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-Tmem176b antibodies, pharmaceutical compositions and uses

Technical field

The invention belongs to the field of biomedicine and relates to an anti-Tmem176b antibody, pharmaceutical compositions and uses. Specifically, the anti-Tmem176b antibody is an anti-Tmem176b monoclonal antibody.

Background technique

Tmem176b is a member of the four-transmembrane structural protein MS4A family. It is located on the membranes of various organelles in cells and has the function of regulating intracellular Ca2 ⁺ transport and thereby regulating immune cell function. Tmem176b is mostly expressed in lymphocytes and monocytes, macrophages, dendritic cells and RORγT ⁺ cells. Studies have shown that reducing the expression of TMEM176B in dendritic cells can activate the Caspase-1/IL-1β signaling pathway and improve the anti-tumor immune function of dendritic cells, thereby improving the body's response to immune checkpoint blockade (ICB) therapy. sensitivity. Another report shows that TMEM176B is highly expressed in human colorectal tumor tissues, has a significant negative correlation with the prognosis of tumor patients, and has a clear correlation with clinical ICB treatment sensitivity.

SHP-1 (also expressed as Shpl in the present invention) is a Src-homology domain 2 (SH2) protein tyrosine phosphatase-1 (SH2-containing protein tyrosine phosphatase, non-receptor type 6 (PTPN6), a tyrosine phosphatase protein mainly expressed in the cytoplasm of hematopoietic cells, is a key factor in regulating intracellular phosphorylation levels. This family has 2 proteins, including SHP-1 and SHP-2. Encoding The SHP-1 gene is located at 12p13 and has two SH2 domains at the N-terminus, a phosphorylation domain and a tyrosine phosphorylation site at the C-terminus. In T lymphocytes, SHP-1 can TCR proximal activation signals such as PLCγ1 and SLP76 are dephosphorylated, down-regulating TCR signals, thereby inhibiting T cell activation, proliferation, and maturation. Studies have shown that peripheral T cells with defective SHP-1 expression also exhibit TCR-induced cell damage. Apoptotic response is enhanced.

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

Contents of the invention

After in-depth research and creative work, the inventor discovered the interaction between Tmem176b and Shp1. Tmem176b inhibits TCR proximal signaling molecules by recruiting Shp1, thereby inhibiting T cell activation, proliferation and anti-tumor function. 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 signaling complex by binding to the extracellular segment of Tmem176b on the surface of T cells, thereby releasing the inhibitory effect of Shp1 on TCR signals. The experimental results show significant tumor inhibitory effect. Combination with PD-1 monoclonal antibody can further improve the anti-tumor effect and has good anti-tumor prospects. The following invention is thereby provided:

One aspect of the invention relates to an anti-Tmem176b antibody or an antigen-binding fragment thereof, the anti-Tmem176b antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 to HCDR3, the light chain variable region The chain variable region contains LCDR1 to LCDR3, where:

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.

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

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.

In some embodiments of the present invention, the anti-Tmem176b antibody or its antigen-binding fragment, 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 the Ig kappa chain C region.

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

The EC ₅₀ of the anti-Tmem176b antibody binding to Tmem176b 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 is measured by Capture ELISA.

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

The KD of the anti-Tmem176b antibody binding to Tmem176b is 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 is measured by Biacore detection method.

In some embodiments of the invention, the anti-Tmem176b antibody or antigen-binding fragment thereof, 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.

The anti-Tmem176b antibody or antigen-binding fragment thereof according to any one of the present invention, which is used to treat or prevent tumors;

Preferably, the tumor is a TMEM176B-positive tumor;

Preferably, the tumor is selected from the group consisting of melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematoma, glioblastoma, lung cancer, prostate cancer, One or more of bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

In the present invention, unless otherwise specified, the Tmem176b (also expressed as TMEM176B) is mouse Tmem176b or human Tmem176b.

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

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

Yet another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of the invention, or the recombinant vector of the invention.

