CN117624352A - anti-Tmem 176b antibody, pharmaceutical composition and application - Google Patents

Abstract

The invention belongs to the field of biological medicine, and relates to an anti-Tmem 176b antibody, a pharmaceutical composition and application thereof. Specifically, the anti-Tmem 176b antibody is a monoclonal antibody to Tmem176 b. In particular, the invention relates to an anti-Tmem 176b antibody or antigen binding fragment thereof, said anti-Tmem 176b antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 and a light chain variable region comprising LCDR1 to LCDR3, wherein: the amino acid sequence of HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 6 and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 7, and the amino acid sequence of LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 9 and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 10. The anti-Tmem 176b antibody has good anti-tumor effect.

Description

anti-Tmem 176b antibody, pharmaceutical composition and application

Technical Field

The invention belongs to the field of biological medicine, and relates to an anti-Tmem 176b antibody, a pharmaceutical composition and application thereof. Specifically, the anti-Tmem 176b antibody is a monoclonal antibody to Tmem176 b.

Background

Tmem176b is one of the four transmembrane structural protein MS4A family members that localizes to a variety of intracellular organelle membranes, with the ability to regulate intracellular Ca2 ⁺ Trafficking and thus regulating immune cell functionActing as a medicine. Tmem176b is overexpressed in lymphocytes, monocytes, macrophages, dendritic cells and RORγT ⁺ And (3) cells. Studies have shown that decreasing TMEM176B expression in dendritic cells activates Caspase-1/IL-1. Beta. Signaling pathway and increases anti-tumor immune function of dendritic cells, thereby increasing sensitivity of the body to immune checkpoint blockade (immune checkpoint blockade, ICB) therapy. In addition, TMEM176B has been reported to be highly expressed in human colorectal tumor tissue, has a remarkable negative correlation with prognosis of tumor patients, and has a clear correlation with clinical ICB treatment sensitivity.

SHP-1 (also denoted SHP1 in the present invention) is a Src homology 2 (SH2) protein tyrosine phosphatase-1 (SH 2-containing protein tyrosine phosphatase, non-receptor type 6 (PTPN 6), a protein of tyrosine phosphatase mainly expressed in the cytoplasm of hematopoietic origin, which is a key factor regulating intracellular phosphorylation levels, 2 proteins including SHP-1 and SHP-2 the gene encoding SHP-1 is located at 12p13, there are two SH2 domains at the N-terminus, one phosphorylation domain and one tyrosine phosphorylation site at the C-terminus in T lymphocytes, SHP-1 can dephosphorylate TCR proximal activation signals such as PLCgamma 1, SLP76, down-regulate TCR signals, and inhibit T cell activation, proliferation and maturation.

There is a need to develop new anti-tumor means.

Disclosure of Invention

The inventors have conducted intensive studies and creative efforts to find the interaction of Tmem176b with Shp 1. Tmem176b achieves inhibition of TCR proximal signaling molecules by recruitment of Shp1, thereby inhibiting T cell activation, proliferation and anti-tumor function, which interactions have important regulatory roles in tumor immune escape. The designed blocking monoclonal antibody can inhibit the Shp1 from approaching the TCR activation signal complex by combining with the extracellular segment of the Tmem176b on the surface of the T cell aiming at the unique mechanism of regulating the T cell activation of the Tmem176b, thereby relieving the inhibition of the Shp1 on the TCR signal. The experimental result shows that the anti-tumor effect can be further improved by combining the anti-tumor agent with the PD-1 monoclonal antibody, and the anti-tumor agent has good anti-tumor prospect. The following invention is thus provided:

one aspect of the invention relates to an anti-Tmem 176b antibody or antigen binding fragment thereof, said anti-Tmem 176b antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 and a light chain variable region comprising LCDR1 to LCDR3, wherein:

the amino acid sequence of HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 7, and

The amino acid sequence of LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 9, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 10.

In some embodiments of the invention, the anti-Tmem 176b antibody or antigen binding fragment thereof, wherein,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

In some embodiments of the invention, the anti-Tmem 176b antibody or antigen binding fragment thereof, wherein the heavy chain constant region of the antibody is an Ig gamma-1chain C region or an Ig gamma-4chain C region; the light chain constant region is Ig kappa chain C region.

In some embodiments of the invention, the anti-Tmem 176b antibody or antigen binding fragment thereof, wherein the anti-Tmem 176b antibody or antigen binding fragment thereof is selected from Fab, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, or chimeric antibody.

In some embodiments of the invention, the anti-Tmem 176b antibody or antigen binding fragment thereof, wherein,

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

In some embodiments of the invention, the anti-Tmem 176b antibody or antigen binding fragment thereof, wherein:

EC in which the anti-Tmem 176b antibody binds to Tmem176b ₅₀ 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 For measurement by Capture ELISA.

In some embodiments of the invention, the anti-Tmem 176b antibody or antigen binding fragment thereof, wherein:

the anti-Tmem 176b antibody binds 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 For measurement by Biacore assay.

