CN116694568A - Medium formulation for activating whole anti-tumor immune system and method for preparing agonist activated whole immune effector cells - Google Patents

Medium formulation for activating whole anti-tumor immune system and method for preparing agonist activated whole immune effector cells Download PDF

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CN116694568A
CN116694568A CN202211690866.0A CN202211690866A CN116694568A CN 116694568 A CN116694568 A CN 116694568A CN 202211690866 A CN202211690866 A CN 202211690866A CN 116694568 A CN116694568 A CN 116694568A
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苏盛
黄劲松
于和鸣
宋亚丽
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Beijing Xibo Biomedical Technology Co ltd
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Abstract

The present application provides a medium formulation for activating the whole anti-tumor immune system comprising polyI: C. CpG ODN and phosphine antigens. Also provided is a method of preparing agonist-activated whole immune effector cells comprising isolating donor autologous PBMCs and using PolyI: C. the CpG ODN and the phosphine antigen are activated and induced, so that the whole immune effector cell activated by the agonist for activating the whole anti-tumor immune system of the organism is obtained. The agonist-activated whole immune effector cells have strong IFN-gamma, granzyme B and perforin secretion capacity, and show tumor cell killing activity in vitro and in vivo.

Description

Medium formulation for activating whole anti-tumor immune system and method for preparing agonist activated whole immune effector cells
Statement of case division
The application is a divisional application of Chinese patent application with the application number of 202210195028.X and the application name of 'a culture medium formula for activating the whole anti-tumor immune system and a method for preparing agonist activated whole immune effector cells', which are filed on 3/2/2022.
Technical Field
The present application relates to immunotherapy of tumors. In particular, the application relates to an immune cell preparation useful for treating tumors.
Background
Tumor immunotherapy has evolved dramatically over a decade, and researchers have developed innovative immunotherapeutic approaches, including chimeric antigen receptor T cell (CAR-T) therapies and neoantigen tumor-infiltrating lymphocyte (TIL) and Cytotoxic T Lymphocyte (CTL) therapies; immune checkpoint inhibitor therapies include PD1/PDL1 inhibitor therapies, CTLA-4 inhibitor therapies, and the like. These treatments have been used clinically to address a number of challenges in tumor treatment, but have also revealed some limitations.
The biggest disadvantage of CAR-T therapy is the severe toxic side effects that occur during the course of treatment. A serious toxic side effect known to date is the Cytokine Release Syndrome (CRS), also known as cytokine storm, which can lead to death of the patient. And the second is neurotoxicity and off-target effect. The neurotoxicity can cause clinical symptoms such as confusion, aphasia, cerebral edema and the like, and the off-target effect can accidentally injure normal tissue cells expressing the same target antigen, so that normal tissue injury or immunodeficiency disease can be caused, and death can be caused when serious. Another disadvantage of CAR-T therapy is that the therapeutic efficacy remains to be improved, which is very pronounced for short term treatment of lymphocytic leukemia and lymphoma, but relapse occurs several months or more after treatment. The third disadvantage is that CAR-T therapy has poor or no therapeutic effect on solid tumors, because CAR-T is difficult to enter the lesions of solid tumors and infiltrate into the tumor; even if it can infiltrate into the inside of solid tumor, it will face the inhibition of tumor microenvironment, making T cells unable to exert anti-tumor effect. The fourth disadvantage is that currently available CAR-T products all use own T cells as starting materials, which is difficult to industrialize, and the price of the product is up to hundreds of thousands of dollars, which is unfavorable for market popularization.
Neoantigens are polypeptides obtained by transcription, translation and processing of nonsensical mutations in tumor cells. Since no tumor antigen is expressed in normal cells, the neoantigen-specific immune reaction does not undergo a central tolerance mechanism and a peripheral tolerance mechanism, so that the neoantigen has application value as a therapeutic target of immunotherapy in theory. With the development of high throughput gene sequencing technology, whole genome and exon sequencing technology can help researchers obtain mutation information (including point mutations, insertional mutations, etc.) on the genome. How to rapidly and accurately identify candidate neoantigens from the data and screen out the neoantigens with high immunogenicity is a urgent problem in the field of tumor immunotherapy. Currently available tools such as pVAC-seq, muPeXI, neopepsee solve the problem of identifying new antigens to some extent, but the effective screening and sequencing of candidate antigens still has a disadvantage. Therefore, in the core technology of preparing new antigen TIL or CTL, there are many technical bottlenecks, which result in low accuracy of target antigen, and the clinical efficacy of the new antigen TIL and CTL therapy is unsatisfactory, and in addition, the preparation period is long, the cost is high, and most patients cannot accept the new antigen TIL or CTL therapy, so that the anti-tumor immune cell therapy cannot be widely used clinically.