Another aspect of the present invention relates to antibody-drug conjugates, which include antibodies or antigen-binding fragments thereof and small molecule drugs, wherein the antibody or antigen-binding fragments thereof are anti-Tmem176b antibodies according to any one of the present invention or Its antigen-binding fragment; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug.

In some embodiments of the present invention, the antibody drug conjugate, wherein the antibody or its antigen-binding fragment is connected to a small molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or peptide bonds;

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.

The antibody drug conjugate according to any one of the present invention is used to treat or prevent tumors;

Preferably, the tumor is a TMEM176B-positive tumor;

Preferably, the tumor is selected from the group consisting of melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematoma, glioblastoma, lung cancer, prostate cancer, One or more of bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

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 also includes one or more pharmaceutically acceptable excipients.

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

In some embodiments of the present invention, the pharmaceutical composition, in which the anti-Tmem176b antibody of the present invention or its antigen-binding fragment or the antibody-drug conjugate of any one of the present invention is the only active ingredient.

In some embodiments of the present invention, the pharmaceutical composition consists of the anti-Tmem176b antibody of the present invention or its antigen-binding fragment or the antibody-drug conjugate of any one of the present invention, and one or more Pharmaceutically acceptable excipients.

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

Preferably, the immune checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR and/or OX40 antibodies;

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.

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 antigen-binding fragment thereof is (1:5) to (5:1 ), preferably (1:2) to (2:1), more preferably 1:1.

A further 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 includes 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;

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

Preferably, the immune checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR and/or OX40 antibodies;

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.

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

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), More preferably, it is 1:1.

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

The first pharmaceutical product and the second pharmaceutical product independently include one or more pharmaceutically acceptable excipients;

Preferably, drug instructions are also included.

Another aspect of the present invention relates to the use 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 in the preparation of drugs for treating or preventing tumors. ;

Preferably, the tumor is a TMEM176B-positive tumor;

Preferably, the tumor is selected from the group consisting of melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematoma, glioblastoma, lung cancer, prostate cancer, One or more of bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

Yet another aspect of the present invention relates to a method for treating or preventing tumors, comprising administering to a subject in need an effective amount of the anti-TMEM176B antibody or antigen-binding fragment thereof according to any one of the present invention or any of the present invention. The steps of the antibody drug conjugate described in one item;

Preferably, the tumor is a TMEM176B-positive tumor;

Preferably, the tumor is selected from the group consisting of melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematoma, glioblastoma, lung cancer, prostate cancer, One or more of bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

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

In some embodiments of the present invention, the method of treating or preventing tumors, wherein,

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

Preferably, it is administered 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 administration method is intravenous drip or intravenous injection.

Yet another aspect of the present invention relates to the use of anti-Tmem176b antibodies or antigen-binding fragments thereof in the preparation of drugs for treating or preventing tumors;

Preferably, the tumor is a TMEM176B-positive tumor;

Preferably, the tumor is selected from the group consisting of melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematoma, glioblastoma, lung cancer, prostate cancer, One or more of bladder cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer;

Preferably, the anti-Tmem176b antibody or antigen-binding fragment thereof can block or inhibit the binding of Tmem176b to Shpl.

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

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

As used herein, the term _EC50 refers to the concentration for 50% ofmaximal effect, the concentration that causes 50% of the maximum effect.

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified into kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, 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 approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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 (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 may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 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 respectively form the antibody binding site. The assignment of amino acids to regions or domains follows Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al. Nature 1989;342:878-883, or IMGT numbering system definition, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT/3Dstructure-DB andIMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, Tcell receptors, MHC, Definition of IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1):D301-D307.

The term "antibody" is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), 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 group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously. A group of identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which 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 hybridoma technology first reported by Kohler et al. ( G, MilsteinC. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256 (5517): 495), but can also be obtained using recombinant DNA technology (eg, see US Patent 4,816,567).