In some embodiments of the invention, the anti-Tmem 176b antibody or antigen binding fragment thereof, wherein,

the heavy chain of the anti-Tmem 176b antibody has an amino acid sequence shown in SEQ ID NO. 3, and the light chain has an amino acid sequence shown in SEQ ID NO. 4.

The anti-Tmem 176b antibody or antigen binding fragment thereof according to any of the invention for use in the treatment or prevention of a tumor;

preferably, the tumor is a TMEM176B positive tumor;

preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchus cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer.

In the present invention, the Tmem176B (also denoted Tmem 176B) is a mouse Tmem176B or a human Tmem176B, if not specified.

Another aspect of the invention relates to an isolated nucleic acid molecule encoding an anti-Tmem 176b antibody or antigen binding fragment thereof of any of the invention.

A further aspect of the invention relates to a recombinant vector comprising an isolated nucleic acid molecule of the invention.

A further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a recombinant vector of the invention.

A further aspect of the invention relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is an anti-Tmem 176b antibody or antigen-binding fragment thereof of any of the invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapeutic.

In some embodiments of the invention, the antibody drug conjugate, wherein the antibody or antigen binding fragment thereof is linked to a 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), for example 1: 2. 1:3 or 1:4.

an antibody drug conjugate according to any one of the invention for use in the treatment or prevention of a tumor;

preferably, the tumor is a TMEM176B positive tumor;

preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchus cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer.

A further aspect of the invention relates to a pharmaceutical composition comprising an effective amount of an anti-Tmem 176b antibody or antigen binding fragment thereof according to any of the invention or an antibody drug conjugate according to any of the invention; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

In some embodiments of the invention, the pharmaceutical composition, wherein the anti-Tmem 176b antibody or antigen binding fragment thereof of the invention or the antibody drug conjugate of any of the invention is an active ingredient (Active Pharmaceutical Ingredient, API).

In some embodiments of the invention, the pharmaceutical composition, wherein the anti-Tmem 176b antibody or antigen binding fragment thereof of the invention or the antibody drug conjugate of any of the invention is the only active ingredient.

In some embodiments of the invention, the pharmaceutical composition consists of an anti-Tmem 176b antibody or antigen binding fragment thereof of the invention or an antibody drug conjugate of any of the invention, and one or more pharmaceutically acceptable excipients.

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

preferably, the immune checkpoint inhibitor is an antibody that targets PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR, and/or OX 40;

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 mab or an anti-PD-L1 blocking mab.

In some embodiments of the invention, the pharmaceutical composition, wherein the mass ratio of the immune checkpoint inhibitor to the anti-TMEM 176B antibody or antigen binding fragment thereof is (1:5) to (5:1), preferably (1:2) to (2:1), more preferably 1:1.

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

the first pharmaceutical product comprises an anti-Tmem 176b antibody or antigen binding fragment thereof of any of the invention or an antibody drug conjugate of any of the invention;

the second pharmaceutical product comprises one or more immune checkpoint inhibitors;

preferably, the immune checkpoint inhibitor is an antibody that targets PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR, and/or OX 40;

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 mab or an anti-PD-L1 blocking mab.

In some embodiments of the invention, the pharmaceutical product combination, wherein,

wherein the mass ratio of the immune checkpoint inhibitor to the anti-Tmem 176b antibody or antigen binding fragment thereof is (1:5) to (5:1), preferably (1:2) to (2:1), more preferably 1:1.

in some embodiments of the invention, the pharmaceutical product combination, wherein,

The first and second pharmaceutical products independently comprise one or more pharmaceutically acceptable excipients;

preferably, the pharmaceutical instructions are also included.

A further aspect of the invention relates to the use of an anti-Tmem 176b antibody or antigen binding fragment thereof according to any of the invention or an antibody drug conjugate according to any of the invention in the manufacture of a medicament for the treatment or prophylaxis of a tumor;

preferably, the tumor is a TMEM176B positive tumor;

preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchus cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer.

A further aspect of the invention relates to a method of treating or preventing a tumor comprising the step of administering to a subject in need thereof an effective amount of an anti-TMEM 176B antibody or antigen binding fragment thereof according to any of the invention or an antibody drug conjugate according to any of the invention;

preferably, the tumor is a TMEM176B positive tumor;

Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchus cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer.

In some embodiments of the invention, the method of treating or preventing a tumor, wherein the administration is prior to or after surgery, and/or prior to or after radiation therapy.

In some embodiments of the invention, the method of treating or preventing a tumor, wherein,

the anti-TMEM 176B antibody or antigen binding fragment thereof is administered in a single dose of 0.1-100mg per kg body weight, preferably 5-50mg or 5-15mg per kg body weight;

preferably, the administration is 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;

preferably, the mode of administration is intravenous drip or intravenous injection.

A further aspect of the invention relates to the use of an anti-Tmem 176b antibody or antigen binding fragment thereof for the manufacture of a medicament for the treatment or prophylaxis of a tumor;

preferably, the tumor is a TMEM176B positive tumor;

Preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchus cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer;

preferably, the anti-Tmem 176b antibody or antigen binding fragment thereof is capable of blocking or inhibiting Tmem176b binding to Shp 1.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In the present invention, the term "blocking monoclonal antibody" refers specifically to monoclonal antibodies for blocking immune checkpoints and their ligands or receptors, e.g. PD-1 and PD-L1 binding sites, for tumor immunotherapy.

