CN113117088A - Use of calcium-activated chloride channel inhibitors in tumor immunotherapy - Google Patents
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Abstract
The invention belongs to the field of biological treatment, and relates to application of a Calcium activated Chloride channel (CaCcs) inhibitor in tumor immunotherapy. The research of the invention shows that the CaCCs inhibitor can enhance the immune infiltration of tumor tissues and can play a role in the synergistic treatment of tumors with immunotherapy such as PD-1 antibody. The invention provides a method for combining a CaCCs ion channel inhibitor with a tumor immunotherapy, which can overcome the defects of low effective rate and limited clinical application of the existing immunotherapy and remarkably improve the tumor treatment effect.
Description
Technical Field
The present invention is in the field of biological therapy. More particularly, it relates to the use of inhibitors of Calcium activated Chloride Channels (CaCCs) in tumor immunotherapy. When the immune therapy is carried out on a tumor patient, the curative effect of the cancer patient can be improved by adding the CaCCs inhibitor, and the defects of low effective rate and limited clinical application of the existing immune therapy can be overcome.
Background
Tumor immunotherapy is an emerging means of tumor therapy. Normally, the immune system of the human body is able to recognize and eliminate allogeneic tumor cells. However, tumor cells adopt different strategies in various stages of anti-tumor immunity for their survival and growth to evade killing by the immune system and generate immune tolerance. Tumor immunotherapy is a method for treating tumors by restoring and maintaining the human immune system and restoring the normal anti-tumor immune response of the human body.
Tumor immunotherapy includes immune checkpoint inhibitors, adoptive T cell therapy, tumor vaccines,
immunotherapy techniques, and the like. (1) Immune checkpoint inhibitors are a mature class of immunotherapeutic drugs under current research. Immune checkpoints are a group of important molecules that mediate immune regulation and play a vital role in the initiation and cessation of immune responses. The immune checkpoint inhibitor has achieved remarkable curative effect in a plurality of clinical studies and is marketed, mainly comprises antibodies CTLA-4, PD-1 and PD-L1, and is the most therapeutically valuable tumor immunotherapy strategy nowadays. (2) Adoptive T cell therapies include CAR-T cell therapy, TIL therapy, TCR-T therapy, and the like. CAR-T technology refers to chimeric antigen receptor T cell therapy technology that allows T cells to recognize tumor antigens by designing, engineering, and expanding them in vitro, and then reinfusing them back to the patient to kill tumor cells; the TCR-T technology utilizes a viral or non-viral vector system to transduce TCR genes which specifically recognize tumor antigens into T cells of peripheral blood sources of patients, and the T cells are returned to the patients after in vitro culture and amplification, so that tumors are killed; TIL therapy refers to the in vitro multiple screening of immune cells in the tumor microenvironment to obtain energyImmune cells that specifically recognize and attack cancer cells. The three technologies are methods for obtaining high-pertinence and high-killing immune cells to be returned to a patient to treat tumors by a certain means. (3) The tumor vaccine is different from the passive immunity mentioned above, belongs to active immunity, can induce or amplify the cell immunity and humoral immunity aiming at the target antigen prestored in the organism by injecting the target antigen related to the tumor, can form long-term immunological memory, and can play a role in reducing the recurrence of the tumor. (4)
The immunotherapy technology is to connect the variable regions of the heavy chains and the light chains of two specific monoclonal antibodies through a connecting peptide by using DNA recombination to form a bispecific antibody, and the technology has a remarkable curative effect in blood tumors at present, such as the CD19/CD3 antibody, and the application in solid tumors is already in the clinical research stage.
The tumor immunotherapy has advanced in the treatment of various tumors, but the problems of low effective rate and primary drug resistance in some patients still exist.
Disclosure of Invention
The invention aims to solve the problems of low effective rate of tumor patients on immunotherapy and limited clinical application. In response, we provide CaCCs inhibitors as agents for enhancing the efficacy of immunotherapy. Our studies show that combining CaCCs inhibitors with immunotherapy can synergistically treat tumors.
