CN108079301B

CN108079301B - Application of active substance for reducing CXCL13 protein activity or expression quantity in preparing medicine for treating malignant tumor metastasis

Info

Publication number: CN108079301B
Application number: CN201611038204.XA
Authority: CN
Inventors: 魏于全; 任骏
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2016-11-23
Filing date: 2016-11-23
Publication date: 2021-03-02
Anticipated expiration: 2036-11-23
Also published as: CN108079301A

Abstract

The invention belongs to the field of medicines, and particularly relates to application of active substances such as antibody protein, nucleic acid sequence and small molecule inhibitor for reducing activity or expression of CXCL13 protein in preparation of a medicine for treating malignant tumor metastasis. The technical problem to be solved by the invention is to provide a targeted therapeutic scheme of immune regulatory molecules aiming at B cells. The invention provides application of an active substance for reducing the activity or expression quantity of CXCL13 protein in preparing a medicament for treating malignant tumor metastasis. Experiments prove that the CXCL13 blocker or blocking antibody can obviously inhibit lung metastasis of malignant tumor; the CXCL13 blocking agent or blocking antibody combined with chemotherapeutic medicine or T cell immune negative regulator has especially obvious effect of inhibiting malignant tumor lung metastasis. Can receive better synergistic effect. The invention provides a new effective choice for treating the malignant tumor metastasis.

Description

Application of active substance for reducing CXCL13 protein activity or expression quantity in preparing medicine for treating malignant tumor metastasis

Technical Field

The invention belongs to the field of tumor immunotherapy, and particularly relates to application of an active substance for reducing CXCL13 protein activity or expression quantity in preparation of a medicine for treating malignant tumor metastasis.

Background

Invasion and metastasis are the most important biological characteristics of life-threatening malignant tumors, and metastatic behavior is one of the most important features that distinguish malignant tumors from benign tumors. Metastasis refers to the whole process of malignant tumor cells breaking away from their primary site, transporting in vivo through various routes, reaching the discontinuous tissue of the primary site to continue proliferation and growth, and forming secondary tumor with the same pathological properties as the primary tumor. The occurrence of metastasis marks the key turning of tumor development, and once distant metastasis occurs, the tumor is usually in a late stage, and the aim of curing is difficult to achieve by local treatment alone. The major pathways for metastasis include: firstly, blood circulation is transferred; lymphatic migration; and thirdly, planting and transferring.

Taking malignant melanoma as an example, metastatic malignant melanoma has a poor prognosis, and statistics show that the median survival period of M1a is 15 months, the B1B period is 8 months, liver and brain metastasis is 4 months, bone metastasis is 6 months, the overall median survival time is 7.5 months, the 2-year survival rate is 15%, and the 5-year survival rate is about 5%. General treatment-based systemic treatments are generally recommended for stage III or metastatic melanoma that cannot be surgically resected, including imatinib, Iplilimumab, Vemurafenib, and high doses of IL-2. Other treatment options include dacarbazine, temozolomide, fotemustine, albumin paclitaxel, paclitaxel ± carboplatin, dacarbazine/temozolomide based combination regimens. In recent years, the treatment of advanced melanoma is favored, and personalized targeted therapy and immune targeted therapy are the hot spots of current research, while chemotherapeutic drugs are still important clinical treatment means.

CXCL13 is a chemokine of the CXC family, also known as B-lymphocyte chemokine, produced by follicular dendritic cells and the like. The receptor Is CXCR5, expressed on the surface of B lymphocytes (K. Mark Ansel et al. CXCL13 Is Required for B1 Cell Homing, Natural Antibody Production, and Body Cavity immunity. immunity, Vol.16, 67-76, January, 2002.). CXCL13-CXCR5 is capable of chemotactic B lymphocytes to migrate to the B zone of the germinal center to form normal lymphoid follicles. The B lymphocyte can secrete IL-10, and has effect of suppressing immune response.

In recent years, targeted therapy based on T cell immune negative regulators such as PD-1 and CTLA-4 has been developed in some tumor therapies, but there is still room for improvement in therapeutic effects.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a tumor targeted therapy scheme aiming at immune regulatory molecules of B cells.

The technical scheme for solving the technical problem is to provide the application of the active substance for reducing the activity or expression quantity of the CXCL13 protein in preparing the medicine for treating the malignant tumor metastasis.

Wherein the active substance is at least one of an antibody protein, a blocking agent, a small molecule inhibitor or a nucleic acid molecule which can reduce the activity or the expression amount of the CXCL13 protein.

Wherein, the antibody protein of the CXCL13 protein is at least one of a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody and a fully human antibody; or at least one of a domain polypeptide that binds to the CXCL13 protein in the above antibody or a recombinant protein comprising a domain polypeptide that binds to the CXCL13 protein in the above antibody.

Wherein, the antagonist of the CXCL13 protein is an antagonist for blocking the CXCL13 protein from being combined with the receptor CXCR5 thereof.

Wherein, the antagonist for blocking the CXCL13 protein from being combined with the receptor CXCR5 thereof is at least one of competitive antagonist or non-competitive antagonist of the CXCL13 protein.

Wherein, the active substance for reducing the CXCL13 expression level is an RNA interference active substance for interfering CXCL13 or an active substance for knocking out a gene encoding CXCL13 protein in a tumor patient.

The RNA interference active substance of CXCL13 is shRNA molecules, siRNA molecules and miRNA molecules aiming at CXCL13 genes or a vector capable of expressing the shRNA molecules, the siRNA molecules or the miRNA molecules.

Wherein, the active substance of the gene for knocking out the CXCL13 protein from the tumor tissue in the body of the patient is cas protein and at least one gRNA small molecule aiming at the CXCL13 gene; or a sequence-specific nuclease for knocking out the CXCL13 gene.

Wherein, the medicine for treating malignant tumor metastasis also contains at least one other anti-tumor active component.

Wherein, the other anti-tumor active components are tumor chemotherapy drugs or targeting antibody anti-cancer drugs.

