CN111789948A - Binding/inhibitors of the membrane protein TRABD2A metalloprotease and uses thereof - Google Patents

Abstract

The present invention relates to the use of binding/inhibitors or gene editing against the membrane protein TRABD2A metalloprotease for the prevention or treatment of tumors and/or viral infections. More specifically, the binding/inhibitor against the membrane protein TRABD2A metalloprotease is an antibody against TRABD2A metalloprotease or a substance such as a small molecule agent that alters the activity of TRABD2A metalloprotease; gene editing is a method of modifying TRABD2A gene to cause deletion, structural change, or expression defect of TRABD2A metalloprotease. The invention also relates to the use of a binding/inhibitor against the membrane protein TRABD2A metalloprotease in combination with an inhibitor against PD-1, such as an anti-PD-1 antibody and/or another therapeutic agent, for the prevention or treatment of tumors and/or viral infections.

Description

Binding/inhibitors of the membrane protein TRABD2A metalloprotease and uses thereof

Technical Field

The present invention is in the field of treatment of viral infections and tumors, including cancer. More specifically, the present invention relates to CD8 through combination/inhibition or gene editing (elimination/mutation)⁺T cell membrane metalloprotease TRABD2A (Trab domain-containing protein 2A) changes its activity/structure to remarkably promote immunity CD8⁺Killing of virus-infected host cells and tumor cells by T Cells (CTLs). Therefore, the invention relates to the application of the combination/inhibitor of the membrane protein TRABD2A metalloprotease to change the activity, or eliminate/mutate the structure, in treating viral infection, tumor such as liver cancer, lung cancer, lymphoma, leukemia and other diseases.

Background

The incidence and mortality of malignant tumors are always on the rise worldwide, and become the main causes of human death. Cancer has begun to become the leading cause of death in china in 2010, severely impacting the average life span of people and impeding the development of socio-economic. About 430 million cancer patients are newly added in 2015, and 1.2 million cancer patients are discovered every day. The high incidence rate of cancer in men is as follows: lung cancer, gastric cancer, esophageal cancer, liver cancer, colon cancer; the female is breast cancer, lung cancer, gastric cancer, colon cancer, and esophageal cancer. 280 thousands of people die from cancer in 2015, the death rate is high and low, and the death rate is lung cancer, gastric cancer, liver cancer, esophagus cancer and colon cancer, and 7500 people die from cancer every day. The treatment method of malignant tumor cancer mainly comprises the following traditional treatment methods with injuries: the comprehensive use scheme comprises surgical excision, chemotherapy and radiotherapy, and the side effects of the comprehensive use scheme are mainly pain of patients, reduced life quality and larger economic burden. It is even more difficult that these therapies rely on early detection. The effect is not obvious for patients with middle and advanced cancer, so finding an effective method for treating malignant tumors including cancer completely is a continuous object of scientific research.

The newly developed tumor immunotherapy method in recent years has the advantages of small damage, wide treatment range, effectiveness for middle and late stage patients and the like. Tumor immunotherapy approaches mainly include the following major categories: the first is a tumor surface specific receptor blocker, such as herceptin drugs, which has the defect of single treatment of lesion; the second type of medicine is the artificial installation of tumor surface specific antigen receptor on CD8⁺On T cells, thereby rendering CD8⁺T cells specifically recognize and attack cancer cells, so-called CAR-T, and cause cancer cell death, however, CAR-T is still in clinical research stage at present, and the operation is complex and not easy to popularize widely; the third class of drugs is tumor site suppression immunization, which utilizes monoclonal antibodies to activate CD8⁺T, results in CD8⁺T cells actively attacking cancer cells, such as monoclonal antibodies to PD-1, have been widely used clinically in cancer therapy as broad-spectrum drugs for cancer therapy, in which CD8 is blocked⁺PD-1 receptor on the surface of T cell can actively promote the killing ability of the T cell to cancer cells. Monoclonal antibodies (monoclonal antibodies) inhibiting PD-1 receptors are excellent new antitumor drugs, are widely used in various countries in the world, and have good effects and further expanded application ranges. Therefore, the anti-tumor drug has a wide spectrum of anti-tumor effects like the anti-PD-1 receptor monoclonal antibody drugs, is effective for middle and late stage patients, and is expected by the majority of patients without injury.

CD8⁺T cells are also effective after viral infectionIt has cell-killing and antiviral effects. During viral infection, dendritic cells confer CD8 by presenting viral antigens⁺T cells are capable of specifically recognizing host cells infected by the virus, and in turn, attacking and killing the infected cells. CD8⁺The antiviral function of T cells plays a positive role in viral infection. However, many times, CD8⁺T cells are confused by tumors or viruses, cannot completely fight viral infection, and also need drug assistance, which is an own negative regulatory mechanism of the immune system, leading to CD8⁺T cells can not be activated continuously, and the immunity of the organism is kept within a normal range. At present, similar to the application of PD-1 in tumor immunotherapy, the immunotherapy of PD-1 can still be applied to eliminate virus-infected cells by blocking CD8 with monoclonal antibodies⁺PD-1 receptor on the surface of T cells, capable of actively promoting CD8⁺T cells continue to kill virus-infected host cells, and PD-1 is therefore considered to be a regulator of the immune response process, acting as a brake.

There remains a need in the art for new agents against viral infections, such as HIV virus, hepatitis virus, and other viral infections that pose serious health risks to humans.

Disclosure of Invention

The invention discovers that CD8 is combined or/and inhibited⁺T cell membrane metalloprotease TRABD2A (Trandomain-stabilizing protein 2A) activity of T cell, and can promote immunity CD8⁺The cell killing activity of T Cells (CTL) enhances the targeted killing of virus-infected host cells and tumor cells.

The inventor finds that the CD8⁺The cell membrane metalloprotease TRABD2A on T cells is a good target for tumor therapy by binding/inhibiting or eliminating/mutating CD8⁺The protein function of the cell membrane metalloprotease TRABD2A on T cells can remarkably promote CD8⁺Killing effect of T cells on tumor cells. The results of the present invention show that CD8 is targeted⁺Binding/inhibitor of TRABD2A or mutation/elimination of TRABD2A gene on T cells significantly promoted CD8⁺T is thinCell depletion patients in vivo isolation of tumor cell lines and isolation of primary cancer tissue cells.

In the invention, the antibody and the small molecule inhibitor aiming at the TRABD2A are also found to be capable of remarkably promoting CD8⁺T cells to eliminate host cells infected by different viruses isolated from the patient.

Thus, the present invention provides a medicament or/and pharmaceutical composition comprising a binding/inhibitor against the cell membrane metalloprotease TRABD2A in combination with a pharmaceutically acceptable carrier, for use in the treatment of tumors and/or viral infections. The binding/inhibitor or gene editing can alter CD8⁺The activity of the T cell membrane metalloprotease TRABD2A (Trab domain-associating protein 2A) protein or the function thereof is eliminated, thereby remarkably promoting the immunity CD8⁺Killing of tumor cells and/or host cells infected with a virus by T cells is effective in treating tumors (including cancer) and/or viral infections.

In one aspect, the invention also provides the use of a binding/inhibitor or gene knockout/mutation to TRABD2A in the manufacture of a medicament for the treatment of tumors (including cancer) and/or viral infections.

In another aspect, the invention also relates to the use of a binding/inhibitor or gene knockout/mutation to TRABD2A in combination with other anti-neoplastic or anti-viral agents for the treatment of tumors (including cancer) and/or viral infections. Accordingly, the present invention also provides a pharmaceutical combination comprising a binding/inhibitor to TRABD2A and an additional therapeutic agent, together with a pharmaceutically acceptable carrier. The invention also provides the use of a binding/inhibitor against TRABD2A in combination with an additional therapeutic agent in the manufacture of a medicament for the treatment of tumours (including cancer) and/or viral infections. In particular, the additional therapeutic agent is an antibody against PD-1, and the combined use of the two can more efficiently kill tumor cells and virus-infected cells, with a more excellent therapeutic effect.

