of the present invention, which is used for treating or preventing tumors;

[0038] Preferably, the tumor is a tumor in which TMEM176B-positive exosomes are present in the patient's blood;

[0039] Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematologic malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0040] Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of the present invention or the antibody-drug conjugate according to any one of the present invention; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0041] In some embodiments of the present invention, the pharmaceutical composition, wherein the anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention or the antibody-drug conjugate of any one of the present invention is the active pharmaceutical ingredient (API).

[0042] In some embodiments of the present invention, the pharmaceutical composition, wherein the anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention or the antibody-drug conjugate described in any one of the present invention is the sole active ingredient.

[0043] In some embodiments of the present invention, the pharmaceutical composition is composed of the anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention or the antibody-drug conjugate described in any one of the present invention, and one or more pharmaceutically acceptable excipients.

[0044] In some embodiments of the present invention, the pharmaceutical composition further comprises one or more immune checkpoint inhibitors;

[0045] Preferably, the immune checkpoint inhibitor is an antibody targeting PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR and / or OX40;

[0046] Preferably, the antibody is a monoclonal antibody or a bispecific antibody;

[0047] Preferably, the antibody is a blocking monoclonal antibody;

[0048] Preferably, the antibody is an anti-PD-1 blocking monoclonal antibody or an anti-PD-L1 blocking monoclonal antibody.

[0049] In some embodiments of the present invention, the pharmaceutical composition, wherein the mass ratio of the immune checkpoint inhibitor to the anti-TMEM176B antibody or its antigen-binding fragment is (1:5) to (5:1), preferably (1:2) to (2:1), and more preferably 1:1.

[0050] In some embodiments of the present invention, the pharmaceutical composition, wherein the mass ratio of the anti-PD-1 blocking monoclonal antibody or the anti-PD-L1 blocking monoclonal antibody to the anti-TMEM176B antibody or its antigen-binding fragment is (1:5) to (5:1), preferably (1:2) to (2:1), and more preferably 1:1.

[0051] In some embodiments of the present invention, the pharmaceutical composition is composed of the anti-Tmem176b antibody or its antigen-binding fragment of the present invention or the antibody-drug conjugate described in any one of the present invention, one or more immune checkpoint inhibitors and one or more pharmaceutically acceptable excipients.

[0052] In some embodiments of the present invention, the pharmaceutical composition is composed of the anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention or the antibody-drug conjugate according to any one of the present invention, an anti-PD-1 blocking monoclonal antibody or an anti-PD-L1 blocking monoclonal antibody, and one or more pharmaceutically acceptable excipients;

[0053] Preferably, the mass ratio of the anti-PD-1 blocking monoclonal antibody or the anti-PD-L1 blocking monoclonal antibody to the anti-TMEM176B antibody or its antigen-binding fragment is (1:5) to (5:1), preferably (1:2) to (2:1), and more preferably 1:1.

[0054] Yet another aspect of the present invention relates to a pharmaceutical product combination comprising a first pharmaceutical product and a second pharmaceutical product, wherein:

[0055] The first drug product comprises the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of the present invention or the antibody-drug conjugate according to any one of the present invention;

[0056] the second drug product comprises one or more immune checkpoint inhibitors;

[0057] Preferably, the immune checkpoint inhibitor is an antibody targeting PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR and / or OX40;

[0058] Preferably, the antibody is a monoclonal antibody or a bispecific antibody;

[0059] Preferably, the antibody is a blocking monoclonal antibody;

[0060] Preferably, the antibody is an anti-PD-1 blocking monoclonal antibody or an anti-PD-L1 blocking monoclonal antibody.

[0061] In some embodiments of the present invention, the pharmaceutical product combination, wherein,

[0062] The mass ratio of the immune checkpoint inhibitor to the anti-Tmem176b antibody or its antigen-binding fragment is (1:5) to (5:1), preferably (1:2) to (2:1), and more preferably 1:1.

[0063] In some embodiments of the present invention, the pharmaceutical product combination, wherein,

[0064] Among them, the mass ratio of the anti-PD-1 blocking monoclonal antibody or anti-PD-L1 blocking monoclonal antibody to the anti-Tmem176b antibody or its antigen-binding fragment is (1:5) to (5:1), preferably (1:2) to (2:1), and more preferably 1:1.

[0065] In some embodiments of the present invention, the pharmaceutical product combination, wherein,

[0066] The first drug product and the second drug product independently comprise one or more pharmaceutically acceptable excipients;

[0067] Preferably, it also contains a drug instruction sheet.