As used herein, the term "humanized antibody" refers to a product in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by the CDR regions of a non-human antibody (donor antibody). Antibodies or antibody fragments, wherein the donor antibody may be a non-human (eg, mouse, rat or rabbit) antibody with desired specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced by amino acid residues of the corresponding non-human antibody, or by 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; 332:323329; Presta, Curr. Op. Struct. Biol. 1992;2:593-596; Clark, Immunol. Today 2000;21:397 402.

As used herein, the term "single chain fragment variable (ScFv)" refers to an antibody comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker. molecular. wherein the VL and VH domains are paired to form a monovalent molecule via a linker that enables production of a single polypeptide chain (see, e.g., Birdet 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: 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 with 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 useful in the present invention are provided 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, described by Kipriyanov et al, J. Mol. Biol. 1999;293:41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

As used herein, the term "isolated" or "isolated" means obtained from the natural state by artificial means. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has 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 the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of. The term "isolated" or "isolated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40). A vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.

As used herein, a cell as a host refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or insect cells such as 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 the reaction between an antibody and the antigen against which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) refers to an antibody that binds to an antigen at a concentration of less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, Binds the antigen with an affinity (K _D ) of 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less.

As used herein, the term " _KD " refers to the dissociation equilibrium constant of a specific 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 the antigen. Typically, the antibody dissociates with a dissociation equilibrium constant (K _D ) of less than about 10 ⁻⁵ M, such as less than about 10 ⁻⁶ M, 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ^{−9 M} , or 10 ⁻¹⁰ M or less. Binds an antigen (e.g., TMEM176B protein). _KD can be determined using methods known to those skilled in the art, such as using the Biacore assay.

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. And in the present invention, amino acids are generally represented by one-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 excipient" or "pharmaceutically acceptable carrier and/or excipient" means pharmacologically and/or physiologically compatible with the subject and the active ingredient Carriers and/or excipients, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by GennaroAR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include but are not limited to: pH adjusters, surface active agents agent, adjuvant, ionic strength enhancer. For example, pH adjusters include but are not limited to phosphate buffer; 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 immune enhancer that, when delivered into the body together with the antigen or in advance, can enhance the body's immune response to the antigen or change the type of immune response. There are many kinds of adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. . Freund's adjuvant is currently the most commonly used adjuvant in animal testing. Aluminum hydroxide adjuvant is commonly used in clinical trials.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease; a disease-treating effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already suffer from the disease. Determining such effective amounts is well within the capabilities 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 overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.

In the present invention, unless otherwise specified, the "first" (for example, the first pharmaceutical product) or the "second" (for example, the second pharmaceutical product) is only for reference and does not have any special order. meaning.

Beneficial effects of the invention

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

(1) The anti-Tmem176b antibody or antigen-binding fragment thereof of the present invention has high affinity to the antigen Tmem176b.

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

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

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

(5) The anti-Tmem176b antibody or antigen-binding fragment thereof 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.

Description of drawings

Figure 1A to Figure 1B: Verification of interaction between Tmem176b and Shp1. in:

Figure 1A: Detection of co-localization of Tmem176b and Shp1. Immunofluorescence analysis of co-localization of overexpressed Tmem176b and Shp1 in EL4 cells.

Figure 1B: Co-immunoprecipitation to detect the interaction between Tmem176b and Shp1 in EL4 cells.

Figure 2: Tmem176b monoclonal antibody 6E8 affinity determination. Capture ELISA method was used to detect the affinity of Tmem176b monoclonal antibody 6E8 with the in vitro expressed and purified antigen Tmem176b.

Figure 3: Determination of the specific binding ability of Tmem176b monoclonal antibody 6E8 to antigen. The Biacore method was used to determine the affinity KD50 value of Tmem176b monoclonal antibody 6E8 and the in vitro expressed and purified antigen Tmem176b.

Figure 4A to Figure 4D: Test of candidate blocking monoclonal antibody 6E8 inhibiting mouse melanoma B16F10 tumor growth. C57B6J mice were subcutaneously inoculated with B16F10 cells to test the tumor growth inhibition of candidate monoclonal antibodies and their combination with α-PD1 antibodies. in:

Figure 4A and Figure 4B: Test of candidate monoclonal antibodies inhibiting the growth and survival of subcutaneously inoculated B16F10 tumors.