In recent years, more and more clinicians have applied immune checkpoint inhibitors to treat a variety of tumors. The immune check point with the most extensive clinical application at present also has the problems of low overall response rate, adverse reaction, drug resistance and the like in the treatment process. For example, PD-1 inhibitors have an effective rate of only 10-30% in non-selected solid tumor patients, but if a treatment subject is selected with tumor gene burden (TMB) as a marker associated with immune checkpoint inhibitor efficacy, a high effective rate of 62% is found for TMB, whereas immune checkpoint inhibitor efficacy-related markers are currently under study, the specific mechanism and accuracy of prediction of which are under verification in large-scale prospective clinical studies. In addition, adverse effects associated with immunotherapy caused by immune checkpoint inhibitors may involve individual organs throughout the body, cause damage to a plurality of organs, and may cause acquired drug resistance after long-term use of immune checkpoint inhibitors.
10 months 2019, oliver Demaria et al published a review on Nature, titled "Harnessing innate immunity in cancer therapy" (tumor treatment with innate immunity). In this review, it was proposed that current immunotherapy of tumors has evolved significantly, but existing therapies (various immunotherapies as described above) are focused on MHC-restricted specific T cells. While these anti-tumor therapies have met with considerable success, only a fraction of patients respond to treatment with a lower overall therapeutic response rate. In recent years, the focus of tumor immunotherapy has gradually shifted to innate immunity.
Disclosure of Invention
The application provides a method for activating innate immunity by using a novel combination of multiple agonists and immunostimulants based on a mode recognition receptor (PRR) theory of targeting innate immunity, thereby activating adaptive immunity and leading to activation of cells of the whole immune system, thereby achieving the purposes of killing and eliminating tumors.
The term "wat" is an english abbreviation for the english "Whole Agonist Stimulation" as used herein, meaning the overall immune response activated by an agonist. Herein, when referring to "wat cells" or "wat effector cells" we mean agonist-activated whole immune effector cells of the application; when referring to "wat precursor cells" it is meant to refer to agonist activated whole immune precursor cells of the application.
In a first aspect, the application provides a culture medium formulation comprising polyI: C (polyinosinic acid), cpG ODN (i.e., oligodeoxynucleotide comprising CpG motifs), and a phosphine antigen.
In a second aspect, the application provides a method of preparing an agonist-activated whole immune effector cell (i.e., a watt effector cell), the method comprising:
(1) Culturing Peripheral Blood Mononuclear Cells (PBMC) to obtain adherent cells and non-adherent suspension cells; collecting adherent cells, and performing cytokine induction culture to obtain immature dendritic cells;
(2) Continuing to induce and culture the immature dendritic cells obtained in step (1) to obtain mature dendritic cells;
(3) Culturing the non-adherent suspension cells obtained in step (1) in a culture medium formulation of the first aspect to obtain agonist-activated whole immune precursor cells (i.e., wat precursor cells);
(4) Mixing and culturing the mature dendritic cells prepared in the step (2) with the agonist-activated whole immune precursor cells prepared in the step (3) to obtain the agonist-activated whole immune effector cells.
In a third aspect, the application provides an agonist-activated whole immune effector cell (i.e., a watt effector cell), wherein the agonist-activated whole immune effector cell has a total T lymphocyte percentage of greater than 90%.
In a fourth aspect, the present application provides the use of an agonist-activated whole immune effector cell of the third aspect for the preparation of an anti-tumour cell preparation.
