Anti-CD209 antibody, product, nucleic acid molecule, vector, cell and application thereof

Abstract

The invention discloses an anti-CD209 antibody, a product, a nucleic acid molecule, a carrier, a cell and application thereof, and relates to the technical field of biomedicine. The invention provides a new therapeutic scheme for accelerating deterioration of tumors by inhibiting T cells through the ligand CD209, the anti-human CD209 monoclonal antibody can specifically recognize CD209 and bind with CD209, so that the human cell surface ligand CD209 is closed, the CD209 cannot inhibit the functions of the T cells, the anti-tumor immunity is enhanced, the tumor growth is obviously inhibited, the lifetime is prolonged, and the anti-human CD209 monoclonal antibody can be used for treating the tumors. The active anti-tumor function is realized in various solid tumors. In addition, the monoclonal antibody provided by the invention can be used for detection, separation and enrichment of human CD209 protein.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to anti-CD209 antibodies, products, nucleic acid molecules, vectors, cells, and their applications. Background Technology

[0002] Cancer has become the second leading cause of death worldwide after cardiovascular disease, posing an extremely serious threat to human life and health. According to the latest data released by the International Agency for Research on Cancer of the World Health Organization, nearly 20 million new cases of cancer were diagnosed worldwide in 2020, and about 10 million people died from cancer [1]. How to reduce the mortality rate of cancer patients and improve their quality of life remains a major problem that urgently needs to be solved.

[0003] By blocking immune checkpoints such as PD-1, CTLA4, and LAG3 with specific antibodies, the crosstalk between tumor cells or suppressor immune cells and CTLs can be blocked, thereby reversing the functional exhaustion level of CTLs and maintaining and enhancing the tumor-killing activity of CTLs. Among the tumor immunotherapy methods, the clinical research of immune checkpoint inhibitors is the most comprehensive and mature, and their application is the most widespread. Several monoclonal antibody drugs targeting CTLA4, PD-1 / PD-L1, and LAG3, such as Ipilimumab, Nivolumab, Pembrolizumab, and Relatlimab, have been approved for the treatment of various cancers such as advanced melanoma, lung cancer, and renal cancer [2]. However, among patients receiving existing immune checkpoint inhibitor therapy, only about 20% of patients achieve durable response and good long-term prognosis, and are often accompanied by varying degrees of immune-related adverse events [3]. Therefore, it is urgent to discover new and more effective immune checkpoints and develop therapeutic drugs targeting them so that more cancer patients can benefit.

[0004] CD209 is mainly expressed in dendritic cells and M2 macrophages. It is a type II transmembrane glycoprotein receptor. Its extracellular domain consists of a neck region and a carbohydrate recognition domain (CRD). The neck region is a 23-amino acid repeat sequence that mainly participates in protein dimerization or oligomerization. The CRD is crucial for CD209 to recognize the mannose structure on specific glycoproteins on pathogens. CD209 has been found to recognize the abnormally glycosylated Lea / Leb glycogen on carcinoembryonic antigen CEA and CEACAM1, thereby mediating the interaction between DCs and colorectal cancer cells. This functional activity may lead to a weakening of the antitumor immune response [4]. The infiltration level of CD209+ tumor-associated macrophages (TAMs) in muscle-invasive bladder cancer and gastric cancer tissues is associated with the patient's sensitivity to immunotherapy and poor prognosis. Studies have shown that CD209+TAMs can lead to impaired CD8+ T cell function, decreased expression of IFN-γ, GZMB and perforin, and increased expression of PD-1 and CTLA4, but the specific mechanism is still unclear [5,6]. Other studies have found that CD209-mediated binding of DCs to Jurkat cells may inhibit DC maturation, and blocking CD209 can improve DC function and T cell differentiation [7].

[0005] Currently, there are very few reports on anti-CD209 antibodies.

[0006] In view of this, the present invention is proposed.

[0007] References:

[0008] 1.Sung H, et al.Global Cancer Statistics 2020: GLOBOCAN Estimates ofIncidence and Mortality Worldwide for 36Cancers in185Countries.CA Cancer JClin.2021May;71(3):209-249.

[0009] 2. Shiravand Y, et al. Immune Checkpoint Inhibitors in CancerTherapy. Curr Oncol. 2022Apr 24; 29(5):3044-3060.

[0010] 3. Siegel RL, et al. Cancer statistics, 2020. CA Cancer J Clin. 2020Jan; 70(1):7-30.

[0011] 4.Nonaka M, et al. Glycosylation-dependent interactions of C-typelectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells. JImmunol. 2008Mar 1; 180(5):3347-56.

[0012] 5.Hu B, et al.Blockade of DC-SIGN+Tumor-Associated MacrophagesReactivates Antitumor Immunity and Improves Immunotherapy in Muscle-InvasiveBladder Cancer.Cancer Res.2020Apr 15;80(8):1707-1719.

[0013] 6.Liu

[0014] 7.Jin Summary of the Invention

[0015] The purpose of this invention is to provide anti-CD209 antibodies, products, nucleic acid molecules, vectors, cells, and their applications to solve the above-mentioned technical problems.

[0016] This invention is implemented as follows:

[0017] In a first aspect, the present invention provides an anti-CD209 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes VH1-CDR1, VH1-CDR2 and VH1-CDR3, and the light chain variable region includes VL1-CDR1, VL1-CDR2 and VL1-CDR3.

[0018] The amino acid sequences of VH1-CDR1, VH1-CDR2 and VH1-CDR3 are shown in SEQ ID NO: 3-5, respectively; or, in sequence, as shown in SEQ ID NO: 3, SEQ ID NO: 11 and SEQ ID NO: 5, respectively.

[0019] The amino acid sequences of VL1-CDR1 and VL1-CDR3 are shown in SEQ ID NO: 6-7, and the amino acid sequence of VL1-CDR2 is WAS.

[0020] Secondly, the present invention also provides the use of an anti-CD209 antibody or its antigen-binding fragment in any of the following:

[0021] (1) Application in detecting CD209 protein in samples, not for the purpose of disease diagnosis or treatment;

[0022] (2) Application in the isolation or enrichment of CD209 protein;

[0023] (3) Application in the preparation of products for detecting CD209 protein;

[0024] (4) Application in the preparation of products containing isolated or enriched CD209 protein;

[0025] (5) Application in the preparation of antitumor drugs, wherein the tumor microenvironment includes: tumor-associated macrophages expressing CD209 surface antigen and / or dendritic cells expressing CD209 surface antigen.

[0026] Thirdly, the present invention also provides a product for detecting CD209 protein, the product having the above-mentioned anti-CD209 antibody or its antigen-binding fragment.

[0027] Fourthly, the present invention also provides a product for isolating or enriching CD209 protein, the product having the above-mentioned anti-CD209 antibody or its antigen-binding fragment.

