WO2020207257A1

WO2020207257A1 - Application of artemisinin compounds in promoting chimeric antigen receptor t cell treatment, and pharmaceutical composition

Info

Publication number: WO2020207257A1
Application number: PCT/CN2020/081343
Authority: WO
Inventors: 刘茂玄; 许晨光; 唐超; 温丽联
Original assignee: 深圳宾德生物技术有限公司
Priority date: 2019-04-08
Filing date: 2020-03-26
Publication date: 2020-10-15
Also published as: CN111789868B; CN111789868A

Abstract

Provided in the present invention is an application of artemisinin compounds in the preparation of formulations that promote chimeric antigen receptor T cell treatment of malignant tumours, autoimmune disease, or AIDS, and also provided is a pharmaceutical composition used for the treatment of malignant tumours, autoimmune disease, or AIDS, comprising chimeric antigen receptor T cells and artemisinin compounds.

Description

Application of artemisinin compounds in promoting chimeric antigen receptor T cell therapy and pharmaceutical composition

The present invention claims the priority of the prior application with the application number 201910276748.7 filed on April 8, 2019 under the title of "Artemisinin Compounds in Promoting Chimeric Antigen Receptor T Cell Therapy and Pharmaceutical Compositions". The content of the previous application is incorporated into this text by way of introduction.

Technical field

The present invention relates to the technical field of biomedicine, in particular to the new use of artemisinin compounds, in particular to the application of artemisinin compounds in promoting chimeric antigen receptor T cell therapy and pharmaceutical compositions.

Background technique

Malignant diseases such as cancer are diseases that seriously endanger human health worldwide. At present, cancer treatment methods include non-specific immunotherapy, active immunotherapy, and adoptive cellular immunotherapy. Among them, adoptive cellular immunotherapy is considered to be a way to completely eliminate cancer cells in existing technology. The chimeric antigen receptor T cell (CAR-T) technology is one of the latest immune cell technologies in the current adoptive cell reinfusion therapy technology. It is to combine genetically engineered T lymphocytes (which can express specific chimeric antigens). The receptor) is returned to the human body to activate T lymphocytes in a manner independent of major histocompatibility complex, thereby specifically killing cancer cells and other malignant target cells. CAR-T technology has shown great application prospects in the treatment of malignant diseases such as cancer due to its targeting, killing activity and durability to malignant cells.

Although CAR-T cell therapy has achieved good results in some clinical trials, it will produce greater side effects (such as cytokine release syndrome, neurotoxicity and off-target effects, etc.) during the treatment process, and it will affect target cells. The killing effect is not strong enough. Therefore, it is necessary to further study CAR-T cell therapy to enhance its killing effect on target cells.

Summary of the invention

In view of this, the technical problem to be solved by the present invention is to provide a new use of artemisinin-based compounds, combining artemisinin-based compounds with CAR-T cell immunotherapy, thereby producing a significant synergistic effect.

In the first aspect, the present invention provides the use of artemisinin compounds in the preparation of preparations for promoting chimeric antigen receptor T (CAR-T) cells to treat malignant tumors, autoimmune diseases or AIDS.

In the present invention, the artemisinin compound includes artemisinin and its derivatives. Among them, artemisinin is a sesquiterpene lactone compound, which has an internal peroxide bridge on its macrocycle. Under the catalysis of ferrous ion or heme, it will release carbon-centric free radicals. . In addition to artemisinin, the artemisinin compounds also include those skilled in the art that can modify the side chain groups on the backbone through simple chemical methods (such as reduction, substitution, etc.), while still retaining the enhanced chimerization. Various derivatives of antigen receptor T cell killing function. The artemisinin derivatives also include various acid and base forms of salts, hydrates, optical isomers and the like.

Optionally, the artemisinin compounds include artemisinin (the structural formula is shown in the following formula (I)), dihydroartemisinin (the structural formula is shown in the following formula (II)), and artemether (the structural formula is shown in the following formula ( Ⅲ) One or more of arteether (the structural formula is shown in the following formula (IV)), and artesunate (the structural formula is shown in the following formula (V)). In some embodiments of the present invention, the artemisinin compound is dihydroartemisinin.