Compared with the existing anti-tumor immunotherapy, the application has the following advantages: (1) The WAST cell preparation consists of immune cells with various different killing mechanisms, and can be used singly or in combination with the existing tumor treatment method and medicine clinically to generate higher effectiveness; (2) Because the used multiple agonists and immunostimulants have good safety and effectiveness, the used main starting materials are autologous PBMC of patients or frozen PBMC conforming to the regulations, and the used cell culture and amplification reagents conform to the GMP regulations, the WAST cell preparation has good safety; (3) WAST cells belong to activated immune cells, so that defects and adverse reactions caused by using various T cells in the prior art can be avoided; (4) WAST cells are broad-spectrum tumor killing agents and can be widely used for treating various solid tumors; (5) The WAST cell preparation does not relate to complex preparation processes such as gene modification, gene recombination and the like, has short preparation period and high safety, and is easy to clinically popularize and apply; (6) The WAST cell preparation can be combined with radiotherapy, chemotherapy and targeted therapy, and the curative effect is improved through synergistic effect; (7) The TNM stage is treated by WAST cell preparation after early operation of stage I or stage II cases, and can reduce recurrence rate.
Drawings
FIG. 1 outlines the preparation of WAST cell preparations of the present application and their anti-tumor mechanisms.
FIG. 2 shows the ratio of cells secreting granzyme B and perforin before and after induction, wherein l represents granzyme B secreting cells, m represents perforin secreting cells, and n represents granzyme B and perforin secreting cells simultaneously; error bars refer to 95% Confidence Intervals (CIs) for various cell measurements.
Figure 3 shows the measurement of the percentage of various cell types before and after induction by agonists and immunostimulants. a: percentage of total T lymphocytes; b: percentage of T helper cells; c: percent killer T cells; e: percentage of total B lymphocytes; f: percentage of NK cells; g: percentage of NKT cells; h: cd25+ cell percentage; i: percentage of monocytes; j: percentage of HLA-DR+ cells; k: vγ9δ2 (CD 4-CD8 low-cd3+) percent; error bars refer to 95% Confidence Intervals (CIs) for various cell measurements.
Detailed Description
The application provides a culture medium formulation comprising polyI: C, cpG ODN and phosphine antigen.
Preferably, the medium formulation further comprises IL-2, IL-15, an anti-CD 3 antibody, an anti-CD 28 antibody, and the phosphine antigen is zoledronic acid;
more preferably, the medium formulation comprises IL-2 at a concentration of 100-500IU/ml, IL-15 at 5-50ng/ml, anti-CD 3 antibody at 0.5-5. Mu.g/ml, anti-CD 28 antibody at 0.5-5. Mu.g/ml, polyI: C at 2-20. Mu.g/ml, cpG ODN at 2-20. Mu.g/ml, zoledronic acid at 10-100. Mu.mol/L;
further preferably, the medium formulation comprises IL-2 at a concentration of 300-500IU/ml, IL-15 at 10-50ng/ml, anti-CD 3 antibody at 1-3 μg/ml, anti-CD 28 antibody at 1-3 μg/ml, polyI: C at 5-15 μg/ml, cpG ODN at 10-75 μmol/L zoledronic acid;
most preferably, the medium formulation comprises IL-2 at a concentration of 300IU/ml, IL-15 at 15ng/ml, anti-CD 3 mab at 1. Mu.g/ml, anti-CD 28 mab at 1. Mu.g/ml, polyI: C at 10. Mu.g/ml, cpG ODN at 10. Mu.g/ml, zoledronic acid at 50. Mu.mol/L.
The application also provides a method of preparing an agonist-activated whole immune effector cell (i.e., a wat effector cell), the method comprising:
(1) Culturing Peripheral Blood Mononuclear Cells (PBMC) to obtain adherent cells and non-adherent suspension cells; collecting adherent cells, and performing cytokine induction culture to obtain immature dendritic cells;
(2) Continuing to induce and culture the immature dendritic cells obtained in step (1) to obtain mature dendritic cells;
(3) Culturing the non-adherent suspension cells obtained in step (1) in a culture medium formulation of the application to obtain agonist activated whole immune precursor cells (i.e., wat precursor cells);
(4) Mixing and culturing the mature dendritic cells prepared in the step (2) with the WAST precursor cells prepared in the step (3) to obtain the agonist activated whole immune effector cells.
In a specific embodiment, the method further comprises the step of isolating peripheral anticoagulants from the donor and isolating Peripheral Blood Mononuclear Cells (PBMCs) therefrom prior to step (1).
The donor is a healthy person or a tumor patient.
In a specific embodiment, the cytokines in step (1) are granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4).
In another embodiment, step (2) is performed using interleukin-2 (IL-2), interleukin-33 (IL-33) and tumor necrosis factor alpha (TNF-alpha) for induction culture.
