A chimeric antigen receptor (car) targeting bcma and its application

A chimeric antigen receptor, targeting technology, applied in the direction of targeting specific cell fusion, peptides containing localization/targeting motifs, applications, etc., can solve the problem of adverse reactions and improve the technology that has not been discussed or verified. effect, reduce adverse reactions and other issues, achieve good anti-tumor effect, simple structure, and reduce the difficulty of operation

Active Publication Date: 2021-08-17
武汉冉谷医疗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the transformation of the intracellular signaling domain has also attracted the attention of researchers, but there are few studies in this area, and most of them are in the preliminary stage. It is difficult to guide how to reduce adverse reactions based on the change of the intracellular signaling domain, such as in the patent document CN111886242A It is disclosed that the CD3ζ polypeptide lacks all or part of the immunoreceptor tyrosine-based activation motif (ITAM), which is beneficial to improve the activation degree of CAR-T cells, and indicates that ITAM1 is essential for the activity of CD3ζ, when it is missing or After the mutation, the tumor killing effect will be greatly reduced, but there is no focus on the improvement of adverse reactions; WO2018183385A1 disclosed that the signal transduction part of the chimeric receptor contains ITAM, such as half-ITAM, but did not verify the specific technical effect

Method used

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  • A chimeric antigen receptor (car) targeting bcma and its application
  • A chimeric antigen receptor (car) targeting bcma and its application
  • A chimeric antigen receptor (car) targeting bcma and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Determination of single domain antibodies targeting BCMA

[0037] 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:

[0038] (1) Alpaca antigen immunization.

[0039] Mix 100 μg of human BCMA protein with 100 μl of Freund’s complete adjuvant to make an emulsified mixture, select healthy adult dromedary camels, and use the emulsified mixture to immunize alpacas by subcutaneous injection at multiple points on the back. The first immunization and the second Freund's complete adjuvant was used for the first immunization, and the subsequent immunization was enhanced with an emulsified mixture of Freund's incomplete adjuvant and human BCMA protein. There were 7-9 immunizations in total, and the immunization interval was 2 weeks. One week after each immunization, collect 100ml of peripheral blood, detect the titer of antiserum by ELISA, coat the detection...

Embodiment 2

[0052] Example 2 CD3ζ intracellular signaling domain structure design

[0053] The wild-type amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQID NO: 12, which includes three ITAM domains, and its structure is YXXL / IX 6-8 YXXL / I, through the corresponding analysis of the sequence structure and its activity in the prior art, it is generally believed that these three ITAM domains are necessary for the CD3ζ chain to exert its physiological activity, especially the phosphorylation of two tyrosine (Y) Plays an integral role in correlation signaling. However, existing research results have shown that in CAR-T cells that use the CD3ζ intracellular signaling domain combined with CD28, due to the high degree of activation of T cells, it often leads to the release of a large number of inflammatory factors, triggering a daunting release of cytokines syndrome. Therefore, the inventors of the present application attempted to modify the ITAM domain based on the...

Embodiment 3

[0059] Example 3 Chimeric Antigen Receptor Structure Design

[0060]Retrieve and obtain the amino acid and nucleotide sequences of signal peptide, CD8 hinge region, CD28 transmembrane region, and CD28 co-stimulatory factor by bioinformatics methods, wherein the amino acid sequence of CD28 transmembrane region is shown in SEQID NO: 6, and the CD8 hinge region The amino acid sequence is shown in SEQID NO:7, and the amino acid sequence of CD28 is shown in SEQID NO:8. In order to facilitate analysis and comparison, four CAR molecules were designed, and their specific structures are as follows: figure 1 As shown, in the above CAR molecules, D1 CD3ζ, D2 CD3ζ, D3CD3ζ and D4 CD3ζ are respectively selected as intracellular signaling domains, wherein the amino acid sequence of the CAR molecule containing D4 CD3ζ is shown in SEQID NO: 10, and its nucleotide sequence is shown in SEQID NO:11 shown.

[0061] In order to verify the influence of co-stimulatory factors, the chimeric antigen ...

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Abstract

The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a chimeric antigen receptor (CAR) targeting BCMA and its application. Transformation of ITAM1 and ITAM3 domains, retaining the original ITAM2, and optimizing part of the amino acid sequence in ITAM2, can improve the survival time of CAR-T endosomes and reduce the expression level of inflammatory factors, which has a good clinical application prospect.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a BCMA-targeted chimeric antigen receptor (CAR) and its application, in particular to a CD3ζ signaling domain modified chimeric antigen receptor and its application. Background technique [0002] Chimeric antigen receptor T cells (CAR-T cells) refer to T cells with a killing effect modified by high-affinity chimeric antigen receptors. The genetically modified T cells can specifically recognize tumor antigens. After recognition, T cells can be activated to proliferate in large numbers and kill tumor cells through immune response. The development of CAR-T cell technology has experienced certain twists and turns and explorations. The first-generation CAR-T cells only have a single signaling molecule, which can specifically recognize the target antigen, but lack co-stimulatory molecules and cannot continuously and effectively kill tumor cells, so they cannot fully Activ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCA61K39/0011A61P35/00C07K14/7051C07K16/2878C07K2317/22C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 吴菲吴凡刘欢杨洋
Owner 武汉冉谷医疗有限公司
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