Application of GPR55 inhibitor in preparation of product for enhancing tumor immune effect

Abstract

The invention provides an application of a GPR55 inhibitor in preparation of a product for enhancing a tumor immune effect, and belongs to the technical field of biological medicines. The product for targeted inhibition of GPR55 can effectively promote infiltration and immune response of CD8 + T cells in tumors and promote killing of the CD8 + T cells on tumor cells.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology. Specifically, this application relates to the use of GPR55 inhibitors in the preparation of products for enhancing tumor immune effects. Background Art

[0002] Radiotherapy and chemotherapy can inhibit tumor growth to a certain extent. However, cancer cells have high-frequency mutations, resulting in resistance to radiotherapy and chemotherapy. At the same time, radiotherapy and chemotherapy have varying degrees of side effects on patients. Due to the limitations of radiotherapy and chemotherapy, currently, technologies based on immunotherapy and cell therapy are booming, and PD-1 and CAR-T therapies have become the new stars among them. However, more than half of tumor patients are difficult to respond to PD-1, and the cost of CAT-T cells is too high for many patients to bear. In addition, the transfused CAR-T cells are difficult to enter the tumor interior and gradually deplete over time. In view of the above situation, it is crucial to develop new immunotherapies and anti-tumor targets.

[0003] Studies have found that the activation of GPR55 inhibits intratumoral T cell infiltration and immune responses, which is not conducive to anti-tumor immunity. Targeted inhibition of GPR55 can effectively promote the infiltration of T cells in tumors, enhance the immune responses and tumor killing of intratumoral infiltrating T cells, and inhibit the growth of the colon cancer cell line MC38. Transfusion of T cells with GPR55 knocked out has stronger anti-tumor activity and significantly inhibits the growth of melanoma. Targeting GPR55 on T cells has an inhibitory effect on both tumor types without obvious side effects. It shows that targeted inhibition of GPR55 promotes the infiltration and functional responses of intratumoral immune cells, solves the current situation that immune cells are difficult to infiltrate and deplete and become dysfunctional in tumors, and provides a new strategy for cell therapy and immunotherapy. Summary of the Invention

[0004] This application aims to solve at least one of the technical problems in the related technologies to some extent. For this purpose, this application proposes the use of GPR55 inhibitors in the preparation of products for enhancing tumor immune effects. After verification, it is found that targeted inhibition of GPR55 can effectively promote the infiltration and immune responses of T cells in tumors and promote their killing of tumor cells.

[0005] This application also proposes an immune cell that can effectively inhibit tumor growth.

[0006] Specifically, the technical solution of this application is as follows:

[0007] In the first aspect, this application proposes the use of GPR55 inhibitors in the preparation of products for enhancing tumor immune effects. In some examples of this application, products containing GPR55 inhibitors can effectively enhance tumor immune effects.

[0008] In some examples of the present application, the GPR55 inhibitor includes at least one of a product that inhibits GPR55 gene expression, a product that inhibits GPR55 protein, and a product that inhibits the functional activity of GPR55 protein.

[0009] In some examples of the present application, the product that inhibits GPR55 gene expression or the product that inhibits GPR55 protein expression includes at least one of polynucleotides, plasmids packaged by lentivirus or retrovirus, viruses, lipids, and inhibitory antibodies against GPR55; the product that inhibits the functional activity of GPR55 protein includes at least one of proteins, polypeptides, enzymes, and small molecule compounds that inhibit GPR55 protein activity.

[0010] In some examples of the present application, the tumors in the product for enhancing tumor immune effect include at least one of breast cancer, lung cancer, colorectal cancer, and melanoma.

[0011] In some examples of the present application, the GPR55 inhibitor is selected from ML-193, CID16020046, or analogs thereof. It has been verified that ML-193 inhibits tumor growth by inhibiting GPR55, and promotes intratumoral T cell functional response and tumor killing.

[0012] In a second aspect, the present application proposes the use of a GPR55 inhibitor in the preparation of a product for enhancing the ability of T cells to kill tumor cells. In some examples of the present application, the product containing the GPR55 inhibitor can inhibit tumor growth by inhibiting GPR55, and promote intratumoral T cell functional response and tumor killing.

[0013] In some examples of the present application, the GPR55 inhibitor is selected from ML-193, CID16020046, or analogs thereof. It has been verified that ML-193 inhibits tumor growth by inhibiting GPR55, and promotes intratumoral T cell functional response and tumor killing.

