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Universal car-t cell and its preparation method and application

A general-purpose, cellular technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, which can solve problems such as missing, low probability of successful HLA matching, and long time consumption.

Active Publication Date: 2020-09-04
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most patients, especially tumor patients, have undergone other treatments before CAR-T cell adoptive therapy, such as chemotherapy and radiotherapy, which leads to less T lymphocytes acquired, and even many patients due to the effect of drugs It is difficult to expand the T cells in vitro, and the T cells from the patient's own source may also have functional attenuation or loss, and it is more difficult to genetically modify T cells, and the therapeutic effect and safety are not easy to control. These obstacles all affect CAT-T Application promotion and development of cell therapy and tumor clinical treatment
[0005] Allogeneic cells refer to cells belonging to the same species but genetically different individuals; although more T lymphocytes can be isolated from the peripheral blood of healthy people, the positive rate of CAR infection is high, and their vitality and function are better than those of patient-derived T lymphocytes. However, allogeneic cell therapy still faces many obstacles: due to the immunogenetic differences between the donor and the recipient, on the one hand, the T lymphocytes of the immunocompetent donor are entering the recipient After the patient has proliferated to a certain extent, the normal cells or tissues of the recipient patient are mistaken for the target to attack, resulting in graft-versus-host response (GVHD). On the other hand, the normal immune system in the recipient as an allogeneic cell may also It will be cleared to produce host-versus-graft reaction (HVGR), which will affect the therapeutic effect
For HVGR, immunosuppressive drugs are mainly used to suppress the activity of the recipient's immune system before treatment, but immunosuppressive drugs will affect the efficacy of reinfused cells during adoptive cell therapy; for GVHD, HLA matching is mainly used for prevention , but the success rate of HLA matching is low, time-consuming and expensive

Method used

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  • Universal car-t cell and its preparation method and application
  • Universal car-t cell and its preparation method and application
  • Universal car-t cell and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1, Chinese population HLA-A typing frequency detection

[0071] 1. SBT (Sequenced Based Typing) detection:

[0072] This method belongs to HLA high-resolution typing technology.

[0073] 4ml of venous blood was extracted from EDTA anticoagulant tubes (blood routine tubes), stored at 4°C for temporary storage, and long-term frozen at -20°C. Then send to BGI for HLA typing test.

[0074] 2. Bioinformatics methods: refer to Boegel, Sebastian, Valesca et al. (2013)

[0075] Using the seq2HLA software, the HLA high-resolution typing (MHC-I and MHC-II, 4 bits) of the sample and the corresponding expression value and p-value were calculated directly from the raw data (fastq format) of RNA-Seq sequencing.

[0076] Experimental results such as figure 1 The 8 HLA-A types with the highest frequency in 8 Chinese are shown, namely A*02, A*11A*24, A*30, A*33, A*03, A*01 and A*26, The gRNA molecules were designed respectively for these 8 subtypes, and the gRNA sequences a...

Embodiment 2

[0077] Example 2, Knockout of TCR and MHC Class I and Class II Molecule Related Gene Vector Construction

[0078] 1. sgRNA molecular design:

[0079] Confirm the genes related to TCR and MHC class 1 and class 2 molecules: B2M, CIITA and TRAC genes are closely related to the functions of genes related to TCR and MHC class 1 and class 2 molecules Confirm the knockout of B2M, CIITA and TRAC genes. B2M, CIITA and TRAC gene sequence numbers were obtained from NCBI to obtain CDS sequence, B2M sequence number AH002619.2, CIITA in NCBI gene ID: 4261, TRAC in NCBI gene ID: 28755, as shown in SEQ ID NO: 1-3. The gRNAs were designed for the identified targeted SEQID NO: 1-3 regions of the CDS region, and the gRNA sequences are shown in Table 1.

[0080] Table 1: gRNA sequences

[0081]

[0082]

[0083] 2. Construction of expression vector for CRISPR / Cas9 gene knockout

[0084] The following primers were designed and synthesized by Nanjing GenScript Biotechnology Co., Ltd. The s...

Embodiment 3

[0099] Example 3. Universal CAR-T cell construction and phenotypic detection

[0100] 1. Confirmation of electroporation conditions

[0101] Under different conditions, 293T cells were used to transfect GFP to optimize the electroporation conditions. After confirming better electroporation conditions, PBMC transfection was carried out. The experimental results are shown in Table 2 and Table 3. The preferred electroporation conditions are PBMC or T cell activation. After 48 hours, the voltage was 1000V, the pulse width was 35ms, and the number of electric shocks was twice for electroporation, and the total number of cells was 2×10 5 , cultured with fresh medium containing IL-2 after electroporation.

[0102] Table 2: Comparison of transfection efficiency and cell viability of transfected 293T cells under different electroporation conditions

[0103] serial number efficiency% Survival % Voltage / V Pulse width / ms Number of shocks 1 0 100 0 1 1 2 ...

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Abstract

The invention belongs to the field of immunotherapy, and relates to a universal CAR-T cell and its preparation method and application. In the universal CAR T cell of the present invention, the functions of T cell antigen receptor (TCR) and major histocompatibility complex (MHC Ⅰ, MHC Ⅱ) in T cells are simultaneously inhibited through multiple gene knockout; The genes of the TCR include TRAC and / or TRBC; the genes encoding the major histocompatibility complex include HLA-A, B2MH and CIITA. The universal CAR-T cells of the present invention can target specific tumor-associated markers and inactivate TCR and MHC functions on the cell surface, which can reduce immune rejection caused by allogeneic cell therapy and safely and effectively eliminate tumor cells in patients. And it is not affected by the patient's own condition or treatment method during use, and can be prepared at any time, and treatment can be given at the best time to ensure the effectiveness of treatment.

Description

technical field [0001] The invention belongs to the field of immunotherapy, and relates to a general-purpose CAR-T cell and its preparation method and application, in particular to a general-purpose T cell, a general-purpose CAR-T cell, its preparation method and its application in medicine. Background technique [0002] Adoptive cell therapy (ACT) is a kind of biotherapy technology, which expands autologous immune cells (mainly T cells) in vitro, and then reintroduces them to tumor patients to achieve therapeutic purposes. It is the fourth treatment after surgery, radiotherapy and chemotherapy, and it is widely used in clinical treatment. [0003] Chimeric antigen receptor, also known as CAR, is an artificial receptor that mimics the function of TCR. When the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal is transmitted into the cell through the hinge region and the transmembrane region, and t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00A61P31/00A61P37/06A61P37/08A61P37/02
CPCA61K35/17C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107A61K39/0011A61K2039/5156
Inventor 张巍李戊玲张茜真单娟娟
Owner CHONGQING PRECISION BIOTECH CO LTD
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