As used herein, the term EC ₅₀ Refers to half maximal effect concentration (concentration for 50%of maximal effect), which refers to a concentration that causes 50% of maximal effect.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is 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 the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises 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 3 domains (CH 1, CH2 and CH 3). 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 may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is prepared from the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to regions or domains follows Bethesda M.d., 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 IMGT numbering system definitions, see Ehrenmann F, kaas Q, lefranc M P. IMGT/3Dstructure-DB and IMGT/Domain GapAlign: a database and a tool for immunoglobulins or antibodies, T cell acceptors, MHC, igSF and MhcSF [ J ]. Nucleic acids research,2009;38 (suppl_1) definition of D301-D307.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different types, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtainable by the hybridoma technique first reported by Kohler et al G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J]Natural, 1975;256 495) but may also be obtained using recombinant DNA techniques (see, e.g., U.S. patent 4,816,567).

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR regions of a human immunoglobulin (recipient antibody) with those of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of the desired specificity, affinity, or reactivity. In addition, some of the amino acid residues of the Framework Regions (FR) of the recipient antibody may also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., jones et al, nature 1986;321:522 525; reichmann et al, nature,1988;332:323329; presta, curr.op. Struct. Biol.1992;2:593-596; clark, immunol. Today 2000;21:397 402.

As used herein, the term "single chain antibody (single chain fragment variable, scFv)" refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) linked by a linker. Wherein the VL and VH domains form monovalent molecules by pairing their linkers that enable production 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 may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, linkers having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used (Holliger et al, proc. Natl. Acad. Sci. USA 1993; 90:6444-6448). Other linkers useful 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, immunology 2001,50 (1): 51-59.

As used herein, the term "isolated" or "isolated" refers to obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" or "separated" does not exclude the presence of substances mixed with artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substances.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may 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. In addition, the vector may also contain a replication origin.

As used herein, cells as hosts refer to cells that can be used to introduce vectors, and include, but are not limited to, prokaryotic cells such as E.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.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, the specific junctionAn antibody that binds (or is specific for) an antigen means that the antibody is present in an amount of less than about 10 ^-5 M, e.g. 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.

As used herein, the term "K _D "refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. Typically, the antibody is present at less than about 10 ^-5 M, e.g. less than about 10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^{- 9} M or 10 ^-10 Dissociation equilibrium constant (K) of M or less _D ) Binding antigen (e.g., TMEM176B protein). K can be determined using methods known to those skilled in the art _D For example using the Biacore assay.

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

As used herein, the term "pharmaceutically acceptable excipients" or "pharmaceutically acceptable carriers and/or excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., remington's Pharmaceutical sciences, edition by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered with an antigen or pre-delivered into an organism, can enhance the organism's immune response to the antigen or alter the type of immune response. There are many adjuvants including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in current animal trials. Aluminum hydroxide adjuvants are used more in clinical trials.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of a disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

In the present invention, the "first" (e.g., first pharmaceutical product) or "second" (e.g., second pharmaceutical product) is merely intended to refer to distinction in a sense and does not have a particular meaning in order, unless specifically indicated.

Advantageous effects of the invention

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

(1) The anti-Tmem 176b antibodies or antigen binding fragments thereof of the invention have a higher affinity for the antigen Tmem176 b.

(2) The anti-Tmem 176b antibody or antigen binding fragment thereof of the invention is capable of effectively competing with Tmem176b for binding to Shp 1.

(3) The anti-Tmem 176b antibody or antigen binding fragment thereof of the present invention is capable of effectively releasing the TCR signals inhibitory effect of Tmem176 b.

(4) The anti-Tmem 176b antibodies or antigen binding fragments thereof of the invention are effective in treating or preventing tumors.

(5) The anti-Tmem 176b antibody or antigen binding fragment thereof of the invention has synergistic anti-tumor effects when used in combination with an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody).

Drawings

Fig. 1A to 1B: tmem176b interacted with Shp 1. Wherein:

fig. 1A: tmem176b co-localizes with Shp 1. Immunofluorescence analysis of co-localization of over-expressed Tmem176b with Shp1 in EL4 cells.

Fig. 1B: co-immunoprecipitation detects the interaction of Tmem176b with Shp1 in EL4 cells.

Fig. 2: tmem176b monoclonal antibody 6E8 affinity assay. Capture ELISA method detects the affinity of Tmem176b monoclonal antibody 6E8 to in vitro expressed purified antigen Tmem176 b.

Fig. 3: tmem176b monoclonal antibody 6E8 was assayed for its ability to specifically bind to antigen. Biacore assay measures Tmem176b monoclonal antibody 6E8 affinity KD50 values for purified antigen Tmem176b expressed in vitro.