[0068] Another aspect of the present invention relates to use of the anti-Tmem176b antibody or antigen-binding fragment thereof described in any one of the present invention or the antibody-drug conjugate described in any one of the present invention in the preparation of a medicament for treating or preventing tumors;

[0069] Preferably, the tumor is a tumor in which TMEM176B-positive exosomes are present in the patient's blood;

[0070] Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematologic malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0071] Another aspect of the present invention relates to a method for treating or preventing tumors, comprising the steps of administering to a subject in need thereof an effective amount of the anti-TMEM176B antibody or antigen-binding fragment thereof, the antibody-drug conjugate, the pharmaceutical composition, or the combination of pharmaceutical products described herein;

[0072] Preferably, the tumor is a tumor in which TMEM176B-positive exosomes are present in the patient's blood;

[0073] Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematologic malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0074] In some embodiments of the present invention, the method for treating or preventing tumors is administered before or after surgery, and / or before or after radiotherapy.

[0075] In some embodiments of the present invention, the method for treating or preventing tumors, wherein:

[0076] The single dosage of the anti-TMEM176B antibody or antigen-binding fragment thereof is 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0077] Preferably, the drug is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every 1 week, every 2 weeks or every 3 weeks;

[0078] Preferably, the administration is by intravenous drip or intravenous injection.

[0079] Another aspect of the present invention relates to the use of an anti-Tmem176b antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating or preventing tumors;

[0080] Preferably, the tumor is a tumor in which TMEM176B-positive exosomes are present in the patient's blood;

[0081] Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer and kidney cancer;

[0082] Preferably, the anti-Tmem176b antibody or antigen-binding fragment thereof can block or inhibit the binding of Tmem176b to Shp1.

[0083] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0084] In the present invention, the term "blocking monoclonal antibody" specifically refers to a monoclonal antibody used to block the binding sites of immune checkpoints and their ligands or receptors, such as PD-1 and PD-L1, for tumor immunotherapy.

[0085] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0086] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The assignment of amino acids to regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the IMGT numbering system as defined by Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0087] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0088] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but it can also be obtained by using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0089] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature 1986; 321: 522 525; Reichmann et al., Nature, 1988; 332: 323 329; Presta, Curr. Op. Struct. Biol. 1992; 2: 593-596; Clark, Immunol. Today 2000; 21: 397 402.

[0090] As used herein, the term "single chain antibody (ScFv)" refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker. The VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242: 423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85: 5879-5883). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al, Protein Eng. 1995; 8: 725-731, Choi et al, Eur. J. Immunol. 2001; 31: 94-106, Hu et al, Cancer Res. 1996; 56: 3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293: 41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59.

[0091] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0092] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0093] As used herein, host cells refer to cells that can be used to introduce vectors, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0094] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D ) binds to the antigen.

[0095] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind with a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 -6 M, 10-7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binding antigen (e.g., TMEM176B protein). K can be determined using methods known to those skilled in the art. D , for example, using a Biacore assay.

[0096] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0097] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0098] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiator that, when delivered to the body together with an antigen or in advance, can enhance the body's immune response to the antigen or change the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0099] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactic effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered treatments.

[0100] In the present invention, unless otherwise specified, the "first" (e.g., the first pharmaceutical product) or "second" (e.g., the second pharmaceutical product) is merely for reference and does not have a particular meaning of order.

[0101] Advantageous Effects of the Invention

[0102] The present invention achieves one or more of the following technical effects (1) to (5):

[0103] (1) The anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention has a high affinity for the antigen Tmem176b.

[0104] (2) The anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention can effectively compete with Tmem176b for binding to Shp1.

[0105] (3) The anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention can effectively relieve the inhibitory effect of Tmem176b on TCR signaling.

[0106] (4) The anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention can effectively treat or prevent tumors.

[0107] (5) The anti-Tmem176b antibody or its antigen-binding fragment of the present invention is used in combination with an immune checkpoint inhibitor (such as an anti-PD-1 antibody) to have a synergistic anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1A-1B: Verification of the interaction between Tmem176b and Shp1.

[0109] Figure 1A: Co-localization of Tmem176b and Shp1. Immunofluorescence analysis of the co-localization of Tmem176b and Shp1 in EL4 cells overexpressing Tmem176b.