Still further, the present invention provides a method (medical device) comprising removing or killing tumor cells or/and virus infected cells by extracorporeal circulation of blood, including dialysis-like extracorporeal procedures, comprising a container containing a binding/inhibiting agent directed to TRABD2A and optionally a container containing, separately or in combination, an additional therapeutic agent. Preferably, the additional therapeutic agent is an antibody against PD-1.

Description of the drawings

FIG. 1 shows that TRABD 2A-specific antibodies significantly enhanced CD8 alone or in combination with anti-PD-1 antibodies⁺Killing effect of T cells on breast cancer cell line MCF7 cells.

FIG. 2 shows that TRABD 2A-specific antibodies significantly enhanced CD8 alone or in combination with anti-PD-1 antibodies⁺Killing effect of T cells on breast cancer cell line MCF-10A cells.

FIG. 3 shows that TRABD 2A-specific antibodies significantly enhanced CD8 alone or in combination with anti-PD-1 antibodies⁺Killing of myeloma cell line K562 cells by T cells.

FIG. 4 shows that TRABD 2A-specific antibodies significantly enhanced CD8 alone or in combination with anti-PD-1 antibodies⁺Killing effect of T cells on primary liver cancer cells.

FIG. 5 shows that TRABD 2A-specific antibodies significantly enhanced CD8 alone or in combination with anti-PD-1 antibodies⁺Killing effect of T cells on primary lung cancer cells.

FIG. 6 shows that TRABD 2A-specific antibodies significantly enhanced CD8 alone or in combination with anti-PD-1 antibodies⁺Killing effect of T cells on primary B cell lymphoma cells.

FIG. 7 shows the isolation and purification of CD8⁺T cells stimulated with Her 2-derived antigenic polypeptides at varying concentrations of Ni²⁺Killing effect on breast cancer cells MCF-7 in the presence of ions.

FIG. 8 shows the isolation and purification of CD8⁺T cells stimulated with Her 2-derived antigenic polypeptides at various concentrations of Co²⁺Killing effect on breast cancer cells MCF-7 in the presence of ions.

FIG. 9 shows the isolation and purification of CD8⁺T cells stimulated with Her 2-derived antigenic polypeptides at various concentrations of TRABD2A protease inhibitor small moleculesKilling effect of compound 1,10-phenanthroline (mu M) on breast cancer cell MCF-7.

FIG. 10 shows HIV-1 infected CD4 isolated from blood of untreated HIV-1 patients⁺And CD8⁺CD4 after T cell mixed culture and treatment with different antibodies for 6 days⁺Survival of T cells.

FIG. 11 shows hepatocytes from 3 untreated HBV three great positive patients and CD8 isolated from lymphocyte population⁺T cells were differentiated according to CD8 cells and hepatocyte 4:1, and determining the survival rate of the liver cells after mixed culture.

FIG. 12 shows hepatocytes obtained from 2 untreated HCV positive patients and CD8 isolated from lymphocyte populations⁺Survival of hepatocytes was determined after mixed culture of T cells.

FIG. 13 shows HIV-1 infected CD4 isolated from 5 untreated HIV-1 patients on Peripheral Blood Mononuclear Cells (PBMCs)⁺And CD8⁺T cells are added with different concentrations of TRABD2A protease inhibitor NiCl during mixed culture₂CD4 measured after (μ M) treatment⁺Survival of T cells.

FIG. 14 shows HIV-1 infected CD4 isolated from Peripheral Blood Mononuclear Cells (PBMCs) from 5 untreated HIV-1 patients⁺And CD8⁺Adding TRABD2A protease inhibitor CoCl into T cells during mixed culture₂(μ M) Virus CD4 assayed after incubation⁺Survival of T cells.

FIG. 15 shows HIV-1 infected CD4 isolated from Peripheral Blood Mononuclear Cells (PBMCs) from 5 untreated HIV-1 patients⁺And CD8⁺The survival rate of virus CD4+ T cells was determined after culturing T cells in mixed culture with TRABD2A protease inhibitor 1,10-phenanthroline (μ M).

The legends in the figures are illustrated as follows:

"viable cell (%)" refers to the measured cancer cell or CD4⁺Percent survival of T cells;

"+", "indicate statistical significance;

"No treatment" refers to a negative control group to which no antibody was added;

"IgG" refers to a negative control for an antibody;

"anti-PD 1" refers to a monoclonal antibody against human PD-1;

"anti-TRABD 2A" refers to a monoclonal antibody against TRABD 2A.

Detailed Description

In the present invention, "a binding/inhibitor against TRABD 2A" means a substance capable of binding to inhibit the biological activity of TRABD2A, including, for example, anti-TRABD2A antibody-like biological substances, and also including small molecule compounds and the like.

The term "antibody" is an immunoglobulin molecule capable of immunospecifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. ("antigen"), through at least one epitope recognition site located in the variable domain of the immunoglobulin molecule. Thus, "antibody" includes not only intact polyclonal or monoclonal antibodies, humanized antibodies, camelized (camelized) antibodies, single chain antibodies, anti-atopic antibodies, and the like, but also mutants thereof, naturally occurring variants, fusion proteins and chimeric antibodies including epitope binding sites having the necessary immunospecificity, and the like. As used herein, an "antigen-binding fragment" of an antibody is an immunoglobulin whose amino acid sequence comprises at least one epitope-binding site of an antibody specific for the antigen, including, for example, fragments (e.g., Fab ', F (ab')₂) Disulfide-linked bispecific fvs (sdfv), and single chain molecules (e.g., scFv).

In one embodiment, the present invention provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a binding/inhibitor to TRABD2A and a pharmaceutically acceptable carrier. In one example, the binding/inhibitor against TRABD2A is an anti-TRABD2A antibody or antigen-binding fragment thereof. anti-TRABD2A antibodies or antigen-binding fragments thereof are known to those of skill in the art and can be prepared by conventional methods such as hybridoma technology, recombinant technology, and the like. In another example, the binding/inhibiting agent against TRABD2A is a small molecule capable of inhibiting TRABD2A activity, such as an inorganic salt such as nickel salt, cobalt salt, or a nickel-containing compound or cobalt-containing compound capable of providing divalent ions, an organic compound binding/inhibiting agent such as 1,10-phenanthroline (1,10-phenanthroline), or the like.

In the present invention, "cobalt-containing compound" means a compound capable of providing Co in vivo and/or in vitro²⁺Any compound of an ion. In one embodiment, wherein the cobalt-containing compound is a compound containing Co2+ ions, such as an organic or inorganic salt of cobalt. More specifically, the cobalt-containing compound is selected from CoCl₂、CoSO₄、CoF₂、CoBr₂And organic cobalt and the like containing Co²⁺Inorganic salts, organic salts, and other cobalt compounds such as macromolecules (cobalt ionization of protein or DNA). In another embodiment, the cobalt-containing compound is in a pharmaceutically acceptable co-crystal form.

In the present invention, "nickel-containing compound" means capable of providing Ni in vivo and/or in vitro²⁺Any compound of an ion. In one embodiment, wherein the nickel-containing compound is Ni-containing²⁺Ionic compounds, such as organic or inorganic salts of nickel. More specifically, the nickel-containing compound is selected from NiCl2, NiSO4, NiF2, NiBr2, organic nickel and the like containing Ni²⁺Inorganic salts, organic salts, and other nickel compounds such as polymers (cobalt-ionized proteins or DNA). In another embodiment, the nickel-containing compound is in a pharmaceutically acceptable co-crystal form.

The pharmaceutical composition of the present invention may comprise the binding/inhibiting agent as the sole active ingredient. Optionally, one or more additional active ingredients may also be included in the pharmaceutical compositions of the present invention. Accordingly, the invention also includes a pharmaceutical composition comprising an effective amount of a binding/inhibitor to TRABD2A and a pharmaceutically acceptable carrier, said pharmaceutical composition further comprising an effective amount of one or more additional therapeutic agents.