[0028] Fifthly, the present invention also provides a composition or antibody-drug conjugate comprising the above-described anti-CD209 antibody or its antigen-binding fragment.

[0029] In a sixth aspect, the present invention also provides an isolated nucleic acid molecule that encodes the aforementioned anti-CD209 antibody or its antigen-binding fragment.

[0030] In a seventh aspect, the present invention also provides an expression cassette or carrier containing the nucleic acid molecule encoding the above-mentioned.

[0031] Eighthly, the present invention also provides a hybridoma cell or recombinant cell, wherein the hybridoma cell secretes the above-mentioned anti-CD209 antibody or its antigen-binding fragment, and the recombinant cell includes the above-mentioned expression cassette or vector.

[0032] In a ninth aspect, the present invention also provides a method for producing the above-mentioned anti-CD209 antibody or its antigen-binding fragment, by culturing the above-mentioned recombinant cells; or by injecting the above-mentioned hybridoma cells into the peritoneal cavity of a non-human mammal and collecting ascites.

[0033] The present invention has the following beneficial effects:

[0034] This invention obtained an anti-CD209 antibody through screening. This antibody specifically recognizes and binds to CD209, thereby blocking the CD209 ligand on the surface of human or other mammalian cells. This prevents CD209 from inhibiting T cell function, allowing T cells to enhance anti-tumor immunity through cytokine secretion, GZMB expression, and perforin, significantly inhibiting tumor growth and exerting a positive anti-tumor function in various solid tumors. Therefore, the anti-CD209 antibody provided by this invention has promising prospects for anti-tumor applications.

[0035] Furthermore, the anti-CD209 antibody provided by this invention exhibits excellent binding activity to the CD209 protein and can be used for the separation, enrichment, and detection of the CD209 protein. It shows promising application prospects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 Statistical results of screening murine monoclonal antibodies against human CD209 to restore T cell function;

[0038] Figure 2 Statistical results of murine monoclonal antibodies against human CD209 inhibiting the growth of MC38 subcutaneous tumors;

[0039] Figure 3Statistical results of the inhibition of LLC subcutaneous tumor growth by murine monoclonal antibody against human CD209;

[0040] Figure 4 Statistical results of murine monoclonal antibody against human CD209 inhibiting lung and liver metastases of melanoma B16F10;

[0041] Figure 5 Statistical results of the inhibition of intracranial growth of neuroblastoma GL261 by murine monoclonal antibodies against human CD209;

[0042] Figure 6 Statistical results of murine monoclonal antibodies against human CD209 inhibiting the growth of ovarian cancer ID8 in the peritoneal cavity;

[0043] Figure 7 Statistical results of murine monoclonal antibodies against human CD209 inhibiting the growth of pancreatic cancer in situ;

[0044] Figure 8 Statistical results of human monoclonal antibodies against human CD209 inhibiting the growth of MC38 subcutaneous tumors;

[0045] Figure 9 Statistical results of human monoclonal antibody against human CD209 inhibiting lung and liver metastases of melanoma B16F10. Detailed Implementation

[0046] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0047] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0048] In a first aspect, the present invention provides an anti-CD209 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes VH1-CDR1, VH1-CDR2 and VH1-CDR3, and the light chain variable region includes VL1-CDR1, VL1-CDR2 and VL1-CDR3.

[0049] The amino acid sequences of VH1-CDR1, VH1-CDR2 and VH1-CDR3 are shown in SEQ ID NO: 3-5, respectively; or, in sequence, as shown in SEQ ID NO: 3, SEQ ID NO: 11 and SEQ ID NO: 5, respectively.

[0050] The amino acid sequences of VL1-CDR1 and VL1-CDR3 are shown in SEQ ID NO: 6-7, and the amino acid sequence of VL1-CDR2 is WAS.

[0051] This invention involves gene synthesis and vector construction to express and purify recombinant human CD209 protein. Mice are immunized with this recombinant protein as an antigen to prepare a monoclonal antibody hybridoma cell line, yielding a mouse anti-human CD209 monoclonal antibody. Screening yields anti-human CD209 monoclonal antibodies capable of recognizing and blocking human CD209. Sequencing revealed that this antibody possesses the aforementioned heavy chain complementarity-determining regions (CDRs) and light chain CDRs. Further humanization of this mouse anti-human CD209 monoclonal antibody is then performed. The aforementioned monoclonal antibody can be used for the detection, isolation, and enrichment of CD209 protein, as well as for the diagnosis, auxiliary diagnosis, prognosis, and efficacy monitoring of diseases using CD209 as a biomarker.

[0052] The sequence of VH1-CDR1, SEQ ID NO: 3 is:

[0053] GFNINVTY;

[0054] The sequence of VH1-CDR2, SEQ ID NO: 4 is:

[0055] IEPANSNI;

[0056] The sequence of VH1-CDR3, SEQ ID NO: 5 is:

[0057] ASGWPWITY;

[0058] The sequence of VL1-CDR1, SEQ ID NO: 6 is: QTLLYSRTRKNY;

[0059] The sequence of VL1-CDR2 is: WAS;

[0060] The sequence of VL1-CDR3, SEQ ID NO: 7 is: QQYFILWT.

[0061] Antibodies with the above amino acid sequence are humanized anti-CD209 monoclonal antibodies.

[0062] Before humanization, the amino acid sequence of VH2-CDR2 of the murine anti-CD209 monoclonal antibody is shown in SEQ ID NO: 10, and the specific sequence is: IDPANGNI.

[0063] The structure of the heavy chain variable region is: FR-H1-CDR1-FR-H2-CDR2-FR-H3-CDR3-FR-H4. The structure of the light chain variable region is: FR-L1-CDR1-FR-L2-CDR2-FR-L3-CDR3-FR-L4.

[0064] In a preferred embodiment of the present invention, the anti-CD209 antibody or its antigen-binding fragment further includes a backbone region, which includes a heavy chain backbone region and / or a light chain backbone region. The heavy chain backbone region includes FR-H1, FR-H2, FR-H3 and FR-H4; the light chain backbone region includes FR-L1, FR-L2, FR-L3 and FR-L4.

[0065] In a preferred embodiment of the present invention, the amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are as shown in SEQ ID NO: 11-14 (humanized), or as shown in SEQ ID NO: 19-22 (mouse-derived).

[0066] The sequence of SEQ ID NO: 11 is as follows: EVQLVQSGAEVKKPGASVKVSCKAS;

[0067] The sequence of SEQ ID NO: 12 is as follows: IHWVRQRPDQGLEWMGR;

[0068] The sequence of SEQ ID NO: 13 is as follows: KFEPNFQDRVTMTADTSTSTVYMELSSLRSEDTAVYYC;

[0069] The sequence of SEQ ID NO: 14 is as follows: WGQGTLVTVSS.

[0070] The sequence of SEQ ID NO: 19 is as follows: EVQLQQSGPDLVKPGASVKLSCTAS.