[0110] Figure 1B: Co-immunoprecipitation detection of the interaction between Tmem176b and Shp1 in EL4 cells.

[0111] Figure 2: Affinity determination of the Tmem176b monoclonal antibody 6E8. Capture ELISA was used to measure the affinity of the Tmem176b monoclonal antibody 6E8 for in vitro expressed and purified Tmem176b antigen.

[0112] Figure 3: Determination of the specific binding ability of Tmem176b monoclonal antibody 6E8 to the antigen. The affinity KD50 value of Tmem176b monoclonal antibody 6E8 to the in vitro expressed and purified antigen Tmem176b was determined by Biacore.

[0113] Figures 4A to 4D show the inhibitory effect of candidate blocking mAb 6E8 on mouse melanoma B16F10 tumor growth. B16F10 cells were subcutaneously inoculated in C57B6J mice to test the inhibitory effect of the candidate mAb and its combination with α-PD-1 antibody on tumor growth.

[0114] Figure 4A and Figure 4B: Test of the candidate mAbs' inhibition of B16F10 subcutaneously inoculated tumor growth and survival.

[0115] Figure 4C and Figure 4D: Test of the inhibition of B16F10 tumor growth and mouse survival by candidate monoclonal antibodies combined with α-PD1.

[0116] Figures 5A to 5D show that the candidate blocking mAb 6E8 inhibits the growth of MC38 colorectal tumors in mice. MC38 cells were subcutaneously inoculated in C57B6J mice to test the inhibitory effects of the candidate mAb and its combination with α-PD-1 antibodies on tumor growth.

[0117] Figure 5A and Figure 5B: Test of the candidate mAbs inhibiting the growth and survival of MC38 subcutaneously inoculated tumors.

[0118] Figure 5C and Figure 5D: Test of the inhibition of MC38 tumor growth and mouse survival by candidate monoclonal antibodies combined with α-PD1.

[0119] Figures 6A and 6B show that the candidate blocking mAb 6E8 inhibits the growth of Hepa1-6 mouse liver cancer tumors. Hepa1-6 cells were orthotopically inoculated into the livers of C57B6J mice to test the inhibitory effect of the candidate mAb on orthotopic liver tumor growth.

[0120] Figure 6A: Orthotopic liver tumor growth in mice.

[0121] Figure 6B: Statistical graph of orthotopic liver tumor volume in mice.

[0122] FIG7A : plv-IRES-C-3×Flag-EGFP vector structure.

[0123] Figure 7B: plv-EGFPL vector structure.

[0124] Figure 7C: plv-EBFPL vector structure. DETAILED DESCRIPTION

[0125] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0126] Example 1: Study on the interaction between Tmem176b and Shp1

[0127] 1. Experimental Materials and Main Reagents

[0128] See Table A below.

[0129] Table A

[0130] NP40 lysis buffer recipe: (20mM tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, 5mM EDTA (pH 8.0), 5mM Na4P2O7, 1mM Na3VO4, 5mM NaF, and protease inhibitor cocktail).

[0131] 2. Experimental Methods

[0132] Stable plasmid construction: The plasmid vector structures for plv-IRES-C-3×Flag-EGFP, plv-EGFPL, and plv-EBFPL are shown in Figures 7A to 7C. First, the plv-IRES-C-3×Flag-EGFP vector was double-digested with XbaI / BamHI to obtain a vector recovery product. The plv-EGFPL and plv-EBFPL vectors were double-digested with BamHI / SalI to amplify the desired Tmem176b and shp1 gene CDS fragments using the primers listed in Table 1 below. The gene fragments were then inserted into the restriction sites of the vector to obtain complete plasmids.

[0133] Table 1: PCR primers

[0134] Preparation of stable virus: 1×10 6293T cells were seeded per well and the cell density reached 70-80%. 2 μg of expression plasmid and packaging plasmids 0.5 μg pMD2.G (Addgene 12259) and 1.5 μg psPAX2 (Addgene 12260) were mixed with 8 μl polyethylenimine (PEI) (1 mg / mL) in 200 μl Opti-MEM. After gentle mixing, the mixture was allowed to stand for 15 minutes and then slowly added to the cells. After 6-8 hours in the cell culture incubator, the culture medium was replaced with prewarmed DMEM complete medium. The supernatant was collected after 24 and 48 hours, centrifuged at 1000 g for 3 minutes, the precipitate was discarded, and the supernatant was collected and stored at -80°C.