Figure 4C and Figure 4D: Candidate monoclonal antibody combined with α-PD1 inhibits B16F10 tumor growth and mouse survival test.

Figure 5A to Figure 5D: Test of inhibiting tumor growth of mouse colorectal tumor MC38 by candidate blocking monoclonal antibody 6E8. C57B6J mice were subcutaneously inoculated with MC38 cells to test the tumor growth inhibition of candidate monoclonal antibodies and their combination with α-PD1 antibodies. in:

Figure 5A and Figure 5B: Test of candidate monoclonal antibodies inhibiting MC38 subcutaneously inoculated tumor growth and survival.

Figure 5C and Figure 5D: Candidate monoclonal antibody combined with α-PD1 inhibits MC38 tumor growth and mouse survival test.

Figure 6A to Figure 6B: Test of candidate blocking monoclonal antibody 6E8 inhibiting mouse liver cancer Hepa1-6 tumor growth. Hepa1-6 cells were orthotopically inoculated into the livers of C57B6J mice to test the inhibitory effect of candidate monoclonal antibodies on the growth of liver orthotopic tumors. in:

Figure 6A: Growth of orthotopic liver tumors in mice.

Figure 6B: Statistical diagram of mouse orthotopic liver tumor volume.

Figure 7A: plv-IRES-C-3×Flag-EGFP vector structure.

Figure 7B: plv-EGFPL vector structure.

Figure 7C: plv-EBFPL vector structure.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Example 1: Study on the interaction between Tmem176b and Shp1

1. Experimental materials and main reagents

As shown in Table A below.

Table A

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

2. Experimental methods

Stable transfer plasmid construction: The plasmid vectors are plv-IRES-C-3×Flag-EGFP, plv-EGFPL, and plv-EBFPL. The vector structures are shown in Figure 7A to Figure 7C. First, use XbaI/BamHI to double-digest the plv-IRES-C-3×Flag-EGFP vector to obtain the vector recovery product. BamHI/SalI double-digest the plv-EGFPL and plv-EBFPL vectors and use the primers in Table 1 below. Amplify the required CDS fragments of Tmem176b and shp1 genes, and insert the gene fragments into the restriction site of the vector to obtain a complete plasmid.

Table 1: PCR primers

Preparation of stably transfected virus: Inoculate 293T cells at 1×10 ⁶ /well in a six-well plate. When the cell density reaches 70-80%, mix 2 μg expression plasmid and packaging plasmid 0.5 μg pMD2.G (Addgene 12259) into 200 μl Opti-MEM. And 1.5μgpsPAX2 (Addgene 12260), 8μl polyethylenimine (PEI) (1mg/mL), mix slightly and let it stand for 15 minutes, then slowly add it to the cells. After 6-8 hours in the cell culture incubator, replace the medium with preheating DMEM complete culture medium, collect the supernatant after 24 and 48 hours, centrifuge at 1000g for 3 minutes, discard the pellet, collect the supernatant and store at -80°C.

Preparation of stably transfected cells: Plate the EL4 suspension cell line in a 24-well plate at 2×10 ⁵ cells/100 μL culture medium per well, then add 400 μL virus supernatant, and add polybrene to the virus liquid (final concentration 10 ng/μL); Centrifuge at 2500 rpm, 37°C for 30 minutes, increase the speed by 6 and decrease the speed by 2; after centrifugation, culture in the incubator for 12 hours, change to fresh medium and continue to culture for 48 hours. The infection efficiency is detected according to the GFP fluorescent marker and the target cell line is sorted.