[0071] The sequence of SEQ ID NO: 20 is as follows: IHWVKQRPDQGLDWIGR.

[0072] The sequence of SEQ ID NO: 21 is as follows: KFDPNFQDRATITADTSSNTAYLQLSSLTPEDTTVYYC.

[0073] The sequence of SEQ ID NO: 22 is as follows: WGQGTLVTVSA.

[0074] In a preferred embodiment of the present invention, the amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 are as shown in SEQ ID NO: 15-18 (humanized) or as shown in SEQ ID NO: 23-26 (mouse-derived).

[0075] The sequence of SEQ ID NO: 15 is as follows: DIVMTQSPDSLAVSLGERATINCKSS;

[0076] The sequence of SEQ ID NO: 16 is as follows: LAWYQQKPGQPPKLLIY;

[0077] The sequence of SEQ ID NO: 17 is as follows: TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC;

[0078] The sequence of SEQ ID NO: 18 is as follows: FGGGTKVEIK.

[0079] The sequence of SEQ ID NO: 23 is as follows: DIVMSQSPSSLAVSAGEKVTMSCKSS;

[0080] The sequence of SEQ ID NO: 24 is as follows: LAWYQQKPGQSPKVLIY;

[0081] The sequence of SEQ ID NO: 25 is as follows: TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC;

[0082] The sequence of SEQ ID NO: 26 is as follows: FGGGTKLEIK.

[0083] In other embodiments, the amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 have at least 80% homology with the sequences shown in SEQ ID NO: 11-14, or at least 80% homology with the sequences shown in SEQ ID NO: 19-22, respectively.

[0084] In other embodiments, the amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 have at least 80% homology with the sequences shown in SEQ ID NO: 15-18, or at least 80% homology with the sequences shown in SEQ ID NO: 23-26, respectively.

[0085] It should be noted that, in other embodiments, the amino acid sequences of each backbone region of the monoclonal antibody provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the corresponding backbone regions described above.

[0086] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region VH of the humanized anti-CD209 antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: 2.

[0087] The sequence of VH1, SEQ ID NO: 1 is:

[0088] EVQLVQSGAEVKKPGASVKVSCKASGFNINVTYIHWVRQRPDQGLEWMGRIEPANSNIKFEPNFQDRVTMTADTSTSTVY MELSSLRSEDTAVYYCASGWPWITYWGQGTLVTVSS.

[0089] The sequence of VL1, SEQ ID NO: 2 is:

[0090] DIVMTQSPDSLAVSLGERATINCKSSQTLLYSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS LQAEDVAVYYCQQYFILWTFGGGTKVEIK.

[0091] The amino acid sequence of the heavy chain variable region VH of the mouse anti-CD209 antibody is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: 9.

[0092] The sequence of SEQ ID NO: 8 is:

[0093] EVQLQQSGPDLVKPGASVKLSCTASGFNINVTYIHWVKQRPDQGLDWIGRIDPANGNIKFDPNFQDRATITADTSSNTAYLQ LSSLTPEDTTVYYCASGWPWITYWGQGTLVTVSA;

[0094] The sequence of SEQ ID NO: 9 is:

[0095] DIVMSQSPSSLAVSAGEKVTMSCKSSQTLLYSRTRKNYLAWYQQKPGQSPKVLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQYFILWTFGGGTKLEIK.

[0096] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region;

[0097] In a preferred embodiment of the present invention, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0098] In a preferred embodiment of the present invention, the heavy chain constant region is selected from any one of IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA, and IgE;

[0099] In a preferred embodiment of the present invention, the light chain constant region is selected from either the kappa (κ) chain or the lambda (λ) chain;

[0100] In a preferred embodiment of the present invention, the species source of the antibody constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans;

[0101] In a preferred embodiment of the present invention, the antibody constant region is derived from human or mouse species. Verification has shown that antibodies composed of human or mouse constant regions can specifically recognize human CD209 and exhibit good anti-tumor effects. In other embodiments, those skilled in the art will readily recognize that replacing the human or mouse constant region with a constant region from another species is also within the scope of protection of the present invention.

[0102] In a preferred embodiment of the present invention, the antibody constant region includes the human Fc region; in a preferred embodiment of the present invention, the antibody constant region includes the Fc region of human IgG4.

[0103] In a preferred embodiment of this invention, the antibody constant region is selected from human IgG4. This invention uses the variable region of a humanized mouse antibody and the constant region of human IgG4 to form a humanized monoclonal antibody with clone number 19H2H7. The results have verified that it enhances anti-tumor immunity, significantly inhibits tumor growth, prolongs survival, and exhibits positive anti-tumor effects in various tumors, including melanoma, lung cancer, colon cancer, pancreatic cancer, neuroblastoma, and ovarian cancer.

[0104] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, and bispecific antibodies. Bispecific antibodies are polymers of monovalent antibodies that recognize different epitopes, capable of binding to different targets or different epitopes of the same target, and possessing higher antigen recognition ability than monovalent antibodies. They can be polymerized together through short linker sequences, thereby converting them into multivalent and multispecific forms.

[0105] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned functional fragments.

[0106] The functional fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by, for example, automated peptide synthesizers sold by Applied BioSystems.

[0107] In one optional embodiment, the anti-CD209 antibody is humanized into a humanized monoclonal antibody or scFv. In another optional embodiment, the light chain variable region and heavy chain variable region of the above antibody are recombined to obtain a single-chain antibody (scFv) with a smaller molecular weight, which can also specifically recognize human CD209.

[0108] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from polypeptides capable of blocking the binding of CD209 to T cells. Any polypeptide capable of blocking the binding of CD209 to T cells and including the aforementioned amino acid sequence is easily implemented by those skilled in the art. For example, linking the CDR sequence in the antigen-binding fragment with a linker peptide (flexible or rigid) and thus possessing the activity of blocking the binding of CD209 to T cells is also within the scope of protection of the present invention.

[0109] Secondly, the present invention also provides the use of anti-CD209 antibody or its antigen-binding fragment in any of the following:

[0110] (1) Application in detecting CD209 protein in samples, not for the purpose of disease diagnosis or treatment;

[0111] (2) Application in the isolation or enrichment of CD209 protein;

[0112] (3) Application in the preparation of products for detecting CD209 protein;

[0113] (4) Application in the preparation of products containing isolated or enriched CD209 protein;

[0114] (5) Application in the preparation of antitumor drugs, wherein the tumor microenvironment includes: tumor-associated macrophages expressing CD209 surface antigen and / or dendritic cells expressing CD209 surface antigen.

[0115] In a preferred embodiment of the present invention, a tumor refers to a tumor that has the following characteristics: the tumor can inhibit the function of T cells through CD209 to accelerate tumor progression.