[0135] Preparation of stable cells: EL4 suspension cells were cultured at 2 × 10 5 Cells / 100 μL culture medium were plated in a 24-well plate, followed by the addition of 400 μL of viral supernatant. Polybrene (final concentration 10 ng / μL) was added to the viral solution. The cells were centrifuged at 2500 rpm and 37°C for 30 min, with a speed increase of 6 and a speed decrease of 2. After centrifugation, the cells were cultured in an incubator for 12 h. Fresh culture medium was then exchanged for an additional 48 h. The infection efficiency was detected based on GFP fluorescent labeling, and the target cell lines were sorted.

[0136] Co-immunoprecipitation (Co-IP): Collect the required EL4 cells, take 1×10 7 The cells were centrifuged at 500 g for 5 min, the supernatant was removed, and the cells were washed once with 1× PBS and centrifuged to remove the supernatant.

[0137] Add 1 mL of NP40 Lysis buffer, pipette to mix, lyse on ice for 30 min, and centrifuge;

[0138] After sonication for a few seconds, the lysed protein suspension was centrifuged at 12,000 rpm and 4°C for 10 min to remove the precipitate. 80 μL of the supernatant was added to 20 μL of 5× SDS loading buffer, mixed, and placed in a 100°C metal bath for 10 min. The suspension was then stored at -20°C as the input.

[0139] Because the stable gene fusion expresses the Flag tag, the Flag in the protein suspension is enriched with M2 Flag beads to obtain the protein complex of the target protein. The remaining protein suspension in the previous step is transferred to the washed M2 Flag beads (1×10 7 cells / 20 μL M2 Flag beads) and vertically mixed at 4°C for 4 h;

[0140] Centrifuge at 8000g, 4℃ for 30s, aspirate the supernatant, add 1ml NP40 Lysis buffer to the beads, mix by inversion, centrifuge at 8000g, 4℃ for 30s, discard the supernatant, and repeat the wash 5-6 times;

[0141] Add 1.5 μL of 3× Flag peptide and 28.5 μL of 2× SDS loading buffer to each tube of beads, mix well, and shake in a metal bath at 25°C at 1200 rpm for 30 min to competitively elute Flag-tagged proteins.

[0142] Centrifuge at 8000g for 30s at 4°C, collect the supernatant, and store at -20°C.

[0143] 3. Experimental Results

[0144] eGFP-Tmem176b and eBFP-Shp1 fusion proteins were simultaneously overexpressed in EL4 cells, and the colocalization of Tmem176b and Shp1 in EL4 cells was observed using confocal microscopy. The results showed that Tmem176b and Shp1 were clearly colocalized in the cytoplasm of EL4 cells (Figure 1A).

[0145] Furthermore, the fusion protein Tmem176b-flag was overexpressed in EL4 cells, and the co-immunoprecipitation (Co-IP) method was used to analyze proteins that may interact with Tmem176b. The results showed that Shp1, a phosphatase that regulates T cell activation in T cells, had a significant interaction with Tmem176b (see Figure 1B).

[0146] Example 2: Design, expression and purification of monoclonal antibodies against Tmem176b

[0147] 1. Experimental Materials and Main Reagents

[0148] 293T cell line (ATCC, CRL-3216)

[0149] Japanese White Rabbit

[0150] DMEM (source culture, L110KJ)

[0151] RIPA 1640 Medium (Source Culture, L210KJ)

[0152] FBS(YOSHI,A1015)

[0153] XY-02a-FC-6His expression vector (Abclonal)

[0154] Tmem176b expressed and purified antigen 142-196aa xy02b-hFc-6His (Abclonal)

[0155] HRP Goat Anti-Rabbit IgG(H+L)(Jackson ImmunoResearch,111-035-045)

[0156] Chromogenic solution (TMB, Thermo Fisher, CAT: 34029)

[0157] Stop solution (2M H2SO4)

[0158] Flow cytometry sorter (BD, Aria III)

[0159] Multifunctional microplate reader (Tecan, Epelx)

[0160] 2. Experimental Methods

[0161] The amino acid fragment 149-209AA of mouse Tmem176b was selected and constructed onto XY02a-FC-6His, and the purified antigen was expressed using the 293FT eukaryotic expression system.