In some embodiments, the additional therapeutic agent is, for example, but not limited to, an alkylating agent (e.g., nitrogen mustard or cisplatin), an angiogenesis inhibitor, an anthracycline (e.g., daunorubicin/daunomycin or doxorubicin)) Antibiotics (e.g., actinomycin D, bleomycin, or amphenicol), antibodies (e.g., anti-VEGF antibodies such as bevacizumab (by Genentech, inc

Sold), anti-EGFR antibodies such as panitumumab (sold by Amgen, inc. as VECTIBIX)^TMSold) or anti-integrin antibodies, such as natalizumab (by Biogen Idee and elan pharmaceuticals, inc

Sold)), antimetabolites (e.g., methotrexate or 5-fluorouracil), antimitotic agents (e.g., vincristine or paclitaxel), cytotoxins (e.g., cytostatics or cytocides), hormonal therapy agents (e.g., selective estrogen receptor modulators (e.g., tamoxifen or raloxifene), aromatase inhibitors, luteinizing hormone-releasing hormone analogs, progestational agents, adrenocorticosteroids, estrogens, androgens, antiestrogens, androgen receptor blockers, etc.), matrix metalloproteinase inhibitors, radioactive elements (e.g., alpha-emitters, gamma-emitters, etc.), or any other chemotherapeutic agent. The additional therapeutic agent may also be, for example, a radiotherapeutic agent. The choice of radiotherapeutic agent is well known to those skilled in the art.

In still other embodiments, the additional therapeutic agent is another antiviral drug, such as, but not limited to, interferon alpha-2 b, interferon alpha-2 a and interferon alpha con-1 (pegylated and non-pegylated), ribavirin, lamivudine (3TC), entecavir, tenofovir, telbivudine (LdT), adefovir, or other emerging antiviral drugs. Preferably, such drugs include antiretroviral drugs such as anti-HIV drugs, drugs that enhance immunity, monoclonal antibody-based drugs.

For the prevention of HIV infection, suitable anti-HIV drugs include, but are not limited to: abacavir, lamivudine, zidovudine, amprenavir, atazanavir, efavirenz, emtricitabine, tenofovir, lopinavir, ritonavir, and the like, or any combination thereof. American food and medicineThe only currently available pre-exposure prophylaxis (PrEP) treatment approved by the regulatory agency is

(emtricitabine/tenofovir DF) for use in the prevention of HIV infection in uninfected persons. An "anti-HIV agent" is any agent that is effective, directly or indirectly, in inhibiting HIV, treating or preventing infection by HIV, and/or treating, preventing or delaying the onset or progression of AIDS or ARC. It is understood that anti-HIV agents are effective in treating, preventing or delaying HIV infection or the onset or progression of AIDS and/or a disease or condition caused thereby or associated therewith. For example, the inhibitors of the present invention may be effectively administered in combination with an effective amount of one or more other anti-HIV drugs selected from HIV antivirals, immunomodulators, anti-infectives, or vaccines useful in the treatment of HIV infection or AIDS, whether during the pre-exposure and/or post-exposure period.

For the prevention or treatment of hepatitis virus infection, the additional therapeutic agent may be, for example, an anti-HBV drug or an anti-HCV drug.

In some embodiments, the anti-HBV agent is, for example, an HBV RNA replication inhibitor, an HBsAg secretion inhibitor, an HBV core protein allosteric modifier, an HBV capsid inhibitor, an antisense oligomer, siRNA, an HBV therapeutic vaccine, an HBV prophylactic vaccine, HBV antibody therapy (monoclonal or polyclonal) and TLR2,3,7,8 and 9 agonists.

In other embodiments, the anti-HCV drug is, for example, an HCV protease inhibitor, an HCV nucleoside or nucleotide polymerase inhibitor, an HCV non-nucleoside polymerase inhibitor, an HCV NS3B inhibitor, an HCV NS4A inhibitor, an HCV NS5A inhibitor, an HCV NS5B inhibitor, an HCV entry inhibitor, a cyclophilin inhibitor, or a combination thereof, non-limiting examples of which include PSI-7977, PSI-938, TMC-435, BMS-790052, BMS-650032, GS-5885, GS-9190, GS-9451, BI-201335, BI-207127, telaprevir, VX-222, mericitabine, and danoprevir.

Preferred HCV protease inhibitors for this purpose include, but are not limited to, telaprevir (Vertex), boceprevir (Merck), BI-201335(Boehringer Ingelheim), GS-9451(Gilead), and BMS-650032 (BMS). Other suitable protease inhibitors include, but are not limited to, ACH-1095(Achillion), ACH-1625(Achillion), ACH-2684(Achillion), AVL-181(Avila), AVL-192(Avila), BMS-650032(BMS), danoprevir (RG7227/ITMN-191, Roche), GS-9132(Gilead), GS-9256(Gilead), IDX-136(Idenix), IDX-316(Idenix), IDX-320(Idenix), MK-5172(Merck), naaprevir (Schrlering-Plough Corp), PHX-1766(Phenomix), TMC-435(Tibotec), vanipredvir (MK-7009, Merck), VBY708(Virobay), VX-500(Vertex), VX-985 (VX-985), or combinations thereof.

Preferred non-nucleoside HCV polymerase inhibitors for use in the present invention include, but are not limited to, GS-9190(Gilead), BI-207127(Boehringer Ingelheim), and VX-222(VCH-222) (Vertex) & ViraChem). Preferred nucleoside HCV polymerase inhibitors include, but are not limited to, PSI-7977(Gilead) and PSI-938 (Gilead). Other suitable and non-limiting examples of suitable HCV polymerase inhibitors include ANA-598(Anadys), BI-207127(Boehringer Ingelheim), BILB-1941(Boehringer Ingelheim), BMS-791325(BMS), filibuvir, GL59728(Glaxo), GL60667(Glaxo), GS-9669(Gilead), IDX-375(Idenix), MK-3281(Merck), tegobrevir, TMC-647055(Tibotec), VCH-759(Vertex and ViraChem), VCH-916(ViraChem), VX-759(Vertex), GS-6620(Gilead), IDX-102 (enIdidIdenx), IDX-189 (Idenix), Inhiex-189 (Inhiex 0608 (Pharctix 0608), GS-6620 (Medix), Vertix-102 (Kliex-64912), Bioxix-MRZ (Bioxix) or combinations thereof. The polymerase inhibitor may be a nucleoside or nucleotide polymerase inhibitor, for example GS-6620(Gilead), IDX-102(Idenix), IDX-184(Idenix), INX-189(Inhibitex), MK-0608(Merck), PSI. -7977(Gilead), PSI-938(Gilead), RG7128(Roche), TMC64912(Medivir), ALS-2200(Alios BioPharma/Vertex), ALS-2158(Alios BioPharma/Vertex), or a combination thereof. The polymerase inhibitor may also be a non-nucleoside polymerase inhibitor, such as PF-00868554 (fevery), ANA-598(Anadys), BI-207127(Boehringer Ingelheim), BILB-1941(Boehringer Ingelheim), BMS-791325(BMS), filibuvir, GL59728(Glaxo), GL60667(Glaxo), GS-9669(Gilead), IDX-375(Idenix), MK-3281(Merck), tegobrevir (Gilead), TMC-647055(Tibotec), VCH-759(Vertex and ViraChem), VCH-916(ViraChem), VX-222(VCH-222) (Vertex and ViraChem), VX-759(Vertex), or combinations thereof.

Preferred NS5A inhibitors include, but are not limited to BMS-790052(BMS) and GS-5885 (Gilead). Non-limiting examples of suitable NS5A inhibitors include GSK62336805(GlaxoSmithKline), ACH-2928(Achillion), AZD2836(Astra-Zeneca), AZD7295(Astra-Zeneca), BMS-790052(BMS), BMS-824393(BMS), GS-5885(Gilead), PPI-1301(Presidio), PPI-461(Presidio) A-831(ArrowTherapeutics), A-689 (ArrowTherapeutics), or combinations thereof.

