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Multi-target chimeric antigen receptor

A chimeric antigen receptor, multi-target technology, applied in cytokine/lymphokine/interferon receptors, hybrid peptides, receptors/cell surface antigens/cell surface determinants, etc.

Inactive Publication Date: 2018-07-06
TIMMUNE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recent studies have shown that the functional activity of CAR-T cells is not only related to its intracellular signal sequence, but also related to the endogenous receptors provided by the target cells. Whether the third-generation CARs are better than the second-generation CARs remains to be clinically verified.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Example 1, Multi-target Chimeric Antigen Receptor Structure and Expression Vector Construction

[0137] 1. The multi-target chimeric antigen receptor (RaceCar) is a protein obtained by polymerizing the main peptide chain X and the auxiliary peptide chain Y;

[0138] The main peptide chain (X) includes the antigen-binding domain (A), the auxiliary peptide chain linking domain (B), the transmembrane domain (C) and the intracellular signaling domain (D), and the auxiliary peptide chain (Y) includes the antigen-binding Domain (E) and main peptide chain connecting domain (F); auxiliary peptide chain connecting domain (B) and main peptide chain connecting domain (F) complement each other to make main peptide chain X and auxiliary peptide chain Y polymerize to form a multi-target chimera Antigen receptor (RaceCar).

[0139] The multi-target chimeric antigen receptor (RaceCar-1) targeting CD19 and PD-L1 positive cells is polymerized from the main peptide chain X1 and the auxil...

Embodiment 2

[0164] Example 2, Flow Cytometry Verification of RaceCar's 293T Cell Expression

[0165] 1. Lenti-X-293T cells (Clontech, article number: 632180, hereinafter referred to as 293T) 1.2*10E 6 Spread in 6-well plate, 37°C, 5% CO 2 Incubate overnight.

[0166] 2. Transfect the vector pFUGW-RaceCar-1 constructed in Example 1 into the cells obtained in the above 1 using lipofectamine3000, at 37°C, 5% CO 2 Cultivate for 48h.

[0167] 3. The 293T cells transfected with pFUGW-RaceCar-1 were digested with trypsin and resuspended in PBS, and the cell density was controlled at 1*10E 6 / mL.

[0168] 4. Add 1.5uL APC Mouse anti-Human CD279 (BD, Cat. No. 558694) and FITC-labeled CD19-FC fusion protein (CD19-FC fusion protein nucleic acid sequence is sequence 35) to 200uL3 treated cells respectively, use FITC After 30 min in ice bath, desalt for labeling, 293F expression), and incubate on ice for 30 min. Centrifuge to remove the supernatant, and resuspend the cells with an equal volume o...

Embodiment 3

[0171] Example 3, Western blot verification of RaceCar expressed in 293T

[0172] 1. Take 5*10E 6 The 293T cells transfected with pFUGW-RaceCar-1 in Example 2 were centrifuged at 1500 R for 10 min and the supernatant was discarded.

[0173] 2. Add 200uL Ripa cell lysate (Biyuntian, Cat. No.: P0013B) and let stand on ice for 30 minutes. Centrifuge at 8000g for 5 minutes to take 20uL of the supernatant, add 5X protein electrophoresis buffer, and place in a metal bath at 95°C for 5 minutes to prepare protein electrophoresis samples.

[0174] 3. Carry out SDS-PAGE. After the electrophoresis is completed, cut off the part of the stacking gel, and transfer to the membrane at 100V, 200mA, and 1h.

[0175] 4. Place the PVDF membrane with the transferred protein in 5% skim milk / PBS solution and block overnight. PBST decolorization shaker washed 3 times, 15min each time.

[0176] 5. Put the PVDF membrane into 1uL antibody / mL1% skimmed milk / PBS solution, in which the antibody is PD1 / ...

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Abstract

The invention discloses a multi-target chimeric antigen receptor. The multi-target chimeric antigen receptor provided by the invention consists of a main peptide chain and an assistant peptide chain.The main peptide chain includes an antigen binding domain A, an assistant peptide chain connection domain B, a transmembrane domain C and an intracellular signal conduction domain D. The assistant peptide chain includes a main peptide chain connection domain F. The antigen binding domain A is a polypeptide with antigen-bonding function. The assistant peptide chain connection domain B and the mainpeptide chain connection domain F are mutually combined. The transmembrane domain C is the transmembrane domain of arbitrary membrane binding protein or the transmembrane domain of transmembrane protein. The intracellular signal conduction domain D includes a primary signal conduction region. The multi-target chimeric antigen receptor provided by the invention can mediate specific cell killing bycombining two antigen binding domains with different antigens. Also a cytokine and cytokine receptor compound are introduced into the multi-target chimeric antigen receptor provided by the invention and can play the role of cytokines.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a multi-target chimeric antigen receptor. Background technique [0002] Chimeric antigen receptor (chimeric antigen receptor, CAR) is usually a kind of transmembrane fusion protein mainly composed of antigen binding domain, transmembrane domain and intracellular signaling domain (patent CN 105392888A), when immune cells Antigen targeting specificity is conferred upon expression of chimeric antigen receptors (eg T cells or NK cells). [0003] The antigen-binding domain of the CAR molecule determines the specific targeting of immune cells. The most typical CAR often uses a single-chain variable region antibody (single-chain variable fragment, scFv) as its antigen-binding domain, which consists of the heavy chain of the antibody The variable region and the light chain variable region are connected by a linker. Because of its simple structure and only one peptide chain, it is very suita...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/078G01N33/68A61K35/17A61P35/00
CPCG01N33/68A61K35/17C12N5/0634C07K14/705C07K14/715C07K16/00C07K16/30C07K2319/03C07K2319/02A61P35/00C07K19/00C12N5/10C12N15/62C12N15/63C07K14/7051C12N5/0636C12N5/0646
Inventor 王磊高斌吴亚松司远吕丹魏卿
Owner TIMMUNE BIOTECH INC
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