Wherein the targeted antibody anticancer drug is at least one of bevacizumab (bevacizumab), aflibercept (aflibercept), Pertuzumab (Pertuzumab), Trastuzumab (Trastuzumab), Cetuximab (Cetuximab), Rituximab (Rituximab), Alemtuzumab (Alemtuzumab) or Panitumumab (Panitumumab).

Wherein, the targeted antibody anticancer drug is an antibody of a T cell immune negative regulatory factor.

Wherein the T cell immune negative regulator is an antibody against CTLA-4 (cytotoxic T lymphocyte-associated antigen-4), PD-1(programmed death receptor-1) or B7-H4.

Wherein, the tumor chemotherapy medicament is a cytotoxic tumor chemotherapy medicament.

Wherein the cytotoxic chemotherapeutic agent for tumor is cyclophosphamide, ifosfamide, mechlorethamine, formononetin, mechlorethamine bicine ethyl ester, melphalan, carmustine, lomustine, semustine, nimustine, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, mechlorethamine, antitumor sinapis, nitramustine, pyrimidine mustard, mechlorethamine, uracil mustard, mannitol mustard, phenylalanine mustard, dibromomannitol, fotemustine, nicarbamustard, 5-fluorouracil, 6-mercaptopurine, cytarabine, methotrexate, 6-thioguanine, actinomycin D, adriamycin, daunorubicin, taxol, vinblastine, colchicine, camptothecin, hydroxycamptothecin, irinotecan, and its salts, At least one of etoposide or teniposide, which is a podophyllotoxin.

Among them, the malignant tumor is a metastatic malignant tumor.

Wherein the malignant tumor easy to metastasize is at least one of melanoma, lung cancer, prostate cancer, breast cancer, rectal cancer, colon cancer, gastric cancer, ovarian cancer, pancreatic cancer or glioma, small cell lung cancer, non-small cell lung cancer, prostate cancer, breast cancer, rectal cancer, colon cancer, gastric cancer, ovarian cancer, pancreatic cancer, glioma, hepatocellular carcinoma, renal cancer, leukemia, sarcoma or lymphoma.

The invention further provides a medicine for treating malignant tumor metastasis. The medicine is prepared by taking an active substance for reducing the activity or expression quantity of CXCL13 protein and at least one other anti-tumor medicine component as active ingredients.

Wherein the active substance in the medicament is at least one of antibody protein, blocking agent, small molecule inhibitor or nucleic acid molecule which can reduce the activity or expression of CXCL13 protein.

The antibody of the CXCL13 protein in the medicine is at least one of a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a structural domain polypeptide which is combined with the CXCL13 protein in the antibody or a recombinant protein which contains the structural domain polypeptide which is combined with the CXCL13 protein in the antibody.

The antagonist of the CXCL13 protein in the medicine is an antagonist for blocking the combination of the CXCL13 protein and a receptor CXCR5 thereof.

The antagonist for blocking the CXCL13 protein from being combined with the receptor CXCR5 of the medicament is at least one of competitive antagonist or non-competitive antagonist of CXCL13 protein.

Wherein the active substance for reducing the CXCL13 expression level in the medicine is an RNA interference active substance for interfering CXCL13 or an active substance for knocking out a gene of a CXCL13 protein in a tumor patient.

Wherein the RNA interference active substance of CXCL13 in the medicine is shRNA molecule, siRNA molecule, miRNA molecule aiming at CXCL13 gene or a carrier capable of expressing the shRNA molecule, siRNA molecule or miRNA molecule.

Wherein, the active substance of the medicine for knocking out the gene for coding the CXCL13 protein from the tumor tissue in the body of the patient is cas protein and at least one gRNA small molecule aiming at the CXCL13 gene; or a sequence-specific nuclease for knocking out the CXCL13 gene.

Wherein, the other anti-tumor active ingredients in the medicine are tumor chemotherapy medicines or targeted antibody anti-cancer medicines.

Wherein the targeted antibody anticancer drug in the above drugs is at least one of bevacizumab (bevacizumab), aflibercept (aflibercept), Pertuzumab (Pertuzumab), Trastuzumab (Trastuzumab), Cetuximab (Cetuximab), Rituximab (Rituximab), Alemtuzumab (Alemtuzumab), or Panitumumab (Panitumumab).

Wherein the targeted antibody anticancer drug in the drug is an antibody of a T cell immune negative regulatory factor.

Wherein the T cell immune negative regulator in the medicine is an antibody against CTLA-4, PD-1 or B7-H4.

Wherein, the tumor chemotherapeutic drug in the drugs is a cytotoxic tumor chemotherapeutic drug.

Wherein the cytotoxic chemotherapeutic drugs for tumor in the above drugs are cyclophosphamide, ifosfamide, mechlorethamine, formononetin, foscarnosine bicine ethyl ester, melphalan, carmustine, lomustine, semustine, nimustine, chlorambucil, hexamethylmelamine, thiotepa, busulfan, methallyl mustard, methicillin, antitumor neomustard, nitragin, pyrimidine mustard, mechlorethamine, uracil mustard, mannitol mustard, phenylalanine mustard, dibromomannitol, fotemustine, alanine nitramustine, 5-fluorouracil, 6-mercaptopurine, cytarabine, methotrexate, 6-thioguanine, actinomycin D, adriamycin, daunorubicin, taxol, vinblastine, vincristine, colchicine, camptothecin, hydroxycamptothecin, vinclokaline, hydroxycamptothecin, methacin, carmustine, lomustine, at least one of irinotecan, etoposide, or teniposide, a podophyllotoxin, and a pharmaceutically acceptable salt thereof.

Wherein, the active substance for reducing the activity or the expression quantity of the CXCL13 protein and other antitumor drugs in the medicine are positioned in the same package or are respectively positioned in mutually independent packages.