Fig. 4A to 4D: candidate blocking mab 6E8 inhibited the mouse melanoma B16F10 tumor growth assay. B16F10 cells were inoculated subcutaneously in C57B6J mice and candidate mab and used in combination with the α -PD1 antibody were tested for tumor growth inhibition. Wherein:

fig. 4A and 4B: candidate mab inhibits B16F10 subcutaneous inoculation tumor growth and survival test.

Fig. 4C and 4D: candidate mab combined with alpha-PD 1 inhibited B16F10 tumor growth and mice survival test.

Fig. 5A to 5D: candidate blocking mab 6E8 inhibited mouse colorectal tumor MC38 tumor growth assay. MC38 cells were inoculated subcutaneously in C57B6J mice and candidate mab and used in combination with the alpha-PD 1 antibody were tested for tumor growth inhibition. Wherein:

fig. 5A and 5B: candidate monoclonal antibodies inhibit MC38 subcutaneous inoculation tumor growth and survival testing.

Fig. 5C and 5D: candidate mab combined with alpha-PD 1 inhibited MC38 tumor growth and mice survival test.

Fig. 6A to 6B: candidate blocking type monoclonal antibody 6E8 inhibits liver cancer Hepa1-6 tumor growth test of mice. In situ inoculation of Hepa1-6 cells into the liver of C57B6J mice, and testing of the inhibition of the candidate monoclonal antibodies on the in situ tumor growth of the liver. Wherein:

fig. 6A: in situ liver tumor growth in mice.

Fig. 6B: in situ liver tumor volume statistics of mice.

Fig. 7A: plv-IRES-C-3 XFlag-EGFP vector structure.

Fig. 7B: plv-EGFPL vector structure.

Fig. 7C: plv-EBFPL vector structure.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: interaction study of Tmem176b with Shp1

1. Experimental materials and primary reagents

Table a below.

Table A

NP40 lysis buffer formula: (20 mM tris-HCl (pH 7.5), 150mM NaCl,1% NP-40,5mM EDTA (pH 8.0), 5mM Na ₄ P ₂ O ₇ ,1mM Na ₃ VO ₄ ,5mM NaF,and protease inhibitor cocktail).

2. Experimental method

Stable transformation plasmid construction: the plasmid vector is plv-IRES-C-3 xFlag-EGFP, plv-EGFPL, plv-EBFPL vector structure as shown in FIGS. 7A-7C. First, the vector recovery product was obtained by double digestion of plv-IRES-C-3 XFlag-EGFP vector with XbaI/BamHI, plv-EGFPL and plv-EBFPL vectors were digested with BamHI/SalI, and the required Tmem176b and shp1 gene CDS fragments were amplified using the primers shown in Table 1 below, and the gene fragments were inserted into the digested sites of the vector to obtain complete plasmids.

Table 1: PCR primer

Preparation of stable transgenic virus: 1X 10 of six-hole plate ⁶ 293T cells were inoculated in wells, after cell density was reached to 70-80%, 200. Mu.l Opti-MEM was mixed with 2. Mu.g of expression plasmid and packaging plasmid 0.5. Mu.g pMD2.G (Addgene 12259) and 1.5. Mu.g psPAX2 (Addgene 12260), 8. Mu. l Polyethylenimine (PEI) (1 mg/mL), gently mixed, left to stand for 15 minutes, slowly added to cells, after 6-8 hours in a cell incubator, the medium was changed to pre-warmed DMEM complete medium, after 24 and 48 hours the supernatant was collected, 1000g centrifuged for 3min, the pellet was discarded, and the supernatant was collected until-80 was stored.

Preparation of stable transfer cells: suspension of EL4 cells at 2X 10 per well ⁵ mu.L of each cell/100. Mu.L of the medium was plated in a 24-well plate, followed by 400. Mu.L of the virus supernatant, and the virus solution was added with polybrene (final concentration 10 ng/. Mu.L); 2500rpm, centrifuging at 37 ℃ for 30min, and increasing the speed by 6 and decreasing the speed by 2; culturing in an incubator for 12h after centrifugation, replacing fresh culture medium for culturing for 48h, detecting infection efficiency according to GFP fluorescent markers, and sorting target cell lines.

Co-immunoprecipitation (Co-IP): the required EL4 cells were collected and 1X 10 cells were taken per group ⁷ Centrifuging 500g for 5min to remove supernatant, washing with 1 XPBS once, and centrifuging to remove supernatant;

adding 1mL NP40 Lysis buffer, blowing and sucking, mixing, cracking on ice for 30min, and centrifuging;

after ultrasonic treatment of the split protein suspension for several seconds, centrifuging at 12000rpm and 4 ℃ for 10min, removing precipitate, adding 80 mu L of supernatant into 20 mu L of 5 multiplied by SDS loading buffer, uniformly mixing, and preserving at-20 ℃ for 10min in a metal bath at 100 ℃ to obtain Input;

because the stable transgenic gene fusion expresses the Flag tag, the Flag in the protein suspension is enriched by using M2Flag beads, and then the protein complex of the target protein is obtained. The remaining protein suspension from the previous step was transferred in its entirety to washed M2Flag beads (every 1X 10) ⁷ Individual cells/20. Mu. L M2Flag beads), were mixed vertically at 4℃for 4h;

centrifuging at 4deg.C for 30s at 8000g, sucking off supernatant, adding 1ml NP40 Lysis buffer to the beads, mixing, centrifuging at 4deg.C for 30s at 8000g, discarding supernatant, and repeating washing for 5-6 times;