[0014] In a third aspect, the present application proposes a product for enhancing tumor immune effect. According to the embodiments of the present application, the product for enhancing tumor immune effect includes: a GPR55 inhibitor. In some examples of the present application, the product containing the GPR55 inhibitor can effectively enhance the immune effect of tumors.

[0015] In some examples of the present application, the GPR55 inhibitor is selected from ML-193, CID16020046, or analogs thereof. It has been verified that ML-193 inhibits tumor growth by inhibiting GPR55, and promotes intratumoral T cell functional response and tumor killing.

[0016] In some examples of the present application, the aforementioned product further includes: pharmaceutically acceptable excipients.

[0017] The aforementioned "pharmaceutically acceptable excipients" may include any solvent, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. Except for the scope where any conventional excipients are incompatible with the bispecific antibody of the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope contemplated by the present application.

[0018] In a fourth aspect, the present application provides an immune cell. According to an embodiment of the present application, the immune cell is selected from T cells with GPR55 gene knockout. In some examples of the present application, the T cells lacking GPR55 have a stronger immune response, and at the same time, their tumor-killing ability is significantly upregulated.

[0019] In a fifth aspect, the present application provides a drug. According to an embodiment of the present application, the aforementioned drug comprises: the immune cell described in the fourth aspect. In some examples of the present application, the aforementioned drug can effectively enhance the immune response of T cells and enhance the tumor-killing ability.

[0020] In some examples of the present application, the aforementioned drug further comprises: pharmaceutically acceptable excipients.

[0021] In some examples of the present application, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0022] In some preferred examples of the present application, the drug is an injection.

[0023] It should be noted that the aforementioned drug includes combinations separated in time and / or space, as long as they can act together to achieve the purpose of the present application. For example, the components contained in the aforementioned drug can be administered to the subject as a whole, or separately. When the components contained in the drug are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0024] The drug of the present application contains a safe and effective amount of the active ingredient of the present application (T cells with GPR55 gene knockout) and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. Generally, the drug formulation should match the administration method, and the dosage form of the drug of the present application is an injection. For example, it is prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The aforementioned drug should be manufactured under aseptic conditions.

[0025] The effective amount of the active ingredient of the present application may vary depending on the mode of administration, the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art based on various factors (e.g., through clinical trials). The foregoing factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment situation, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0026] In the present application, "administration" refers to introducing a predetermined amount of a substance into a patient by a suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous injection, intramuscular injection, subcutaneous injection, etc., but the present invention is not limited to the exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous injection or subcutaneous injection.

[0027] In the present application, "treatment" is used to refer to obtaining the desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting the disease, e.g., arresting the development of the disease; or (c) alleviating the disease, e.g., reducing the symptoms associated with the disease. As used herein, "treatment" covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the disease of the individual, including but not limited to administering a drug containing the compound described herein to an individual in need thereof.

[0028] In a sixth aspect, the present application provides a method for treating tumors. According to an embodiment of the present application, the foregoing method includes: administering an effective dose of a GPR55 inhibitor or immune cells to a patient, wherein the immune cells are selected from T cells knocked out of the GPR55 gene. The use of a GPR55 inhibitor or immune cell therapy can effectively enhance the tumor killing ability, control the growth rate of tumors, reduce side effects, and prolong the survival time of patients / animals.

[0029] In some examples of the present application, the foregoing effective dose of the GPR55 inhibitor or immune cells can be determined based on the patient's weight, tumor severity or tumor stage.

[0030] In the present application, "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0031] Additional aspects and advantages of the present application will be given in part in the following description, will become apparent in part from the following description, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0033] Figure 1 Schematic diagram of the tumor immunity results of GPR55 gene knockout mice provided for an embodiment of the present application;

[0034] Figure 2 Schematic diagram of the tumor immunity results of GPR55 inhibitor ML-193 provided for an embodiment of the present application;

[0035] Figure 3 Schematic diagram of the tumor immunity results of GPR55 inhibitor CID16020046 provided for an embodiment of the present application;

[0036] Figure 4 Schematic diagram of the tumor immunity results of GPR55 inhibitor ML-193 on GPR55 gene knockout mice and wild-type mice provided for an embodiment of the present application;

[0037] Figure 5 Schematic diagram of the tumor immunity results of adoptively transferred GPR55 gene knockout CD8 + T cells provided for an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The following details the embodiments of the present invention, and the examples of the embodiments are shown in the drawings. Wherein the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0039] During the description of the present invention, the relevant terms in this article are explained and described. These explanations and descriptions are only for the convenience of understanding the solution and should not be regarded as a limitation to the protection solution of the present invention.