The dosage form of the drug of the present invention may be various preparation types in the art suitable for the drug to exert its pharmacological effect. Obviously, since the drug for reducing the activity or expression level of CXCL13 protein and the other anti-tumor drug are separately packaged in separate packages, they may be in the same dosage form or different dosage forms.

In another aspect of the invention, a method of treating metastasis from a malignant tumor is provided. The method is to treat metastasis of a malignant tumor by administering to the patient an effective amount of an active substance and/or therapeutic method that reduces the activity or expression of a CXCL13 protein.

Wherein the active substance in the method is at least one of an antibody protein, a blocking agent, a small molecule inhibitor or a nucleic acid molecule which can reduce the activity or the expression amount of the CXCL13 protein.

The antibody of the CXCL13 protein in the above method is at least one of a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a domain polypeptide in the antibody that binds to the CXCL13 protein, or a recombinant protein containing a domain polypeptide in the antibody that binds to the CXCL13 protein.

Wherein, the antagonist of CXCL13 protein in the method is an antagonist for blocking CXCL13 protein from binding with a receptor CXCR5 thereof.

Wherein the antagonist for blocking the binding of CXCL13 protein and its receptor CXCR5 in the above method is at least one of competitive antagonist or non-competitive antagonist of CXCL13 protein.

Wherein the active substance for reducing the expression level of CXCL13 in the method is an RNA interference active substance for interfering CXCL13 or an active substance for knocking out a gene encoding CXCL13 protein in a tumor patient.

Wherein the RNA interference active substance of CXCL13 in the method is an shRNA molecule, an siRNA molecule, an miRNA molecule aiming at CXCL13 gene or a vector capable of expressing the shRNA molecule, the siRNA molecule or the miRNA molecule.

Wherein, the active substances for knocking out the gene coding CXCL13 protein from the tumor tissues in the body of the patient are cas protein and at least one gRNA small molecule aiming at CXCL13 gene; or a sequence-specific nuclease for knocking out the CXCL13 gene.

Further, the above methods are administered to the patient during the period of time of an effective amount of a medicament and/or method of treatment that reduces the activity or expression of a CXCL13 protein; at least one additional antineoplastic agent component and/or method of treatment is also administered to the patient in an effective amount.

The other anti-tumor active components are tumor chemotherapeutic drugs or targeting antibody anti-cancer drugs.

Wherein the targeted antibody anticancer drug in the above drugs is at least one of bevacizumab (bevacizumab), aflibercept (aflibercept), Pertuzumab (Pertuzumab), Trastuzumab (Trastuzumab), Cetuximab (Cetuximab), Rituximab (Rituximab), Alemtuzumab (Alemtuzumab) or Panitumumab (Panitumumab).

In the present invention, the administration of an active substance or a drug means that the drug is introduced into a patient to act by injection, oral administration, mucosal administration, or the like.

The treatment of malignant tumor metastasis in the above embodiments of the present invention refers to a process of reducing or preventing malignant tumor cells from continuing to grow from a primary site, via lymphatic channels, blood vessels, body cavities, or other pathways, to other sites.

The malignant tumor is a metastatic malignant tumor. Specifically, the malignant tumor susceptible to metastasis is at least one of melanoma, small cell lung cancer, non-small cell lung cancer, prostate cancer, breast cancer, rectal cancer, colon cancer, gastric cancer, ovarian cancer, pancreatic cancer, glioma, hepatocellular carcinoma, renal cancer, leukemia, sarcoma, or lymphoma.

It is understood by those skilled in the art that the active substance for reducing the activity or expression level of CXCL13 protein is capable of reducing the activity or expression level of human CXCL13 protein, and the treatment of malignant tumor metastasis is for treating human malignant tumor metastasis.

The invention has the beneficial effects that: the invention creatively discovers that the reduction of CXCL13 gene expression or the blocking of the activity of CXCL13 protein can better treat tumor metastasis, and develops a new technical scheme for treating the tumor metastasis on the basis. The medicine and the method for treating tumor metastasis provided by the invention have good effects through the verification of metastasis models of various tumors, and more unexpectedly, the medicine and the method are combined with chemotherapeutic medicines or blocking antibody medicines aiming at a T cell immune negative regulatory factor PD-1 to achieve more ideal treatment effects, provide a new strategy for treating tumor metastasis by targeting B cells, and have good application prospects.

Drawings

FIG. 1 shows experimental pulmonary metastasis in leukemia 14 days after knockout of CXCL13 gene. Wherein, the figure 1-A is a lung gross image, the figure 1-B is the statistics of the number of tumor metastasis nodules on the lung, and the figure 1-C is the H & E staining of lung pathological sections. The results show that the tumor node number of melanoma blood metastasis to lung is obviously reduced after the CXCL13 gene is knocked out.

FIG. 2 shows experimental pulmonary metastasis in blood for 21 days after knockout of CXCL13 gene. Wherein FIG. 2-A is a lung gross image, FIG. 2-B is a statistics of tumor metastasis nodules on the lung, and FIG. 2-C is H & E staining of lung pathological sections. The results show that the tumor node number of melanoma blood metastasis to lung and lung metastasis after knockout of the CXCL13 gene is significantly reduced.

The first and second graphs demonstrate that CXCL13 is knocked out, and the process of melanoma lung metastasis can be obviously inhibited.

FIG. 3 shows experimental pulmonary metastasis with Lewis lung cancer treated by knocking-out CXCL13 gene for 21 days. Wherein, FIG. 3-A is the lung gross picture, FIG. 3-B is the lung and the metastasis weighing, and FIG. 3-C is the H & E staining of the lung pathological section. The result shows that the blood circulation metastasis of the Lewis lung cancer cell can be obviously reduced after CXCL13 is knocked out.

FIG. 4 shows spontaneous lung metastasis of Lewis lung cancer treated after knockout of the CXCL13 gene. Wherein, FIG. 4-A is a lung gross image, FIG. 4-B is a tumor metastasis nodule number statistic on lung, and FIG. 4-C is lung pathological section H & E staining. After the CXCL13 gene is knocked out, spontaneous transfer of Lewis lung cancer cells is obviously inhibited.