[0162] Experimental-grade Japanese white rabbits were immunized four times with the expressed purified antigen. Serum was collected for ELISA analysis, and spleens were harvested for B cell isolation. Single B cell clones were obtained and cultured. Supernatants from these clones were collected and analyzed by ELISA. Positive B cell clones were selected, and RNA was extracted and amplified to identify the LEM sequence (Linear Expression Module). The candidate LEM sequence was used to construct a recombinant antibody expression plasmid, and a recombinant rabbit anti-Tmem176b monoclonal antibody, designated 6E8, was expressed and purified.

[0163] 2. Experimental Results

[0164] The sequence information of Tmem176b monoclonal antibody 6E8 is as follows:

[0165] Heavy chain variable region:

[0166] Light chain variable region:

[0167] Heavy chain full length:

[0168] Light chain full length:

[0169] The six CDRs of Tmem176b mAb 6E8 are defined using the IMGT numbering system as follows:

[0170] HCDR1: GFSLSSYA (SEQ ID NO: 5)

[0171] HCDR2: ISIRGNT (SEQ ID NO: 6)

[0172] HCDR3:ARNTLYSSGWGGSDL(SEQ ID NO:7)

[0173] LCDR1: QSVYNNNN (SEQ ID NO: 8)

[0174] LCDR2: YAS (SEQ ID NO: 9)

[0175] LCDR3:QGEFSCSSADCNA(SEQ ID NO:10)

[0176] The sequence of the heavy chain HCDR1 is shown in SEQ ID NO: 5, the sequence of HCDR2 is shown in SEQ ID NO: 6, and the sequence of HCDR3 is shown in SEQ ID NO: 7;

[0177] The sequence of the light chain LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9, and the sequence of LCDR3 is shown in SEQ ID NO:10.

[0178] Example 3: Affinity experiment of monoclonal antibody and TMEM176B

[0179] 1. Experimental Materials and Main Reagents

[0180] α-Tmem176b monoclonal antibody (6E8)

[0181] Tmem176b expressed and purified antigen 142-196aa xy02b-hFc-6His (Abclonal)

[0182] HRP Goat Anti-Rabbit IgG(H+L)(Jackson ImmunoResearch,111-035-045)

[0183] Chromogenic solution (TMB, Thermo Fisher, CAT: 34029)

[0184] Stop solution (2M H2SO4)

[0185] Multifunctional microplate reader (Tecan, Epelx)

[0186] 2. Experimental Methods

[0187] Capture ELISA was used to detect the affinity of Tmem176b monoclonal antibody 6E8 to in vitro expressed and purified antigen Tmem176b.

[0188] Three candidate monoclonal antibodies including antibody 6E8 were diluted with PBS at 2 μg / mL, 25 μl / well, and coated on 384-well plates (Corning, CAT: 3700) at 4°C overnight; washing solution (self-prepared) was used at 75 μl / well for 5 times; nonspecific binding sites were blocked with 50 μl / well blocking solution (self-prepared) and incubated at room temperature for 1 hour; washing solution (self-prepared) was used at 75 μl / well for 5 times; purified antigen 142-196aa xy02b-hFc-6His expressed by diluted Tmem176b (0.1 μg / ml as the starting concentration, 3-fold gradient dilution for 9 steps, 25 μl / well, incubated at room temperature for 1 hour); washing solution (self-prepared) was used at 75 μl / well for 5 times; secondary antibody (HRP Goat Anti-Rabbit IgG (H+L), Jackson ImmunoResearch, 111-035-045) was diluted with dilution buffer at a ratio of 1:5000, 25 μl / well was added to a 384-well plate, and incubated at room temperature in the dark for 1 hour; washing solution (self-prepared) was used at a ratio of 75 μl / well and washed 5 times; color development solution (TMB, Thermo Fisher, CAT: 34029) was diluted at a ratio of 1:5, 25 μl / well was used, and color was developed at room temperature in the dark for 3 minutes; 10 μl of stop solution (2 M H2SO4) was added to each well to terminate the reaction; the plate was read at 450 nm\630 nm, and the 630 nm background was subtracted from 450 nm.

[0189] 3. Experimental Results

[0190] As shown in Table 2 and Figure 2.

[0191] Table 2

[0192] Note: Antibodies 1F9 and 4F8 are rabbit monoclonal antibodies against mouse Tmem176b.

[0193] The results showed that the EC of antibody 6E8 and Tmem176b antigen 50 The value was 0.000893, indicating that the monoclonal antibody had a strong affinity for binding to the antigen.