Co-immunoprecipitation (Co-IP): Collect the required EL4 cells, take 1×10 ⁷ cells from each group, centrifuge at 500g for 5 minutes to remove the supernatant, wash once with 1×PBS, and centrifuge to remove the supernatant;

Add 1mL NP40 Lysis buffer, mix by pipetting, lyse on ice for 30 minutes, and centrifuge;

Ultrasonicate the lysed protein suspension for a few seconds, centrifuge at 12000 rpm and 4°C for 10 min, remove the precipitate, add 80 μL of the supernatant to 20 μL of 5×SDS loading buffer, mix well, and store in a metal bath at 100°C for 10 min, then store at -20°C. Input;

Because the stably transduced gene fusion expresses the Flag tag, M2 Flag beads are used to enrich the Flag in the protein suspension, and then obtain the protein complex of the target protein. Transfer all the remaining protein suspension from the previous step to the washed M2 Flag beads (1 × 10 ⁷ cells/20 μL M2 Flag beads), and mix vertically at 4°C for 4 hours;

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

Add 1.5 μL 3×Flag peptide and 28.5 μL 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;

Centrifuge at 8000g for 30 seconds at 4°C, take the supernatant and store at -20°C.

3.Experimental results

The 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 had obvious co-localization in the cytoplasm of EL4 cells (results shown in Figure 1A).

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 T cell activation regulator Phosphatase, a significant interaction occurred with Tmem176b (results shown in Figure 1B).

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

1. Experimental materials and main reagents

293T cell line (ATCC, CRL-3216)

Japanese White Rabbit

DMEM (Yuanpei, L110KJ)

RIPA 1640Medium (Yuanpei, L210KJ)

FBS(YOSHI,A1015)

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

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

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

Chromogenic solution (TMB, Thermo Fisher, CAT: 34029)

Stop solution (2M H ₂ SO ₄ )

Flow cytometry sorter (BD, Aria III)

Multifunctional microplate reader (Tecan, E pelx)

2. Experimental methods

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

The expressed and purified antigen was immunized against experimental-grade Japanese white rabbits for a total of 4 times. The serum was collected for ELISA detection, and the spleen of the rabbit was taken for B cell sorting. A single B cell clone of the immunized rabbit was obtained. Single cell culture was performed, and the cultured B cells were collected and cultured. Clear and conduct ELISA test. Select positive B cell clones, extract RNA and amplify to obtain the LEM sequence (Linear expression module). The candidate LEM sequence was used to construct a recombinant antibody expression plasmid to express and purify the recombinant rabbit-derived anti-Tmem176b monoclonal antibody, named 6E8.

2.Experimental results

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

Heavy chain variable region:

METGLRWLLLVAVLKGVQCQSVEESGGRLLKPDETLTLTCTVS GFSLSSYA MSWVRQAPGKGLEWIGI ISIRGNT YYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFC ARNTLYSSGWGGSDL WGQGTLVTVSS(SEQ IDNO:1)

Light chain variable region:

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQAS QSVYNNNN LAWYQQKPGQPPKLLIY YAS TLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYC QGEFSCSSADCNA FGGGTEVVVK(SEQ IDNO:2)

Full length of heavy chain:

METGLRWLLLVAVLKGVQCQSVEESGGRLLKPDETLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIISIRGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARNTLYSSGWGGSDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNV AHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTS EWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:3)

Full length of light chain:

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNNNLAWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLT LTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:4)

The 6 CDRs of Tmem176b monoclonal antibody 6E8 are defined through the 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 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;

The sequence of 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.

Example 3: Affinity experiment between monoclonal antibodies and TMEM176B

1. Experimental materials and main reagents

α-Tmem176b monoclonal antibody (6E8)

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

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

Chromogenic solution (TMB, Thermo Fisher, CAT: 34029)

Stop solution (2M H ₂ SO ₄ )

Multifunctional microplate reader (Tecan, E pelx)