1.5 mu L of 3 xFlag peptides and 28.5 mu L of 2 x SDS loading buffer are added into each tube of the beads, and after uniform mixing, metal bath oscillation is performed at 25 ℃ for 1200rpm for 30min, and the proteins with the Flag labels are competitively eluted;

8000g, centrifuging at 4℃for 30s, collecting supernatant, and preserving at-20 ℃.

3. Experimental results

eGFP-Tmem176b and eBFP-Shp1 fusion proteins were simultaneously overexpressed in EL4 cells, and the co-localization of Tmem176b and Shp1 in EL4 cells was observed using confocal microscopy. The results show that Tmem176b and Shp1 have a distinct co-localization in the cytoplasm of EL4 cells (see figure 1A for results).

Further, the fusion protein Tmem176b-flag was overexpressed in EL4 cells and analyzed for proteins that may have interactions with Tmem176b using Co-immunoprecipitation (Co-IP) methods, showing that: shp1, a phosphatase that regulates T cell activation in T cells, has significant interactions with Tmem176B (see FIG. 1B for results).

Example 2:anti-cancer agentDesign and expression of monoclonal antibody of Tmem176bAnd purifying

1. Experimental materials and primary reagents

293T cell line (ATCC, CRL-3216)

Japanese white rabbit

DMEM (Yuanpei, L110 KJ)

RIPA 1640Medium (Yuanpei L210 KJ)

FBS(YOSHI,A1015)

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

Tmem176b expresses purified antigen 142-196aa xy02b-hFc-6His (Abclonal)

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

Color development liquid (TMB, thermo Fisher, CAT: 34029)

Termination liquid (2M H) ₂ SO ₄ )

Flow cell sorter (BD, aria III)

Multifunctional enzyme label instrument (Tecan E pelx)

2. Experimental method

The amino acid fragment 149-209AA of the mouse Tmem176b is selected, constructed on XY02a-FC-6His, and the 293FT eukaryotic expression system is adopted to express and purify the antigen.

And immunizing an experimental Japanese white rabbit expressing the purified antigen for 4 times, taking blood serum for ELISA detection, taking a rabbit spleen for B cell sorting, obtaining single B cell clone of the immunized rabbit, carrying out single cell culture, collecting culture B cell supernatant, and carrying out ELISA detection. Positive-response B cell clones were selected, RNA extracted and amplified to obtain LEM sequences (Linear expression module). The candidate LEM sequence is used for constructing a recombinant antibody expression plasmid, expressing and purifying the recombinant rabbit anti-Tmem 176b monoclonal antibody, and is named as 6E8.

2. Experimental results

The sequence information for Tmem176b mab 6E8 is as follows:

heavy chain variable region:

METGLRWLLLVAVLKGVQCQSVEESGGRLLKPDETLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIISIRGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARNTLYSSGWGGSDLWGQGTLVTVSS(SEQ ID NO:1)

light chain variable region:

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNNNLAWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCNAFGGGTEVVVK(SEQ ID NO:2)

heavy chain full length:

METGLRWLLLVAVLKGVQCQSVEESGGRLLKPDETLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIISIRGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARNTLYSSGWGGSDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:3)

full length of light chain:

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNNNLAWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:4)

the 6 CDRs of Tmem176b mab 6E8 are defined by IMGT numbering system as follows:

HCDR1：GFSLSSYA(SEQ ID NO:5)

HCDR2：ISIRGNT(SEQ ID NO:6)

HCDR3：ARNTLYSSGWGGSDL(SEQ ID NO:7)

LCDR1：QSVYNNNN(SEQ ID NO:8)

LCDR2：YAS(SEQ ID NO:9)

LCDR3：QGEFSCSSADCNA(SEQ ID NO:10)

the sequence of the heavy chain HCDR1 is shown as SEQ ID NO. 5, the sequence of the HCDR2 is shown as SEQ ID NO. 6, and the sequence of the HCDR3 is shown as SEQ ID NO. 7;

the sequence of the light chain LCDR1 is shown as SEQ ID NO. 8, the sequence of the LCDR2 is shown as SEQ ID NO. 9, and the sequence of the LCDR3 is shown as SEQ ID NO. 10.

Example 3: monoclonal antibody and TMEM176BA kind of electronic deviceAffinity experiments

1. Experimental materials and primary reagents

alpha-Tmem 176b monoclonal antibody (6E 8)

Tmem176b expresses purified antigen 142-196aa xy02b-hFc-6His (Abclonal)

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

Color development liquid (TMB, thermo Fisher, CAT: 34029)

Termination liquid (2M H) ₂ SO ₄ )

Multifunctional enzyme label instrument (Tecan E pelx)

2. Experimental method

Capture ELISA method detects the affinity of Tmem176b monoclonal antibody 6E8 to in vitro expressed purified antigen Tmem176 b.