The application also provides an agonist-activated whole immune effector cell (i.e., wat effector cell), characterized in that the percentage of total T lymphocytes in said wat effector cell is greater than 90%.
In a specific embodiment, the WAST effector cells have a killer T cell percentage of 45% to 70%, a NKT cell percentage of 7.5% to 25%, and a V gamma 9 delta 2 cell percentage of 5% to 50%.
In particular, the percentage of total T lymphocytes in the watt effector cells is 99.01%, 98.8%, 98.78%, 99.12%, 96.98%, 93.89%;
in particular, the percentage of killer T cells in the watt effector cells is 67.42%, 47.34%, 45.35%, 48.31%, 46.37%, 69.07%;
in particular, the percentage of NKT cells in the watt effector cells is 17.89%, 19.92%, 24.63%, 10%, 7.5%, 10.55%;
in particular, the percentage of vγ9δ2 cells in the watt effector cells is 38.39%, 11.02%, 12.08%, 5.68%, 47.57%, 19.8%;
in yet another specific embodiment, the WAST effector cells have a percent of granzyme B secreting cells of 15% -55%, perforin secreting cells of 15% -50%, and granzyme B and perforin secreting cells of 10% -45%.
In particular, the percentage of cells secreting granzyme B in the wat effector cells is 24.95%, 41.16%, 25.92%, 52.93%, 15.62%, 23.77%;
in particular, the percentage of cells secreting perforin in the watt effector cells is 48.34%, 39.07%, 39.66%, 48.72%, 15.35%, 20.1%;
in particular, the percentage of cells that secrete granzyme B and perforin simultaneously in the watt effector cells is 21.35%, 31.6%, 21.25%, 43.7%, 10.8%, 15.1%.
The application also provides application of the WAST effector cell in preparing an anti-tumor cell preparation. Preferably, the tumor is a solid tumor; more preferably, the solid tumor is lung cancer, liver cancer or breast cancer.
In the present application, the agonists PolyI: C, cpG ODN and the immunostimulant phosphine antigen are used in combination.
The present application will be described in detail with reference to the following examples. The experimental methods in the following examples were carried out under the conditions described in the "ATCC cell culture Manual" according to the conventional experimental conditions without specifying the specific technique or conditions, and the conditions suggested by the specification were referred to when the specifications of the reagent company are explicitly described in the examples. The reagents or instruments used are not specific to the manufacturer and are commercially available in conventional form.
Agonists and immune inducing agents used in the examples of the present application: (1) AIM-V cell culture medium available from ThermoFisher company; (2) Human lymphocyte separator tube (ready-to-use), available from daceae as company; (3) IL-4, GM-CSF, TNF- α, IL-2, IL-15, IL-33, anti-CD 3 mab, anti-CD 28 mab, all available from Peprotech; (4) PolyI C, cpG ODN (5'-tcgtcgttttcgcg-3'), all available from InvivoGen; (5) zoledronic acid, purchased from the national drug group.
EXAMPLE 1 extraction and cultivation of Peripheral Blood Mononuclear Cells (PBMC)
(1) The donated peripheral anticoagulants from healthy people were isolated at 50ml. 25ml of peripheral blood was added to each of the two 50ml separation tubes.
(2) The centrifuge tube was trimmed, slowly raised and lowered at 1700rpm with a horizontal centrifuge, and centrifuged at room temperature for 20min.
(3) Excess plasma was aspirated from the uppermost portion. The lymphocyte layer was gently aspirated with a 2ml sterile pipette, transferred into a new 50ml centrifuge tube, and the lymphocyte layer in all tubes was aspirated into the same 50ml centrifuge tube. 0.9% sterile physiological saline was added to 50ml, and thoroughly mixed.
(4) Balancing a centrifuge tube, rapidly lifting and descending the centrifuge tube at 1700rpm by using a horizontal centrifuge, and centrifuging the centrifuge tube at room temperature for 10min; the tube was removed and the supernatant was completely discarded.
(5) The preheated AIM-V medium was removed from the 37℃incubator, 5ml of the cell pellet was taken therefrom, thoroughly mixed, carefully handled to avoid the generation of air bubbles, and then AIM-V medium was added to a cell concentration of 1X 106 cells/ml, and the cells were mixed.