[0194] Example 4: Specificity experiment of monoclonal antibody and TMEM176B

[0195] 1. Experimental Materials and Main Reagents

[0196] NTA Series S Sensor Chip CM5 (GE, BR100530)

[0197] Amine Coupling Kit (GE, 100050)

[0198] α-Tmem176b monoclonal antibody (6E8)

[0199] Tmem176b expressed and purified antigen 142-196AA (Abclonal)

[0200] 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 75mg / ml)

[0201] N-hydroxysuccinimide(NHS,115mg / ml)

[0202] Ethanolamine (1M)

[0203] NaOH (50 mM)

[0204] 70% glycerin

[0205] Sodium acetate-acetic acid buffer solution (10 mM), pH gradient is 5.5, 5.0, 4.5, 4.0, etc. The solution must be filtered through a 0.22 μm filter membrane.

[0206] Glycine-hydrochloric acid buffer solution (10 mM), pH gradient is 1.5, 2.0, 2.5, 3.0, etc. 4 gradients, the solution must be filtered through a 0.22 μm filter membrane.

[0207] 2. Experimental Methods

[0208] Biacore was used to detect the binding specificity of the prepared monoclonal antibody to the antigen.

[0209] The expressed and purified Tmem176b antigen was dissolved in PBS and used for protein chip coupling.

[0210] The NTA sensor chip module was inserted into a BIAcore instrument. A mixture of 75 μl of NHS and 75 μl of EDC was passed through a flow cell at a flow rate of 5 μl / min. The surface was activated by injection of 35 μl of the NHS / EDC mixture. Then, 35 μl of in vitro expressed and purified Tmem176b antigen was injected onto the activated surface. Excess reactive groups were inactivated by injection of 35 μl of ethanolamine. A 10 μl injection of 20 mmol / L HCl was performed, followed by removal of non-covalently bound material with Extradean. The in vitro expressed and purified Tmem176b antigen was diluted to 1 μg / mL in PBS and coupled for 60 seconds at a flow rate of 10 μL / min. The analyte, antibody 6E8, was analyzed at concentrations of 1.5625, 3.125, 6.25, 12.5, 25, and 50 nM at a flow rate of 30 μL / min, with binding for 120 seconds and dissociation for 200 seconds. The binding and dissociation parameters of monoclonal antibody 6E8 and antigen Tmem176b were measured. The experiment was performed in triplicate.

[0211] 3. Experimental Results

[0212] As shown in Table 3 and Figure 3.

[0213] Table 3

[0214] The results showed that the KD value of monoclonal antibody 6E8 and Tmem176b antigen was 9.59×10 -10 M (the average value of 3 parallel experiments), showing strong specific binding properties.

[0215] Example 5: Antitumor Experiment (1)

[0216] 1. Experimental Animals and Samples

[0217] C57B6J mice, B16F10 (mouse melanoma) cells.

[0218] α-Tmem176b monoclonal antibody (6E8)

[0219] α-isotype control monoclonal antibody (Abclonal, AC042)

[0220] α-PD1 blocking monoclonal antibody (anti-PD-1 blocking monoclonal antibody; MCE, RMP1-14).

[0221] 2. Experimental Methods

[0222] To test the inhibitory effect of the candidate monoclonal antibody on tumor growth, the inhibitory effect of the candidate monoclonal antibody on tumor growth was tested in a wild-type C57B6J mouse subcutaneous inoculation tumor model.

[0223] Wild-type C57B6J mice were subcutaneously inoculated with 2×105 B16F10 cells, wait until the tumor has grown for 10 days or the volume reaches 100 mm 3 , for treatment, the experiment was divided into PBS control group, isotypte control group, 6E8 treatment group, α-PD1 treatment group and 6E8 and α-PD1 combination group; 6 mice in each group. The dosage of 6E8 treatment group, isotype control group and α-PD1 treatment group was 10 mg / kg / time, administered once every 3 days, for 3 consecutive times, and the administration method was intraperitoneal injection. The combined dosage of 6E8 and α-PD1 blocking monoclonal antibody was 10 mg / kg / time (6E8 10 mg / kg / time, α-PD110 mg / kg / time, twice injected at the same time), administered once every 3 days, for 3 consecutive times, and the administration method was intraperitoneal injection. Tumor growth was measured every 2 days. Until the tumor volume reached 2000mm 3 .

[0224] 3. Experimental Results

[0225] The results showed that the candidate monoclonal antibody could effectively inhibit the growth of B16F10 subcutaneous tumors compared with the control isotype antibody (Figure 4A, Figure 4B).