2. Experimental methods

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

Dilute three candidate monoclonal antibodies, including antibody 6E8, with PBS at 2 μg/mL, 25 μl/well, coat a 384-well plate (Corning, CAT: 3700), and leave overnight at 4°C; use washing solution (self-prepared) at 75 μl /well, wash 5 times; use 50μl/well blocking solution (self-prepared) to block non-specific binding sites, and incubate at room temperature for 1 hour; use washing solution (self-prepared) at 75μl/well, wash 5 times; add the diluted Tmem176b Express and purify the antigen 142-196aa xy02b-hFc-6His (0.1 μg/ml as the starting concentration, 9 gradients of 3-fold dilution, 25 μl/well, incubation at room temperature for 1 hour); use washing solution (self-prepared) at 75 μl/well, Wash 5 times; the secondary antibody (HRP Goat Anti-Rabbit IgG (H+L), Jackson ImmunoResearch, 111-035-045) is diluted with dilution buffer at a dilution ratio of 1:5000, and 25 μl/well is added to the 384-well plate. Incubate at room temperature in the dark for 1 hour; use washing solution (self-prepared) at 75 μl/well, wash 5 times; use chromogenic solution (TMB, Thermo Fisher, CAT: 34029), dilute at a dilution ratio of 1:5, use 25 μl/well at room temperature Protect from light and develop color for 3 minutes; add 10 μl stop solution (2M H ₂ SO ₄ ) to each well to stop the reaction; read the plate at 450nm\630nm, and use 450nm to subtract the 630nm background.

3.Experimental results

As shown in Table 2 and Figure 2.

Table 2

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

Note: Antibodies 1F9 and 4F8 are rabbit-derived monoclonal antibodies directed against mouse Tmem176b.

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

Example 4: Specificity experiment of monoclonal antibody and TMEM176B

1. Experimental materials and main reagents

NTA chip Series S Sensor Chip CM5 (GE, BR100530)

Amine Coupling Kit (GE, 100050)

α-Tmem176b monoclonal antibody (6E8)

Tmem176b expressed 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% glycerin

Sodium acetate-acetic acid buffer solution (10mM), the pH gradient is 4 gradients such as 5.5, 5.0, 4.5, and 4.0. The solution must be filtered through a 0.22μm filter membrane.

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

2. Experimental methods

Use Biacore detection to prepare monoclonal antibodies with antigen-binding specificity.

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

Embed the NTA sensor chip module into the BIAcore instrument, mix 75 μl of NHS and 75 μl of EDC, and flow through a flow cell at a flow rate of 5 μl/min. Inject 35 μl of NHS/EDC mixture to activate the surface, inject 35 μl of in vitro expressed and purified Tmem176b antigen protein onto the activated surface, and inject 35 μl of ethanolamine to inactivate excess reactive groups. Rapidly inject 10 μl of 20 mmol/LHCl, and then use Extradan to remove non-covalently bound material. In vitro expressed and purified Tmem176b antigen was diluted to 1 μg/mL in PBS and coupled at a flow rate of 10 μL/min for 60 seconds. The analyte antibody 6E8 was analyzed at concentrations of 1.5625, 3.125, 6.25, 12.5, 25, and 50nM, with a flow rate of 30 μL/min, binding for 120s, and dissociation for 200s. Detection of binding and dissociation parameters of monoclonal antibody 6E8 and antigen Tmem176b. The experiment was carried out three times in parallel.

3.Experimental results

As shown in Table 3 and Figure 3.

table 3

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

Example 5: Anti-tumor experiment (1)

1. Experimental animals and experimental samples

C57B6J mouse, B16F10 (mouse melanoma) cells.

α-Tmem176b monoclonal antibody (6E8)

α-isotype control monoclonal antibody (Abclonal, AC042)

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

2. Experimental methods

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 the subcutaneous inoculation tumor model of wild-type C57B6J mice.

Wild-type C57B6J mice were subcutaneously inoculated with 2×10 ⁵ B16F10 cells. Treatment was performed after the tumor had grown for 10 days or reached a volume of 100 mm ³ . The experiment was divided into PBS control group, isotypte control group, 6E8 treatment group, α-PD1 treatment group and combined Use groups, 6 mice in each group. The dose of isotype in the experimental group 6E8 and the control group was 10 mg/kg/time, once every 3 days, and for 3 consecutive times. The administration method was intraperitoneal injection. The dosage of α-PD1 and 6E8 combined with α-PD1 blocking monoclonal antibody is 10 mg/kg/time, once every 3 days, for 3 consecutive times, and the administration method is intraperitoneal injection. Tumor growth was measured every 2 days. until the tumor volume reaches 2000mm ³ .