(6) The homogenized cell suspension was transferred to 75cm 2 The cell culture flask is gently shaken and mixed uniformly, and placed in a 37 ℃ incubator for culture.
EXAMPLE 2 preparation of Dendritic Cells (DCs)
(1) PBMC obtained in example 1 were resuspended in AIM-V medium and added at 75cm 2 In a cell culture flask, 37 ℃,5% CO 2 Culturing.
(2) After 3 hours, the flask was removed, gently shaken, and the suspended cells were aspirated.
(3) AIM-V medium containing 500IU/ml IL-4 and 500IU/ml GM-CSF was added to the flask.
(4) Half-volume changes were made on day 3 and day 5 after the culture, respectively, and AIM-V medium containing 500IU/ml IL-4 and 500IU/ml GM-CSF was added.
(5) AIM-V cell culture medium was added at day 7 with final concentration of 10ng/ml TNF- α, 300IU/ml IL-2, 10ng/ml IL-33.
(6) Mature dendritic cells were harvested on day 8.
Example 3 preparation of WAST effector cells
(1) Suspension cells were obtained by step (2) of example 2, resuspended in AIM-V medium, and the cell concentration was adjusted to 1X 106 cells/ml, the suspension being enriched for T lymphocytes. To this suspension was added IL-2 at a final concentration of 300IU/ml, IL-15 at 15ng/ml, anti-CD 3 mab at 1. Mu.g/ml, anti-CD 28 mab at 1. Mu.g/ml, polyI: C at 10. Mu.g/ml, cpG ODN at 10. Mu.g/ml, zoledronic acid at 50. Mu.mol/L, 37℃C, 5% CO 2 Culturing for 8 days, and changing liquid every 3 days to obtain WAST precursor cells;
(2) On day 8, the mature dendritic cells prepared in example 2 were mixed with wat precursor cells in a ratio of mature dendritic cells to wat precursor cells = 1:10 and then cultured for 2 days;
(3) Collecting cells on day 10 to obtain WAST effector cells.
Example 4WAST effector cell phenotype assay
6 healthy human WAST effector cells were obtained according to the preparation method of WAST effector cells described in examples 1-3. Cell phenotyping was performed using a flow cytometer to determine the change in percentage of each type of cells before and after induction (before induction: PBMC, respectively, 1 to 6; after induction: WAST effector cells, respectively, 1 'to 6') using the medium formulations of the present application, and the results are shown in the following table.
Paired T-test statistical data analysis was performed on the data using data analysis software SPSS19.0, all tests were double sided tests, test level α=0.05. The analysis results are shown in FIG. 3.
As can be seen from fig. 3, the percentage of cells associated with tumor killing in wat effector cells obtained after induction was significantly increased. Taking sample 3 as an example: for the total T lymphocyte percentage, 57.83% before induction and 98.78% after induction; for the percentage of killer T cells, 13.97% before induction and 45.35% after induction; for the percentage of NKT cells, 2.54% before induction and 24.63% after induction; for the percentage of vγ9δ2 (CD 4-CD8 Low-cd3+) cells, 3.42% before induction and 12.18% after induction. These cells are the main killer cells in watt effector cells, with a significant increase in proportion after induction.
Example 5 Granzyme B and Perforin (Perforin) secretion Capacity assay of WAST effector cells
The ability of wat effector cells prepared in example 4 to secrete granzyme B, perforin was examined by flow cytometry. The following table illustrates the change in secretion capacity of cells before and after induction (before induction: PBMC, respectively denoted by 1 to 6; after induction: WAST effector cells, respectively denoted by 1 'to 6').
Paired T-test statistical data analysis was performed on the data using data analysis software SPSS19.0, all tests were double sided tests, test level α=0.05. The analysis results are shown in FIG. 2.
As shown in fig. 2, the number of cells that secreted granzyme B or perforin or both granzyme B and perforin after induction was significantly increased, indicating that wat effector granzyme B, perforin secretion was stronger than PBMC.
Example 6 determination of the in vitro direct killing ability of WAST effector cells against 3 different tumor cell lines
In this example, WAST effector cells were obtained according to the preparation method in examples 2-3, respectively, using 3 exception PBMC (all from Shanghai Miao Biotechnology Co.) as starting material, and designated WAST-121061203C, WAST-121030702C, WAST-121050801C, respectively. The three cells are taken as effector cells, A549 (human non-small cell lung cancer), MCF-7 (human breast cancer cells) and HepG2 (human liver cancer cells) are taken as target cells, and the in vitro direct killing test is carried out with the effective target ratio of 20:1 and 30:1 respectively.
