Application of Tim-3 and autologous RAK cell culture method

Abstract

The invention relates to the technical field of adoptive cellular immunotherapy, in particular to application of Tim-3 and an autologous RAK cell culture method. The invention provides application of Tim-3 as an inhibition target to improvement of the anti-tumor effect of RAK cells, and provides an autologous RAK cell culture method and a tumor treatment method. According to the invention, by closing the Tim-3 antigen on the autologous RAK cell, the immunosuppression effect of Tim-3 and the ligand thereof is relieved, so that the killing ability of the autologous RAK cell to the positive tumor of the Tim-3 ligand is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of adoptive cell immunotherapy, and in particular to the application of Tim-3 and an autologous RAK cell culture method. Background Art

[0002] Cancer refers to malignant tumors, including carcinoma, sarcoma and carcinosarcoma. It is caused by malignant cell proliferation, is invasive and can metastasize. Cancer is one of the important causes of human death. The basic means of treating cancer include surgical resection, radiotherapy, and radiotherapy. However, due to the different types of cancer, different tissues and organs involved, different stages of the disease, and different responses to various treatments, most patients need comprehensive treatment. The so-called comprehensive treatment is to use surgery, chemotherapy, radiotherapy, immunotherapy, traditional Chinese medicine treatment, interventional therapy, microwave therapy and other means according to the patient's physical condition, the pathological type of the tumor, the scope of invasion, etc., in order to significantly improve the cure rate and improve the patient's quality of life.

[0003] Adoptive cellular immunotherapy is a treatment method that infuses autologous or allogeneic immune effector cells activated and expanded in vitro into the patient's body. Autologous RAK cells (Retronectinactived killer cells) are a new type of anti-tumor adoptive immunotherapy cells. They are immune cells with high proliferation and killing abilities obtained by culturing T lymphocytes through stimulation with multiple cytokines such as IL-2, IFN-γ, and CD3mAb. In 2014, the RAK cell Phase I clinical trial approval (2014L01920) issued by NMPA was obtained. In 2016, autologous RAK cells were used in Phase I / II clinical studies in four types of cancer (metastatic renal cancer, metastatic non-small cell lung cancer, metastatic castration-resistant prostate cancer, and primary liver cancer). The study fully demonstrated the safety and effectiveness of autologous RAK cells. In 2022, NMPA issued two Phase II clinical trial approvals for RAK cells to treat advanced renal cancer and prevent postoperative recurrence of liver cancer (No.: CXSL2200445 and CXSL2200446). On this basis, the field is still looking forward to further improving the therapeutic effect of RAK cells. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an application of Tim-3 and an autologous RAK cell culture method, which improves the anti-tumor activity of RAK cells by blocking Tim-3.

[0005] The present invention provides: use of a substance that inhibits the level and / or activity of Tim-3 in preparing a reagent for improving the tumor inhibition effect of RAK cells.

[0006] Tim-3 is a member of the T cell immunoglobulin and mucindomain (Tim) molecule family and is one of the important molecules that mediate T cell exhaustion. Currently, it has been found that Tim-3 has four ligands, including Galectin-9 (the main ligand), Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM-1), High Mobility Group Box 1 (HMGB1), and Phosphatidylserine (PtdSer). Studies have shown that the above four ligands are widely expressed in a variety of solid tumors. The present invention found that Tim-3 is highly expressed in autologous RAK cells, CD4 + Tim-3 accounts for 40%-80% of T cells, CD8 + It accounts for 65% to 90% of T cells. This molecule binds to the ligand and activates the inhibitory signaling pathway in T cells, which leads to T cell dysfunction and reduces the anti-tumor efficacy of autologous RAK cells. Inhibiting the level and / or activity of Tim-3 can significantly enhance the ability of RAK cells to kill tumor cells.

[0007] In the present invention, the methods of inhibiting Tim-3 level and / or activity include: TIM-3 gene knockout, Galectin-9 monoclonal antibody and other Tim-3 immunosuppression relief technologies combined with RAK ​​cell therapy, and small molecule targeted drugs for the four Tim-3 ligands (Galectin-9, CEACAM-1, PtdSer, HMGB1) combined with RAK ​​cell therapy and other technologies. In some specific embodiments, the substance that inhibits Tim-3 level and / or activity is Tim-3 antibody.