[0116] In a preferred embodiment of the present invention, the tumor is selected from at least one of melanoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, neuroblastoma, ovarian cancer, breast cancer, liver cancer, non-small cell lung cancer, gastric cancer, adrenocortical carcinoma, renal cell carcinoma, cervical squamous cell carcinoma, bile duct cancer, esophageal cancer, head and neck squamous cell carcinoma, thyroid cancer, thymoma, bladder cancer, and endometrial cancer.

[0117] In one alternative implementation, the colorectal tumor includes one or more of the following types: polypoid, stricture, and ulcerative.

[0118] In one alternative implementation, breast cancer includes, but is not limited to, breast epithelial carcinoma;

[0119] Preferably, the breast cancer is ductal carcinoma or lobular carcinoma of the breast.

[0120] Breast cancer, preferably stage II to IV and / or poorly differentiated invasive ductal carcinoma, comedo carcinoma and medullary carcinoma (preferably grade 2).

[0121] Ovarian cancer, serous and mucinous carcinoma (preferably stage Ic to IIIb), granulosa cell tumors, surface epithelial-stromal tumors (adenocarcinomas), cystadenocarcinomas, and endometrioid tumors.

[0122] Uterine cancer, preferably including endometrioid adenocarcinoma (preferably stage I to IIIc).

[0123] Bladder cancer, preferably including transitional cell carcinoma (preferably stage II to IV).

[0124] Lung cancer, preferably including small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma), and large cell lung cancer.

[0125] In a preferred embodiment of the present invention, the tumor is an in situ tumor or a metastatic tumor.

[0126] In a preferred embodiment of the present invention, the above-mentioned drug also includes a pharmaceutically acceptable carrier, including but not limited to fillers, lubricants, disintegrants, binders, flow aids, etc.

[0127] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, dicalcium phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, and indigo.

[0128] The methods of drug administration include, but are not limited to: injection, gavage, oral administration, etc.

[0129] In a preferred embodiment of the present invention, the product for detecting CD209 protein is selected from at least one of the following: columns, reagents, nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, test tubes, tubes, microplate wells, membranes, and chips.

[0130] The chips include, but are not limited to, chromatin immunoprecipitation chips, immunoprecipitation chips, immunoprecipitation chips, or CD209 fusion protein precipitation chips.

[0131] In a preferred embodiment of the present invention, the product for detecting CD209 protein is selected from a kit.

[0132] In a preferred embodiment of the present invention, the kit is a chemiluminescent immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit, a fluorescence immunoassay kit, or a time-resolved fluorescence immunoassay kit.

[0133] Application (3) includes: using products that detect CD209 protein for any of the purposes of disease diagnosis, auxiliary diagnosis, prognosis and efficacy monitoring using CD209 as a marker.

[0134] Accordingly, the addition of appropriate excipients or additives to test reagents and kits is also within the scope of protection of this invention. For example, adding protective agents to extend the shelf life, and adding solubilizers and stabilizers to respectively solubilize and stabilize protein components.

[0135] Furthermore, in order to better integrate with ELISA plates or detection chips, the pretreatment and modification of the surface of ELISA plates or detection chips to include active groups that readily bind proteins are also within the scope of protection of this invention.

[0136] In a preferred embodiment of the present invention, the product for separating or enriching CD209 protein in application (4) is selected from at least one of the following: column, reagent, nanoparticle, magnetic bead, agarose gel microsphere, silica microsphere, latex microsphere, test tube, tube, microplate concave well, membrane and chip.

[0137] For example, anti-CD209 antibodies or their antigen-binding fragments can be coated onto a vector. CD209 can specifically bind to the anti-CD209 antibody or its antigen-binding fragment, thereby separating the CD209 protein from the sample. To improve separation efficiency, a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the antibody can also be used to separate the CD209 protein. Tags can be selected from His tags, Epitope tags, Protease cleavage sites, Destabilizing domains, Detection tags, Purification tags, Signal peptides, Cell-penetrating peptides, Fluorescent genes, Linkers, Regulatory proteins, CAR-T proteins, TRX, GST, DSBA, MyC tags, HA tags, etc.

[0138] Products for isolating or enriching CD209 protein include, but are not limited to: enriched or purified products of fusion proteins with anti-CD209 antibodies and protein tags; products for isolating or purifying interacting proteins with anti-CD209 antibodies and protein tags; or products for isolating or purifying anti-CD209 antibody-protein tag-DNA complexes. The above includes: a carrier and an antibody on the carrier; preferably, the carrier is selected from nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, chips, or porous materials.

[0139] Thirdly, the present invention also provides a product for detecting CD209 protein, the product having the above-mentioned anti-CD209 antibody or its antigen-binding fragment.

[0140] In a preferred embodiment of the present invention, the anti-CD209 antibody or its antigen-binding fragment is labeled with a detectable marker;

[0141] Detectable markers refer to substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc. These properties enable qualitative or quantitative detection of the corresponding target.

[0142] In a preferred embodiment of the present invention, the detectable marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

[0143] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0144] In optional embodiments, fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy...). 5. Cy5.5, Cy3, etc. or similar), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0145] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0146] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0147] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0148] In optional embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0149] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0150] In optional embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0151] In a preferred embodiment of the present invention, the product for detecting CD209 protein is selected from at least one of the following: columns, reagents, nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, test tubes, tubes, microplate wells, membranes, and chips.

[0152] In a preferred embodiment of the present invention, the product for detecting CD209 protein is selected from a kit.

[0153] The kit is a magnetic microparticle chemiluminescence detection kit; preferably, the chip is a chromatin immunoprecipitation chip, immunoprecipitation chip, immunoprecipitation chip or CD209 fusion protein precipitation chip.

[0154] Products for isolating or enriching CD209 protein include, but are not limited to: enriched or purified products of fusion proteins with anti-CD209 antibodies and protein tags; products for isolating or purifying interacting proteins with anti-CD209 antibodies and protein tags; or products for isolating or purifying anti-CD209 antibody-protein tag-DNA complexes. The above includes: a carrier and an antibody on the carrier; preferably, the carrier is selected from nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, chips, or porous materials.

[0155] Fourthly, the present invention also provides a product for isolating or enriching CD209 protein, the product having the above-mentioned anti-CD209 antibody or its antigen-binding fragment.

[0156] In a preferred embodiment of the present invention, the product for separating or enriching CD209 protein is selected from at least one of the following: column, reagent, nanoparticle, magnetic bead, agarose gel microsphere, silica microsphere, latex microsphere, test tube, tube, microplate concave well, membrane, and chip.

[0157] Fifthly, the present invention also provides a composition or antibody-drug conjugate comprising the above-described anti-CD209 antibody or its antigen-binding fragment;

[0158] In a preferred embodiment of the present invention, the composition is a pharmaceutical composition;

[0159] In a preferred embodiment of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients;

[0160] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from at least one of fillers, disintegrants, lubricants, flavoring agents, binders, suspending agents, and flavorings.