Non-limiting examples of suitable cyclophilin inhibitors include alisporivir (Novartis & Debiopharm), NM-811(Novartis), SCY-635(Scynexis), or combinations thereof.

Non-limiting examples of suitable HCV entry inhibitors include ITX-4520(iTherx), ITX-5061(iTherx), or combinations thereof.

In the present invention, specific examples of such additional antiviral agents include, but are not limited to, AP-H005, A-831(Arrow Therapeutics) (NS5A inhibitor), A-689(Arrow) Therapeutics) (NS5A inhibitor), INX08189 (inhibitor of polymerase), ITMN-191(Intermune/Roche) (NS3/4A protease inhibitor), VBY-376 (protease inhibitor) (Virobay), ACH-1625(Achillion, protease) inhibitor), IDX136(Idenix, protease inhibitor), IDX316(Idenix, protease inhibitor), VX-813(Vertex), SCH900518(Schering-Plough), TMC-435(Tibotec), ITMN-191(Intermune, Roche), MK-7009(Merck), IDX-PI (Nortvais), R7128(Roche), Pf-868554 (Pf-PF-868554) (Idienx-PF-184), non-PF-184 (Idenix-4878691), IDX-375(Idenix, NS5B polymerase inhibitor), PPI-461(Presidio), BILB-1941(Boehringer Ingelheim), GS-9190(Gilead), BMS-790052(BMS), CTS-1027(Conatus), GS-9620(Gilead), PF-4878691 (pfeiri), RO5303253 (Roche), ALS-2200(Alios BioPharma/Vertex), ALS-2158(Alios BioPharma/Vertex), GSK62336805(GlaxoSmithKline), or any combination thereof.

Preferably, the binding/inhibitors of the invention against TRABD2A may be used alone or/and in combination with anti-PD-1 antibodies. anti-PD-1 antibodies that can be used in the present invention include those known in the art or those that can be produced by one of skill in the art according to conventional methods, such as the antibodies known in the art as nivolumab (US8008449), pembrolizumab (US8354509), MEDI0608(US8609089), pidilizumab (US8686119), or any anti-PD-1 antibody as described in US patent nos. 6808710, 7488802, 8168757, and the like. Optionally, the pharmaceutical composition of the invention may comprise an additional third therapeutic agent in addition to the binding/inhibitor against TRABD2A and the anti-PD-1 antibody. The third therapeutic agent may be selected from the ranges as described above for the additional therapeutic agents.

The therapeutic agents of the present invention may be administered simultaneously (as a single formulation or separate formulations) or sequentially with other therapies.

The pharmaceutical compositions of the present invention may be administered by any suitable route or method of administration, including but not limited to oral, parenteral, or using implantable compositions or devices. The frequency of administration can be 1-3 times daily, or can be administered in a dosage regimen of twice weekly, once biweekly, twice monthly or once monthly for the treatment or prevention of tumors.

As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, proteins, nucleic acid carriers, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Further examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffering agents which increase the shelf-life or efficacy of the medicament.

The compositions of the present invention may also be presented in a variety of forms, such as liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form depends on the desired mode of administration and therapeutic application. Typical compositions exist in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. In one instance, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In another case, the agent is administered by intravenous infusion or injection. In another instance, the agent is administered by intramuscular or subcutaneous injection. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with or without an added preservative. The compositions may take such forms as suspensions, liquids or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be presented in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The medicaments of the invention may be administered, for example, orally (e.g., by tablets or capsules), parenterally (including subcutaneous, intravenous, intramuscular, or intrasternal injection, or other infusion techniques), or by inhalation spray, in the form of one or more unit dose pharmaceutical compositions containing an effective amount of the inhibitor of the invention, alone or in combination, and conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles for the treatment or prevention of cancer. The compounds may also be administered parenterally by implantable drug delivery compositions or devices adapted to provide an effective amount of the inhibitors of the invention over an extended period of time.

Solid formulations suitable for oral administration (e.g., powders, pills, capsules, and tablets) can be prepared according to techniques known in the art, and can use solid excipients such as starches, sugars, kaolin, lubricants, binders, disintegrants, and the like. Liquid preparations suitable for oral administration (e.g., suspensions, syrups, elixirs and the like) can be prepared according to techniques known in the art and can employ any of the usual media, such as water, glycols, oils, alcohols and the like. For oral administration, solid dosage forms, in particular tablets, are preferred.

Parenteral compositions of the compounds of the invention may be prepared according to techniques known in the art and typically employ sterile water as a carrier and optionally other ingredients such as stabilizers and/or dissolution aids. Injectable solutions or suspensions may be prepared according to methods known in the art, for example, in which the carrier comprises saline solution, dextrose solution, or a solution containing a mixture of saline and dextrose. Implantable compositions can also be prepared according to methods known in the art, for example, wherein the carrier comprises the active chemical ingredient and a suitable excipient (e.g., a polymer), or using an implantable device for drug delivery.

Further description of processes suitable for preparing Pharmaceutical compositions containing cobalt-containing compounds of The invention and ingredients suitable for use in such compositions are provided in Remington's Pharmaceutical Sciences, 18 th edition (edited by a.r. gennaro, Mack Publishing co., 1990) and Remington-The Science and practice of Pharmacy), 22 th edition (published by Pharmaceutical Press and philiadelphia color of Pharmaceutical University of The Sciences, 2012, ISBN 978085711-062-6), and The like.

The preferred method or route of administration may also vary depending on the time interval between doses in the dosage regimen. For example, an effective amount of a cobalt-containing compound for prophylactic use can be administered orally at intervals such as, but not limited to, once daily, twice weekly, once biweekly, twice monthly, or once monthly. While one unit dose is preferably administered orally at each dosing interval, one or more oral unit doses can be administered at each dosing interval as needed to deliver the appropriate amount of active agent.

Alternatively, an effective amount of a cobalt-containing compound of the invention for prophylactic use may be administered parenterally, for example, but not limited to, once a week, once every two weeks, twice a month, once a quarter, twice a year, once a year, or at longer intervals, for example, but not limited to, once every 18 months or once every two years. The longer the interval between each administration of the active agent, the larger the active dose that may be required for each administration. Thus, one or more unit doses may be administered as needed to deliver an appropriate amount of active agent, e.g., one or more injections or infusions of a compound of the invention, or one or more implant compositions or devices, at each dosing interval.

Any dosage regimen for prophylactic use may be a continuous dosage regimen or an intermittent dosage regimen.

For prophylactic treatment, the amount of cobalt-containing compound per unit dose can be from 0.1mg to 500 mg; or 0.1 to 400mg or more, for use in longer interval dosage regimens. The dosage of each unit dose will vary depending upon the time interval between doses in the dosage regimen.

The medicaments of the invention may be administered parenterally in the form of unit doses of pharmaceutical compositions, including subcutaneous injections, intravenous, intramuscular, or intrasternal injections, or other infusion techniques (one or more injections or infusions may be administered as required to deliver the appropriate amount of active agent at each dosing interval). The inhibitors of the present invention may also be administered parenterally by implantable drug delivery compositions or devices adapted to provide an effective amount of the inhibitor over an extended period of time.

The unit dose of an effective amount of an inhibitor of the present invention may be administered parenterally at intervals such as, but not limited to, once a week, once every two weeks, twice a month, once a quarter, twice a year, or once a year, or at longer intervals such as, but not limited to, once every 18 months or once every two years.

The compositions may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an inhibitor of the present invention. "therapeutically effective amount" means an amount effective, at dosages and for periods of time required, to achieve the desired therapeutic result. The therapeutically effective amount of the inhibitor may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the compound to elicit a desired response in the individual. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time required, to achieve the desired prophylactic result. Typically, because the prophylactic dose is administered to the subject prior to or at an early stage of the disease, the prophylactically effective amount can be lower than the therapeutically effective amount.