Optionally, the CAR-T cells include CAR-T cells that target one or more of CD19, CD20, CD22, CD33, CD38, CD317, BCMA, EGFR, Mesothelin, DR5, c-met, OX40, and HER2. T cells.

Wherein, the T cells in the CAR-T cells are derived from autologous T cells, allogeneic T cells, or iPSC-induced T cells.

Wherein, the malignant tumors include leukemia, lymphoma, multiple myeloma, glioma, liver cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer and prostate cancer. One or more, but not limited to this.

Wherein, the autoimmune disease includes one of systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis, ulcerative colitis and Hashimoto's thyroiditis or Many, but not limited to this.

The applicant of the present invention has discovered that artemisinin compounds can significantly enhance the effect of CAR-T cells in treating malignant tumors, and the combination of the two shows a synergistic effect. Therefore, the combined use of artemisinin-based compounds and CAR-T cells can reduce the amount of CAR-T cells to reduce the side effects caused by them under the premise of ensuring the efficacy, and at the same time, it can also improve the treatment of malignant tumors by CAR-T cells. Effect. In addition, artemisinin compounds are small-molecule organic compounds, which are cheap and can reduce the cost of CAR-T cell immunotherapy.

In the second aspect, the present invention also provides a pharmaceutical composition for the treatment of malignant tumors, autoimmune diseases or AIDS, the pharmaceutical composition comprising chimeric antigen receptor T (CAR-T) cells and artemisinin Class compound.

Optionally, the pharmaceutical composition further includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is preferably chemically inert to the artemisinin compound of the present invention, and has no harmful side effects or toxicity under the conditions of use.

The pharmaceutically acceptable carriers, for example, excipients, stabilizers, suspending agents or diluents, etc., are well known by those skilled in the art and are publicly available.

In the pharmaceutical composition, the CAR-T cell and the artemisinin compound may exist alone or in a mixture. When used as a combination of drugs, the two can be administered at the same time, or sequentially or in different modes of administration.

For example, the CAR-T cell is administered parenterally, for example, intravenous injection, arterial injection, subcutaneous injection, intradermal injection, intrathecal injection, or intramuscular injection. The CAR-T cells can be dissolved or suspended in a solution suitable for parenteral administration in an acceptable carrier, including isotonic sterile aqueous injection solutions and non-aqueous solutions. The administration methods of artemisinin compounds include oral administration, intravenous injection, subcutaneous injection, intramuscular injection, sublingual administration, and nasal spray administration. When the artemisinin compound is administered by intravenous injection, subcutaneous injection, intramuscular injection, etc., it can be made into an injection preparation.

In one embodiment of the present invention, the CAR-T cell is administered intravenously, and the artemisinin compound is administered orally. Further, in the pharmaceutical composition, the dosage ratio of the artemisinin compound to the CAR-T cells is (0.5-1.0 nmol): (0.25×105-1×105 cells). For example, the dosage ratio of the two is 0.5nmol: 0.25×105 cells, 0.5nmol: 0.5×105 cells, 0.1mmol: 1×105 cells, 1.0nmol: 0.25×105 cells, 1.0nmol: 0.5×105 Cells, 1.0nmol: 1×105 cells.

Optionally, the density of CAR-T cells used is 0.5×10 6 to 5×10 6 cells/mL. For example, 1×106, 2×106, or 0.5×106 cells/mL.

In the third aspect, the present invention also provides the application of the pharmaceutical composition in the preparation of preparations for the treatment of malignant tumors, autoimmune diseases or AIDS.

In addition, the present invention also provides a method for treating malignant tumors, autoimmune diseases or AIDS, which is to administer the pharmaceutical composition of the present invention to test subjects with malignant target cells. The malignant target cell is the cell corresponding to the malignant tumor, autoimmune disease or AIDS.

Optionally, the ratio of the number of the CAR-T cells to the malignant target cells is (2-10):1. For example, it is 2.5:1, 5:1 or 10:1.