[0161] Pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically acceptable carriers, excipients, or solvents. Pharmaceutically acceptable excipients include a variety of organic or inorganic carriers and / or excipients because they are commonly used for pharmaceutical purposes, particularly for solid drug formulations. Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylene pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, hydrolyzed starch, carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, talc, and sodium lauryl sulfate; and flavorings such as citric acid, menthol, and glycine. Orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (e.g., methylparaben, ethylparaben, propylparaben, butylparaben); stabilizers, such as citric acid, sodium citrate, acetic acid, and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0162] In a preferred embodiment of the present invention, the dosage form of the drug is tablet, pill, powder, suspension, gel, emulsion, cream, granule, nanoparticle, capsule, suppository, injection or spray.

[0163] In an alternative embodiment, the aforementioned drug is a liquid pharmaceutical preparation (such as an injectable formulation), such as a solution, suspension, or gel, which typically contains a liquid carrier, such as water, and / or a pharmaceutically acceptable organic solvent. Furthermore, such liquid preparations may also contain pH adjusters, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (e.g., methylcellulose), dyes, and / or flavoring agents, as defined above. The drugs may be isotonic, i.e., they may have the same osmotic pressure as blood. The isotonicity of the drug can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickener, such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the reagent selected.

[0164] In a preferred embodiment of the invention, the drug is formulated for oral or injectable administration. Injectable administration includes, but is not limited to, subcutaneous injection, intramuscular injection, intravenous injection, and intradermal injection.

[0165] In a preferred embodiment of the present invention, the toxin, radioactive isotope, and radionuclide are used.

[0166] Preferably, the antibody-drug conjugate further comprises additional molecules conjugated directly or via a spacer to the anti-CD209 antibody or its antigen-binding fragment;

[0167] Preferably, the additional molecule is selected from labeled groups. Preferably, the labeled group is selected from at least one of labeled peptides, fluorescent groups, enzyme groups, chemiluminescent groups, biotin groups, and metal particles.

[0168] In a sixth aspect, the present invention also provides an isolated nucleic acid molecule that encodes the aforementioned anti-CD209 antibody or its antigen-binding fragment.

[0169] Considering the degeneracy of codons, the gene sequence encoding the above-mentioned (e.g., Fab segment) antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0170] In a seventh aspect, the present invention also provides an expression cassette or carrier containing the nucleic acid molecule encoding the above-mentioned.

[0171] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged into a virus) into the host cell through transformation, transfection, or transduction to express the target gene.

[0172] One type of vector is the plasmid, a circular double-stranded DNA molecule, which ligates the target DNA fragment into the plasmid circle. Another type of vector is the viral vector, which ligates and packages the target DNA fragment into the viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, and oncolytic virus). Once these vectors enter the host cell, they can express the target gene.

[0173] Eighthly, the present invention also provides a hybridoma cell or recombinant cell, wherein the hybridoma cell secretes the above-mentioned anti-CD209 antibody or its antigen-binding fragment, and the recombinant cell includes the above-mentioned expression cassette or vector.

[0174] In one alternative implementation, the recombinant cells are selected from at least one of prokaryotic host cells, eukaryotic host cells, and bacteriophages;

[0175] In one alternative implementation, the prokaryotic host cell is Escherichia coli, Streptomyces, Bacillus subtilis, or Mycobacterium.

[0176] In one alternative implementation, the eukaryotic host cell is an animal cell, a plant cell, or a fungus;

[0177] In one alternative embodiment, the animal cells are selected from mammalian cells, insect cells, or Caenorhabditis elegans.

[0178] The mammalian cells are selected from any one of the following: 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, HeLa cells, MDCK cells, Hep-2 cells, and Per6 cells. Among them, the 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for the production of antibodies or recombinant proteins and are well known to those skilled in the art.

[0179] In one alternative embodiment, the fungus is selected from any one of Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces hansenii, Candida albicans, Kluyveromyces lactis, Aspergillus nidus, Schizosoma saccharizoides, and Keratophyton lipolytica. Candida albicans, for example, is selected from Candida albicans or Candida glabrata.

[0180] Ninthly, the present invention also provides a method for producing the above-described anti-CD209 antibody or its antigen-binding fragment, comprising culturing the above-described recombinant cells; or injecting the above-described hybridoma cells into the peritoneal cavity of a non-human mammal and collecting ascites fluid. The ascites fluid is then subjected to separation and purification of the target antibody.

[0181] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0182] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0183] In previous studies, this invention obtained a recombinant protein (hCD209-Fc) of the extracellular domain (59-404aa) of human CD209 using a eukaryotic expression system. In vitro studies showed that hCD209-Fc significantly inhibited the activation of human Jurkat cells and mouse CD4+ and CD8+ T cells mediated by CD3 / CD28 antibodies. A specific antibody targeting human CD209 (anti-hCD209 mAb) was prepared, and corresponding CD209 humanized mice were constructed to verify its antitumor activity. Anti-hCD209 mAb was found to reverse the inhibitory effect of hCD209 on T cell activation in vitro, and to inhibit the growth of MC38, LLC and GL261 subcutaneous tumors in vivo, as well as inhibit B16 lung and liver metastases. It also inhibited the growth of ID8 tumors, primary colon cancer and intracranial neuroblastoma. After humanization, Anti-hCD209 mAb was also found to inhibit the growth of MC38 subcutaneous tumors and pancreatic cancer in vivo, and inhibit B16 lung or liver metastases. CD209 may become a promising new target for anti-tumor immunotherapy.

[0184] Example 1

[0185] Preparation of recombinant human CD209 protein and mouse anti-human CD209 monoclonal antibody.

[0186] 1. Preparation of recombinant human CD209 protein

[0187] Human CD209 gene expression vector was constructed using genetic engineering techniques. Human CD209 cDNA was incorporated into a pATX2 (EcoRI / NotI restriction site) expression vector, transformed, cultured, and endotoxin-free plasmids were extracted. The plasmid (500 μg) was transfected into 500 ml of 293F cells. When cell viability decreased to approximately 30%, the cell culture supernatant was collected, centrifuged at 1500 rpm for 5 minutes to remove cells and cell debris, and then centrifuged again at 10000 rpm for 5 minutes. The cell culture supernatant was then filtered through a 0.45 μM filter. The human CD209-Fc protein was purified by protein G affinity chromatography.

[0188] 2. Preparation of mouse anti-human CD209 monoclonal antibody

[0189] (1) Immunizing mice with recombinant human CD209-Fc protein

[0190] Four C57 mice were immunized with the prepared human CD209-Fc recombinant protein. Immunization was performed by subcutaneous injection of 1 mg / 0.025 ml antigen into the axilla, paw, and groin. A primary immunization was administered, followed by 2-4 booster immunizations, each 14 days apart. Seven days after the final booster immunization, serum titers were measured. If the titer met the standard, a pulse immunization was performed by intraperitoneal injection of 0.5 ml of 200 ug / ml antigen solution. Three days later, spleen cells were harvested and fused with myeloma cells.