Dosage regimens may be adjusted to provide the best desired response (e.g., therapeutic or prophylactic response). For example, a bolus injection may be administered, several divided doses may be administered over a period of time or the dose may be reduced or increased accordingly as indicated by the urgency of the treatment state. Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units of a single dose for a mammalian subject to be treated; each unit containing a predetermined amount of an inhibitor of the present invention calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

Illustrative non-limiting ranges of therapeutically or prophylactically effective amounts of the present invention directed against an inhibitor of the cell membrane metalloprotease TRABD2A are 0.025 to 50mg/kg, 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. In one case, the inhibitor is administered in the formulation as a sterile aqueous solution. It is noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, the particular dosage regimen should be adjusted over a period of time according to the individual need and the professional judgment of the person administering or directing the administration of the composition, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or administration of the composition.

In another aspect, the invention provides the use of a binding/inhibitor against the cell membrane metalloprotease TRABD2A in the manufacture of a medicament for the treatment or prevention of tumours (including cancer) and/or viral infections. In one embodiment, the binding/inhibitor is an antibody directed against the cell membrane metalloprotease TRABD 2A. In another embodiment, the binding/inhibitor is a small molecule compound, such as a cobalt-containing compound or a nickel-containing compound, or an organic small molecule binding/inhibitor, such as 1,10-phenanthroline, capable of inhibiting the activity of the cell membrane metalloprotease TRABD 2A. The cobalt-containing compound may be an organic salt of cobalt orInorganic salts capable of producing Co after being administered into the body²⁺Ions. For example, the cobalt-containing compound may be selected from CoCl₂、CoSO₄、CoF₂、CoBr₂Containing Co or the like²⁺Or any combination thereof. The Ni-containing compound may be an organic or inorganic salt of nickel, which is capable of generating Ni upon administration into the body²⁺Ions. For example, the nickel-containing compound may be selected from NiCl₂、NiSO₄、NiF₂、NiBr₂Containing Ni²⁺Or any combination thereof.

The present invention also provides a method for preventing or treating tumors (including cancer) and/or viral infections or delaying or inhibiting tumor growth in an individual in need thereof, which method comprises administering to said individual a binding/inhibitor of the invention against the cell membrane metalloprotease TRABD 2A. The binding/inhibitor against the cell membrane metalloprotease TRABD2A may be an antibody against the cell membrane metalloprotease TRABD2A, a cobalt-containing compound, a nickel-containing compound, or a small molecule inhibitor such as 1,10-phenanthroline, or any combination thereof. The method also includes administering to the individual a gene edit of the invention directed to the cell membrane metalloprotease TRABD 2A. The gene editing for the cell membrane metalloprotease TRABD2A includes: gene silencing of small RNA of the plasmid vector, gene treatment methods such as CRISPR/Cas9 gene mutation, gene knockout and the like. Optionally, administering to the individual an additional anti-cancer agent in combination. The additional anti-cancer agent may be as described above for the pharmaceutical composition.

Preferably, in the methods of the invention, a therapeutically or prophylactically effective amount of a binding/inhibitor of the invention against the cell membrane metalloprotease TRABD2A and an anti-PD-1 antibody are administered in combination to the individual. The anti-PD-1 antibody is as described above. Optionally, a third therapeutic agent is also used in combination in the methods of the invention, as described above for the pharmaceutical compositions.

The binding/inhibiting agents of the present invention against the cell membrane metalloprotease TRABD2A may be administered orally by any means that causes the active agent to come into contact with the site of action of the agent. The compounds may be administered orally by conventional means of pharmaceutical administration, as individual therapeutics or combinations of therapeutics. It may be administered alone, but is typically administered with an orally administered pharmaceutical carrier of choice, containing an effective amount of the compound and one or more conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. Solid formulations suitable for oral administration, such as, but not limited to, tablets, capsules, powders, pills, can be prepared according to techniques known in the art, and solid excipients, such as starches, sugars, kaolin, lubricants, binders, disintegrants and the like can be used. Liquid preparations suitable for oral administration (e.g., suspensions, syrups, elixirs and the like) can be prepared according to techniques known in the art and can employ any of the usual media, such as water, glycols, oils, alcohols and the like. For oral administration, solid dosage forms, in particular tablets, are preferred.

Examples of dosing regimens of orally administered inhibitors that may be used in the method include twice weekly dosing, once every two weeks dosing, twice monthly dosing, and once monthly dosing. The selected dosing regimen will use a dosage appropriate to provide treatment or prevention of cancer during the time interval from each administration to the next.

In certain embodiments, the methods of the invention comprise administering an inhibitor in combination with radiation therapy to produce a long lasting anti-tumor response and/or enhance survival of cancer patients. In some embodiments, the methods of the invention comprise administering radiation therapy to a cancer patient prior to, concurrently with, or after administration of the inhibitor. For example, after administration of one dose or multiple doses of antibody, radiotherapy may be administered to a tumor lesion in one dose or multiple doses. In some embodiments, radiation therapy can be administered locally to a tumor lesion to enhance local immunogenicity of the patient's tumor (adjuvant radiotherapy) and/or to kill tumor cells after systemic administration of an inhibitor (ablative radiotherapy). In certain embodiments, the antibody may be administered in combination with radiotherapy and a chemotherapeutic agent (e.g., temozolomide or cyclophosphamide) or a VEGF antagonist (e.g., aflibercept).

A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing a mutant virus, receptor-mediated endocytosis (see, e.g., Wu et al, 1987, J.biol.chem.262: 4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous layers (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active substances.

The pharmaceutical compositions of the present invention may be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, pen delivery devices are readily used to deliver the pharmaceutical compositions of the present invention as opposed to subcutaneous delivery. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable tubing string containing the pharmaceutical composition. Once all of the pharmaceutical composition within the column has been administered and the column is empty, the empty column can be easily discarded and replaced with a new column containing the pharmaceutical composition. The pen delivery device may then be reused. In a disposable pen delivery device, there is no replaceable tubing string. In contrast, disposable pen delivery devices are pre-filled with a pharmaceutical composition contained in a reservoir inside the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

In some cases, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials may be used. In yet another embodiment, the controlled release system may be placed close to the target of the composition, thus requiring only a fraction of the systemic dose.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, instillation, and the like. These injectable preparations can be formulated by known methods. For example, injectable preparations may be formulated, for example, by dissolving, suspending or emulsifying the antibody or salt thereof described above in a sterile aqueous or oleaginous medium conventionally used for injection. As an aqueous medium for injection, there is, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents and the like, which can be combined with the use of a suitable solubilizing agent such as ethanol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant [ e.g., polysorbate-80, HC0 — 50 (polyoxyethylene (50mol) adduct of hydrogenated castor oil) ], and the like. As the oily medium, there can be used, for example, sesame oil, soybean oil and the like which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol and the like. The injection thus prepared is preferably filled in a suitable ampoule.

Advantageously, the pharmaceutical composition for oral or parenteral use described above is formulated in a dosage form in a unit dose suitable for matching the dose of active principle. Dosage forms in unit dose form include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like.

The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease state, and the individual undergoing therapy. In some cases, depending on the potency or individual response of the compound, it may be desirable to deviate upwards or downwards from a given dose. The amount and frequency of administration can be adjusted at the discretion of the attending clinician taking these factors into account.

The term "individual" or "patient" as used herein refers to an animal, preferably a mammal, such as a non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or primate (e.g., monkey, such as cynomolgus monkey, human, etc.), most preferably a human, that has been or will be the subject of treatment. The treatment includes prophylactic treatment, observation or experiment. In one embodiment of the invention, in each of the methods, pharmaceutical compositions, medicaments, uses, combinations, aspects and other embodiments described and/or claimed herein, the subject is a human subject.