3.Experimental results

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

The results also show that the combined use of α-PD1 blocking monoclonal antibodies can further inhibit the growth of subcutaneous tumors, and the tumor inhibitory effect after combined use is stronger than the effect of using candidate monoclonal antibodies or PD1 monoclonal antibodies alone. Statistical analysis has a significant difference, indicating that The combination of anti-TMEM176B antibody and anti-PD1 antibody has a certain synergistic effect (Figure 4C, Figure 4D). It is suggested that this candidate monoclonal antibody can be combined with existing immunotherapy regimens and has the potential to further improve the therapeutic efficacy.

Example 6: Anti-tumor experiment (2)

1. Experimental animals and experimental samples

C57B6J mouse, MC38 (mouse colorectal cancer) cells.

α-Tmem176b monoclonal antibody (6E8)

α-isotype control monoclonal antibody (Abclonal, AC042)

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

2. Experimental methods

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 tumor model of subcutaneous inoculation of wild-type C57B6J mice.

Wild-type C57B6J mice were subcutaneously inoculated with 1×10 ⁶ MC38 cells. Treatment was carried out after the tumor had grown for 12 days or reached a volume of 100 mm ³ . The experiment was divided into PBS control group, isotypte control group, 6E8 treatment group, α-PD1 treatment group and combined Use groups, 6 mice in each group. The dose of isotype in the experimental group 6E8 and the control group was 10 mg/kg/time, once every 3 days, and for 3 consecutive times. The administration method was intraperitoneal injection. The dosage of α-PD1 and 6E8 combined with α-PD1 blocking monoclonal antibody is 10 mg/kg/time, once every 3 days, for 3 consecutive times, and the administration method is intraperitoneal injection. Tumor growth was measured every 4 days. Until the tumor volume reaches 2000mm ³ .

3.Experimental results

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

The results also show that the combined use of α-PD1 blocking monoclonal antibodies can further inhibit the growth of subcutaneous tumors (Figure 5C, Figure 5D), and the tumor inhibitory effect after combined use is significantly stronger than the effect of using monoclonal antibodies or PD1 monoclonal antibodies alone. Statistical analysis showed significant differences, indicating that the combination of anti-TMEM176B antibody and anti-PD1 antibody has a certain synergistic effect. It is suggested that this candidate monoclonal antibody can be combined with existing immunotherapy regimens and has the potential to further improve the therapeutic efficacy.

Example 7: Anti-tumor experiment (3)

1. Experimental animals and experimental samples

C57B6J mice, Hepa1-6 (mouse liver cancer) cells.

α-Tmem176b monoclonal antibody (6E8)

α-isotype control monoclonal antibody (Abclonal, AC042)

2. Experimental methods

In order to test the inhibitory effect of the candidate monoclonal antibody on the growth of liver cancer, the inhibitory effect of the candidate monoclonal antibody on the tumor growth was tested in the Hepa1-6 tumor model of wild-type C57B6J mouse liver inoculation.

Wild-type C57B6J mice were subcutaneously inoculated with 2×10 ⁶ Hepa1-6 cells. After 10 days of tumor growth, the subcutaneous tumors were removed and cut into 1 mm ³ tumor tissue blocks. The C57B6J mice to be inoculated were surgically anesthetized, and the liver of each mouse was wrapped. Two tumor masses were buried and surgically sutured. After 3 weeks, the tumor-bearing C57B6J mice were treated. The experiment was divided into PBS control group, isotype control group and 6E8 antibody treatment group, with 5 mice in each group. The dosage of 6E8 in the experimental group and Isotype in the control group was 10 mg/kg/time, once every 3 days, and 3 times in a row. The administration method was intraperitoneal injection. After 6 weeks of tumor growth, samples were taken for tumor size measurement and analysis.

3.Experimental results

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

Although specific embodiments of the invention have been described in detail, they will be understood by those skilled in the art. According to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are within the protection 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.