The above purpose of the invention is realized by the following technical scheme:
calcium-activated chloride channels (CaCCs) are a class of cell membrane-expressed calcium-activated chloride channels that mediate a series of important physiological functions, of which the TMEM16A protein/ANO 1 gene is representative. The CaCcs inhibitors are of a wide variety of types, including small molecule inhibitors and naturally extracted components. The small molecule inhibitor comprises CaCcinh-A01, T16Ainh-A01, MONNA, 9-phenonthrol, Ani9, AACTs, NFA, DIDS, NPPB and the like, and the natural extract components comprise ginsenoside Rb1, resveratrol, tannic acid, gallotannin, eugenol, alkannin, andrographolide, flavonoids compounds and the like. Among them, small molecule inhibitors T16Ainh-A01 and CaCcinh-A01 are representative more specific inhibitors of CaCcs. T16Ainh-A01 is a class of aminophenylthiazoles, is an inhibitor of TMEM16A protein/ANO 1 gene, and inhibits the current of chloride ions mediated by TMEM 16A. The CaCcinh-A01 is an inhibitor of TMEM16A protein/ANO 1 gene and is also an inhibitor of CaCcs. Given the wide variety of CaCCs inhibitors, two representative molecules were selected for study as CaCCs inhibitors.
Three representative murine tumor cell lines, namely an intestinal cancer cell line MC38, a melanoma cell line B16-OVA and a breast cancer cell line 4T1, are respectively used for constructing a subcutaneous tumor model in a C57BL/6J mouse with healthy immunity. When the tumor grew to a certain size, IgG control treatment, PD-1 antibody treatment, CaCcs inhibitor (T16Ainh-A01 or CaCcinh-A01) treatment, and PD-1 antibody and CaCcs inhibitor (T16Ainh-A01 or CaCcinh-A01) combination treatment were administered, respectively. The results show that at a given dose, the CaCCs inhibitor alone has no significant effect on tumor growth, while the CaCCs inhibitor in combination with the PD-1 antibody can significantly promote the curative effect of the PD-1 antibody.
Further, we analyzed changes in the immune microenvironment in tumor tissues following administration of IgG control treatment, PD-1 antibody treatment, CaCCs inhibitor treatment, and combination PD-1 antibody and CaCCs inhibitor treatment. Flow cytometry results show that CaCCs inhibitors can significantly increase infiltration of activated killer T cells in tumor tissues. The results show that the CaCCs inhibitor promotes the curative effect of immunotherapy by activating the body's own immune system, and the CaCCs inhibitor can improve the curative effect of tumor immunotherapy.
Accordingly, the invention claims:
the application of the CaCcs inhibitor in preparing a tumor immunotherapy synergist or in preparing a tumor treatment drug.
And the application of the inhibitor of TMEM16A protein/gene ANO1 in the preparation of a synergist for tumor immunotherapy or the application in the preparation of a tumor treatment drug.
And the application of T16Ainh-A01 in preparing tumor immunotherapy synergist or preparing tumor treatment drugs.
And the application of CaCcinh-A01 in preparing tumor immunotherapy synergist or tumor treatment medicine. Preferably, the inhibitor comprises a small molecule inhibitor or a monoclonal antibody inhibitor.
In addition, the tumor comprises an advanced solid tumor. The tumors include endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, squamous cell carcinoma of the anogenital region, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, lung squamous cell carcinoma, gastric cancer, bladder cancer, gallbladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland carcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematologic cancer, and combinations thereof.
In addition, the above-mentioned tumor immunotherapy refers to a therapy for combating tumor cells using the body's immune system, and includes immune checkpoint inhibitor therapy, adoptive T cell therapy, tumor vaccine therapy or
Immunotherapy.
The immune checkpoint inhibitors include, but are not limited to, CTLA4 antibodies, PD-1 antibodies, PD-L1 antibodies.
Such adoptive T cell therapies include, but are not limited to, CAR-T cell therapy, TIL therapy, TCR-T therapy.
The tumor vaccine includes but is not limited to cell vaccine prepared by tumor cells or dendritic cells, and molecular vaccine prepared by protein, polypeptide, DNA, RNA and virus.
