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Universal chimeric antigen receptor-T (CAR-T) cell as well as preparation method and application thereof

A universal, cellular technology, applied in the field of preparation, universal CAR-T cells, to save time and cost, shorten preparation time, and reduce preparation cost

Inactive Publication Date: 2018-11-13
武汉圣惠康生物医药科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Autoantibodies are closely related to kidney diseases. At present, the key autoantibodies and their antigens in various kidney diseases have been identified, such as PLA2R, Gd-IgA1, and anti-dsDNA. These kidney diseases are essentially an autoantibody-mediated autoantibody Immune diseases, studies have proved that a variety of autoimmune diseases are caused by self-reactive B cells, so far, there is no CAR-T cell for targeting and killing self-reactive B cells

Method used

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  • Universal chimeric antigen receptor-T (CAR-T) cell as well as preparation method and application thereof
  • Universal chimeric antigen receptor-T (CAR-T) cell as well as preparation method and application thereof
  • Universal chimeric antigen receptor-T (CAR-T) cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Preparation of universal CAR-T cells (svCAR-T)

[0039] (1) Synthesizing the anti-GCN single-chain antibody region, the sequence of which is shown in SEQ ID NO:1;

[0040](2) Obtaining of the Hinge-TM-CD137-CD3ζ gene sequence: design and synthesize the Hinge-TM-CD137-CD3ζ expression cassette of the chimeric antigen receptor according to the CD8α, CD137 and CD3ζ gene sequences, including the signal peptide (carrying the gene sequence such as SEQ IDNO: 6), hinge region (carrying gene sequence as shown in SEQ ID NO: 5), CD3ζ region (carrying gene sequence as shown in SEQ ID NO: 2), transmembrane region (carrying gene sequence as shown in SEQ ID NO: 4 shown), the co-stimulatory region (carrying the gene sequence shown in SEQ ID NO:3) and the anti-GCN single-chain antibody region (carrying the sequence of SEQ ID NO:1) were gene synthesized, and the obtained plasmid was named pCD-HTCC.

[0041] (3) Hinge-TM-CD137-CD3ζ was connected with vector pCDH-CMV-MCS to obtain vector p...

Embodiment 2

[0047] Preparation of GCN-Autoantibody Affinity Peptide Fusion Polypeptide (GCN-sP) Leader

[0048] 1) The polypeptide GCN is derived from the 14 amino acid peptides of the yeast transcription factor GCN4, which can bind with high affinity to the front end of svCAR anti-GCNscFV, and can guide and bridge the combination of svCAR-T and target autoreactive B cells. Its sequence is shown in SEQ ID NO: 7.

[0049] 2) The linker is used to connect the polypeptide GCN and the autoantibody affinity peptide, and its sequence is shown in SEQ ID NO:8.

[0050] 3) Candidate autoantibody affinity peptides are identified autoimmune nephropathy-related autoantigen epitope peptides or mimetic peptides:

[0051] IMN affinity peptide Pp: the sequence is shown in SEQ ID NO:19;

[0052] IgAN affinity peptide Pa: the sequence is shown in SEQ ID NO:20;

[0053] LN affinity peptide Pd: the sequence is shown in SEQ ID NO:21;

[0054] MPO-AAV affinity peptide Pma: the sequence is shown in SEQ ID N...

Embodiment 3

[0069] Affinity Test of GCN-sP Series Peptides and Autoantibodies by Enzyme-Linked Immunosorbent Assay

[0070] 1) Preparation of GCN-sP and control peptides: The affinity of autoantibodies between 10 kinds of GCN-sP synthesized in Example 2 and 5 kinds of control peptides (Pp, Pa, Pd, Pma, Ppa) was detected.

[0071] 2) Negative and positive serum preparation:

[0072] 30 cases of serum from LN patients confirmed to contain anti-dsDNA antibodies by clinical tests;

[0073] ●Thirty cases of serum from IMN patients confirmed to contain anti-PLA2R antibody by clinical tests;

[0074] 30 cases of sera from IgAN patients with positive Gd-IgA1 confirmed by human Gd-IgA1 detection kit (27600, Japan IBL);

[0075] ●Thirty cases of serum from MPO-AAV patients confirmed to contain MPO-ANCA antibody by clinical tests;

[0076] ●Thirty cases of serum from PR3-AAV patients confirmed to contain PR3-ANCA antibody by clinical tests;

[0077] ·Negative control serum is normal human serum....

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Abstract

The invention provides a universal chimeric antigen receptor-T (CAR-T) cell as well as a preparation method and application thereof. The universal CAR-T cell can recognize polypeptide GCN and GCN-autoantibody affinity peptide fusion polypeptides, and can recognize and kill different self-reactive b cells in a targeted way under the guidance and regulation of different GCN-autoantibody affinity peptide fusion polypeptide guiders. The intensity or complete closure of an in vivo immune effect of the CAR-T cell can be very conveniently controlled, so that the reduction of other risks or side effects such as cytokine storms in general CAR-T cell therapy is facilitated; the universal CAR-T cell is different from conventional CAR-T cells need to be designed with different CARs aiming at differenttargets; therefore, time and cost are greatly reduced, and the success rate is increased.

Description

technical field [0001] The invention belongs to the field of immunotherapy, and specifically relates to a universal CAR-T cell, a preparation method and an application. Background technique [0002] CAR-T cell technology is to recombine the single-chain antibody (scFv) that recognizes tumor-associated antigens and the intracellular signaling domain "immunoreceptor tyrosine activation motif (ITAM, usually CD3ζ)" in vitro to generate recombinant The plasmid is then transfected into natural T cells in vitro, and the purified and expanded T cells express chimeric antigen receptors (CAR), and the T cells expressing CAR are called CAR-T cells. CAR-T cells can bind tumor antigens in an antigen-dependent, non-MHC-restricted manner, initiate and activate specific tumor-killing effects. [0003] At present, this technology has made great progress in the field of tumor treatment research. In 2011, June CH et al. reported for the first time that a patient with chronic lymphocytic leuke...

Claims

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Application Information

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IPC IPC(8): C12N5/10A61K35/17A61P37/02A61P13/12A61P9/00
CPCA61P9/00A61P13/12A61P37/02A61K35/17C07K14/7051C07K16/18A61K2039/5156C07K2319/33C07K2319/02C12N2510/00
Inventor 王惠明刁波王刚
Owner 武汉圣惠康生物医药科技有限责任公司
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