[0008] In the present invention, the Tim-3 antibody is a monoclonal antibody or a polyclonal antibody, which can specifically recognize Tim-3 only, or can be a bispecific antibody or a multispecific antibody that recognizes Tim-3. The present invention is not limited to this. Any antibody that can bind to Tim-3 on the surface of RAK cells can achieve the technical effect of the present invention.

[0009] In the present invention, the tumor is a Tim-3 ligand-positive tumor. In some embodiments, the tumor includes but is not limited to gastric cancer, lung cancer, renal clear cell carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma or breast cancer.

[0010] Furthermore, the present invention also provides a reagent for improving the tumor inhibition effect of RAK cells, which comprises Tim-3 antibody and a solvent.

[0011] In the reagent of the present invention, the solvent is PBS (phosphate) buffer solution or 0.9% NaCl aqueous solution (physiological saline).

[0012] In some embodiments, the pH value of the agent for enhancing the tumor inhibition effect of RAK cells is 6.5-7.5.

[0013] In some embodiments, the amount of Tim-3 antibody in the reagent is 0 to 5 μg / 10 6 In some specific embodiments, the concentration of Tim-3 antibody in the reagent is 0.532-2.128 μg / 10 6 More specifically, in the reagent, the concentration of Tim-3 antibody is 0.532 μg / 10 6 RAK cells, 1.064 μg / 10 6 RAK cells, 1.596μg / 10 6 RAK cells or 2.128 μg / 10 6 RAK cells.

[0014] Furthermore, the present invention also provides a method for improving the tumor inhibition effect of RAK cells, which comprises mixing RAK cells with the above-mentioned reagent and then incubating them.

[0015] In the embodiment of the present invention, every 1×10 6 cells are mixed with 0.1-10 μg of Tim-3 antibody. In the incubation system of the present invention, the density of RAK cells is ~1×10 7 / mL, the concentration of Tim-3 antibody is 1-25 μg / mL. In some specific embodiments, in the incubation system, the cell density is 1×10 7 / mL, the concentration of Tim-3 antibody was 0.532~2.128μg / 10 6 More specifically, in the incubation system, the cell density is 1×10 7 / mL, the concentration of Tim-3 antibody was 0.532μg / 10 6 RAK cells, 1.064 μg / 10 6 RAK cells, 1.596μg / 10 6RAK cells or 2.128 μg / 10 6 RAK cells.

[0016] In the embodiment of the present invention, the incubation is performed at a temperature greater than 0° C. for not less than 5 minutes.

[0017] In some embodiments, the incubation temperature is 0°C to 40°C, for example, the incubation temperature is 0°C to 10°C, 10°C to 25°C or 18°C ​​to 37°C, specifically, the incubation temperature is 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C or 40°C.

[0018] In some embodiments, the incubation time is 5 min to 60 min, for example, the incubation time is 5 to 15 min, 15 to 30 min, 20 to 60 min or 30 to 60 min, specifically, the incubation time is 5 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min.

[0019] The RAK cells described in the present invention are RAK cells of autologous origin, and can also be RAK cells of allogeneic origin. In the embodiment of the present invention, the preparation of RAK cells includes collecting 50-60 mL of venous blood, separating mononuclear cells (PBMCs) by density gradient separation fluid, stimulating and culturing for about 14 days with multiple cytokines such as IL-2 and CD3mAb, and obtaining immune cells with high proliferation and killing abilities.

[0020] Furthermore, the present invention also provides RAK cells prepared by the method described above.

[0021] Furthermore, the present invention also provides an anti-tumor preparation, which includes: RAK cells prepared by the method described above. The anti-tumor agent also includes a buffer and a stabilizer. The buffer includes phosphate buffer (PBS) buffer and 0.9% NaCl (normal saline), and the stabilizer includes a protein stabilizer or a carbohydrate stabilizer, such as albumin and trehalose.

[0022] Furthermore, the present invention also provides an anti-tumor method, which comprises administering the anti-tumor preparation of the present invention, wherein the administration method comprises intravenous infusion.