[0226] The results also showed that the combined use of α-PD1 blocking monoclonal antibodies can further inhibit subcutaneous tumor growth, and the tumor suppression effect of the combined use is stronger than that of the candidate monoclonal antibody or PD1 monoclonal antibody alone. The statistical analysis showed significant differences, indicating that the combination of anti-TMEM176B antibodies and anti-PD1 antibodies has a certain synergistic effect (Figure 4C, Figure 4D). This suggests that the candidate monoclonal antibody can be combined with existing immunotherapy regimens to further enhance therapeutic efficacy.

[0227] Example 6: Antitumor Experiment (2)

[0228] 1. Experimental Animals and Samples

[0229] C57B6J mice, MC38 (mouse colorectal cancer) cells.

[0230] α-Tmem176b monoclonal antibody (6E8)

[0231] α-isotype control monoclonal antibody (Abclonal, AC042)

[0232] α-PD1 blocking monoclonal antibody (MCE, RMP1-14).

[0233] 2. Experimental Methods

[0234] To test the inhibitory effect of the candidate monoclonal antibody on colorectal tumor growth, the inhibitory effect of the candidate monoclonal antibody on tumor growth was tested in a wild-type C57B6J mouse subcutaneous inoculation tumor model.

[0235] Wild-type C57B6J mice were subcutaneously inoculated with 1×10 6 MC38 cells, wait until the tumor has grown for 12 days or reaches a volume of 100 mm 3 , for treatment, the experiment was divided into PBS control group, isotypte control group, 6E8 treatment group, α-PD1 treatment group and 6E8 and α-PD1 combination group, with 6 mice in each group. The dosage of 6E8 treatment group, isotype control group and α-PD1 treatment group was 10 mg / kg / time, administered once every 3 days, for 3 consecutive times, and the administration method was intraperitoneal injection. The combined dosage of 6E8 and α-PD1 blocking monoclonal antibody was 10 mg / kg / time (6E8 10 mg / kg / time, α-PD1 10 mg / kg / time, injected twice at the same time), administered once every 3 days, for 3 consecutive times, and the administration method was intraperitoneal injection. Tumor growth was measured every 4 days. Until the tumor volume reached 2000mm 3 .

[0236] 3. Experimental Results

[0237] The results showed that the monoclonal antibody could effectively inhibit the growth of MC38 subcutaneous tumors compared with the control isotype antibody ( Figure 5A, Figure 5B ).

[0238] The results also showed that combined use with an α-PD1 blocking monoclonal antibody further inhibited subcutaneous tumor growth (Figures 5C and 5D). The tumor suppression effect of this combination was significantly stronger than that of either monoclonal antibody or PD1 monoclonal antibody alone, with statistically significant differences. This suggests that the combination of anti-TMEM176B and anti-PD1 antibodies has a synergistic effect. This suggests that this candidate monoclonal antibody can be combined with existing immunotherapy regimens to further enhance therapeutic efficacy.

[0239] Example 7: Antitumor Experiment (3)

[0240] 1. Experimental Animals and Samples

[0241] C57B6J mice, Hepa1-6 (mouse hepatocarcinoma) cells.

[0242] α-Tmem176b monoclonal antibody (6E8)

[0243] α-isotype control monoclonal antibody (Abclonal, AC042)

[0244] 2. Experimental Methods

[0245] To test the inhibitory effect of the candidate monoclonal antibody on liver cancer growth, the inhibitory effect of the candidate monoclonal antibody on tumor growth was tested in a Hepa1-6 tumor model inoculated into the liver of wild-type C57B6J mice.

[0246] Wild-type C57B6J mice were subcutaneously inoculated with 2×10 6 After 10 days of tumor growth, Hepa1-6 cells were harvested and cut into 1 mm 3 Tumor tissue blocks were obtained by surgically anesthetizing C57B6J mice to be inoculated. Two tumor blocks were embedded in the liver of each mouse and surgically sutured. Three weeks later, the tumor-bearing C57B6J mice were treated with PBS, isotype, and 6E8. Each group consisted of five mice. The 6E8-treated and isotype-treated groups received 10 mg / kg of steroids every three days for three consecutive doses via intraperitoneal injection. Tumors were sampled after six weeks of growth for tumor size measurement and analysis.

[0247] 3. Experimental Results

[0248] The results showed that the candidate monoclonal antibody could effectively inhibit the growth of Hepa1-6 orthotopic liver tumors compared with the isotype control antibody (Figure 6A, Figure 6B), indicating that the antibody of the present invention has the potential to inhibit liver tumor growth.