By combining CAR-T cells and artemisinin compounds with synergistic effects, the present invention can reduce the side effects caused by CAR-T cells by reducing the dose of CAR-T cells under the premise of ensuring the curative effect. Enhance the therapeutic effect of CAR-T cell therapy.

Description of the drawings

Figure 1 shows the effector cells: target cells = 10:1, the killing rate of each experimental group to target cells;

Figure 2 shows the effector cells: target cells = 5:1, the killing rate of each experimental group to target cells;

Figure 3 shows the effector cell: target cell = 2.5:1, the killing rate of each experimental group to the target cell;

In each figure, CAR-T is a CAR-T cell targeting CD19; DHArt is dihydroartemisinin.

detailed description

The following are the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also considered This is the protection scope of the present invention.

In addition to artemisinin, dihydroartemisinin, artemether, artemether, and artesunate, the "artemisinin compounds" of the present invention may also include their corresponding tautomers, Pharmaceutically acceptable salts or prodrugs, and can also include those skilled in the art can modify the side chain groups on the backbone through simple chemical methods (such as reduction, substitution, etc.), while still retaining the enhanced chimerization Various derivatives of antigen receptor T cell killing function. The synthesis methods of these derivatives can refer to textbooks in the prior art. At the same time, artemisinin and its derivatives also include various acid-base salts, hydrates, and optical isomers.

In the following examples, CAR-T cells targeting CD19 are taken as an example to verify the synergistic effect between CAR-T cells and artemisinin compounds.

The reagents used in the embodiments of the present invention are all common commercial products, and all are available in the market.

Example one

1. Resuscitate the CD19-targeting CAR-T cells cryopreserved in liquid nitrogen, and culture the CAR-T cells in a complete medium without interleukin IL overnight. Raji cells were cultured overnight in DMEM medium containing 10% FBS and 1% double antibody.

2. Collect CAR-T cells and target cells Raji cells cultured overnight, wash them with PBS, centrifuge (300g, 4min), and discard the supernatant.

3. Resuspend CAR-T cells and Raji cells with KBM 581 basal medium without interleukin IL and 1% fetal bovine serum to adjust their cell density. The cell density of CAR-T was adjusted to 2×10 ⁶ cells/mL, 1×10 ⁶ cells/mL and 5×10 ⁵ cells/mL, and the cell density of target cell Raji was 2×10 ⁵ cells/mL.

4. Take a U-shaped 96-well plate and place 50μL of CAR-T cells of different densities, 50μL of Raji cells, and 0.5μL of DMSO or different concentrations of dihydroartemisinin dissolved in DMSO in each well (the final concentrations are respectively 5μM, 10μM). At the same time, increase the group with only basic medium, only CAR-T cells, only target cells, only CAR-T cells and dihydroartemisinin, and only target cells and dihydroartemisinin as controls. The experiment was repeated three times. The layout of the 96-well plate is shown in Table 1 below:

Table 1 Experimental layout

(Note: In Table 1, CAR-T: CAR-T cells targeting CD19; Target: target cell Raji cells; DHArt: dihydroartemisinin, Media represents KBM without interleukin IL and containing 1% fetal bovine serum 581 Basal Medium)

5. Put the drug-added 96-well plate into an incubator at 37°C for 4 hours. According to the instructions of the BioLegend Lactate Dehydrogenase (LDH) Cell Killing Detection Kit, during the period of incubation to 3.5 hours, 10 μL of lysis buffer was added to the H7-12 well as a control (maximum release group of target cells).

6. Centrifuge the 96-well plate cultured for 4 hours (250g, 2min), transfer 50μL of supernatant from each well to a new transparent flat-bottom 96-well plate, add 50μL of the kit working solution (assay buffer) to each well, at room temperature Avoid light for 30min.

7. After the reaction in the dark for 30 minutes, read the OD ₄₉₀ values in all wells on a full-wavelength reader.

8. The background average value (H1-H3) is subtracted from all experimental groups and control groups, and the value after subtracting the background is used to calculate the killing efficiency, as shown in the following formula (1):

Cell killing rate (%) = [(experimental group release-spontaneous release of effector cells-spontaneous release of target cells) / (maximum release of target cells-spontaneous release of target cells)] × 100% Formula (1)

Among them, the maximum release of target cells should be (the average value of H10 to H12 after removing the background) minus the lysis volume correction group (the average value of H7 to H9 after removing the background).