The release assay was tested using CCK8 (cell counting kit (Cell Counting Kit) -8) to test the ability of wat cells to kill a549, MCF-7, hepG2 in vitro, and the method was performed as follows:
(1) Adjusting the target cell concentration to 5X 10 4 Individual/ml;
(2) The following control groups were set up: blank control, only 200. Mu.l AIM-V medium per well; target cell control group, without effector cells, add 100 μl AIM-V medium per well, and then add 100 μl target cells; effector cell control group, without target cells, 100. Mu.l AIM-V medium was added to each well, followed by 100. Mu.l effector cells; three duplicate wells were set for each group and were all at 37 ℃,5% co 2 Culturing for 24 hours;
(3) For the experimental group, target cells were transferred at 100. Mu.l/wellMoving to 96-well plate, setting three compound wells for each group, and setting 5% CO at 37 DEG C 2 Culturing for 24 hours (at which time the number of target cells increased to 10000 per well); then adding 100 μl of effector cells to each experimental well, wherein the ratio of effector cells to target cells (i.e. the effective target ratio) is 20:1 and 30:1 respectively;
(4) The control group and the experimental group were treated with 5% CO at 37℃respectively 2 Culturing for 4-6 hours;
(5) Mu.l of CCK8 reagent is added to each well and the mixture is cultured for 2 hours at 37 ℃;
(6) The enzyme label instrument measures the absorbance value OD450 of each hole, and the killing activity is calculated by taking the average value A of the compound holes, wherein the calculation formula is as follows:
the killing rate (%) = [1- (a experimental group-a effector cell control group)/(a target cell control group-a blank control group) ]x100%.
The following tables list the absorbance OD450 and calculated killing rates measured in each control group and experimental group in vitro killing experiments using a549 (human non-small cell lung cancer), MCF-7 (human breast cancer cell), hepG2 (human liver cancer cell) as target cells, respectively.
Results of WAST effector cells killing lung cancer cell line A549 in vitro
Results of WAST effector cell killing breast cancer cell line MCF-7 in vitro
Results of WAST effector cells killing liver cancer cell line HepG2 in vitro
(1) Direct killing results of WAST effector cells on A549 (human non-small cell lung cancer)
WAST-121061203C has killing rates of 41.94% and 53.18% respectively at effective target ratios of 20:1 and 30:1;
WAST-121030702C has killing rates of 36.37% and 47.36% respectively at effective target ratios of 20:1 and 30:1;
WAST-121050801C has effective target ratio of 20:1, 30:1 of 80.37% and 96.33%.
(2) Results of direct killing of MCF-7 (human breast cancer cells) by WAST effector cells
WAST-121061203C has killing rates of 9.17% and 17.20% respectively at effective target ratios of 20:1 and 30:1;
WAST-121030702C has killing rates of 17.83% and 32.67% respectively at effective target ratios of 20:1 and 30:1;
WAST-121050801C has effective target ratio of 20:1, 30:1 of 11.94% and 20.82%.
(3) Results of direct killing of WAST effector cells on HepG2 (human hepatoma cell)
WAST-121061203C has killing rates of 17.81% and 28.90% respectively at effective target ratios of 20:1 and 30:1;
WAST-121030702C has killing rates of 19.33% and 31.98% respectively at effective target ratios of 20:1 and 30:1;
WAST-121050801C has effective target ratios of 20:1 and 30:1 of 25.51 percent and 44.24 percent respectively.
The in vitro direct killing results of the three WAST effector cells on 3 different tumor cell lines show that the WAST effector cells have in vitro killing effects on tumor cells, and the anti-tumor activity of the WAST effector cells is proved.
EXAMPLE 7 efficacy experiment of WAST cells in mice killing solid tumors (taking non-Small cell lung cancer tumor-bearing model as an example)
In this example, NPI immunodeficient mice (carried out by Beijing Ai Dema Biotechnology Co., ltd.) were used as CDX lung cancer A549 tumor-bearing mice model, with a week age of 6-8 weeks and a body weight of 20-30g. When the tumor in the mouse grows to 80-150mm 3 At this time, human WAST cells were injected via tail vein and intratumoral tumor periphery in time-point fractions, and tumor growth was observed and tumor size was measured.