[0191] (2) Cell fusion

[0192] Mice that underwent shock immunization and whose serum titers met the requirements were selected. Blood was collected from the eyeballs for use as a positive control for hybridoma screening. The mice were then euthanized by cervical dislocation and disinfected in 75% alcohol for at least 30 seconds. The spleens of the mice were collected to prepare a suspension of spleen cells and counted. SP2 / 0 myeloma cells were also collected and counted. The spleen cells and myeloma cells were mixed in a 5:1 ratio in a 50ml centrifuge tube and centrifuged at 1000rpm for 5min. The supernatant was discarded, and the centrifuge tube wall was gently tapped to loosen the cell pellet. The centrifuge tube was placed in a 37℃ water bath, and preheated PEG was added to the cell pellet at a uniform rate (1ml over 1min). During the addition of PEG, the centrifuge tube was rotated while gently stirring with the tip of a pipette. The mixture was allowed to stand for 90s. Add preheated 1640 basal medium at a constant rate (first addition), 1 ml over 1 minute, while gently stirring. Add preheated 1640 basal medium at a constant rate (second addition), 2 ml over 1 minute, while gently stirring. Add preheated 1640 basal medium at a constant rate (third addition), 9 ml over 3 minutes, while gently stirring. Add preheated 1640 basal medium at a constant rate, while gently stirring until 40 ml remains. Place the centrifuge tube in a 37°C water bath and let it stand for 3 minutes. Centrifuge the confluent cell suspension at 800 rpm for 5 minutes, discard the supernatant, gently tap the centrifuge tube wall to loosen the cell pellet, add an appropriate amount of HAT medium according to the number of spleen cells, mix well by pipetting, and seed into a 96-well cell culture plate. Maintain in HAT selection medium for 7–10 days, then replace with HT medium. During the selection culture period, when hybridoma cells cover 1 / 10 of the bottom area of ​​the well, collect the cell culture supernatant to start detecting specific antibodies, and screen out the wells containing positive antibodies.

[0193] (3) ELISA detection of antibodies

[0194] Coat a 96-well microplate with 100 μl (2 μg / ml) of CD209 recombinant protein antigen solution and incubate overnight at 4°C. The next day, remove the coating solution, add 300 μl / well of 5% skim milk, and block at room temperature for 1 hour. Remove the blocking solution, wash three times with PBST, and pat dry on absorbent paper. Add 100 μl / well of primary antibody and incubate at 37°C for 60 minutes. Remove the primary antibody, wash three times with PBST, and pat dry on absorbent paper. Add 100 μl / well of secondary antibody and incubate at 37°C for 30 minutes. Remove the secondary antibody, wash three times with PBST, and pat dry on absorbent paper. Add 100 μl / well of TMB chromogenic buffer and incubate at 37°C for 5-20 minutes, observing the color development. Add 50 μl / well of 2M hydrochloric acid as stop solution, and read the absorbance using a microplate reader at a wavelength of 450-620 nm.

[0195] (4) Hybridoma subcloning

[0196] Subcloning of hybridoma cells in positive wells was performed using the limiting dilution method. The hybridoma cells to be cloned were resuspended from the culture wells and counted to a concentration of 10 cells / ml. 100 μl of diluted cells was added to each well, and the wells were incubated at 37°C with 8% CO2 for 8-9 days. The culture supernatant was collected to detect antibody activity. Positive wells with well-grown monoclonal hybridomas were selected and transferred to 24-well plates for further subcloning or expansion culture.

[0197] (5) Production of mouse anti-human CD209 monoclonal antibody

[0198] Mice were intraperitoneally injected with 0.5 ml of Freund's incomplete adjuvant 7-21 days prior to the event, and hybridoma cells were collected and their density adjusted to 2 × 10⁻⁶. 6 2 × 10⁶ hybridoma cells / ml were injected intraperitoneally into mice that had previously been injected with incomplete adjuvant. 6 / 0.5ml / mouse. Ascites fluid was collected from mice 7-12 days after cell injection. The ascites fluid was purified by Protein G affinity chromatography, eluted with 0.1M glycine solution (pH 2.5), and neutralized with 1M Tris-HCl solution (pH 9.0). The pH of the eluent was tested to confirm its neutrality. The solution was then filtered through a 50KD ultrafiltration column. The antibody was replaced in PBS, and the antibody concentration was determined. At the same time, 2ug of antibody was taken for gel staining to verify antibody purity.

[0199] (6) Screening mouse anti-human CD209 monoclonal antibodies that restore the function of CD209 in inhibiting T cells.

[0200] Activation of human Jurkat cell lines and primary CD4+ and CD8+ T cells from humans and mice was induced by CD3 (2 μg / mL) and CD28 (2 μg / mL) antibodies (anti-CD3 / CD28). The level of IL-2 in the cell culture supernatant was detected by ELISA as a marker of T cell activation and proliferation. To assess the effect of hCD209-Fc on T cell activation, anti-CD3 / CD28-coated plates were seeded with T cells after being coated with hCD209-Fc. Fc protein was used as a control. The regulatory effect of CD209 on gene transcription during T cell activation was analyzed at the whole-cell level by transcriptome sequencing.

[0201] The obtained research results show that hCD209-Fc has a significant inhibitory effect on IL-2 levels in the supernatant of anti-CD3 / CD28-induced human Jurkat, mouse CD4+ and CD8+ T cells, suggesting that CD209 can inhibit T cell activation.

[0202] To screen for monoclonal strains capable of restoring CD209's inhibitory effect on T cells, hCD209-Fc (30 μg / mL) was premixed with the supernatant of the monoclonal strain and coated overnight at 4°C with CD3 (2 μg / mL) and CD28 (2 μg / mL) at room temperature for 2 hours. After the coating solution was discarded, T cells were seeded. IL-2 levels were detected by ELISA after 48 hours. One monoclonal strain (clone number 19H2H7) was found to specifically recognize and bind to CD209, thus blocking the human cell surface ligand CD209. This prevented CD209 from inhibiting T cell function, allowing the anti-CD3 / CD28 antibody to normally induce activation of human Jurkat cell lines and primary CD4+ and CD8+ T cells in humans and mice, restoring the T cell's IL-2 secretion function. Figure 1 As shown.

[0203] Through cell function experiments, this embodiment screened out a mouse anti-human CD209 monoclonal antibody that can restore the function of T cells in secreting IL-2: clone number 19H2H7.

