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Chimeric antigen receptor (CAR) targeting GPC3 and anticancer applications

A chimeric antigen receptor, targeting technology, applied in the direction of peptides containing localization/targeting motifs, antibody medical components, DNA/RNA fragments, etc., can solve the high risk of side effects, cytokine storm, and poor targeting. It can achieve the effect of prolonging the life cycle, inhibiting overexpression, and reducing the expression level.

Active Publication Date: 2020-06-30
BEIJING DAXI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the above studies, although CAR-T cells targeting GPC3 have been proposed and prepared, and useful attempts have been made in the expression of cytokines and the selection of bispecific targets, etc., they still face weak targeting and high risk of side effects , easily lead to technical problems such as cytokine storms, which limit the application of CAR-T cells to a certain extent, especially in the field of treatment of refractory and relapsed liver cancer

Method used

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  • Chimeric antigen receptor (CAR) targeting GPC3 and anticancer applications
  • Chimeric antigen receptor (CAR) targeting GPC3 and anticancer applications
  • Chimeric antigen receptor (CAR) targeting GPC3 and anticancer applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtaining of monoclonal antibody targeting GPC3.

[0035] Anti-human GPC3 monoclonal antibody was obtained by immunizing BALB / c mice with human GPC3 protein. Specifically: use recombinant human GPC3 as the immunogen, dissolve the recombinant antigen in PBS, mix it with Freund's agent in equal volume, immunize 6-week-old BALB / c mice, kill the mice by neck dislocation after 2 weeks, take out the spleen, and prepare a single spleen cell suspension. At the same time, mouse myeloma cells were revived with 1640 complete medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Cultivate in an incubator, and subculture for 2-3 times, so that the myeloma cells are in a state of vigorous growth, and then digest the cells and prepare the myeloma cell suspension. The prepared myeloma cell suspension and spleen cell suspension were fully mixed at a ratio of 1:1, centrifuged and co-precipitated, and cell fusion was performed with PEG as a fusion agent in a water bath at ...

Embodiment 2

[0037] Example 2 Determination of the affinity of monoclonal antibodies targeting GPC3.

[0038] The binding reaction curves of 5 concentrations of human GPC3 antigens and 15 concentrations of targeting GPC3 antibodies were established by indirect ELISA, and the affinity constants of targeting GPC3 antibodies and human GPC3 antigens were calculated accordingly, as shown in Table 1. In the present invention Six monoclonal antibodies targeting GPC3 with higher affinity were screened. According to literature reports (Broad-spectrum anti-tumor efficacy of EGFR family medium-affinity chimeric antigen receptor modified T cells, Zhang Hao) selection of antigen-binding domains with low-to-medium levels of affinity to construct CAR-T cells seems to be more effective in reducing off-target effects. Normal cells that express the target antigen at a low level can effectively exert the immune effect of specifically killing tumor cells. Therefore, in the present invention, the No. 3 monoclo...

Embodiment 3

[0041] Example 3 Cloning of variable region genes of monoclonal antibodies targeting GPC3.

[0042] Based on the 5'RACE technology, the VL and VH genes of the anti-GPC3 monoclonal antibody were cloned through degenerate primers. The nucleic acid fragment was introduced into the restriction site and enriched by PCR, and then purified using the PCR product purification kit (OMEGA) , the purified product was connected to the pUC19 cloning vector, transformed into DH5α Escherichia coli competent cells, positive clones were screened by ampicillin antibiotics, and the positive clones were sent for sequencing verification, and according to NCBIIgBLAST (http: / / www.ncbi.nlm.nih .gov / ) Immunoglobulin gene comparison analysis results, and screen out the results of functional antibody variable region sequences, in which the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are shown as SEQ ID NO.1 and SEQ ID NO. 2. As shown in SEQ ID NO.3, the amino acid s...

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Abstract

The invention belongs to the technical field of biological pharmacy, and specifically relates to a chimeric antigen receptor (CAR) targeting GPC3 and anticancer applications. The chimeric antigen receptor includes signal peptide, an antigen binding region targeting GPC3, a hinge region, a transmembrane region, a costimulating factor and an intracellular signal domain. The growth of tumors in vivoand in vitro can be effectively inhibited by selecting the antigen binding region targeting the GPC3 and having medium affinity and adjusting the connection sequence of variable regions of a light chain and heavy chain, so that the secretion levels of immune factors can be reduced; and the chimeric antigen receptor has good clinical application prospects.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a chimeric antigen receptor (CAR) targeting GPC3 and its anticancer application. Background technique [0002] Tumor is a malignant disease that seriously threatens human health, and liver cancer is one of the most common malignant tumors in the world. Its morbidity and mortality are increasing year by year, seriously threatening human health. Studies have shown that the incidence of liver cancer It ranks fifth in the world among malignant tumors, and ranks third in death rate. In China, due to the presence of serious cancer-causing risk factors such as hepatitis B virus (HBV) infection, the incidence and mortality of liver cancer are particularly severe. More than 100,000 people die of liver cancer and its complications every year. Although medical workers have conducted in-depth research on liver cancer, carried out a large number of clinical practice activities, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10C12N15/62A61K39/00A61P35/00
CPCA61K39/001111A61K2039/5156A61K2039/5158A61P35/00C07K14/7051C07K2319/02C07K2319/03C12N5/0636C12N2510/00
Inventor 刘欢杨洋
Owner BEIJING DAXI BIOTECHNOLOGY CO LTD
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