The above-mentioned
Immunotherapeutic techniques include bispecific antibodies designed against cell membrane molecules of tumor cells and immune cells, in particular, also bispecific antibodies against cell membrane molecules of CaCCs and immune cells。
Based on the above results, the present invention can provide a tumor treatment drug comprising an inhibitor of Calcium activated Chloride Channels (CaCCs), preferably, a drug for tumor immunotherapy, as described above (e.g., PD-1 antibody).
Preferably, as an alternative, the CaCCs inhibitor is an inhibitor of TMEM16A protein/gene ANO 1.
Preferably, as an alternative, the CaCcs inhibitor is T16Ainh-A01 or CaCcinh-A01.
The medicament may further comprise one or more pharmaceutically acceptable carriers, excipients and/or vehicles.
In the tumor combination treatment regimens of the invention, the CaCCs inhibitor and the tumor immunotherapy can be administered simultaneously, sequentially or alternately.
The invention has the following beneficial effects:
the invention provides a novel combined therapy for guiding immunotherapy, which can enhance the curative effect of tumor immunotherapy by using CaCCs inhibitors and overcome the defects of low effective rate and limited clinical application of immunotherapy. The tumor combined treatment scheme is based on the existing immunotherapy, and the mature molecular drugs are simultaneously, sequentially or alternately combined for administration, so that the tumor combined treatment scheme can be rapidly and clinically applied.
Drawings
FIG. 1 shows that PD-1 antibodies and CaCcs inhibitors (T16Ainh-A01 and CaCcinh-A01) have combined therapeutic effects.
FIG. 2 shows that CaCCs inhibitors enhance infiltration of activated killer T cells in tumor tissue.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 combination of PD-1 antibody and CaCcs inhibitor (T16Ainh-A01 and CaCcih-A01)
1. Experimental materials:
(1) tumor cell lines: the cell line comprises a murine intestinal cancer cell line MC38, a murine melanoma cell line B16-OVA and a murine breast cancer cell line 4T 1.
(2) Experimental mice: c57BL/6J mice.
(3) Medicine preparation: murine PD-1 antibody, clone No. G4C 2;
(4) medicine preparation: CaCcs inhibitors, T16Ainh-A01 and CaCcinh-A01.
2. The experimental steps are as follows:
(1) will be 1 × 106MC38 cell line, 5X 105B16-OVA cell line, 2X 1054T1 cell lines were injected subcutaneously into immunocompromised C57BL/6J mice to construct a tumor model.
(2) The tumor models were each divided into 6 groups for treatment by administration:
1) control group: administering an IgG control treatment;
2) treatment group 1: administering a murine PD-1 antibody treatment;
3) treatment group 2: administering a T16Ainh-A01 treatment;
4) treatment group 3: administration of CaCcinh-A01 for treatment;
5) treatment group 4: administering a murine PD-1 antibody in combination with T16Ainh-a01 treatment;
6) treatment group 5: administering a murine PD-1 antibody in combination with CaCCinh-a01 treatment;
wherein IgG is administered on days 5, 8 and 11, respectively, at a dose of 250 μ g; the PD-1 antibody is respectively administered on days 5, 8 and 11, and the dosage is 250 mug;
t16Ainh-A01(10mg/kg) or CaCcinh-A01(20mg/kg) was administered once every 2 days beginning on day 5.
(3) Tumor size was measured every 3 days and tumor volume was calculated as length x width/2.
3. The results describe:
the treatment results are shown in FIG. 1, the growth curve of single tumor (graph A) and the final volume statistics (graph B) of the subcutaneous tumor formation model of (A & B) MC38 cell line, (C & D) the growth curve (graph C) and the final volume statistics (graph D) of the subcutaneous tumor formation model of (B & D) B16-OVA cell line.
The results show that the T16Ainh-A01 or CaCcinh-A01 has no obvious effect when used alone, the PD-1 antibody is effective when used singly, and the combined effect of the PD-1 antibody and the CaCcs inhibitor (T16Ainh-A01 and CaCcinh-A01) is obviously better than that of the PD-1 antibody when used singly (figure 1), which indicates that the combined treatment scheme has a synergistic effect.
Example 2CaCCs inhibitors enhance infiltration of activated killer T cells in tumor tissue
1. Experimental materials:
(1) tumor cell lines: murine intestinal cancer cell line MC 38.