Three candidate monoclonal antibodies, including antibody 6E8, were diluted with PBS and coated in 384 well plates (Corning, CAT: 3700) at 2. Mu.g/mL, 25. Mu.l/well, overnight at 4 ℃; washing 5 times with wash solution (self-priming) at 75 μl/well; blocking the non-specific binding sites using 50 μl/well blocking solution (self-assembly), incubation for 1 hour at room temperature; washing 5 times with wash solution (self-priming) at 75 μl/well; after dilution Tmem176b expressed purified antigen 142-196aa xy02b-hFc-6His (0.1. Mu.g/ml as starting concentration, 3-fold gradient 9 gradients, 25. Mu.l/well, incubation at room temperature for 1 h); washing 5 times with wash solution (self-priming) at 75 μl/well; secondary antibodies (HRP coat Anti-Rabbit IgG (h+l), jackson ImmunoResearch, 111-035-045) were diluted 1:5000 with dilution buffer, 25 μl/well was added to 384 well plates and incubated for 1H at room temperature in the absence of light; washing 5 times with wash solution (self-priming) at 75 μl/well; the color development solution (TMB, thermo Fisher, CAT: 34029) was diluted in a dilution ratio of 1:5, and developed for 3min at room temperature in the absence of light at 25 μl/well; mu.l of stop solution (2M H) was added to each well ₂ SO ₄ ) Terminating the reaction; 450 nm/630 nm read plate, using 450nm minus 630nm background.

3. Experimental results

As in table 2 and fig. 2.

TABLE 2

Clone	1F9	4F8	6E8
				EC 50 (μg/ml)	0.0007322	0.002399	0.000893

Remarks: antibodies 1F9 and 4F8 are rabbit monoclonal antibodies to murine Tmem176 b.

The results showed that the EC of antibody 6E8 with Tmem176b antigen ₅₀ The value was 0.000893, suggesting that the monoclonal antibody has a strong affinity for antigen binding.

Example 4: specificity experiment of monoclonal antibody and TMEM176B

1. Experimental materials and primary reagents

NTA chip Series S Sensor Chip CM (GE, BR 100530)

Amino coupling kit (Amine Coupling Kit, GE, 100050)

alpha-Tmem 176b monoclonal antibody (6E 8)

Tmem176b expresses purified antigen 142-196AA (Abclonal)

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

N-hydroxysuccinimide(NHS,115mg/ml)

Ethanolamine (1M)

NaOH(50mM)

70% glycerol

Sodium acetate-acetic acid buffer (10 mM), pH gradient 5.5, 5.0, 4.5, 4.0, etc. 4 gradients, the solution was filtered through 0.22 μm filter.

Glycine-hydrochloric acid buffer (10 mM) with pH gradient of 1.5, 2.0, 2.5, 3.0, etc. 4 gradients, the solution was filtered through 0.22 μm filter.

2. Experimental method

Monoclonal antibodies were prepared for antigen binding specificity using Biacore assays.

Expression purified Tmem176b antigen was dissolved in PBS for protein chip coupling.

NTA sensor chip module was inserted into BIAcore instrument, and mixed with 75. Mu.l NHS and 75. Mu.l EDC, and flowed through a flow cell at a flow rate of 5. Mu.l/min. Surface activation was performed by injection of 35. Mu.l of NHS/EDC mixture, surface activation was performed by injection of 35. Mu.l of purified Tmem176b antigen protein expressed in vitro, and excess reactive groups were inactivated by injection of 35. Mu.l of ethanolamine. 10 μl of 20mmol/LHCl was injected rapidly and then the non-covalently bound material was removed with Extradean. Expression of purified Tmem176b antigen in vitro was diluted to 1 μg/mL in PBS and coupled at a flow rate of 10 μl/min for 60 seconds. Analyte antibody 6E8 was assayed at 1.5625, 3.125, 6.25, 12.5, 25, 50nM concentration, 30 μL/min flow rate, binding 120s, dissociation 200s. The binding dissociation parameters of monoclonal antibody 6E8 with antigen Tmem176b were detected. Experiments were performed 3 times in parallel.

3. Experimental results

As in table 3 and fig. 3.

TABLE 3 Table 3

The results showed that the KD value of the monoclonal antibody 6E8 and Tmem176b antigen is 9.59X10-10M (average of 3 parallel experiments), and shows strong specific binding property.

Example 5: antitumor experiment (1)

1. Experimental animal and experimental sample

C57B6J mice, B16F10 (mouse melanoma) cells.

alpha-Tmem 176b monoclonal antibody (6E 8)

Alpha-isotype control mab (Abclonal, AC 042)

alpha-PD 1 blocking type monoclonal antibodies (anti-PD-1 blocking type monoclonal antibodies; MCE, RMP 1-14).

2. Experimental method

To test the inhibition of tumor growth by the candidate monoclonal antibodies, the inhibition of tumor growth by the candidate monoclonal antibodies was tested in a wild type C57B6J mouse subcutaneously vaccinated tumor model.