Tumors (including cancers) that may be treated, prevented and/or alleviated by the methods and compositions of the invention include, but are not limited to, the following types: acute myeloid leukemia, adrenal tumors, AIDS-related cancers, soft tissue alveolar sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumors, breast cancer, carotid tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe cell renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrocytoma of the skin, desmoplastic small round cell tumor, ependymal tumor, Ewing's tumor, extraosseous myxoid chondrosarcoma, incomplete bone fibrogenesis, dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, malignant glioma, islet cell tumor, Kaposi's sarcoma, renal cancer, leukemia, lipoma/benign lipoma, liposarcoma/malignant lipoma, lipoma/malignant lipoma, Hepatoma, lymphoma, lung carcinoma, medulloblastoma, melanoma, meningioma, malignant mesothelioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, non-small cell lung carcinoma, ovarian carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, parathyroid tumor, pediatric carcinoma, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate carcinoma, retrouveal melanoma, rare hematological diseases, renal cell carcinoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin carcinoma, soft tissue sarcoma, squamous cell carcinoma, gastric carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid metastatic cancer, ovarian cancer, and uterine cancer. Preferably, the method or composition is used for treating, preventing and/or alleviating lung cancer, gastric cancer, esophageal cancer, liver cancer, colon cancer, breast cancer and the like.

The methods and compositions of the present invention may be used for the treatment, prevention, inhibition and/or amelioration of the following types of viruses, including, but not limited to, one or more of the following: herpes Simplex Virus (HSV) -1, HSV-2, Varicella Zoster Virus (VZV), Cytomegalovirus (CMV), Human Herpes Virus (HHV) -6, HHV-7, Kaposi's sarcoma-associated herpes virus (KSHV), JC virus, BK virus, parvovirus b19, adeno-associated virus (AAV) and adenovirus. In some embodiments, the virus comprises one or more of the following viruses: adenovirus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papilloma virus, BK virus, JC virus, smallpox virus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, Coxsackie virus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, Human Immunodeficiency Virus (HIV) (including HIV-1 and HIV-2), influenza virus, citrulline virus, Junin virus, lassa virus, Marburg virus, Sabiya virus, Crimiya-Congo hemorrhagic fever virus, Var fever, Var, ebola virus, marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, hendra virus, nipah virus, rabies virus, hepatitis delta virus, rotavirus, circovirus, collivirus, and Banna virus.

The cobalt-containing compound or nickel-containing compound may be administered in the form of a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal. The terms "pharmaceutically acceptable salt" and "pharmaceutically acceptable co-crystal" refer to salts or co-crystals that are not biologically or otherwise undesirable (e.g., are neither toxic nor otherwise deleterious to the recipient thereof). In the present invention, the cobalt-containing compound and/or nickel-containing compound may be used in the form of an acid addition salt thereof with an inorganic or organic acid, such as, but not limited to, a salt with hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, trifluoroacetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, valeric acid, succinic acid, and the like. Alternatively, in the present invention, the cobalt-containing compound and/or nickel-containing compound may be used in the form of an acid co-crystal thereof with an inorganic or organic acid, such as, but not limited to, a salt with benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, trifluoroacetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, valeric acid, succinic acid, or the like. The salts and co-crystals may be obtained from cobalt-containing compounds and/or nickel-containing compounds by conventional methods known to those skilled in the art, for example by combination with organic or inorganic acids or bases in solvents or dispersants, or by ion exchange with other salts.

As used herein, the term "composition" is intended to encompass the products comprising the inhibitors of the invention, as well as any product which results from combining the specified ingredients. Ingredients suitable for inclusion in a pharmaceutical composition are pharmaceutically acceptable ingredients, which means that the ingredients must be compatible with each other and not deleterious to the recipient thereof.

In a further aspect, the invention also provides a kit comprising a binding/inhibitor against the cell membrane metalloprotease TRABD2A and instructions for use, and optionally an inhibitor against PD-1 and/or one or more other anti-cancer agents.

The present invention is described in detail below with reference to specific examples in order to provide the public with a better understanding of the contents of the present invention. It is to be understood that this description is made only by way of example and not as a limitation on the scope of the invention. It will be apparent to those skilled in the art that various modifications, adaptations, and variations of the present invention can be made in accordance with the same or similar principles. Such improvements, modifications and changes are intended to be within the scope of the appended claims.

Examples

The invention is described in detail below with reference to specific embodiments for the purpose of enabling the public to better understand the technical content without limiting the technical content, and in fact, modifications made on the same or similar principles are within the scope of the claims as claimed.

Example 1

Collecting 50-100mL of blood from healthy human, separating and purifying CD8+ T cells, and stimulating with Her 2-derived antigen polypeptide at 2 × 10⁶Density culture in/mL for 4 daysActivating in vitro, and adding 5 × 10⁵/mL breast cancer cells MCF-7 were co-cultured for 3 days. After that, the survival rate of MCF-7 breast cancer cells was determined by adding different antibodies for 5 days. The results show that specific anti-TRABD2A antibodies as TRABD2A binding/inhibitors significantly enhanced CD8⁺The killing rate of the T cells to the tumor cells can obtain stronger killing effect when the anti-PD-1 antibody is used in combination. The results are shown in FIG. 1.

Example 2

Extracting 50-100mL of blood from healthy people, and separating and purifying CD8⁺T cells, stimulated with Her 2-derived antigenic polypeptide, according to 2X 10⁶Density culture in/mL for 4 days for in vitro activation, and addition of 5X 10⁵The breast cancer cells MCF-10A are cultured for 3 days in a total volume. After which various antibodies (5ug/mL anti-TRABD2A monoclonal antibody, purchased from RD, USA) were added for 5 days, and survival of MCF-10A breast cancer cells was determined. The results show that specific anti-TRABD2A antibodies as TRABD2A binding/inhibitors significantly enhanced CD8⁺The killing rate of T cells to the breast cancer cell line MCF-10A cells can obtain stronger killing effect when an anti-PD-1 antibody is used in combination. The results are shown in FIG. 2.

Example 3

Extracting 50-100mL of blood from healthy people, and separating and purifying CD8⁺T cells, stimulated with VEGF antigen polypeptide, according to 2X 10⁶Density culture in/mL for 4 days for in vitro activation, and addition of 6X 10⁵the/mL bone marrow cancer cell line K5620A cells were co-cultured for 3 days. After 5 days of treatment with different antibodies, the survival of K562 cancer cells was determined. The results show that the TRABD 2A-specific antibody, alone or in combination with an anti-PD-1 antibody, significantly enhanced CD8⁺Killing effect of T cells on bone marrow cancer cell line K562 cells. The results are shown in FIG. 3.

Example 4

Grinding and purifying the cancer tissue excised from the patient with liver cancer to form cancer cell colony, removing lymphocytes mixed in the cancer cell colony by using CD43 and CD19 biomarkers, purifying primary liver cancer cells and using DMEM + 10% FSB according to the proportion of 2.5 multiplied by 10⁵Culture in/mL. At the same time, from the frontExtracting separated lymphocyte from cancer tissue, purifying CD8+ T cell, activating in vitro by using fixed cancer cell antigen and IL-2 for 6 days, mixing with primary liver cancer cell according to the ratio of 4:1, treating for 10 days by using different antibodies, and determining the survival rate of cancer cells. The results show that the TRABD 2A-specific antibody, alone or in combination with an anti-PD-1 antibody, significantly enhanced CD8⁺Killing effect of T cells on primary liver cancer cells. The results are shown in FIG. 4.

Example 5

Recovering cancer tissue of a patient, grinding and disrupting the tissue, obtaining a lung cancer cell suspension by a corresponding method, removing lymphocytes mixed therein with biomarkers CD43 and CD19, purifying primary lung cancer cells and using DMEM + 10% FSB at 2.5X 10⁵Culture in/mL. Simultaneously, CD8 was purified from lymphocytes previously isolated from cancer tissues⁺T cells are stimulated and activated for 6 days by using fixed same tumor cell antigens and IL-2, mixed with primary lung cancer cells according to the ratio of 4:1, and treated by different antibodies for 10 days, and then the survival rate of the cancer cells is determined. The results show that the TRABD 2A-specific antibody, alone or in combination with an anti-PD-1 antibody, significantly enhanced CD8⁺Killing effect of T cells on primary lung cancer cells. The results are shown in fig. 5.

