CN110819703A

CN110819703A - Method for evaluating safety of CART cells

Info

Publication number: CN110819703A
Application number: CN201810888439.0A
Authority: CN
Inventors: 刘雅容; 史子啸
Original assignee: Shanghai Hrain Biotechnology Co Ltd
Current assignee: Shanghai Hrain Biotechnology Co Ltd
Priority date: 2018-08-07
Filing date: 2018-08-07
Publication date: 2020-02-21

Abstract

The invention provides a method for evaluating the preclinical toxicology of CART cells. The system systematically expounds toxicology detection indexes of CART cells before entering clinic, and the evaluation is divided into in vivo experiment evaluation and in vitro experiment evaluation.

Description

Method for evaluating safety of CART cells

Technical Field

The invention relates to the field of medical biology, and in particular relates to a preparation method of CART cells.

Background

Chimeric Antigen Receptor-T cell (CAR-T) T cell refers to a T cell that is genetically modified to recognize a specific Antigen of interest in an MHC non-limiting manner and to continuously activate expanded T cells. The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors besides surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. CAR-T cell back-infusion therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of studies show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and remarkably improve the survival condition of patients.

Chimeric Antigen Receptors (CARs) are a core component of CAR-T, conferring on T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of the CAR includes a tumor-associated antigen (TAA) binding region (usually derived from scFV fragment of the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.

Before clinical experiments, the CART cells need systematic evaluation of drug effect, pharmacokinetics, pharmacology and toxicology, and the safety and effectiveness of the CART cells are comprehensively researched. The invention systematically describes a CART preclinical safety assessment method.

Disclosure of Invention

One aspect of the invention provides a method for assessing the safety of CD19-CART cells, including in vitro assay assessment methods and in vivo assay assessment methods.

A second aspect of the invention provides an in vitro assay evaluation method comprising:

analyzing and detecting a gene insertion site;

(II) soft agar cell growth test;

(III) in vitro culture amplification test.

Preferably, gene insertion site analysis detects retroviral insertion sites on the human genome by high throughput sequencing.

Preferably, the soft agar cell growth assay detects whether the CART cells form clones. .

Preferably, the in vitro culture expansion test is to observe the cell state and cell proliferation under culture conditions of different IL-2 concentrations.

A third aspect of the invention provides an in vivo experimental evaluation method comprising:

(I) single administration mode;

(II) repeat dosing model;

(III) a vascular stimulation model;

(IV) formulation safety model.

Preferably, the single-dose model is a single injection of the CART cell model in immunodeficient and tumor-bearing immunodeficient mice.

Preferably, the repeated administration model is a cynomolgus monkey and normal C57BL mouse multiple injection CART cell model.

Preferably, the vascular stimulation model is a New Zealand rabbit intravenous auricular injection CART cell model.

Preferably, the preparation safety model is to inject CART frozen stock solution into mice and observe whether or not the organs are abnormal.

The invention has the following beneficial effects: the invention relates to a method for comprehensively evaluating the safety of CART cells.

Drawings

FIG. 1 model diagram of repeated administration

Detailed Description

Examples

The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The in vitro detection method of the present invention is a conventional experimental method, i.e., high throughput sequencing, clone formation experiment, cell proliferation detection experiment, which is not described in this example. In the examples in vivo evaluation experiments are presented.

Example 1 Single dose model of NOG mice

NOG mice, female, 6 weeks old. The group was divided into NT cell control group and CAR-T group. Each group contained 30 mice.

2. The tail vein was injected with the corresponding solvent or CAR-T injection in a volume of 200. mu.l per mouse. The number of cells per mouse was 2.5X 10⁸A CAR⁺Cells/kg. The day of dosing was marked 0D.

3. The survival time of each group of mice was observed and a survival curve was plotted.

The results of this example are shown in the figure, the CART group significantly extended the survival of tumor-bearing mice. .

Example 3 repeat dosing model

C57BL mice, half female and half, 6 weeks old. Divided into a vehicle cell control group 1, a vehicle cell control group 2, a CAR-T group 1, a CAR-T group 2, and a CAR-T group 2, with 50 mice per group.

2. The tail vein was injected with the corresponding solvent or CAR-T injection in a volume of 200. mu.l per mouse. Group 1 and 2 were given physiological saline as vehicle control; group 3 and 4 administration of anti-mouse CD19T cells 1X 10 for the first administration⁷One cell/one; secondary administration 5X 10⁶Two doses of 1X 10 cells/cell, group 5 anti-mouse CD19T cells⁶One cell/one. Pretreatment is carried out 1 day before the two times of administration in the groups 2, 3 and 4, and the pretreatment method is to inject 200mg/kg of cyclophosphamide into the abdominal cavity.

4. The survival time of each group of mice was observed and a survival curve was plotted.

The results of this example show that the animals of each group were not abnormal in clinical observation, body weight, food intake, body temperature, blood cell count, biochemical index of blood, organ weight, gross anatomy, etc.

Example 4 vascular stimulation model

1. New Zealand rabbits, male and female 2 each, marginal ear vein infusion CAR⁺T cell 15.1X 10⁶The injection dosage form is 90% normal saline and 10% human serum albumin;

2. new Zealand rabbits, male and female 2 each, marginal ear vein infusion CAR⁺T cells 5.4X 10⁶CAR⁺The injection volume is 10ml/kg, and the injection dosage form is CART cell infusible frozen stock solution.

The results of this example show that neither of the two formulations of CART cell injection has a significant irritant effect on the marginal veins of rabbits.

Example 5 formulation safety model

ICR mice, male and female half, total 30. The groups were divided into 3 groups: 1X 10⁷Total viable cells/individual; negative control group: 0.9% sodium chloride injection; vehicle control group: and (5) freezing and storing the liquid. Each group contained 10 mice.

2. The tail vein was injected with the corresponding solvent or CAR-T injection at a dose of 10ml/kg per mouse.

4. Each group of mice was observed for 2 weeks.

The results of this example show that no significant abnormality was observed in the major organs (heart, liver, spleen, lung, kidney, stomach, brain) of mice in the general caesarean section vehicle control group, negative control group and test sample group, indicating that the formulation meets the safety requirements.

Claims

1. A method for evaluating the safety of CD19-CART cells comprises an in vitro experiment evaluation method and an in vivo experiment evaluation method.

2. The in vitro assay of claim 1 wherein the assay comprises gene insertion site assay, soft agar cell growth assay, in vitro culture amplification assay.

3. The in vivo experimental evaluation as claimed in claim 1, wherein the in vivo experimental evaluation comprises a single administration model, a repeated administration model, a vascular stimulation model, and a preparation safety model.

4. The gene insertion site assay of claim 2 wherein the retroviral insertion site is detected on the human genome by high throughput sequencing.

5. The soft agar cell growth assay of claim 2, which detects whether CART cells form clones.

6. The in vitro culture amplification assay of claim 2, wherein the cell status and cell proliferation are observed under culture conditions with different IL-2 concentrations.

7. The single-dose model of claim 3, which is a single injection of the CART cell model in normal immunodeficient and tumor-bearing immunodeficient mice.

8. The immunodeficient mouse of claim 3 is a NOG mouse.

9. The repeated administration model of claim 3 is a multiple injection CART cell model in cynomolgus monkeys and normal C57 mice.

10. The vascular stimulation model of claim 3 is a New Zealand rabbit ear vein injection CART cell model.

11. The safety model of the preparation according to claim 3 is a mouse injected with CART frozen stock solution to observe whether or not the organs are abnormal.