The specific operation method is as follows:
(1) The WAST effector cell WAST-121030702C in example 6was suspended in physiological saline for use;
(2) WAST effector cell injection frequency was 1 time per week for 3 weeks, and WAST effector cell injection was performed 3 times on day 0 (D0), day 7 (D7), and day 14 (D14), respectively, with the first injection as day 0; the experiments were divided into 3 groups, respectively: saline group, intratumoral tumor peri-injection group (i.t.) (the group was injected with wat-121030702C cells), intravenous injection group (i.v.) (the group was injected with wat-121030702C cells), 3 CDX tumor-bearing mice per group;
(3) Determining the injected dose of wat cells: WAST cells were used at a clinically expected dose of 5X 10 based on a 60kg adult 8 -5×10 9 Individual cells (i.e. 0.8X10) 7 -0.8×10 8 Individual cells/kg). Based on the CDX mice weighing 30g, the normal maximum tolerated dose is 1X 10 7 The individual cells (this dose is the highest dose used in the mouse animal test of the current cell products) are the high dose group;
(4) Injection dose: physiological saline group, 200 μl of physiological saline was injected into each mouse; intratumoral injection group, 1×10 per injection per mouse 7 A cell; intravenous injection group, 1×10 per injection per mouse 7 A cell;
after 3 injections were completed, the tumor-bearing volume of the mice was measured on day 25. The caliper is used for measuring the long diameter and the short diameter of the tumor, and the calculation formula of The Volume (TV) of the tumor is as follows: tv=0.5a×b 2 (a=long diameter length, b=short diameter length). Tumor volume measurements for the 3 groups of mice were as follows:
project Physiological saline group Intratumoral injection group Intravenous injection group
Tumor mean volume (mm) 3 ) 363.14 275.30 248.80
Median volume (mm) 3 ) 418.01 311.22 248.80
From the above table, after continuously injecting wat effector cells, tumor-bearing volumes of mice were measured on day 25, and the average tumor volumes of mice in the intratumoral injection group and intravenous injection group were smaller than those of the normal saline group.
In addition, on day 17, immunocytophenotype flow assays were performed on mouse peripheral blood via a flow cytometer, wherein the results of cd8+cd69+ flow assays on mouse peripheral blood were as follows:
project Physiological saline group Intratumoral injection group Intravenous injection group
CD8 + CD69 + Percentage (%) 0 11.92 74.11
The results of the phenotype flow type detection of the peripheral blood immune cells of the mice on the 17 th day show that the CD8 of the intravenous injection group + CD69 + The percentage was 74.11%, while the physiological saline group CD8 + CD69 + Percentage was 0%, CD8 in intratumoral peri-injection group + CD69 + The percentage was 11.92%. Depending on the route of administration, the intravenous injection group and the intratumoral peri-intratumoral injection group differ in the state in which immune cells are activated.
Conclusion: WAST effector cells are activated in mice in the intravenous injection group and the intratumoral peritumoral injection group, and after 3 times of WAST effector cell injection, the analysis of the results shows that the WAST effector cells can inhibit tumors whether the intratumoral peritumoral injection or the intravenous injection WAST effector cells are injected.
EXAMPLE 8B-NDG mice intravenous WAST effector cell preparation toxicity animal test for Single administration
Purpose of test
The WAST cell preparation is an unmodified immune cell, is clinically used for treating solid tumors, and is administrated by intravenous infusion. The experiment shows that the property, the degree and the reversibility of toxic reaction possibly caused by WAST cell preparation are observed 14 days after the WAST cell preparation is singly administrated to the B-NDG mouse through veins, the target tissue of a toxic target organ is presumed, and reference information is provided for clinical safety. The receiving units of the experiment are as follows: hebei medicine Co., ltd (GLP) of Guozai.
SPF grade B-DNG mice (purchased from Biotechnology of Biotechnology Inc. of the Bai Osai Jiangsu Gene) were used for this experiment. The weight of female mice is 18.84g-22.01g, the weight of male mice is 26.14g-28.89g, and the weight of individuals with the same sex is within +/-20% of the average weight. Mice were approximately 7 weeks of age at the time of group administration.