Example 6

Collecting 50-100mL of blood from patients with Diffuse Large B-cell Lymphoma (DLBCL), separating and purifying PBMCs from peripheral blood mononuclear cells, and respectively purifying CD8⁺T cells and B cancer cells. CD8⁺T cells stimulated with isolated cancer tissue antigens at 2X 10⁶Activation was performed at a density of 4/mL for 4 days, followed by 4:1 in admixture with isolated primary B cancer cells. B cancer cell viability was determined 5 days after treatment with different antibodies. The results show that the TRABD 2A-specific antibody, alone or in combination with an anti-PD-1 antibody, significantly enhanced CD8⁺The T cells have obvious killing effect on primary B cell lymphoma cells.

Example 6

Extracting punctured bone marrow of a patient, separating and purifying lymphocytes, culturing in vitro, adding different cancer antigens to stimulate and culture, and simultaneously culturing for 10 days by using an anti-TRABD2A monoclonal antibody or an anti-PD 1 monoclonal antibody to detect the survival rate of B lymph cancer cells.

Example 7

Extracting 50-100mL of blood from healthy people, and separating and purifying CD8⁺T cells, stimulated with Her 2-derived antigenic polypeptide, according to 2X 10⁶Density culture in/mL for 4 days, and adding 5X 10⁵McF-7/mL Breast cancer cells and varying concentrations of Ni²⁺The ions were cultured for 5 days, and then the survival rate of MCF-7 cancer cells was measured. The results showed that purified CD8 was isolated⁺T cells stimulated with Her 2-derived antigenic polypeptides at varying concentrations of Ni²⁺Has obvious killing effect on breast cancer cells MCF-7 in the presence of ions (mu M). The results are shown in FIG. 7.

Example 8

Extracting 50-100mL of blood from healthy people, and separating and purifying CD8⁺T cells, stimulated with Her 2-derived antigenic polypeptide, according to 2X 10⁶Activation was performed by density culture in/mL for 4 days, followed by addition of 5X 10⁵McF-7/mL Breast cancer cells and Co at various concentrations²⁺Ion culture for 5 days, and then determining the survival rate of MCF-7 cancer cells. The results show that isolated and purified CD8+ T cells were stimulated with Her 2-derived antigenic polypeptides at different concentrations of Co²⁺Has obvious killing effect on breast cancer cells MCF-7 in the presence of ions (mu M).

Example 9

Extracting 50-100mL of blood from healthy people, and separating and purifying CD8⁺T cells, stimulated with Her 2-derived antigenic polypeptide, according to 2X 10⁶Activation was performed by density culture in/mL for 4 days, followed by addition of 5X 10⁵MCF-7 and TRABD2A combined small molecule compound 1,10-phenanthroline in different concentrations are cultured for 5 days, and then the survival rate of MCF-7 cancer cells is measured. The results showed that purified CD8 was isolated⁺T cells after stimulation with Her 2-derived antigenic polypeptides in the presence of varying concentrations of TRABD2A protease in combination with the small molecule compound 1,10-phenanthroline (. mu.M)Has obvious killing effect on breast cancer cells MCF-7. The results are shown in Table 9.

Example 10

50-100mL of blood was taken from each of 5 untreated HIV-1 patients, 1-2mL of the blood was isolated and the viral load in the plasma was determined, Peripheral Blood Mononuclear Cells (PBMC) were extracted from the remaining blood using Ficoll, and HIV-1 infected CD4 was purified from the PBMC, respectively⁺And CD8⁺T cells, CD4⁺T cells were as per 3.5X 10⁶The cells were cultured in RPMI1640 medium containing 10% FBS serum and activated with PHA and IL2 for two days. At the same time, CD8 is added⁺T cells were as per 3.5X 10⁶The cells were cultured in RPMI1640 medium containing 10% FBS serum, and supplemented with Gag polypeptide population and IL 2. Separately culturing CD4⁺And CD8⁺After 3 days of T cells, the two were combined at a ratio of 4:1, and TRABD2A or blocking antibody to PD-1 and control IgG were added, and after further 5 days of continuous culture, CD4 was measured⁺Survival of T cells. The results show that specific anti-TRABD2A antibodies as TRABD2A inhibitors significantly enhanced CD8⁺CD4 of T cells against HIV-1 infection⁺The killing rate of T cells can obtain stronger killing effect when an anti-PD-1 antibody is used in combination. The results are shown in FIG. 10.

Example 11

Liver tissue was harvested by puncturing 3 untreated HBV patients with three-plus-one HBV, and after milling, lymphocyte populations were removed therefrom using CD43 and CD19 biomarkers, and hepatocytes were obtained using DMEM and 10% FBS serum at 1.0X 10⁵Culture in/mL. Simultaneously, CD8 was further isolated from the lymphocyte population⁺T cells, and converting CD8⁺After T cells were cultured in vitro with HBV-specific antigen and IL2 for 5 days, CD8 was added⁺T cells and hepatocytes the ratio of 4:1, and after 10 days of treatment with different antibodies, the survival rate of hepatocytes was determined. The results show that specific anti-TRABD2A antibodies as TRABD2A binding/inhibitors significantly enhanced CD8⁺The killing rate of T cells to liver cells from HBV patients with three-plus-1 HBV can obtain stronger killing effect when an anti-PD-1 antibody is used in combination. The results are shown in FIG. 11.

Example 12

Liver tissue was harvested by puncturing 2 untreated HCV positive patients and after milling, lymphocyte populations were removed from them using CD43 and CD19 biomarkers and hepatocytes were obtained using DMEM and 10% FBS serum at 1.0X 10⁵Culture in/mL. Simultaneously, CD8 was further isolated from the lymphocyte population⁺T cells, and converting CD8⁺After 5 days of in vitro T cell culture stimulated with HCV specific antigen and IL2, CD8 was added⁺T cells and hepatocytes the ratio of 4:1, and after 10 days of treatment with different antibodies, the survival rate of hepatocytes was determined. The results show that specific anti-TRABD2A antibodies as TRABD2A binding/inhibitors significantly enhanced CD8⁺The killing rate of T cells against hepatocytes from HCV-positive patients, in combination with anti-PD-1 antibodies, resulted in a stronger killing effect. The results are shown in FIG. 12.

Example 13

Extracting 50-100mL of blood from each of 5 untreated HIV-1 patients, collecting 1-2mL of the blood, separating plasma, measuring the virus load in the plasma, extracting Peripheral Blood Mononuclear Cells (PBMC) from the remaining blood with Ficoll, purifying CD4+ and CD8+ T cells from the PBMC, and separating CD4⁺T cells were as per 3.5X 10⁶The cells were cultured in RPMI1640 medium containing 10% FBS serum and activated with PHA and IL2 for two days. At the same time, CD8 is added⁺T cells were as per 3.5X 10⁶The cells were cultured in RPMI1640 medium containing 10% FBS serum, and supplemented with Gag polypeptide population and IL 2. Separately culturing CD4⁺And CD8⁺3 days after T cells, the two were combined at a ratio of 4:1 and the inhibitory ion Ni of TRABD2A protease was added²⁺(μM，NiCl₂) After further 5 days of continuous culture, CD4 was measured⁺Survival of T cells. The results showed that purified CD8 was isolated⁺T cells after activation with PHA and IL2, Ni at various concentrations²⁺CD4 for HIV-1 infection in the presence of ions (. mu.M)⁺T cells have obvious killing effect. The results are shown in FIG. 13.