[0040] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0041] In this text, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0042] Embodiments of the present application will be described in more detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present application, and should not be construed as a limitation of the present application. Reagents or instruments for which the manufacturer is not indicated are all conventional products that can be obtained commercially.

[0043] The materials used in the following examples are as follows:

[0044] Wild-type (WT) mice: purchased from Jiangsu Genscript Biotech Co., Ltd.;

[0045] GPR55 - / - Mice: developed and constructed by the Animal Center of the University of Science and Technology of China;

[0046] MC38: donated by the research group of Professor Wang Yucai of the University of Science and Technology of China;

[0047] B16F10: donated by the research group of Professor Wang Yucai of the University of Science and Technology of China;

[0048] ML-193: MCE, HY-110125;

[0049] CID16020046: MCE, HY-16697

[0050] AF700 anti-mouse CD8 antibody: Biolegend, 100730;

[0051] PE / Cy7 anti-mouse IFN-γ antibody: Biolegend, 505826;

[0052] PE anti-mouse perforin: Biolegend, 154306;

[0053] APC anti-mouse Granzyme B: Biolegend, 372204;

[0054] Example 1: GPR55 inhibits the infiltration and activation of T cells

[0055] Animal model:

[0056] Use GPR55 - / -Mice and their littermate controls of GPR55 + / + Mice were subcutaneously implanted with 1×10 MC38 tumor cells.

[0057] Tumor tissue collection and processing:

[0058] On the 24th day after tumor implantation, the tumor tissues of the mice were harvested, and the lymphocytes infiltrating into the tumor were isolated. Flow cytometry was used to label T cells and their functional molecules to analyze the proportion and functional responses of T cells.

[0059] Result analysis:

[0060] The presence of GPR55 inhibits the function of T cells and restricts their infiltration into the tumor microenvironment. Knockout (KO) of GPR55 significantly promoted the infiltration of T cells into the tumor, while enhancing their functional responses and anti-tumor ability. The results are as Figure 1 shown, indicating that GPR55 restricts the infiltration and immune response of T cells in the tumor. Knockout of GPR55 in mice promotes the infiltration of T cells into the tumor and IFN-γ response, promotes their anti-tumor immunity, and knockout of GPR55 significantly inhibits tumor growth.

[0061] Example 2: GPR55 inhibitor ML-193 significantly inhibits tumor growth

[0062] Animal model:

[0063] GPR55 + / + mice were subcutaneously implanted with 1×10 6 MC38 tumor cells.

[0064] Treatment protocol:

[0065] On the 9th day after tumor implantation, the mice were evenly divided into two groups according to tumor volume and given the following treatments respectively:

[0066] CTRL group: Injected with 10 μL DMSO + 40 μL PEG300 + 5 μL Tween 80 + 45 μL normal saline.

[0067] 5 mg / kg ML-193 group: Injected with 2 μL ML-193 + 8 μL DMSO + 40 μL PEG300 + 5 μL Tween 80 + 45 μL normal saline.

[0068] Treat once every 3 days until the 25th day after tumor implantation.

[0069] Tumor tissue collection and analysis:

[0070] Harvest the tumor tissue, isolate the lymphocytes infiltrating into the tumor, and use flow cytometry to label CD8 + T cells and their functional molecules.

[0071] Result analysis:

[0072] As Figure 2 shown, ML-193 significantly inhibited tumor growth, promoted the infiltration of intratumoral T cells and tumor killing ability, indicating that by inhibiting GPR55, the anti-tumor function of T cells can be enhanced.

[0073] Example 3: GPR55 inhibitor CID16020046 significantly inhibits tumor growth

[0074] Animal model:

[0075] Using GPR55 + / + 1×10 6 MC38 tumor cells were subcutaneously implanted in mice.

[0076] Treatment regimen:

[0077] On the 9th day after tumor inoculation, the mice were divided into two groups according to tumor volume and given the following treatments respectively:

[0078] CTRL group: Injected with 10 μL DMSO + 40 μL PEG300 + 5 μL Tween 80 + 45 μL normal saline.

[0079] 5 mg / kg ML-193 group: Injected with 2 μL ML-193 + 8 μL DMSO + 40 μL PEG300 + 5 μL Tween 80 + 45 μL normal saline.

[0080] 5 mg / kg CID16020046 group: Injected with 2 μL CID16020046 + 8 μL DMSO + 40 μL PEG300 + 5 μL Tween 80 + 45 μL normal saline.

[0081] Treated once every 3 days until the 23rd day after tumor inoculation.

[0082] Tumor tissue collection and analysis:

[0083] Harvested tumor tissues, isolated the infiltrated lymphocytes in the tumor, and used flow cytometry to label CD8 + T cells and their functional molecules.