[0023] The present invention provides an application of Tim-3 as an inhibitory target in improving the anti-tumor effect of RAK cells, and provides an autologous RAK cell culture method and a tumor treatment method. The present invention blocks the Tim-3 antigen on the autologous RAK cells, relieves the immunosuppressive effect of Tim-3 and its ligand, and thereby enhances the killing ability of autologous RAK cells against Tim-3 ligand-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown are the expression levels of immune regulatory points in 6 autologous RAK cell samples;

[0025] Figure 2 The Tim-3 expression levels of 7 autologous RAK cell samples are shown;

[0026] Figure 3 It shows the effect of Tim-3 antibody dosage on the blocking effect;

[0027] Figure 4 It shows the stability of Tim-3 antibody blocking RAK cell efficiency;

[0028] Figure 5 It showed that there was no free Tim-3 monoclonal antibody remaining in the supernatant of autologous RAK cells after blocking;

[0029] Figure 6 The results showed that the killing ability of RAK cells in vitro after Tim-3 blockade was significantly higher than that of untreated RAK cells. DETAILED DESCRIPTION

[0030] The present invention provides the application of Tim-3 and the method for culturing autologous RAK cells. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0031] The present invention has found that Tim-3 is highly expressed in autologous RAK cells. This molecule binds to the ligand and activates the inhibitory signaling pathway in T cells, which in turn leads to T cell dysfunction and reduces the anti-tumor efficacy. The present invention obtains immune cells that can be used for the treatment of tumors expressing Tim-3 ligands by blocking Tim-3 in RAK cells, thereby relieving the immunosuppression mediated by Tim-3, obtaining RAK cells with higher anti-tumor activity, and achieving a highly effective anti-tumor effect.

[0032] The test materials used in the present invention are all common commercial products, and can be purchased on the market. Among them, each antibody is commercially available.

[0033] The present invention will be further described below in conjunction with embodiments:

[0034] Example

[0035] 1. Experimental Materials

[0036] (1) Cell samples: clinical samples of autologous RAK cells, detection dosage 1×10 6 / Experimental tube;

[0037] (2) Reagents: anti-CD3, APC antibody, anti-CD4, PercP / cy5.5 antibody, anti-CD8, FITC antibody, anti-Tim-3, APC antibody, isotype control, APC, anti-CTLA-4, APC / cy7 antibody, anti-LAG-3, PE / cy7 antibody, anti-PD-1, PE antibody, blocking anti-CD366 antibody, PBS buffer

[0038] (3) Instruments: desktop centrifuge, flow cytometer, microplate reader, cell counter

[0039] 2. Experimental Procedure

[0040] Detection of autologous RAK cell immune examination

[0041] (1) Autologous RAK cell samples were obtained, with a cell number of 1×10 6 / Experimental tube;

[0042] (2) The cells of each sample were equally divided into 1.5 ml EP tubes, with a cell volume of 1×10 6 , the system is 100ul PBS buffer;

[0043] (3) Add 8ul of CD4, 8ul of CD8, 3ul of CD366, 3ul of CTLA-4, 3ul of LAG-3, and 3ul of PD-1 fluorescent antibodies to each EP tube. Take another 7 1.5ml EP tubes containing cells from the same source, one tube as a negative control, and add CD3, CD4, CD8, CD25, CTLA-4, and LAG-3 to the other 6 tubes as single staining tubes. Incubate at 4°C in the dark for 30 min.

[0044] (4) After the fluorescent antibody incubation, add 1 ml of PBS buffer to each EP tube, mix by inverting, and centrifuge at 1500 rpm for 5 min;

[0045] (5) After centrifugation, discard the supernatant, re-add 1 ml of PBS buffer to resuspend, and detect using a flow cytometer.

[0046] Detection of the optimal amount of Tim-3 blocking antibody

[0047] (1) Clinical samples were selected, and the cell volume of each sample tube was 1×10 6 cells, set 5 antibody dosage concentration gradients: 0ug / 10 6 cell,0.532ug / 10 6 cell,1.064ug / 10 6 cell,1.596ug / 10 6 cell,2.128ug / 10 6 Cells were incubated at room temperature for 30 min.

[0048] (2) After incubation at room temperature, add 1 ml of PBS buffer to elute the free antibodies, then resuspend the cell pellet with 100 ul of PBS buffer, stain with fluorescent antibodies (CD4, CD8, Tim-3), and incubate at 4°C for 30 min;

[0049] (3) Add 1 ml of PBS buffer and centrifuge at 1500 rpm for 5 min to elute, resuspend in 1 mL of PBS, and detect using a flow cytometer to select the antibody concentration with the highest blocking efficiency;