Example 14

50-100mL of blood was drawn from each of 5 untreated HIV-1 patients,taking 1-2mL of the separated plasma, determining the virus load in the plasma, extracting Peripheral Blood Mononuclear Cells (PBMC) from the residual blood by using Ficoll, and respectively purifying CD4 from the PBMC⁺And CD8⁺T cells, CD4⁺T cells were as per 3.5X 10⁶The cells were cultured in RPMI1640 medium containing 10% FBS serum and activated with PHA and IL2 for two days. At the same time, CD8 is added⁺T cells were as per 3.5X 10⁶The cells were cultured in RPMI1640 medium containing 10% FBS serum, and supplemented with Gag polypeptide population and IL 2. Separately culturing CD4⁺And CD8⁺After 3 days of T cells, the two were combined at a ratio of 4:1 and the inhibitory ion Co of TRABD2A protease was added²⁺(μM，CoCl₂) After further 5 days of continuous culture, CD4 was measured⁺Survival of T cells. The results showed that purified CD8 was isolated⁺T cells after activation with PHA and IL2, Co at various concentrations²⁺CD4 in the presence of ions (. mu.M) in HIV-1 infected subjects⁺T cells have obvious killing effect. The results are shown in FIG. 14.

Example 15

50-100mL of blood was drawn from each of 5 untreated HIV-1 patients, 1-2mL of which was taken to separate plasma and determine the viral load in the plasma, Peripheral Blood Mononuclear Cells (PBMC) were extracted from the remaining blood with Ficoll, and CD4 was purified from the PBMC, respectively⁺And CD8⁺T cells, CD4⁺T cells were as per 3.5X 10⁶The cells were cultured in RPMI1640 medium containing 10% FBS serum and activated with PHA and IL2 for two days. At the same time, CD8 is added⁺T cells were as per 3.5X 10⁶The cells were cultured in RPMI1640 medium containing 10% FBS serum, and supplemented with Gag polypeptide population and IL 2. Separately culturing CD4⁺And CD8⁺3 days later, the T cells were combined at a ratio of 4:1, and 1,10-phenanthroline (μ M), which is an inhibitor of TRABD2A protease, was added thereto, and after further 5 days of continuous culture, CD4 was measured⁺Survival of T cells. The results showed that purified CD8 was isolated⁺T cells, after activation with PHA and IL2, were directed to CD4 in HIV-1 infected patients in the presence of various concentrations of 1,10-phenanthroline⁺T cells have obvious killing effect. The results are shown in FIG. 15.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (21)

1. A pharmaceutical composition comprising a binding/inhibitor against the membrane protein TRABD2A metalloprotease together with a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, further comprising an additional therapeutic agent.

3. The pharmaceutical composition of claim 2, wherein the additional therapeutic agent is an anti-PD-1 antibody.

4. Use of an inhibitor against the membrane protein TRABD2A metalloprotease for the manufacture of a medicament for the prevention or treatment of a tumor or viral infection.

5. The pharmaceutical composition according to any one of claims 1-3 or the use according to claim 4 wherein the binding agent is an antibody or antigen binding fragment thereof specific for TRABD2A metalloprotease, such as monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, receptors and chimeras and the like.

6. The pharmaceutical composition according to any one of claims 1-3 or the use according to claim 4, wherein the binding inhibitor is a specific small molecule agent inhibitor of TRABD2A metalloprotease, for example an inorganic compound inhibitor such as a nickel compound or a cobalt compound, or an organic compound inhibitor such as 1, 10-phenanthroline.

7. The use according to any one of claims 4-6, wherein the formulation is used in combination with a binding/inhibitor specific for PD-1, such as an anti-PD-1 antibody.

8. A pharmaceutical composition comprising a binding/inhibitor against the membrane protein TRABD2A metalloprotease and an anti-PD-1 antibody, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8 wherein the binding/inhibitor against the membrane protein TRABD2A metalloprotease is an antibody or antigen binding fragment thereof specific for TRABD2A metalloprotease, e.g. monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody, receptor and chimera or the like.

10. The pharmaceutical composition according to claim 8, wherein the binding/inhibitor against the membrane protein TRABD2A metalloprotease is a specific small molecule preparation of TRABD2A metalloprotease, e.g. an inorganic preparation such as a nickel containing compound or a cobalt containing compound, or an organic preparation such as 1, 10-phenanthroline.

11. The pharmaceutical composition of claim 8, wherein the inhibitor specific for PD-1 is an anti-PD-1 antibody or an antigen-binding fragment thereof, such as a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, or the like.

12. A kit comprising in separate containers or in the same container a binding/inhibitor for the membrane protein TRABD2A metalloprotease and an anti-PD-1 antibody, together with instructions for use.

13. The kit of claim 12 wherein the binding/inhibitor against the membrane protein TRABD2A metalloprotease is an antibody or antigen-binding fragment thereof specific for TRABD2A metalloprotease, e.g. monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody, receptor and chimera or the like.

14. The kit according to claim 13, wherein the binding/inhibitor against the membrane protein TRABD2A metalloprotease is a specific small molecule preparation of TRABD2A metalloprotease, e.g. an inorganic preparation such as a nickel containing compound or a cobalt containing compound, or an organic preparation such as 1, 10-phenanthroline.

15. The kit of claim 13, wherein the inhibitor specific for PD-1 is an anti-PD-1 antibody or an antigen-binding fragment thereof, such as a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, or the like.

16. The pharmaceutical composition or kit according to the preceding claims, wherein the pharmaceutical combination or kit is for use in the prevention or treatment of a tumor or a viral infection.

17. A method of treating or preventing a tumor or viral infection comprising administering to a subject in need thereof a binding/inhibitor to the membrane protein TRABD2A metalloprotease, and optionally an inhibitor specific for PD-1, such as an anti-PD-1 antibody or antigen-binding fragment thereof, and/or an additional therapeutic agent.

18. The method according to claim 17 wherein the binding/inhibitor against the membrane protein TRABD2A metalloprotease is an antibody or antigen binding fragment thereof specific for TRABD2A metalloprotease, e.g. a monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody etc., or a specific small molecule preparation of TRABD2A metalloprotease, e.g. an inorganic preparation such as a nickel compound or a cobalt compound, or an organic preparation such as 1,10-phenanthroline, or any combination thereof.

19. A method of editing the metalloprotease gene of the membrane protein TRABD2A which results in deletion or structural alteration of TRABD2A metalloprotease.

20. The use according to any one of claims 4-7, the pharmaceutical composition or kit of claim 16 or the method of any one of claims 17-18, wherein the tumor is selected from the following types: acute myeloid leukemia, adrenal tumors, AIDS-related cancers, soft tissue alveolar sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumors, breast cancer, carotid tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe cell renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrocytoma of the skin, desmoplastic small round cell tumor, ependymal tumor, Ewing's tumor, extraosseous myxoid chondrosarcoma, incomplete bone fibrogenesis, dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, malignant glioma, islet cell tumor, Kaposi's sarcoma, renal cancer, leukemia, lipoma/benign lipoma, liposarcoma/malignant lipoma, lipoma/malignant lipoma, Hepatoma, lymphoma, lung carcinoma, medulloblastoma, melanoma, meningioma, malignant mesothelioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, non-small cell lung carcinoma, ovarian carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, parathyroid tumor, pediatric carcinoma, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate carcinoma, retrouveal melanoma, rare hematological diseases, renal cell carcinoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin carcinoma, soft tissue sarcoma, squamous cell carcinoma, gastric carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid metastatic cancer, ovarian cancer, and uterine cancer.

21. The use according to any one of claims 4-7, the pharmaceutical composition or kit of claim 16, or the method of any one of claims 17-18, wherein the virus is selected from one or more of the following viruses: adenovirus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papilloma virus, BK virus, JC virus, smallpox virus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, Coxsackie virus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, Human Immunodeficiency Virus (HIV) (including HIV-1 and HIV-2), influenza virus, citrulline virus, Junin virus, lassa virus, Marburg virus, Sabiya virus, Crimiya-Congo hemorrhagic fever virus, Var fever, Var, ebola virus, marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, hendra virus, nipah virus, rabies virus, hepatitis delta virus, rotavirus, circovirus, collivirus, and Banna virus.

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