[0249] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-Tmem176b antibody or an antigen-binding fragment thereof, wherein the anti-Tmem176b antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, and The amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

10.

2. The anti-Tmem176b antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

2.

3. The anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein: The heavy chain constant region of the antibody is Ig gamma-1 chain C region or Ig gamma-4 chain C region; the light chain constant region is Ig kappa chain C region.

4. The anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The anti-Tmem176b antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

5. The anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The antibody includes non-CDR regions, and the non-CDR regions are from species other than murine, such as human antibodies or rabbit antibodies.

6. The anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein: The anti-Tmem176b antibody binds to the EC of Tmem176b 50 is less than or equal to 0.003 μg / mL, less than or equal to 0.002 μg / mL, or less than or equal to 0.001 μg / mL; Preferably, the EC 50 It was measured by Capture ELISA method.

7. The anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein: The anti-Tmem176b antibody binds to Tmem176b with a KD of less than or equal to 5E-8, less than or equal to 1E-8, or less than or equal to 1E-9; Preferably, the EC 50 It was measured by Biacore detection method.

8. The anti-Tmem176b antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain of the anti-Tmem176b antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain is shown in SEQ ID NO:

4.

9. An isolated nucleic acid molecule encoding the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A recombinant vector comprising the isolated nucleic acid molecule of claim 9.

11. A host cell comprising the isolated nucleic acid molecule of claim 9 or the recombinant vector of claim 10.

12. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein: The antibody or antigen-binding fragment thereof is the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug.

13. The antibody-drug conjugate according to claim 12, wherein: The antibody or antigen-binding fragment thereof is connected to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond; Preferably, the molar ratio of the antibody or antigen-binding fragment thereof to the small molecule drug is 1:(2-4).

14. A pharmaceutical composition comprising an effective amount of the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the antibody-drug conjugate according to any one of claims 12 to 13; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

15. The pharmaceutical composition according to claim 14, further comprising one or more immune checkpoint inhibitors; Preferably, the immune checkpoint inhibitor is an antibody targeting PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR and / or OX40; Preferably, the antibody is a monoclonal antibody or a bispecific antibody; Preferably, the antibody is a blocking monoclonal antibody; Preferably, the antibody is an anti-PD-1 blocking monoclonal antibody or an anti-PD-L1 blocking monoclonal antibody; Preferably, the mass ratio of the immune checkpoint inhibitor to the anti-Tmem176b antibody or its antigen-binding fragment is (1:5) to (5:1), preferably (1:2) to (2:1), and more preferably 1:

1.

16. A drug product combination comprising a first drug product and a second drug product, wherein: The first drug product comprises the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the antibody-drug conjugate according to any one of claims 12 to 13; the second drug product comprises one or more immune checkpoint inhibitors; Preferably, the immune checkpoint inhibitor is an antibody targeting PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR and / or OX40; Preferably, the antibody is a monoclonal antibody or a bispecific antibody; Preferably, the antibody is a blocking monoclonal antibody; Preferably, the antibody is an anti-PD-1 blocking monoclonal antibody or an anti-PD-L1 blocking monoclonal antibody.

17. The pharmaceutical product combination according to claim 16, wherein: in, The mass ratio of the immune checkpoint inhibitor to the anti-Tmem176b antibody or its antigen-binding fragment is (1:5) to (5:1), preferably (1:2) to (2:1), and more preferably 1:1; Preferably, the first drug product and the second drug product independently comprise one or more pharmaceutically acceptable excipients; Preferably, it also contains a drug instruction sheet.

18. Use of the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the antibody-drug conjugate according to any one of claims 12 to 13 in the preparation of a drug for treating or preventing tumors; Preferably, the tumor is a tumor in which TMEM176B-positive exosomes exist in the patient's blood; Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer and kidney cancer.

19. The anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the antibody-drug conjugate according to any one of claims 12 to 13, for use in treating or preventing tumors; Preferably, the tumor is a tumor in which TMEM176B-positive exosomes exist in the patient's blood; Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer and kidney cancer.

20. A method for treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of the anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the antibody-drug conjugate according to any one of claims 12 to 13, the pharmaceutical composition according to any one of claims 14 to 15, or the pharmaceutical product combination according to any one of claims 16 to 17; Preferably, the tumor is a tumor in which TMEM176B-positive exosomes exist in the patient's blood; Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer and kidney cancer.

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