[0050] Stability of RAK cells blocked by Tim-3 antibody

[0051] (1) After obtaining clinical samples, RAK cells from the same source were placed in 6 1.5 ml EP tubes, numbered 1, 2, 3, 4, 5, and 6, with 1 × 10 cells in each tube. 6 cells, tube 1 was the control tube before blocking, and tubes 2-6 were added with blocking Tim-3 monoclonal antibody, respectively, and incubated at room temperature for 30 min;

[0052] (2) After incubation at room temperature, 1 ml of PBS buffer was added to elute the free antibodies, and the cell pellet was resuspended in 100 ul of PBS buffer, stained with fluorescent antibodies (CD4, CD8, Tim-3), and incubated at 4°C for 30 min;

[0053] (3) Add 1 ml of PBS buffer and centrifuge at 1500 rpm for 5 min to elute. Add 1 mL of PBS to tube 1 and tube 2 respectively to resuspend and detect using a flow cytometer.

[0054] (4) 1 ml of RAK cell culture medium (T551) was added to tubes 2-6 respectively, and the tubes were placed in a 37°C 5% CO2 incubator for 1 h, 2 h, 4 h, and 24 h, respectively, and then stained with fluorescent antibodies (CD4, CD8, Tim-3) and incubated at 4°C for 30 min;

[0055] (5) Add 1 ml of PBS buffer and centrifuge at 1500 rpm for 5 min to elute. Add 1 mL of PBS to tube 1 and tube 2 respectively to resuspend and detect using a flow cytometer;

[0056] Detection of free antibodies after Tim-3 antibody blocking

[0057] (1) After obtaining clinical samples, RAK cells from the same source were placed in seven 1.5 ml EP tubes, numbered 1, 2, 3, 4, 5, 6, and 7, with 1 × 10 cells in each tube. 6 cells, add blocking Tim-3 monoclonal antibody to each tube and incubate at room temperature for 30 min;

[0058] (2) After incubation at room temperature, add 1 ml of PBS buffer to elute the free antibodies, then resuspend the cell pellet with 100 ul of PBS buffer and stain with fluorescent antibodies (CD4, CD8, Tim-3). The amount of Tim-3 fluorescent antibody added to tubes 1-7 is 5 ul, 4 ul, 3 ul, 2 ul, 1 ul, and 0 ul, respectively. The cell supernatant after blocking the antibody. Incubate at 4°C for 30 min;

[0059] (3) Add 1 ml of PBS buffer and centrifuge at 1500 rpm for 5 min to elute, and then detect by flow cytometer;

[0060] In vitro killing assay

[0061] (1) Prepare target cells (gastric adenocarcinoma cell line GSE-1). Plate 5000 target cells / well in a 96-well plate overnight.

[0062] (2) Prepare effector cells (autologous RAK cells) with effector-target ratios of 20:1, 40:1, 60:1, 80:1, and 100:1, respectively. Set up five replicate wells for each effector-target ratio. The control group refers to the instructions of the Cytotoxicity LDH Assay Kit-WST@(CK12), with five replicate wells for each control group.

[0063] (3) Culture in a 37°C CO2 incubator for 30 min;

[0064] (4) Add Working Solution to each well and react at room temperature for 30 min in the dark;

[0065] (5) After adding Stop Solution to each well, immediately measure the absorbance at 490 nm using a microplate reader;

[0066] (6) The amount of solution in each well is shown in Table 1.

[0067] Result calculation: Cell damage rate (%) = [(ABE) / (CDE)] × 100%

[0068] A: absorbance of E+T well - absorbance of background Blank

[0069] B: Absorbance of low control well E - absorbance of background Blank

[0070] C: Absorbance of T high control well - absorbance of background Blank well

[0071] D: Absorbance of T high control Blank - absorbance of background Blank

[0072] E: Absorbance of low T control - absorbance of background Blank

[0073] Table 1 Solution volume in each well (one-step method)

[0074] E+T E low control T high control T High Control Blank T low control BackgroundBlank Culture medium - 50μL 50μL 100μL 50μL 100μL Effector cell suspension 50μL 50μL - - - - Target cell suspension 50μL - 50μL - 50μL - Lysis Buffer - - 10μL 10μL - - Working Solution 100μL 100μL 100μL 100μL 100μL 100μL Stop Solution 50μL 50μL 50μL 50μL 50μL 50μL

[0075] *E: effector cells, T: target cells

[0076] 3. Results description

[0077] (1) High expression of Tim-3 antigen in autologous RAK cells ( Figure 1 , Figure 2 ).

