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Chimeric antigen receptor (CAR) targeting BCMA and application thereof

A chimeric antigen receptor and targeting technology, which can be used to target specific cell fusion, polypeptides containing positioning/targeting motifs, applications, etc., can solve the symptoms of exacerbation, limit the clinical application of CAR-T cells, etc. problems, to achieve the effects of optimizing the preparation method, suppressing the immune factor storm, and being easy for genetic manipulation

Active Publication Date: 2020-11-13
佑仁细胞工程(浙江)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are also some problems in the use of the above-mentioned CAR-T cells, including that with the increase of the dose of CAR-T cells, the symptoms of cytokine release syndrome (CRS) in patients aggravated, fever and tachycardia, central The occurrence of side effects such as granulocytopenia, thrombocytopenia, hypotension and acute kidney injury to varying degrees limits the clinical application of CAR-T cells

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Determination of single domain antibodies targeting BCMA

[0038] Human BCMA protein is used to immunize alpacas, and phage display library is used to screen single-domain antibodies targeting BCMA. The specific methods include:

[0039] (1) Antigen immunization. Select healthy adult dromedary camels, mix human BCMA protein at a ratio of 15 μg / Kg and Freund’s adjuvant at a ratio of 1:1, and immunize with multiple subcutaneous injections on the back, immunizing 6-8 times in total, with an interval of 2 weeks . Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the remaining immunizations. After the immunization, 100 mL of peripheral blood from the neck of the dromedary camel was collected every week, and the titer of BCMA antibody in the serum of the alpaca was detected by ELISA. When the concentration of the BCMA antibody reached 128K or more, it met the requirements for the construction of an antibo...

Embodiment 2

[0046] Example 2 Determination of chimeric antigen receptor gene sequence

[0047] Retrieve and obtain the amino acid and nucleotide sequences of signal peptide, CD28 transmembrane region, 4-1BB co-stimulatory factor, CD3ζ intracellular signal domain, human CD28 hinge region by bioinformatics method, wherein the amino acid sequence of CD28 transmembrane region is as SEQID As shown in NO:2, the amino acid sequence of 4-1BB is shown in SEQID NO:4, the amino acid sequence of signal peptide is shown in SEQID NO:5, and the amino acid sequence of CD3ζ intracellular signal domain is shown in SEQID NO:3. In order to facilitate analysis and comparison, the targeted BCMA ssFv fragment with a common antibody structure maintained by our laboratory was selected as a positive control for subsequent experiments, and a chimeric antigen receptor gene sequence was constructed by total gene synthesis, which has a single-domain antibody antigen binding The CAR-VHH structure of domain functional e...

Embodiment 3

[0048] Example 3 Preparation of plasmid vector carrying chimeric antigen receptor gene

[0049] 3.1 Digestion of the target product and linearization of the plasmid vector

[0050] The CAR-VHH and CAR-AN gene sequences were amplified and obtained by PCR, and Xba I and BamH I restriction sites were added at both ends of the sequences. The target gene fragment and the lentiviral vector plasmid pCDH-CMV-MCS-EF1-GFP-T2A-Puro (purchased from Addgene) were subjected to Xba I and BamH I double enzyme digestion reaction. The enzyme digestion reaction conditions were: 37°C, enzyme digestion 40min.

[0051] 3.2 Recovery of digested products

[0052] Electrophoresis was performed on 1.5% agarose gel containing CAR-VHH, CAR-AN fragments and pCDH-CMV-MCS-EF1-GFP-T2A-Puro DNA fragments. After electrophoresis, the agarose containing the above nucleic acid fragments were respectively The strips were cut and placed in two clean EP tubes. The DNA in the agarose gel was purified and concentr...

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Abstract

The invention belongs to the technical field of biological pharmacy, in particular to a chimeric antigen receptor (CAR) targeting BCMA and an application thereof. According to the chimeric antigen receptor, a single-domain antibody targeting BCMA is used as an antigen binding domain; in the process of preparing the CAR-T cells, optimization is carried out by adjusting the proportion of CD4<+> andCD8<+> T cells, the obtained novel CAR-T cells can effectively kill tumor cells, cell factors such as IFN-gamma, IL-10 and IL-6 are controlled within an acceptable range, and the product 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 BCMA and an application thereof. Background technique [0002] According to statistics, millions of patients die every year due to malignant tumors and related diseases. Malignant tumors are one of the diseases that seriously threaten human health. The second most common hematological malignancy, it accounts for about 1% of neoplastic diseases and about 13% of hematological malignancies, with an incidence ranging from 1 to 4 per 100,000, occurring The rate is higher in men than in women. The survival period of multiple myeloma is very short. If no treatment is performed, the median survival period of patients with advanced MM is only 6 months, and even if they receive chemotherapy and other treatments, the median survival period will not exceed 3 years. Only A quarter of patients can survive for more than 5 years, so MM i...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K14/70521C07K14/70532C07K14/70578C07K16/2878C07K2317/569C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 刘欢杨洋
Owner 佑仁细胞工程(浙江)有限公司
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