Figure 5 is the activation of anti-tumor immunity in the tumor metastasis microenvironment after knock-out CXCL13 gene therapy.

Wherein FIG. 5-A is a reduction in metastases of regulatory B cells capable of down-regulating anti-tumor immunity by suppressing T cell activation in the tumor microenvironment via secretion of interleukin-10. Fig. 5-B is an increase in activated CD4 positive helper T cells in the tumor microenvironment and fig. 5-C is an increase in activated CD8 positive cytotoxic T cells, demonstrating that down-regulation of regulatory B cells in fig. 5-a enhances the tumor-killing T cell immune response in the tumor microenvironment, explaining the reason for reduced tumor metastasis following CXCL13 knockout. FIGS. 5-D, 5-E, 5-F, and 5-G show the changes in mRNA levels of transforming growth factor (TGF-. beta.), Perforin (Perforin), Interferon-. gamma.and Granzyme B (Granzyme-B) in the metastases, respectively, by real-time fluorescent quantitative PCR. The down regulation of transforming growth factor can effectively enhance the anti-tumor immunity of T cells. Meanwhile, perforin, granzyme B and interferon-gamma are factors secreted by T lymphocytes in tumor killing, and the enhancement of the expression of the factors indicates the enhancement of anti-tumor immunity.

FIG. 6 shows that CXCL13 gene knockout is combined with cyclophosphamide as a chemotherapeutic agent to treat melanoma metastasis. Wherein FIG. 6-A is a lung gross image, FIG. 6-B is a statistics of tumor metastasis nodules on the lung, and FIG. 6-C is H & E staining of lung pathological sections. The result shows that the knockout of the CXCL13 gene or the single use of cyclophosphamide can inhibit the blood circulation metastasis of melanoma, but the knockout of the CXCL13 gene and the combined use of the chemotherapy drug cyclophosphamide can achieve very good synergistic effect and remarkably inhibit the lung metastasis of melanoma.

FIG. 7 shows that CXCL13 gene knockout and monoclonal antibody anti-PD-1 are combined to treat melanoma metastasis in blood circulation. Wherein FIG. 7-A is a lung gross image, FIG. 7-B is a statistics of tumor metastasis nodules on the lung, and FIG. 7-C is H & E staining of lung pathological sections. The results show that CXCL13 knockout or the administration of an anti-PD-1 antibody alone significantly inhibited melanoma hematogenous metastasis. The CXCL13 knockout effect can be very good after the anti-PD-1 antibody is combined. CXCL13 was knocked out to down-regulate inhibitory B cells, which, when combined with anti-PD-1 antibodies, synergistically amplified the effective anti-tumor T cell response.

FIG. 8 is a schematic representation of the treatment of melanoma hematogenous metastasis with the combination of the CXCL13 antibody and a monoclonal anti-PD-1 antibody. The results show that the anti-CXCL13 antibody or the anti-PD-1 antibody has obvious effect of inhibiting the melanoma metastasis alone, the combined medication can inhibit the melanoma metastasis more obviously, and the efficacy of the anti-CXCL13 antibody is proved in the prior art.

Detailed Description

In exploring the course of treatment of immune diseases, the present inventors noted infiltration of B lymphocytes in some metastases of malignant tumors and an increase in the B lymphocyte chemokine CXCL13 in peripheral blood. Some B lymphocytes are capable of secreting IL-10 and thus act to down-regulate the immune response, and this population of cells generally appears to be CD19 and IL-10 positive. Based on a large amount of creative work, the inventor of the invention finds that the effect of effectively inhibiting malignant tumor lung metastasis after CXCL13 gene knockout or CXCL13 protein activity blocking can be realized, and corresponding antagonist medicines can be developed to treat malignant tumor metastasis based on the effect.

A large number of further research experiments are carried out, and the CXCL13 gene is knocked out to obviously inhibit the hematogenous metastasis of malignant tumors, including experimental lung metastasis and spontaneous lung metastasis models of melanoma and Lewis lung cancer. And the method for down-regulating or knocking out the CXCL13 gene or blocking the CXCL13 protein can be combined with chemotherapeutic drugs of cyclophosphamide and PD-1 blocking antibody drugs for application, and can achieve obviously better curative effect. Particularly, in one embodiment of the invention, the monoclonal antibody of the CXCL13 protein and the monoclonal antibody of PD-1 are used in combination, and the effect of resisting tumor metastasis is greatly enhanced. Therefore, the skilled person has good reason to believe that the technical scheme of the invention has obvious curative effect in the treatment of various malignant tumor metastasis, and especially has good prospect in the treatment of malignant tumor which is easy to metastasize, such as melanoma, small cell lung cancer, non-small cell lung cancer, prostate cancer, breast cancer, rectal cancer, colon cancer, gastric cancer, ovarian cancer, pancreatic cancer, glioma, hepatocellular carcinoma, renal carcinoma, leukemia, sarcoma or lymphoma, and the like.

On the basis of the creative work, the first aspect of the invention provides the application of an active substance for reducing the activity or expression quantity of CXCL13 protein in preparing a medicine for treating malignant tumor metastasis. The active substances are antibody protein, a blocking agent, a small molecule inhibitor and a nucleic acid sequence which can reduce the activity or the expression quantity of CXCL13 protein. After reading the present disclosure, those skilled in the art will readily appreciate that the antibody protein, blocking agent, small molecule inhibitor and nucleic acid sequence capable of reducing the activity or expression level of CXCL13 protein can be used for preparing and obtaining a good application for treating malignant tumor metastasis.

For example, at least one of antibody protein of CXCL13 protein, such as monoclonal antibody, polyclonal antibody, single-chain antibody or recombinant protein containing the structure domain of the antibody combined with CXCL13 protein can be used for preparing the medicine for treating malignant tumor metastasis and has good effect.