Wild type C57B6J mice were inoculated subcutaneously 2X 10 ⁵ B16F10 cells, until tumor growth for 10 days or volume to 100mm ³ The treatment was performed in a PBS control group, an isotopte control group, a 6E8 treatment group, an α -PD1 treatment group, and a combination group, each group having 6 mice. The dose of the isotype administration of the experimental group 6E8 and the control group is 10 mg/kg/time, the administration is carried out once in 3 days, and the administration is carried out for 3 times continuously, and the administration mode is intraperitoneal injection. The combined dosage of the alpha-PD 1 and the 6E8 and the alpha-PD 1 blocking monoclonal antibody is 10 mg/kg/time, the administration is carried out once in 3 days, and the continuous administration is carried out for 3 times, and the administration mode is intraperitoneal injection. Tumor growth was measured every 2 days. Until the tumor volume reaches 2000mm ³ 。

3. Experimental results

The results showed that the candidate monoclonal antibodies were effective in inhibiting B16F10 subcutaneous tumor growth compared to the control isotype antibodies (fig. 4A, fig. 4B).

The results also show that the combined use of the alpha-PD 1 blocking monoclonal antibodies can further inhibit the growth of subcutaneous tumors, and the tumor inhibition effect after the combined use is stronger than that of the single use of the candidate monoclonal antibodies or the PD1 monoclonal antibodies, and the statistical analysis has significant differences, so that the anti-TMEM 176B antibody and the anti-PD 1 antibody have a certain synergistic effect (figures 4C and 4D). The candidate monoclonal antibodies are suggested to be useful in combination with existing immunotherapeutic regimens, with the potential to further enhance therapeutic efficacy.

Example 6: antitumor experiment (2)

1. Experimental animal and experimental sample

C57B6J mice, MC38 (mouse colorectal cancer) cells.

alpha-Tmem 176b monoclonal antibody (6E 8)

Alpha-isotype control mab (Abclonal, AC 042)

alpha-PD 1 blocking monoclonal antibodies (MCE, RMP 1-14).

2. Experimental method

To test the inhibition of colorectal tumor growth by the candidate monoclonal antibodies, the inhibition of tumor growth by the candidate monoclonal antibodies was tested in a wild-type C57B6J mouse subcutaneously vaccinated tumor model.

Subcutaneous inoculation of wild-type C57B6J mice with 1X 10 ⁶ MC38 cells, when tumor grows for 12 days or volume to 100mm ³ The treatment was performed in a PBS control group, an isotopte control group, a 6E8 treatment group, an α -PD1 treatment group, and a combination group, each group having 6 mice. The dose of the isotype administration of the experimental group 6E8 and the control group is 10 mg/kg/time, the administration is carried out once in 3 days, and the administration is carried out for 3 times continuously, and the administration mode is intraperitoneal injection. The combined dosage of the alpha-PD 1 and the 6E8 and the alpha-PD 1 blocking monoclonal antibody is 10 mg/kg/time, the administration is carried out once in 3 days, and the continuous administration is carried out for 3 times, and the administration mode is intraperitoneal injection. Tumor growth was measured once every 4 days. Until the tumor volume reaches 2000mm ³ 。

3. Experimental results

The results showed that the monoclonal antibodies were effective in inhibiting MC38 subcutaneous tumor growth compared to control isotype antibodies (fig. 5A, 5B).

The results also show that the combined use of the alpha-PD 1 blocking type monoclonal antibodies can further inhibit the growth of subcutaneous tumors (fig. 5C and 5D), and the tumor inhibition effect after the combined use is obviously stronger than that of the monoclonal antibodies or the PD1 monoclonal antibodies, and the statistical analysis has obvious difference, so that the anti-TMEM 176B antibody and the anti-PD 1 antibody have a certain synergistic effect. The candidate monoclonal antibodies are suggested to be useful in combination with existing immunotherapeutic regimens, with the potential to further enhance therapeutic efficacy.

Example 7: antitumor experiment (3)

1. Experimental animal and experimental sample

C57B6J mice, hepa1-6 (mouse liver cancer) cells.

alpha-Tmem 176b monoclonal antibody (6E 8)

Alpha-isotype control mab (Abclonal, AC 042)

2. Experimental method

To test the inhibition of the growth of liver cancer by the candidate monoclonal antibody, the inhibition of the growth of tumor by the candidate monoclonal antibody is tested in a liver inoculation Hepa1-6 tumor model of a wild type C57B6J mouse.

Wild type C57B6J mice were inoculated subcutaneously 2X 10 ⁶ Hepa1-6 cells, after 10 days of tumor growth, subcutaneous tumors were removed and excised to 1mm ³ Tumor tissue blocks, C57B6J mice to be vaccinated were surgically anesthetized, and 2 tumor blocks were embedded per mouse liver and surgically sutured. Tumor-bearing C57B6J mice were treated 3 weeks later and the experiment was divided into PBS control group, isotype control group and 6E8 antibody-treated group, 5 mice per group. The Isotype doses of the experimental group 6E8 and the control group are 10 mg/kg/time, and are administered once in 3 days and 3 times continuously, and the administration mode is intraperitoneal injection. Samples were taken 6 weeks after tumor growth for tumor size measurement and analysis.