[0084] Result analysis:

[0085] As Figure 3 shown, ML-193 and CID16020046 significantly inhibited tumor growth.

[0086] Example 4: ML-193 inhibits tumor growth through GPR55

[0087] Animal model:

[0088] Using GPR55 + / + Mice and their littermate control GPR55 - / - Mice were subcutaneously implanted with 1×10 6 MC38 tumor cells.

[0089] Treatment regimen:

[0090] On the 9th day after tumor implantation, the mice were evenly divided into four groups according to tumor volume, and the treatment regimens were as follows:

[0091] CTRL group: Injected with 10 μL DMSO + 40 μL PEG300 + 5 μL Tween 80 + 45 μL normal saline.

[0092] 5 mg / kg ML-193 group: Injected with 2 μL ML-193 + 8 μL DMSO + 40 μL PEG300 + 5 μL Tween 80 + 45 μL normal saline.

[0093] Treated once every 3 days until the 25th day after tumor implantation.

[0094] Tumor tissue collection and analysis:

[0095] Harvested tumor tissue, weighed and photographed.

[0096] Result analysis:

[0097] The results were as Figure 4 shown, ML-193 significantly inhibited tumor growth in GPR55 + / + mice, while having no significant effect on GPR55 - / - mice, indicating that ML-193 inhibits tumor growth through the GPR55 pathway.

[0098] Example 5: T cells with knocked-out GPR55 have stronger anti-tumor ability

[0099] Animal model:

[0100] Using Rag1 - / - mice were subcutaneously implanted with 5×10 5 B16F10 tumor cells.

[0101] CD8 + T cell transfer:

[0102] On the 3rd day after tumor implantation, 3×10 6 CD8 - / - T cells from GPR55 + / + mice and their littermate control GPR55 + mice were respectively transferred via the tail vein.

[0103] On the 18th day after tumor inoculation, tumor tissues were harvested, and CD8+ T cells infiltrating into the tumor were isolated. Flow cytometry was used to label CD8+ T cells and their intracellular functional molecules, and the proportion of CD8 + T cells in the tumor and the intensity of immune response were analyzed.

[0104] Result analysis:

[0105] The transferred GPR55 - / - CD8+ T cells had a stronger immune response, and at the same time, their tumor-killing ability was significantly upregulated. The results were as Figure 5 shown, indicating that GPR55 knockout promoted the function and activation of T cells, and transferring T cells with GPR55 knockout had better anti-tumor ability and significantly inhibited tumor growth.

[0106] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0107] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0108] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. Application of GPR55 inhibitors in the preparation of products for enhancing tumor immunity effects.

2. The use according to claim 1, characterized in that: The GPR55 inhibitor includes at least one of a product that inhibits the expression of the GPR55 gene, a product that inhibits the GPR55 protein, and a product that inhibits the functional activity of the GPR55 protein.

3. The use according to claim 2, characterized in that: The product for inhibiting the expression of the GPR55 gene or the product for inhibiting the expression of the GPR55 protein includes: at least one of a polynucleotide, a plasmid packaged by a lentivirus or a retrovirus, a virus, a lipid and an inhibitory antibody against GPR55; the product for inhibiting the functional activity of the GPR55 protein includes at least one of a protein, a polypeptide, an enzyme and a small molecule compound that inhibits the activity of the GPR55 protein.

4. The use according to claim 1, characterized in that: The tumor in the product for enhancing tumor immunity effect includes at least one of breast cancer, lung cancer, liver cancer, pancreatic cancer, colorectal cancer and melanoma.

5. The use according to any one of claims 1 to 4, characterized in that: The GPR55 inhibitor is selected from ML-193, CID16020046 or analogs thereof.

6. Use of GPR55 inhibitors in the preparation of products for enhancing the ability of T cells to kill tumor cells; Preferably, the GPR55 inhibitor is selected from ML-193, CID16020046 or analogs thereof.

7. A product for enhancing tumor immunity, characterized in that: The products used to enhance tumor immunity effects include: GPR55 inhibitors; Preferably, the GPR55 inhibitor is selected from ML-193, CID16020046 or an analog thereof; Optionally, it further comprises: pharmaceutically acceptable excipients.

8. An immune cell, characterized in that The immune cells are selected from GPR55 gene knockout T cells.

9. A drug, characterized in that include: The immune cell according to claim 8.

10. The drug according to claim 9, characterized in that Further including: Pharmaceutically acceptable excipients; Optionally, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers; Preferably, the drug is an injection.

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