The monoclonal antibody may be a specific antibody that recognizes a particular determinant of an antigenic molecule. The monoclonal antibody also includes genetically engineered antibodies and the like. Also included are antibody fragments that have biological activity. For example, antibodies raised against mouse or other species may be humanized to replace some of the human-derived fragments, and thus antibodies raised against human CXCL13 protein are within the scope of the invention.

For example, antagonists of CXCL13 protein, especially antagonists for blocking CXCL13 protein from binding with its receptor CXCR5, can be used for preparing medicaments for treating malignant tumor metastasis and have good effects. And these antagonists may be selected from competitive antagonists or non-competitive antagonists of CXCL13 protein.

For example, an active substance capable of reducing the expression level of CXCL13, such as an RNA interference sequence for encoding a CXCL13 protein gene or an active substance for knocking out a CXCL13 protein gene from a patient body, can be used for preparing a medicament for treating malignant tumor metastasis and has a good effect. Such as shRNA, siRNA, miRNA molecules directed against CXCL13 protein-encoding genes, and various expression vectors that can express interfering molecules.

The knockout of the CXCL13 protein gene-encoding active substance from a patient, particularly from tumor tissue, can be a criprpr/cas system for knockout of the CXCL13 gene, and generally includes one or more gRNA small molecules and cas proteins (e.g., cas9 protein) for the CXCL13 gene. It may also be an active substance used in other various means for targeting gene editing.

The knockout of the active substance encoding the CXCL13 protein gene in a patient may also be a sequence-specific nuclease for knocking out the CXCL13 gene. For example, sequence-specific nucleases such as ZFN (zinc finger nuclease), TALEN and the like designed and expressed for knocking out the CXCL13 gene are currently available for preparing drugs for treating malignant tumor metastasis and achieving good effects.

Furthermore, based on the above inventive studies, those skilled in the art will recognize that the above-mentioned medicament comprising an active substance that reduces the activity or expression level of CXCL13 protein may further comprise at least one other anti-tumor active ingredient.

And the other anti-tumor active ingredients can be anti-tumor drugs of tumor chemotherapy drugs or targeting antibodies.

There are currently a number of targeted antibody anti-cancer drugs in the art, such as bevacizumab (bevacizumab), aflibercept (aflibercept), Pertuzumab (Pertuzumab), Trastuzumab (Trastuzumab), Cetuximab (Cetuximab), Rituximab (Rituximab), Alemtuzumab (Alemtuzumab), Panitumumab (Panitumumab), and the like. The medicines can be used together with active substances for reducing the activity or expression quantity of CXCL13 protein, so that a better effect of treating malignant tumor metastasis is achieved.

In particular, the targeting antibody anti-cancer drugs can be selected from antibodies of T cell immune negative regulators. For example, antibodies against CTLA-4, PD-1 or B7-H4.

The tumor chemotherapy drugs have more choices, and particularly, cytotoxic tumor chemotherapy drugs can be selected.

The cytotoxic tumor chemotherapy drug can be selected from cyclophosphamide, ifosfamide, mechlorethamine, formononetin, fosetyl diglycine ethyl ester of phosphoramide mechlorethamine, melphalan, carmustine, lomustine, semustine, nimustine, chlorambucil, hexamethylmelamine, thiotepa, Marilan, carmustine, mechlorethamine, antitumor sinapis, nitragin, pyrimidine mustard, mechlorethamine, uracil mustard, mannitol mustard, phenylalanine mustard, dibromo mannitol, fotemustine, alanine nitramustine, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside and other drugs.

Yet another aspect of the present invention is to provide a medicament for treating metastasis of malignant tumor. The medicament comprises the active substance for reducing the activity or expression of CXCL13 protein and at least one other anti-tumor medicament component as active ingredients.

It is understood that, due to the differences in the properties of the drugs themselves and the differences in the administration routes, dosage amounts, administration times, etc., the active substance for reducing the activity or expression amount of the CXCL13 protein and at least one other anti-tumor drug in the drug of the present invention may be mixed together to form a formulation, or may be separately formed into formulations; they may be in the same package or in separate packages. This is because their respective dosage forms may be the same or different. This is adjustable and selectable by the person skilled in the art as a function of the circumstances.

In a third aspect of the invention, a method of treating metastasis from a malignant tumor is provided. The method is effective in reducing the activity or expression level of CXCL13 protein. One skilled in the art will appreciate that the method includes, but is not limited to, administering to a patient an effective amount of a medicament that reduces the activity or expression of a CXCL13 protein.

Further, the methods of the invention are performed during the administration of an effective amount of a medicament and/or method of treatment that reduces the activity or expression of a CXCL13 protein; at least one additional antineoplastic agent component and/or method of treatment is also administered to the patient in an effective amount.

The treatment of malignant tumor metastasis in the above technical solutions of the present invention refers to a process of reducing or blocking malignant tumor cells from growing from the primary site, passing through the lymphatic channel, blood vessels or body cavities, and reaching other sites to continue growing. The malignant tumor is easy-to-transfer malignant tumor. However, currently recognized metastatic malignancies are represented by: melanoma, small cell lung cancer, non-small cell lung cancer, prostate cancer, breast cancer, rectal cancer, colon cancer, gastric cancer, ovarian cancer, pancreatic cancer, glioma, hepatocellular carcinoma, renal cancer, leukemia, sarcoma, lymphoma, etc.

The following examples are intended to illustrate specific embodiments of the present invention. The experimental procedures, reagents and instruments used in the following examples were all routinely selected unless otherwise specified.

The reagents and equipment used in the examples were as follows:

first, main reagent

1. Real-time fluorescent quantitative PCR reagents, consumables (BIO-Rad, USA) and primers (purchased from Huada gene): interferon-gamma (IFN-gamma), Perforin (Perforin), Granzyme-b (Granzyme-b) transforming growth factor-beta 1 (TGF-beta 1), 18s RNA as reference. RNA extraction kit (Axygen, usa); reverse transcription kit (Takara Co.).