[0204] 3. Hybridoma cell sequencing / variable region gene sequencing

[0205] Hybridoma cells (clone number 19H2H7) were collected after reaching the logarithmic growth phase. Total RNA was extracted from the hybridoma cells, and first-strand cDNA complementary to the full-length mRNA was obtained by RT-PCR using 3'RACE and 5'RACE techniques. Using the synthesized cDNA as a template, the antibody heavy and light chain variable region genes were amplified by PCR. The antibody variable region genes were ligated into a T vector, transformed, and positive clones were selected for sequencing. Bioinformatics analysis of the sequencing results yielded the antibody variable region gene sequences, as shown in SEQ ID NO: 8-9. The 19H2H7 antibody light and heavy chain V region sequences (SEQ ID NO: 1, SEQ ID NO: 2) were then submitted to the IMGT online server, where the CDR and FR components were identified.

[0206] Example 2

[0207] Preparation of humanized CD209 antibody.

[0208] 1. Construction of humanized antibody plasmids

[0209] The constant regions of the heavy and light chains of the human IgG4 antibody were cloned into the pCAGGS vector. The variable regions of the heavy and light chains of the obtained mouse anti-human CD209 antibody were then humanized. These modified variable regions were inserted into the pCAGGS expression vector containing the constant region of the human IgG4 antibody, thus constructing a humanized antibody expression vector. This vector was then transfected into 293F mammalian cells for recombinant antibody expression (500ug / 500ml). The humanized monoclonal antibody with clone number 19H2H7 consists of the humanized variable region of the mouse antibody and the constant region of the human IgG4 antibody.

[0210] The constant region CH amino acid sequence of IgG4 (SEQ ID NO: 27):

[0211] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0212] The constant region CL amino acid sequence of IgG4 (SEQ ID NO: 28):

[0213] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGECS.

[0214] 2. Expression and purification of recombinant antibodies

[0215] After transfecting 293F cells, continue culturing for 5-6 days, collect the cell culture supernatant, and purify the humanized antibody against human CD209 in the supernatant using a Protein A affinity chromatography column.

[0216] Example 3

[0217] Characterization of murine and humanized antibodies against human CD209

[0218] 1. Mouse experiment on the treatment of subcutaneous colorectal cancer MC38 tumors with murine monoclonal antibody against human CD209.

[0219] 1×10 6MC38 cells were subcutaneously inoculated into CD209 humanized mice. The mice were divided into two groups and administered Control mAb (Beyotime drug number A7050) and 19H2H7 mAb (the murine anti-CD209 antibody from Example 1) intraperitoneally, starting on day 5, at a dose of 200 μg / mouse, three times a week. Tumor growth was monitored during this period. Mice were euthanized by dislocation on day 18 after tumor implantation, and the tumors were harvested to determine their size and weight. It was found that 19H2H7 mAb significantly inhibited tumor growth compared to Control mAb. Figure 2 As shown.

[0220] 2. Mouse experiment on the treatment of subcutaneous tumors of LLC cells in lung cancer using a murine monoclonal antibody against human CD209.

[0221] 5×10 5 LLC cells were subcutaneously inoculated into CD209 humanized mice. The mice were divided into two groups and administered Control mAb and 19H2H7 mAb, respectively, via intraperitoneal injection at a dose of 200 μg / mouse, starting on day 5, three times a week. Tumor growth was monitored during this period. Mice were euthanized by dislocation on day 34 after tumor implantation, and tumors were harvested to determine tumor size and weight. The results showed that 19H2H7 mAb significantly inhibited tumor growth compared to Control mAb. Figure 3 As shown.

[0222] 3. Mouse experiments using murine monoclonal antibodies against human CD209 to treat lung and liver metastases of melanoma B16F10.

[0223] 3×10 5 B16F10 cells were intravenously injected into CD209 humanized mice. The mice were divided into two groups and administered Control mAb and 19H2H7 mAb, respectively, via intraperitoneal injection at a dose of 200 μg / mouse starting on day 1, three times a week. Mice were euthanized by dislocation on day 18 after tumor implantation. Lungs and livers were harvested after perfusion to observe melanoma metastasis. The results showed that, compared to Control mAb, 19H2H7 mAb significantly inhibited melanoma metastasis to the lungs and liver. Figure 4 As shown.

[0224] 4. Mouse experiment on the treatment of intracranial neuroblastoma GL261 with murine monoclonal antibody against human CD209.

[0225] 3×10 5GL261-LUC cells were intracranially inoculated into CD209 humanized mice. The mice were divided into two groups and administered Control mAb and 19H2H7 mAb, respectively, via intraperitoneal injection at a dose of 500 μg / mouse starting on day 7. Ultrasound irradiation reversibly disrupts the blood-brain barrier in the brain. Administration was performed 1 hour before ultrasound and twice weekly. During this period, in vivo imaging was used to observe tumor growth by monitoring fluorescence intensity. All mice died on day 29 after tumor implantation. Compared to Control mAb, 19H2H7 mAb significantly inhibited tumor growth and prolonged the survival of mice. Figure 5 As shown.

[0226] 5. Mouse experiment on the treatment of ID8 ovarian cancer with murine monoclonal antibody against human CD209.

[0227] 3×10 6 ID8 cells were intraperitoneally injected into CD209 humanized mice. The mice were divided into two groups and administered Control mAb and 19H2H7 mAb, respectively, intraperitoneally at a dose of 200 μg / mouse starting on day 1, three times a week. On day 32 after tumor implantation, a large amount of ascites was observed in the mice. Compared to Control mAb, 19H2H7 mAb significantly inhibited ID8 tumor growth and suppressed ascites production in the peritoneal cavity. Figure 6 As shown.

[0228] 6. Mouse experiments using murine monoclonal antibodies against human CD209 to treat pancreatic cancer in situ.

[0229] 1.5×10 5 KPC cells were inoculated into the pancreas of CD209 humanized mice. The mice were divided into two groups and administered Control mAb and 19H2H7 mAb, respectively, via intraperitoneal injection at a dose of 200 μg / mouse starting on day 5, three times a week. It was found that compared to Control mAb, 19H2H7 mAb significantly inhibited the growth of pancreatic tumors. Figure 7 As shown.

[0230] 7. Mouse experiment on the treatment of MC38 subcutaneous tumors with humanized monoclonal antibody (hIgG) against human CD209.

[0231] 1×10 6MC38 cells were subcutaneously inoculated into CD209 humanized mice. Mice were divided into two groups and administered Control hIgG and 19H2H7 hIgG, respectively, intraperitoneally at a dose of 200 μg / mouse starting on day 5, three times a week. Tumor growth was monitored during this period. Mice were sacrificed by dislocation on day 18 after tumor bearing, and tumor size and weight were measured. Compared to Control hIgG (Selleck product code: A2052), 19H2H7 hIgG significantly inhibited tumor growth and prolonged the survival curve of tumor-bearing mice. Figure 8 As shown.

[0232] 8. Mouse experiment on the treatment of melanoma B16F10 liver metastases with humanized monoclonal antibody (hIgG) against human CD209.