[0078] The study selected 6 autologous RAK cell samples and detected the expression of 4 immune checkpoints in RAK cells according to the technical plan. After fluorescent antibody staining, flow cytometry can divide RAK cells into several subgroups, including CD4 + Tim-3 accounts for 40%-80% of T cells ( Figure 1 Middle A), CD8 + T cells account for 65% to 90% ( Figure 1 (B).

[0079] To further verify the accuracy of the results, 7 autologous RAK cell samples were selected and the expression of Tim-3 was detected using the same method. The results were consistent ( Figure 2 ).

[0080] (2) The optimal amount of blocking Tim-3 antibody ( Figure 3 )

[0081] The study selected two clinical samples and blocked autologous RAK cells according to the technical plan. After blocking, they were labeled with fluorescent antibodies and detected by flow cytometry. 6 The amount of blocking antibody required for each cell is 1.33ug (e.g. Figure 3 In Figure A, LIGU and CHQU represent two different samples. To ensure the accuracy of the experimental results, the inventors reselected 6 autologous RAK cell samples from different sources (CLQJ, LIGU, GHRU, ZHXI, YLPI and DSHU) and divided them into 1×10 6 1.33ug of Tim-3 blocking antibody was added to each cell, and after blocking, the cells were labeled with fluorescent antibodies and detected by flow cytometry. The results showed that the blocking effect was significant, with a blocking rate of more than 90% (e.g. Figure 3 (B).

[0082] (3) Stability of Tim-3 antibody blocking RAK cell efficiency ( Figure 4 )

[0083] The study selected two clinical samples (LXYI and WRCH) and blocked autologous RAK cells according to the technical solution process. After blocking, they were placed for different time periods and tested by flow cytometry using fluorescent antibody labeling. The results showed that the blocking efficiency was stable within 24 hours (e.g. Figure 4 A in 4 and B in 4).

[0084] (4) No free Tim-3 monoclonal antibody remained in the supernatant of autologous RAK cells after blocking ( Figure 5 )

[0085] The study selected two clinical samples (ZGQI and WALI) and blocked autologous RAK cells according to the technical plan. After blocking, different amounts of Tim-3 fluorescent antibodies were added to 6 tubes, and 1 tube was added to the blocked cell supernatant. The results showed that the blocked cell supernatant did not contain free Tim-3 monoclonal antibodies (such as Figure 5 A in 5 and B in 5).

[0086] (5) The killing ability of RAK cells after Tim-3 blockade was significantly higher than that of untreated RAK cells ( Figure 6 )

[0087] Study Selection Cytotoxicity LDH Assay Kit-WST @ The (CK12) kit was used to design different effector-target ratios for in vitro killing experiments using gastric adenocarcinoma cell line (GSE-1) as target cells. The results showed that the killing ability of RAK cells blocked by Tim-3 on target cells (especially when the effector-target ratio was 100:1) was significantly higher than that of untreated RAK cells (such as Figure 6 ).

[0088] The above results fully prove that the tumor-killing ability of RAK cells blocked by Tim-3 is significantly enhanced.

[0089] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a substance that inhibits the level and / or activity of Tim-3 in the preparation of a reagent for enhancing the tumor inhibition effect of RAK cells.

2. The use according to claim 1, characterized in that: The substance that inhibits the level and / or activity of Tim-3 is a Tim-3 antibody.

3. The use according to claim 1, characterized in that: The tumor is a Tim-3 ligand-positive tumor.

4. The use according to claim 3, characterized in that: The tumor is gastric cancer, lung cancer, renal clear cell carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma or breast cancer.

5. A reagent for improving the tumor inhibition effect of RAK cells, characterized in that: Includes Tim-3 antibody and solvent.

6. The reagent according to claim 5, characterized in that in: The solvent is phosphate buffer or physiological saline; the concentration of the Tim-3 antibody is 0.1-10 μg / mL.

7. A method for improving the tumor inhibition effect of RAK cells, characterized in that: The RAK cells are mixed with the reagent according to claim 5 or 6 and then incubated.

8. The method according to claim 7, characterized in that Each 1×10 6 The cells were mixed with 0.1-10 μg of Tim-3 antibody; The incubation temperature is greater than 0°C and the incubation time is not less than 5 minutes.

9. RAK cells obtained by the method of claim 7 or 8.

10. An antitumor preparation, characterized in that: include: The RAK cell or in vitro cultured immune cell according to claim 9.

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