2. DMEM medium (Gibco, usa), fetal bovine serum (Gibco, usa), physiological saline (korn pharmaceuticals).

3. Medicine preparation: monoclonal anti-PD-1mab (BioXcell, USA), cyclophosphamide CTX (Melphalan), monoclonal anti-mouse CXCL13mab (R & D, USA)

4. Flow-through antibody: APC-labeled CD45(BD Pharmingen, usa), PE-labeled CD19(BD Pharmingen, usa), FITC-labeled IL-10(BD Pharmingen, usa), FITC-labeled CD8 α (BD Pharmingen, usa), APC-labeled CD4(BD Pharmingen, usa), PE-labeled CD69(BD Pharmingen, usa), percp. cy5.5-labeled CD3e (BioLegend, usa).

5. Flow intracellular staining kit: BD Cytofix/Cytoperm Plus fire/Permeabilization Kit (With BD Golgi protein transport in inhibitor relating monensin) (BD Bioscience, USA).

Second, instrument

1. Superclean benches (sujing group);

2. flow cytometer BD FACS calibur (BD Bioscience, usa);

3.15ml centrifuge tubes (BD Filcon, usa), 50ml centrifuge tubes (BD Filcon, usa), 100mm cell culture plates (lying macrobiosis);

4. inverted microscope (Olympus, japan); upright microscope (Leica, germany);

5. a micro-adjustable pipette (Eppendorf corporation, germany);

6. liquid nitrogen tanks, refrigerators;

constant temperature of 7.37 ℃ and 5% CO₂Cell culture chambers (Thermo corporation, usa);

8. ophthalmic scissors, ophthalmic forceps;

9. horizontal centrifuges (Thermo corporation, usa);

quantitative Real-Time PCR CFX96(BioRad, USA).

Third, experimental animal

1. Female C57BL/6 mice (purchased from Vantotony, Beijing) for 6-7 weeks;

CXCL 13-/-mice (purchased from the Jackson laboratory, USA)

Example I Experimental lung metastasis experiment for melanoma

1. Tumor cell culture and establishment of a metastatic tumor model:

(1) culturing melanoma B16 cells in DMEM medium (containing 10% fetal calf serum), digesting the cells in the plate at 37 ℃ for about 2 minutes when the cells grow to about 50-70%, neutralizing the culture medium, blowing and beating the residual adherent cells, transferring the cells into a 15ml conical centrifuge tube, centrifuging the cells for 3 minutes at 1500 rpm, and discarding the supernatant.

(2) Serum-free DMEM medium was used to resuspend the washed cells and centrifuged for 3 minutes at 1500 rpm. And repeating the steps once.

(3) Cell counts, resuspension concentration 1x10⁶Per mL, 2X 10 per old rat tail vein ⁵200 μ L of tumor cells.

(4) Mice were sacrificed by cervical dislocation 14 or 21 days after tumor cell inoculation, lungs were removed by opening the chest cavity, and blood clots were washed away with normal saline and serum-free medium. And (5) photographing, counting the number of lung metastasis nodules or weighing.

2. Paraffin section:

(1) the metastatic tumor tissue was placed in an embedding cassette and fixed by immersion in 4% paraformaldehyde solution for 48 hours.

(2) After fixation, the cells were rinsed overnight with tap water.

(3) Gradient dehydration: dehydration in 75% alcohol for 1 hour, dehydration in 85% alcohol for 1 hour, dehydration in 95% alcohol for 1 hour, and dehydration in 100% alcohol for one hour.

(4) Immersed in xylene for one hour.

(5) The paraffin was soaked for two hours.

(7) The embedding cassette was placed in fresh wax and embedded with an embedding machine.

(8) After the wax block was cooled, it was placed on ice and sliced on a microtome. The slice thickness was 3 microns. The temperature of the bleaching piece is 42 ℃, and the temperature of the spreading piece is 65 ℃.

(9) And after redundant paraffin on the slices is baked, placing the slices on a slice copper frame, and dewaxing and hydrating. Soaking in xylene for 20 min, 100% ethanol for 2 min, 95% ethanol for 2 min, 85% ethanol for 2 min, and 75% ethanol for 2 min, and rinsing in distilled water for 10 min.

H & E staining:

(1) hematoxylin staining was performed for 1 minute.

(2) The basket was rinsed with tap water for 10 minutes.

(3) Soaking in 75% alcohol solution of hydrochloric acid for 10 sec.

(4) Tap water was flushed for 30 seconds.

(5) Eosin stain for 40 seconds.

(6) Rinse with tap water for 30 seconds.

(7) After slicing and air drying, the neutral gum is sealed. And (4) observing under a light mirror.

4. Flow cytometry analysis:

(1) fresh metastatic tumor tissue was harvested, minced with an ophthalmic scissors, and digested with 10mL collagenase (1mg collagenase/mL serum-free 1640 medium) at 37 ℃ for 2 hours.

(2) The digested tissue cell suspension was filtered through a 70-micron screen to prepare a single cell suspension.

(3) Centrifuge for 3 minutes at 1500 rpm and discard the supernatant. 5mL of erythrocyte lysate was added to lyse the erythrocytes for 3 minutes.

(4) Centrifuge for 3 minutes at 1500 rpm and discard the supernatant. Cells were washed twice with PBS.

(5) After the cells were reselected with PBS, they were dispensed into flow tubes. Each tube 1X10⁶And (4) cells.

(6) Marker staining on cell membrane: add 1. mu.L of fluorescence labeled antibody into each tube, mix them well, and react for 30 minutes at 4 ℃ in the dark. Add 1mL PBS to each tube and centrifuge at 1500 rpm for 3 minutes. The supernatant was discarded. And repeating the steps. Add 100. mu.L PBS to each tube, and test on the machine.