[0233] 3×10 5 B16F10 cells were intravenously injected into CD209 humanized mice via the tail vein. The mice were divided into two groups and administered Control hIgG and 19H2H7 hIgG, respectively, via intraperitoneal injection at a dose of 200 μg / mouse starting on day 1, three times a week. Mice were euthanized by dislocation on day 18 after tumor bearing. Liver tissue was harvested after perfusion, and melanoma metastasis was observed. Compared to Control hIgG, 19H2H7 hIgG significantly inhibited melanoma metastasis to the liver and prolonged the survival curve of tumor-bearing mice. Figure 9 As shown.

[0234] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An anti-CD209 antibody or its antigen-binding fragment, characterized in that, It includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes VH1-CDR1, VH1-CDR2 and VH1-CDR3, and the light chain variable region includes VL1-CDR1, VL1-CDR2 and VL1-CDR3. The amino acid sequences of VH1-CDR1, VH1-CDR2 and VH1-CDR3 are as shown in SEQ ID NO: 3-5, respectively; or, as shown in SEQ ID NO: 3, SEQ ID NO: 11 and SEQ ID NO: 5, respectively. The amino acid sequences of VL1-CDR1 and VL1-CDR3 are shown in SEQ ID NO: 6-7, and the amino acid sequence of VL1-CDR2 is WAS.

2. The anti-CD209 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-CD209 antibody or its antigen-binding fragment further includes a backbone region, which includes a heavy chain backbone region and / or a light chain backbone region. The heavy chain backbone region includes FR-H1, FR-H2, FR-H3, and FR-H4; the light chain backbone region includes FR-L1, FR-L2, FR-L3, and FR-L4. Preferably, the amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 have at least 80% homology with the sequences shown in SEQ ID NO: 11-14, or at least 80% homology with the sequences shown in SEQ ID NO: 19-22. Preferably, the amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 have at least 80% homology with the sequences shown in SEQ ID NO: 15-18, or at least 80% homology with the sequences shown in SEQ ID NO: 23-26. Preferably, the amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are as shown in SEQ ID NO: 11-14, or as shown in SEQ ID NO: 19-22, respectively; Preferably, the amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 are as shown in SEQ ID NO: 15-18, or as shown in SEQ ID NO: 23-26, respectively; Preferably, the antibody or its antigen-binding fragment further includes a constant region; Preferably, the constant region includes a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region is selected from any one of IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA, and IgE; Preferably, the light chain constant region is selected from either the kappa (κ) chain or the lambda (λ) chain; Preferably, the species source of the antibody constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Preferably, the species source of the antibody constant region is human or mouse; Preferably, the antibody constant region includes the human Fc region; more preferably, the antibody constant region includes the Fc region of human IgG4. Preferably, the antibody constant region is selected from human IgG4; Preferably, the antigen-binding fragment is selected from any one of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2 and bispecific antibodies; Preferably, the antigen-binding fragment is selected from polypeptides capable of blocking the binding of CD209 to T cells.

3. The use of the anti-CD209 antibody or its antigen-binding fragment as described in any one of claims 1-2 in any of the following: (1) Application in the detection of CD209 protein in samples, wherein the application is not for the purpose of diagnosis or treatment of disease; (2) Application in the isolation or enrichment of CD209 protein; (3) Application in the preparation of products for detecting CD209 protein; (4) Application in the preparation of products containing isolated or enriched CD209 protein; (5) Application in the preparation of antitumor drugs, the tumor microenvironment includes: Tumor-associated macrophages expressing CD209 surface antigen and / or dendritic cells expressing CD209 surface antigen; Preferably, the tumor refers to a tumor that has the following characteristics: the tumor can inhibit the function of T cells through CD209 to accelerate tumor progression; Preferably, the tumor is selected from at least one of melanoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, neuroblastoma, ovarian cancer, breast cancer, liver cancer, non-small cell lung cancer, gastric cancer, adrenocortical carcinoma, renal cell carcinoma, cervical squamous cell carcinoma, bile duct cancer, esophageal cancer, head and neck squamous cell carcinoma, thyroid cancer, thymoma, bladder cancer, and endometrial cancer; Preferably, the tumor is an in situ tumor or a metastatic tumor; Preferably, the product for detecting CD209 protein is selected from at least one of the following: columns, reagents, nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, test tubes, tubes, microplate wells, membranes, and chips. Preferably, the product for detecting CD209 protein is selected from a kit; Preferably, the kit is a chemiluminescent immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit, a fluorescence immunoassay kit, or a time-resolved fluorescence immunoassay kit. Preferably, the application (3) includes: using the product for detecting CD209 protein for any one of the purposes of disease diagnosis, auxiliary diagnosis, prognosis and efficacy monitoring using CD209 as a marker; Preferably, the product for separating or enriching CD209 protein in the application (4) is selected from at least one of columns, reagents, nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, test tubes, tubes, microplates, membranes, and chips.

4. A product for detecting CD209 protein, characterized in that, The product has the anti-CD209 antibody or its antigen-binding fragment as described in any one of claims 1-2; Preferably, the anti-CD209 antibody or its antigen-binding fragment is labeled with a detectable marker; Preferably, the detectable marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers; Preferably, the product for detecting CD209 protein is selected from at least one of the following: columns, reagents, nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, test tubes, tubes, microplate wells, membranes, and chips. Preferably, the product for detecting CD209 protein is selected from a kit.

5. A product for isolating or enriching CD209 protein, characterized in that, The product has the anti-CD209 antibody or its antigen-binding fragment as described in any one of claims 1-2; Preferably, the product for isolating or enriching CD209 protein is selected from at least one of the following: columns, reagents, nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, test tubes, tubes, microplates, membranes, and chips.

6. A composition or antibody-drug conjugate, characterized in that, It includes the anti-CD209 antibody or its antigen-binding fragment as described in any one of claims 1-2; Preferably, the composition is a pharmaceutical composition; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients; Preferably, the drug in the antibody-drug conjugate is a toxin, a radioactive isotope, or a radionuclide; Preferably, the antibody-drug conjugate further comprises additional molecules conjugated directly or via a spacer to the anti-CD209 antibody or its antigen-binding fragment; Preferably, the additional molecules are selected from labeled groups.

7. An isolated nucleic acid molecule, characterized in that, It encodes the anti-CD209 antibody or its antigen-binding fragment as described in any one of claims 1-2.

8. An expression box or carrier, characterized in that, It contains the nucleic acid molecule encoding the claim 7.

9. A hybridoma cell or recombinant cell, characterized in that, The hybridoma cells secrete the anti-CD209 antibody or its antigen-binding fragment as described in any one of claims 1-2, and the recombinant cells include the expression cassette or vector as described in claim 8.

10. A method for producing the anti-CD209 antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, Cultivate the recombinant cells as described in claim 9; or inject the hybridoma cells as described in claim 9 into the peritoneal cavity of a non-human mammal and collect ascites.

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