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Chimeric antigen receptor (CAR) and application thereof

A chimeric antigen receptor, recombinant vector technology, applied in applications, antibody medical components, antibody mimics/scaffolds, etc., can solve serious adverse reactions, patient death and other problems, achieve strong anti-tumor effects, inhibit tumor growth, The effect of suppressing serious adverse reactions

Active Publication Date: 2022-02-08
博威尔斯(北京)商贸有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CD19 is also expressed in normal B cells, making CART cells targeting it attack normal cells, leading to serious adverse reactions and even patient death

Method used

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  • Chimeric antigen receptor (CAR) and application thereof
  • Chimeric antigen receptor (CAR) and application thereof
  • Chimeric antigen receptor (CAR) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Chimeric Antigen Receptor Molecular Design

[0028] The inventor screened antibodies targeting human ROR1 protein in the early stage, obtained multiple monoclonal antibodies, screened out the antibody molecule with the highest affinity, and selected its ScFv fragment containing heavy chain and light chain as the chimeric antigen receptor shown in the present invention. Antigen binding domain, the antigen binding domain can be combined with the target antigen at a high level, and its affinity can reach 1.51E-09nM.

[0029] In order to obtain safe and effective CART cells, the present invention provides two chimeric antigen receptor structures, such as figure 1 as shown, figure 1 The structure shown in A is the classic second-generation CAR structure, including signal peptide, anti-human ROR1 scFv, hinge region, transmembrane region, 4-1BB intracellular domain and CD3ζ intracellular domain; figure 1 The Fc fragment was introduced into the structure shown in B...

Embodiment 2

[0033] Embodiment 2: CART cell preparation

[0034] According to the amino acid sequence of the above elements, its nucleotide sequence was obtained by reverse transcription, nucleic acid amplification, etc., and the corresponding gene fragment was inserted into the lentiviral expression vector pLent-EF1a-FH-CMV-RFP-P2A-Puro vector. The above expression vector was introduced into DH5αE.coli by electrotransformation, and after the sequencing was correct, the plasmid was extracted and purified using a plasmid extraction kit (purchased from QIAGEN).

[0035] 5% CO at 37°C 2 Cultivate 293T cells in an incubator at a constant temperature so that the growth density reaches 70%-80% before transfection, and replace with fresh medium for later use; respectively use liposomes, and the above-mentioned purified plasmids and pLP / VSVG, pLP1, pLP2 plasmid mixtures Dilute the serum-free DMEM-HG medium, then mix the two evenly, and let stand at room temperature for 15 minutes to form a liposo...

Embodiment 3

[0037] Example 3 In vitro killing effect of CART cells on tumor cells

[0038] According to reports, the ROR1 antibody has inhibitory effects on various hematological tumors and solid tumors, and according to the preliminary experimental results of the present invention, the HCDR provided by the present invention contains HCDR shown in SEQ ID NO: 1-3 and SEQ ID NO: 4 Monoclonal antibodies in CDR regions such as LCDR shown in -6 have strong recognition ability and killing effect on hematopoietic tumor cells. Therefore, this example mainly verifies the inhibitory effect of related CART cells on hematopoietic tumor cells.

[0039] ROR1 is expressed in various hematopoietic tumors, and NB4 (acute myeloid leukemia cell line), K562 (chronic myeloid leukemia cell line), Raji (lymphoma cell line) and U266 (multiple myeloma cell line) are selected for use in the present invention. ) as the research object, and the killing effect was detected by the CCK-8 method. Specific methods inclu...

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Abstract

The invention relates to a chimeric antigen receptor (CAR) and application thereof. The invention provides an ROR1 specific chimeric antigen receptor which comprises a signal peptide, an anti-human ROR1scFv, a hinge region, a transmembrane region, a 4-1BB intracellular domain and a CD3 zeta intracellular domain, the anti-human ROR1scFv can be combined with a target antigen in a high affinity mode, and off-target benefits are prevented; furthermore, an Fc fragment is introduced into the chimeric antigen receptor, so that the tumor killing effect can be improved, immune factor storm can be prevented, the safety and effectiveness of treatment can be improved, and a new scheme is provided for development of related medicines and tumor immunotherapy.

Description

technical field [0001] The invention belongs to the field of tumor immunotherapy and the field of biotechnology, and specifically provides a chimeric antigen receptor (CAR) and an application thereof. Background technique [0002] Tumor has become one of the most serious diseases threatening human health because of its refractory, high mortality, and low clinical manifestations. For this reason, researchers have tried a variety of tumor treatment methods and techniques, including surgical treatment, Radiation therapy, chemical drug therapy, gene therapy, etc., but no effective treatment method has been found so far. With the deepening of biotechnology and immunology research, tumor immunotherapy and anti-tumor antibodies have been valued by the industry. Since the 1980s, hundreds of antibodies have been approved for tumor treatment worldwide, and immune checkpoints The new generation of immunotherapy technology represented by inhibitors and chimeric antigen receptor T cells...

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
CPCC07K16/2803C07K14/7051C12N15/86C12N5/0686A61K39/001111A61P35/00A61P35/02C07K2319/02C07K2319/03C07K2317/622C07K2317/56C07K2317/565C07K2319/30C12N2510/00C12N2740/15043A61K2039/5156A61K2039/804
Inventor 张克礼雷林均
Owner 博威尔斯(北京)商贸有限公司
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