3. Experimental results

The results showed that the candidate monoclonal antibodies were effective in inhibiting Hepa1-6 liver in situ tumor growth compared to isotype control antibodies (fig. 6A, fig. 6B). The antibodies of the invention are suggested to have the potential to inhibit liver tumor growth.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (21)

1. An anti-Tmem 176b antibody or antigen binding fragment thereof, said anti-Tmem 176b antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 and a light chain variable region comprising LCDR1 to LCDR3, wherein:

the amino acid sequence of HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 7, and

the amino acid sequence of LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 9, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 10.

2. The anti-Tmem 176b antibody or antigen binding fragment thereof of claim 1, wherein,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

3. The anti-Tmem 176b antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein the heavy chain constant region of the antibody is an Ig gamma-1chain C region or an Ig gamma-4chain C region; the light chain constant region is Ig kappa chain C region.

4. The anti-Tmem 176b antibody or antigen-binding fragment thereof of any of claims 1-3, wherein the anti-Tmem 176b antibody or antigen-binding fragment thereof 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-Tmem 176b antibody or antigen-binding fragment thereof of any of claims 1-4, wherein,

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

6. The anti-Tmem 176b antibody or antigen binding fragment thereof of any one of claims 1-5, wherein:

EC in which the anti-Tmem 176b antibody binds to Tmem176b ₅₀ 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 For measurement by Capture ELISA.

7. The anti-Tmem 176b antibody or antigen binding fragment thereof of any one of claims 1-6, wherein:

the anti-Tmem 176b antibody binds 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 For measurement by Biacore assay.

8. The anti-Tmem 176b antibody or antigen binding fragment thereof of claim 1, wherein,

the heavy chain of the anti-Tmem 176b antibody has an amino acid sequence shown in SEQ ID NO. 3, and the light chain has an amino acid sequence shown in SEQ ID NO. 4.

9. An isolated nucleic acid molecule encoding the anti-Tmem 176b antibody or antigen binding fragment thereof of any of claims 1-8.

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 antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is the anti-Tmem 176b antibody or antigen-binding fragment thereof of any 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 chemotherapeutic.

13. The antibody drug conjugate of claim 12, wherein the antibody or antigen binding fragment thereof is linked to a 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-Tmem 176b antibody or antigen binding fragment thereof of any of claims 1-8 or the antibody drug conjugate of any of claims 12-13; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

15. The pharmaceutical composition of claim 14, further comprising one or more immune checkpoint inhibitors;

preferably, the immune checkpoint inhibitor is an antibody that targets PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR, and/or OX 40;

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 mab or an anti-PD-L1 blocking mab.

16. The pharmaceutical composition of claim 15, wherein the mass ratio of the immune checkpoint inhibitor to the anti-Tmem 176b antibody or antigen binding fragment thereof is from (1:5) to (5:1), preferably from (1:2) to (2:1), more preferably 1:1.

17. A pharmaceutical product combination comprising a first pharmaceutical product and a second pharmaceutical product, wherein:

the first pharmaceutical product comprises the anti-Tmem 176b antibody or antigen binding fragment thereof of any of claims 1 to 8 or the antibody drug conjugate of any of claims 12 to 13;

the second pharmaceutical product comprises one or more immune checkpoint inhibitors;

preferably, the immune checkpoint inhibitor is an antibody that targets PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR, and/or OX 40;

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 mab or an anti-PD-L1 blocking mab.

18. The pharmaceutical product combination according to claim 17, wherein,

wherein the mass ratio of the immune checkpoint inhibitor to the anti-Tmem 176b antibody or antigen binding fragment thereof is (1:5) to (5:1), preferably (1:2) to (2:1), more preferably 1:1.

19. the pharmaceutical product combination according to any one of claims 17 to 18, wherein,

The first and second pharmaceutical products independently comprise one or more pharmaceutically acceptable excipients;

preferably, the pharmaceutical instructions are also included.

20. Use of an anti-Tmem 176b antibody or antigen binding fragment thereof of any of claims 1 to 8 or an antibody drug conjugate of any of claims 12 to 13 in the manufacture of a medicament for the treatment or prophylaxis of a tumor;

preferably, the tumor is a TMEM176B positive tumor;

preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchus cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer.

21. Use of an anti-Tmem 176b antibody or antigen binding fragment thereof in the manufacture of a medicament for the treatment or prophylaxis of a tumor;

preferably, the tumor is a TMEM176B positive tumor;

preferably, the tumor is one or more selected from melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchus cancer, lymphoma, ovarian cancer, esophageal cancer, hematological tumor, glioblastoma, lung cancer, prostate cancer, bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer;

Preferably, the anti-Tmem 176b antibody or antigen binding fragment thereof is capable of blocking or inhibiting Tmem176b binding to Shp 1.