(2) Experimental mice: c57BL/6J mice.
(3) Medicine preparation: murine PD-1 antibody, clone No. G4C 2; t16 Ainh-A01.
(4) Reagent: granzyme B-PE flow antibody, CD45-PC5.5 flow antibody, IFN- γ -BV421 flow antibody, Zombie UV flow antibody, CD8-BV605 flow antibody, TCR β -BV785 flow antibody, and other conventional reagents for flow cytometry.
2. The experimental steps are as follows:
(1) will be 1 × 106An MC38 cell line was injected subcutaneously into C57BL/6J mice to construct a tumor model.
(2) The tumor models were divided into 4 groups:
1) control group: administering an IgG control treatment;
2) treatment group 1: administering a murine PD-1 antibody treatment;
3) treatment group 2: administering a T16Ainh-A01 treatment;
4) treatment group 3: administering a murine PD-1 antibody in combination with T16Ainh-a01 treatment;
wherein IgG is administered on days 5 and 8, respectively, at a dose of 250 μ g; the PD-1 antibody is respectively administered on days 5 and 8, and the dosage is 250 mug;
t16Ainh-A01(10mg/kg) was administered once daily beginning on day 5.
(3) On day 10, tumor tissue was removed for flow analysis.
3. The results describe:
the results of the treatment are shown in FIG. 2, in which A shows an example of analysis of the results of flow cytometry, and B shows IFN γ of tumor tissue in a subcutaneous tumor formation model of MC38 cell line+CD8+T cells account for the proportion of live CD45 positive immune cells.
The results show that T16Ainh-A01 can significantly increase IFN gamma compared with the control group+CD8+Proportion of T cells, and IFN γ in the PD-1 antibody in combination with T16Ainh-A01 treatment group+CD8+The proportion of T cells was significantly greater than the PD-1 antibody treated group (fig. 2), suggesting that CaCCs inhibitors may enhance infiltration of activated killer T cells in tumor tissues.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The application of Calcium activated Chloride channel (CaCcs) inhibitor in preparing tumor immunotherapy synergist or tumor treatment medicine is provided.
The application of the inhibitor of TMEM16A protein/gene ANO1 in preparing a synergist for tumor immunotherapy or preparing a drug for tumor treatment.
The application of T16Ainh-A01 in preparing tumor immunotherapy synergist or preparing tumor treatment drugs.
The application of CaCcinh-A01 in preparing tumor immunotherapy synergist or tumor treatment medicine.
5. The use of claim 1 or 2, wherein the inhibitor comprises a small molecule inhibitor or a monoclonal antibody inhibitor.
6. The use of any one of claims 1-4, wherein the tumor is selected from the group consisting of endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, squamous cell carcinoma of the anogenital area, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, lung squamous cell carcinoma, gastric cancer, bladder cancer, gallbladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland carcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, and combinations thereof.
7. The use of claims 1-4, wherein the tumor immunotherapy is a therapy against tumor cells using the body's immune system, and comprises immune checkpoint inhibitor therapy, adoptive T cell therapy, tumor vaccine therapy or
Immunotherapy;
wherein preferably the immune checkpoint inhibitor includes, but is not limited to, CTLA4 antibody, PD-1 antibody, PD-L1 antibody; such adoptive T cell therapies include, but are not limited to, CAR-T cell therapy, TIL therapy, TCR-T therapy; the tumor vaccine includes but is not limited to cell vaccine prepared by tumor cells or dendritic cells, molecular vaccine prepared by protein, polypeptide, DNA, RNA and virus; the above-mentioned
Immunotherapeutic techniques include bispecific antibodies designed against cell membrane molecules of tumor cells and immune cells, and in particular also those against CaCCs and immune cells.
8. A medicament for the treatment of tumours, comprising an inhibitor of Calcium activated Chloride Channels (CaCCs), preferably together with a medicament for the immunotherapy of tumours.
9. The agent for treating tumor according to claim 8, which is an inhibitor of TMEM16A protein/gene ANO 1.
10. The agent for treating tumor according to claim 8, which comprises T16Ainh-A01 or CaCcinh-A01, and is used for immunotherapy of tumor.
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