(7) Intracellular cytokine staining: after the marker on the cell membrane is dyed, 250 mu L of fixed perforating fluid is added into each tube, and the tube is protected from light at 4 ℃ for 30 minutes. 1mL of wash solution was added to each tube, centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the procedure was repeated once. Add 1. mu.L of fluorescence labeled antibody into each tube, mix them well, and react for 30 minutes at 4 ℃ in the dark. 1mL of wash solution was added to each tube and centrifuged at 1500 rpm for 3 minutes. The supernatant was discarded. And repeating the steps. Add 100. mu.L PBS to each tube, and test on the machine. The fixed punch solution and wash solution come from a flow intracellular staining kit: BD Cytofix/Cytoperm Plus fire/Permeabilization Kit (With BD Golgi protein transport in inhibitor relating Monensin) (BD Bioscience).

The experiment was repeated 3 times in total.

5. Real-time fluorescent quantitative PCR:

(1) total RNA extraction:

a. tumor tissue was ground to a powder in liquid nitrogen.

b. When tissue RNA is extracted, 1ml of Trizol reagent is used for cracking the tissue every 50-100 mg of the tissue; when extracting cell RNA, firstly centrifuging to precipitate cells, and centrifuging at each 5-10 gamma 10⁶After adding 1ml of Trizol to each cell, repeatedly blowing with a gun or vigorously shaking to lyse the cells;

c. transferring the Trizol lysate of the tissues or cells into an EP tube, and placing the Trizol lysate at room temperature (15-30 ℃) for 5 minutes;

d. adding chloroform into the EP tube according to the amount of 0.2ml of chloroform added into 1ml of TRIZOL, covering a cover of the EP tube, shaking the mixture in hands for 15 seconds, standing the mixture at room temperature (15-30 ℃) for 2-3 minutes, and centrifuging 12000g (2-8 ℃) for 15 minutes;

e. placing the upper water phase in a new EP tube, adding 0.5ml of isopropanol into 1ml of TRIZOL, placing for 10 minutes at room temperature (15-30 ℃), and centrifuging for 10 minutes at 12000g (2-8 ℃);

f. discarding the supernatant, washing by adding 1ml of 75% ethanol into 1ml of TRIZOL, mixing by vortex, centrifuging for 5 minutes at 7500g (2-8 ℃), and discarding the supernatant;

g. allowing the precipitated RNA to dry naturally at room temperature;

h. the RNA pellet was dissolved with RNase-free water.

(2) Sampling, reverse transcription and fluorescent real-time quantitative PCR are carried out according to the kit operation manual

6. And (4) analyzing results:

the result shows that the CXCL13 gene knockout can remarkably inhibit the hematogenous pulmonary metastasis of melanoma, the number of nodules transferred to the lung by the melanoma is greatly reduced, and meanwhile, paraffin sections and H are cut&The large reduction of tumor cells in the lung was also seen in the E section under light (see FIGS. 1 and 2). Flow cytometry analyzed the relevant changes of lymphocytes in the tumor microenvironment (see figure 5). Flow cytometry detection finds a reduction in metastases of inhibitory B cells, which are able to suppress T cell activation in the tumor microenvironment by secreting interleukin-10, thereby down regulating anti-tumor immunity. Activated CD4⁺Helper T cell and activated CD8⁺The increase of killer T cells proves that the T cell immune response of killing tumors in the tumor microenvironment is enhanced after the down regulation of inhibitory B cells, and explains the reason of the reduction of tumor metastasis after the CXCL13 is knocked out. Real-time fluorescent quantitative PCR was performed to detect the changes in mRNA levels of transforming growth factor (TGF-. beta.), Perforin (Perforin), Interferon-. gamma. (Interferon-. gamma.) and Granzyme B (Granzyme-B) in the metastases, as shown in FIGS. 5-D, 5-E, 5-F and 5-G. Transforming growth factor is an immunoregulatory molecule, the down-regulation of which is effective in enhancing the anti-tumor immunity of T cells. Meanwhile, perforin, granzyme B and interferon-gamma are factors secreted by T lymphocytes during tumor killing, the interferon gamma can further amplify effective anti-tumor reaction, and the perforin and granzyme participate in killing and cracking of tumor cells. Therefore, the reduction of infiltration of the inhibitory B cells in a tumor microenvironment is caused after the CXCL13 is knocked out, so that the activation of effective anti-tumor T cells in the tumor microenvironment is increased, the anti-tumor immune response is enhanced, and the tumor metastasis is obviously reduced.

EXAMPLE II Experimental pulmonary metastasis experiments in Lewis lung carcinoma

1. Tumor cell culture and establishment of a metastatic tumor model:

(1) the Lewis lung cancer LL2 cells are cultured in a DMEM medium (containing 10% fetal calf serum), when the cells grow to about 50-70% in a plate, the cells are digested for about 1 minute at 37 ℃, residual adherent cells are blown and beaten after the cells are neutralized in the medium, the cells are transferred into a 15ml conical centrifuge tube, the cells are centrifuged for 3 minutes at 1500 revolutions, and the supernatant is discarded.

Mice were sacrificed by cervical dislocation 21 days after tumor cell inoculation, lungs were removed by opening the chest cavity, and blood clots were washed away with saline and serum-free medium. And taking a picture and weighing.

2. Paraffin sections and H & E staining. (same as in example 1)

The experiment was repeated 3 times in total.

3. And (4) analyzing results: after the CXCL13 gene is knocked out, the hematogenous metastasis of the Lewis lung cancer can be obviously inhibited. Thus, treatment based on blocking or silencing CXCL13 is not melanoma-specific or certain tumor-specific. It is based on the principle of down-regulating the amount of infiltrating inhibitory B cells in metastases, so that the therapeutic effect is applicable to a variety of tumor metastases.

EXAMPLE III Lewis Lung cancer spontaneous Lung metastasis experiment

1. Tumor cell culture and spontaneous metastasis model establishment:

(3) Cell counts, resuspension concentration 1x10⁷/mL, 1x10 subcutaneous administration per mouse⁶Tumor cells, 100. mu.L.

(4) Subcutaneous tumors were surgically removed 14 days after tumor cell inoculation.

