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Chimeric antigen receptor and application thereof in preparation of product for treatment of tumors

A chimeric antigen receptor and single-chain antibody technology, applied in the field of biomedicine, can solve problems such as toxic side effects, insufficient sustained viability, and reducing the safety of CAR-T therapy

Active Publication Date: 2020-11-06
CARBIOGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing CAR-Ts generally show the defect of insufficient survival ability in vivo
Blindly increasing the dose of CAR-T cells can easily cause strong toxic side effects, such as inflammatory factor storm and central nervous system toxicity, which in turn reduces the safety of CAR-T therapy

Method used

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  • Chimeric antigen receptor and application thereof in preparation of product for treatment of tumors
  • Chimeric antigen receptor and application thereof in preparation of product for treatment of tumors
  • Chimeric antigen receptor and application thereof in preparation of product for treatment of tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1. Preparation of CAR-T cells

[0141] 1. Construction of retroviral vector

[0142] 1. Optimization of the full-length cDNA sequence of wild-type human IFNα2b gene

[0143] The full-length sequence of wild-type human IFNα2b gene cDNA is called nIFNα2b. In order to make nIFNα2b more suitable for expression in human cells, under the condition that the amino acid sequence encoded by nIFNα2b remains unchanged, the codon optimization of nIFNα2b sequence was carried out on the website http: / / sg.idtdna.com / site to obtain oIFNα2b, The nucleotide sequence of oIFNα2b is shown at positions 1570-2133 of SEQ ID NO.1.

[0144] 2. Design and synthesis of LILRB4-CAR-IFN gene sequence

[0145] The LILRB4-CAR-IFN gene sequence sequentially includes the coding gene sequence of human CD8 leader peptide, the coding gene sequence of LILRB4 scFv, the coding gene sequence of human CD8 hinge transmembrane region, the coding gene sequence of human 4-1BB intracellular region, the codi...

Embodiment 2

[0178] Example 2, CFSE labeling method to detect the specific killing effect of CAR-T cells on tumor cells

[0179] CFSE (CFDA-SE) is a cell staining reagent that can fluorescently label living cells. It can easily penetrate the cell membrane, covalently bind to intracellular proteins in living cells, and release green fluorescence after hydrolysis. The principle of CFSE labeling living cells can be used to label and quantify tumor cells, so as to detect the killing efficiency of CAR-T cells on tumor target cells. The specific method is: the target cells are equally divided into two groups and adjusted to the same cell density. Stained with low concentration and high concentration of CFSE respectively, wherein high concentration stained target cells and non-stained immune cells were co-cultured according to a certain ratio. After a period of incubation, the high-concentration stained tube of target cells (along with immune cells) was mixed in equal volume with the low-concent...

Embodiment 3

[0194] Example 3. Tumor transplantation model to detect the tumor killing effect of CAR-T cells in animals

[0195] 1. Construction of K562 tumor cells overexpressing LILRB4 gene

[0196] K562 cells that do not express LILRB4 were used as the starting cells, and the LILRB4 gene was overexpressed in K562 cells to realize its function as LILRB4-positive target cells. The specific construction method is as follows: insert the synthetic LILRB4 sequence (see SEQ ID NO.8) into the AgeI and EcoRI restriction sites of the pRV-Luc-GFP vector, and obtain the pRV-Luc-GFP-LILRB4 recombinant expression vector after sequencing verification ( Figure 4 ). The pRV-Luc-GFP-LILRB4 recombinant expression vector verified by sequencing was transfected into K562 cells to obtain K562 cells overexpressing the LILRB4 gene (referred to as K562-LILRB4). LILRB4 antibody flow cytometry was used to detect whether LILRB4 was correctly expressed on the surface of K562 cells.

[0197] 2. Tumor transplanta...

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Abstract

The invention discloses a chimeric antigen receptor and an application thereof in preparation of a product for treatment of tumors. The chimeric antigen receptor sequentially comprises a lead peptide,an anti-LILRB4 single-chain antibody, a human CD8 hinge transmembrane region, a human 4-1BB intracellular region, a human CD3 zeta intracellular region, a self-cleaving peptide and human IFN full length. A new generation of CAR-T cell treatment product is designed and developed by using LILRB4 as an antigen target for the first time, and a gene-optimized human full-length interferon (IFN) fragment is added to the C terminal of the LILRB4-CAR. Compared with a CAR-T cell which only expresses the LILRB4-CAR, the CAR-T cell which expresses the LILRB4-CAR-IFN has higher tumor killing capacity, andthe safety and effectiveness of the CAR-T cell in tumor treatment are further improved.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a chimeric antigen receptor targeting LILRB4 and its application, in particular to a chimeric antigen receptor containing LILRB4-CAR and secreting cytokines and its application. Background technique [0002] Acute myeloid leukemia (AML) is the most common type of leukemia in adults with the highest mortality rate, and it still faces challenges in treatment. At present, the treatment of acute myeloid leukemia is still dominated by chemotherapy, and the patient is bridging to allogeneic hematopoietic stem cell transplantation (allo-HSCT) after achieving complete remission. This method can make some AML patients achieve complete remission, and for AML patients receiving allo-HSCT, the 5-year disease-free survival (Disease-free survival, DFS) can reach 40-50%. However, about 43% of patients relapse after receiving the first chemotherapy, and only half of relapsed patient...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02
CPCA61K2039/5156A61P35/00A61P35/02A61K39/001111A61K39/001141A61K2039/804C07K14/56C07K14/565C07K14/7051C07K14/70517C07K14/70578C07K16/2803C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 朱建高杨文君
Owner CARBIOGENE THERAPEUTICS CO LTD
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