Specifically, taking the experimental group A7-A9 as an example, the combined application of CAR-T cells and dihydroartemisinin kills Raji cells (%)=[(A7 to A9 average after background removal-A10 to A12 Average after background-H4 to H6 average after background removal)/(Maximum release of target cells-average after H4 to H6 background removal)]×100%. Take the experimental group A4-A6 as an example, using CAR-T cells alone to kill Raji cells (%)=[(A4 to A6 average after background removal-A1 to A3 average after background removal-H4 to H6 Average value after background)/(Maximum release of target cells-average value of H4 to H6 after background removal)]×100%.

The results of killing tumor cells are shown in Figures 1-3. As can be seen from Figure 1-3, 5μM and 10μM dihydroartemisinin have almost no killing effect on tumor cells (Raji), but when it is combined with CAR-T cells, it can significantly enhance the killing of CAR-T cells effect. In addition, when the ratio of effector cells to target cells is 10:1, 5:1, 2.5:1, dihydroartemisinin can significantly enhance the killing effect of CAR-T cells.

The above-mentioned embodiments only express several embodiments of the present invention, and the descriptions are relatively specific and detailed, but they should not be interpreted as limiting the scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

Application of artemisinin compounds in preparing preparations for promoting chimeric antigen receptor T cells to treat malignant tumors, autoimmune diseases or AIDS.
The application according to claim 1, wherein the artemisinin compound includes one or more of artemisinin, dihydroartemisinin, artemether, artemether, and artesunate.
The application according to claim 1, wherein the malignant tumors include leukemia, lymphoma, multiple myeloma, glioma, liver cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, breast cancer , One or more of ovarian cancer, cervical cancer and prostate cancer;

The autoimmune disease includes one or more of systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis, ulcerative colitis and Hashimoto's thyroiditis .
A pharmaceutical composition, characterized in that the pharmaceutical composition comprises a chimeric antigen receptor T cell and an artemisinin compound.
The pharmaceutical composition of claim 4, wherein the chimeric antigen receptor T cell targets CD19, CD20, CD22, CD33, CD38, CD317, BCMA, EGFR, Mesothelin, DR5, c-met A chimeric antigen receptor T cell of one or more of OX40, HER2.
The pharmaceutical composition of claim 4, wherein the T cells used in the chimeric antigen receptor T cells are derived from autologous T cells, allogeneic T cells, or iPSC-induced T cells.
The pharmaceutical composition according to claim 4, wherein the artemisinin compound is one or more of artemisinin, dihydroartemisinin, artemether, artemether and artesunate Kind.
The pharmaceutical composition according to any one of claims 4-7, wherein the chimeric antigen receptor T cell and the artemisinin compound exist alone or in a mixed state.
The pharmaceutical composition according to any one of claims 4-7, wherein the chimeric antigen receptor T cell and the artemisinin compound are administered simultaneously, sequentially or in different ways. The mode of administration is given.
The pharmaceutical composition of claim 9, wherein the chimeric antigen receptor T cells are administered by intravenous injection, arterial injection, subcutaneous injection, intradermal injection, intrathecal injection, and intramuscular injection; The administration modes of the artemisinin compounds include oral administration, intravenous injection, subcutaneous injection, intramuscular injection, sublingual administration, spray nasal administration and the like.
The pharmaceutical composition according to claim 10, wherein the chimeric antigen receptor T cell is administered intravenously, and the artemisinin compound is administered orally.
The pharmaceutical composition of claim 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
The use of the pharmaceutical composition according to any one of claims 4-12 in the preparation of preparations for treating malignant tumors, autoimmune diseases or AIDS.
The application according to claim 13, wherein the malignant tumors include leukemia, lymphoma, multiple myeloma, glioma, liver cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, breast cancer , Ovarian cancer, cervical cancer and prostate cancer; the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis One or more of ulcerative colitis and Hashimoto’s thyroiditis.