Dose design
The maximum dose method is adopted in the test, WAST effector cells are WAST-121030702C, and the low and high dose groups of WAST effector cells are respectively designed to have 1×10 dose concentration 7 Cell/ml, 4.25X10 7 Cells/ml, administration volume of 20ml/kg body weight, and administration dose of 2×10, respectively 8 Cell/kg body weight, 8.5X10 8 The cell/kg body weight is about 2.4-10 times of the clinical human dose. The solvent control group was given an equal dose of a cell cryopreservation protection premix (purchased from Nanjing Sansheng Biotechnology Co., ltd., main ingredients: dextran, glucose, process water).
Experimental method
90B-DNG mice were randomly divided into 3 groups of 30 animals each, each half: solvent control group, WAST effector cell low dose group (2X 10) 8 Cell/kg), WAST effector cells high dose group (8.5X10) 8 Cells/kg). The administration volume of each group was 20ml/kg. For WAST effector cell low dose group, administration was 1X 10 by tail vein injection 7 Cells/ml; for the WAST effector cell high dose group, 4.25X10 were administered by tail vein injection 7 Cells/ml; the solvent control group was given an equal volume of cytoprotective solution by tail vein injection. The day of administration was day 1 of the experiment (i.e., D1).
After administration, the general state of each group of mice is observed every day, and the weight and the feeding amount are respectively measured; at the end of the observation period at D15, each group of mice was euthanized after weighing, and was subjected to hematology, hemagglutination, biochemical blood test, general anatomic observation, and main organ weighing, and histopathological examination was performed on abnormal organs found by general anatomic observation.
Results
Under the test conditions, the B-DNG mice were single intravenous injection of WAST effector cells and recovered from drug withdrawal for 14 days. During the trial, none of the mice in each group had seen the obvious abnormal changes in general status, body weight, and food intake associated with the injection of wat effector cells. No animal death and severe toxic reactions were seen. The Maximum Tolerated Dose (MTD) of mice to WAST effector cells was 8.5X10 8 Cells/kg.
EXAMPLE 9 preliminary clinical pre-testing of WAST cell preparations on tumor patients
To observe the safety of wat cell formulations in treating tumors and their possible effectiveness, the following conditions were used: 1. animal experiments prove that the safety is high; 2. patient volunteers request wat cell preparation treatment; 3. no fee is charged for treatment.
The following 5 tumor patients were treated with WAST effector cells for 1-2 courses. Clinical pathology and TNM staging for 5 patients were as follows:
case number Clinical grade pathological diagnosis TNM staging
Example 1 Malignant tumor for craniocerebral orbital communication Stage IV B
Example 2 HBV-associated hepatocellular carcinoma Stage III A
Example 3 HBV-associated hepatocellular carcinoma Stage III B
Example 4 After breast cancer surgery Stage III B
Example 5 After breast cancer surgery Stage IV B
The specific treatment process is as follows:
(1) Peripheral anticoagulants from the above 5 patients were isolated, and WAST effector cells from each patient were prepared and stored for later use as described in examples 1-3.
(2) Each patient was administered the prepared individual wat effector cells.
The WAST effector cells were administered to each patient by intravenous infusion at a dose of 5X 10 during the course of treatment 8 -5×10 9 Cells, were administered 3-6 times consecutively to WAST effector cells. No adverse reactions were observed during the treatment. All cases observed for 6-12 months showed stable disease conditions, and reduced tumor, cancer cells or metastatic cancer numbers, up-regulated quality of life. Clinical studies are currently underway.

Claims (6)

1. A wat effector cell, wherein the percentage of total T lymphocytes in said wat effector cell is greater than 90%.
2. The wat effector cell of claim 1, wherein the wat effector cell has a killer T cell percentage of 45% to 70%, a NKT cell percentage of 7.5% to 25%, and a vγ9δ2 cell percentage of 5% to 50%.
3. The wat effector cell of claim 1 or 2, wherein the wat effector cell has a percentage of cells that secrete granzyme B of 15% -55%, a percentage of cells that secrete perforin of 15% -50%, and a percentage of cells that secrete granzyme B and perforin of 10% -45%.
4. Use of a wat effector cell according to any one of claims 1-3 for the preparation of an anti-tumor cell preparation.
5. The use of claim 4, wherein the tumor is a solid tumor.
6. The use of claim 5, wherein the solid tumor is lung cancer, liver cancer or breast cancer.
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