(5) 11 days after the surgical removal of subcutaneous tumors, the mice were sacrificed by cervical dislocation, the thoracic cavity was opened to remove the lungs, and the blood clots were washed away with physiological saline and serum-free medium. And (5) photographing and counting the number of the lung metastasis nodules.

2. Paraffin sections and H & E staining. As in example 1.

3. And (4) analyzing results: the spontaneous metastasis model better simulates the process of blood-based metastasis from the primary site to the lung during tumor metastasis. On the other hand, the potential value of treatment based on blocking or silencing CXCL13 was demonstrated. Knockout of CXCL13 significantly inhibited spontaneous hematogenous metastasis of lewis lung cancer cells, as shown in fig. 3.

EXAMPLE four treatment of melanoma metastasis with cyclophosphamide in combination with chemotherapy

Cyclophosphamide is the most commonly used alkylating antineoplastic agent. Cyclophosphamide has no antitumor activity in vitro, andafter entering the body, it passes through the liver firstMicroparticlesFunction(s)Oxidase enzymeConverted into aldehyde phosphoramide. The aldehydic amide is unstable and is decomposed into amide nitrogen mustard and acrolein in tumor cells, and the amide nitrogen mustard has cytotoxic effect on the tumor cells. Cyclophosphamide is a bifunctional alkylating agent andcell cycle non-specific drugsIt can interfere DNA and RNA functions, especially has larger influence on DNA, cross-links with DNA, inhibits DNA synthesis, and has most obvious effect on S phase.

1. Tumor cell culture and metastasis model establishment:

2. Treatment with cyclophosphamide:

(1) after inoculation of tumor cells day0, day2, day4, 2mg of cyclophosphamide was intraperitoneally injected into each mouse. The control group was given solvent PBS.

(2) Mice were sacrificed by cervical dislocation 14 days after tumor cell inoculation, lungs were removed by opening the chest cavity, and blood clots were washed away with saline and serum-free medium. And (5) photographing and counting the number of the lung metastasis nodules.

3. Paraffin sections and H & E staining. (same as in example 1)

4. And (4) analyzing results: chemotherapy drugs often have the problems of limited curative effect, easy generation of drug resistance, incapability of radically treating tumors and the like, and have strong toxicity. After CXCL13 is knocked out, the excellent treatment effect can be achieved by combining cyclophosphamide treatment, and the tumor load of tumor metastasis is reduced to a very small level, as shown in figure 6. When cyclophosphamide kills tumor cells, the down regulation of inhibitory B cells and the subsequent amplification of effective anti-tumor T cell immune response are caused after CXCL13 is knocked out, and the two synergistic effects inhibit the hematogenous metastasis of melanoma.

EXAMPLE V treatment of melanoma metastasis with combination of the monotherapy anti-PD-1

PD-1(programmed death 1) programmed death receptor 1, an important immunosuppressive molecule, is expressed on the surface of T cells. Tumor cells evade immune surveillance often bind to PD-1 on the surface of T cells via PD-L1 on their surface, thereby inhibiting killing of the tumor by T cells. The anti-PD-1 antibody has already made a certain progress in malignant melanoma and non-small cell lung cancer, but the treatment effect of the anti-PD-1 antibody still has a great space for improvement.

1. Tumor cell culture and metastasis model establishment:

2. anti-PD-1mab treatment:

(1) after inoculation of tumor cells, day1 and day3 were intraperitoneally injected with anti-PD-1 monoclonal antibody at 200. mu.g per mouse, and control IgG2a control antibody was administered at 200. mu.g per mouse. And (5) carrying out intraperitoneal injection.

3. Paraffin sections and H & E staining. (same method as in example 1)

4. And (4) analyzing results: the results show that CXCL13 knockout or the administration of an anti-PD-1 antibody alone significantly inhibited melanoma hematogenous metastasis. The anti-PD-1 monoclonal antibody can block the combination of PD-L1 on the surface of tumor cells and PD-1 on the surface of T cells, so that the contact of tumor cells can inhibit the response of effective anti-tumor T cells. The CXCL13 knockout effect can be very good after the anti-PD-1 antibody is combined. After CXCL13 is knocked out, inhibitory B cells are down-regulated, and after the CXCL13 is combined with an anti-PD-1 antibody, effective anti-tumor T cell reaction is synergistically amplified.

EXAMPLE sixthly, anti-CXCL13 combination with single drug resistant anti-PD-1 for the treatment of melanoma metastasis

1. Tumor cell culture and metastasis model establishment:

2. Antibody therapy:

(1) after tumor cell inoculation, antibody drugs anti-CXCL13, anti-PD-1 and anti-CXCL13 are administered for combined treatment. anti-PD-1 doses were 200. mu.g per mouse, inoculated with tumor cells day1, day 3; the dose of anti-CXCL13 was 250 μ g per mouse, administered twice a week after tumor cell inoculation, i.p. injection.

3. And (4) analyzing results: the result shows that the anti-CXCL13 antibody blocks CXCL13 protein at the protein level, and can obviously inhibit melanoma blood circulation lung metastasis; at the same time, the combination of anti-PD-1 antibody can obtain better synergistic therapeutic effect, as shown in figure 8. The anti-CXCL13 antibody has good drug property and clinical application value.

Claims

1. The application of the active substance for reducing the activity or expression quantity of the CXCL13 protein in preparing the medicine for treating the malignant tumor metastasis: the medicine for treating malignant tumor metastasis also contains at least one other anti-tumor active ingredient; the other anti-tumor active component is an antibody of a T cell immune negative regulator; the antibody of the T cell immune negative regulator is an anti-PD-1 antibody; the active substance for reducing the activity of the CXCL13 protein is an antibody for inhibiting the activity of the CXCL13 protein; the malignant tumor is melanoma.

2. Use according to claim 1, characterized in that: the antibody protein capable of inhibiting the CXCL13 protein is at least one of a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody and a fully human antibody thereof.