Chimeric antigen cell receptor and application thereof

A technology of cell receptors and chimeric antigens, applied in the direction of receptors/cell surface antigens/cell surface determinants, hybrid peptides, anti-tumor drugs, etc., can solve the problems of low TCR receptor affinity, screening, and restriction of TCR receptors Application and other issues

Inactive Publication Date: 2018-11-13
安诺柏德生物医药科技(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TCR receptors have a high degree of specificity, and the affinity of spontaneously formed TCR receptors in the natural state is generally low, and the recognition process with protein antigen peptide-MHC complexes is limited by the type of MHC molecules, and the human TCR receptors that have been identified so far There are more than 300 types of MHC molecules, and it is almost impossible to screen a specific TCR for each MHC molecule and corresponding antigenic peptide, which severely limits the application of TCR receptors in the field of adoptive immune cell therapy

Method used

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  • Chimeric antigen cell receptor and application thereof
  • Chimeric antigen cell receptor and application thereof
  • Chimeric antigen cell receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] In this example, taking targeting human CD19 protein as an example, the structure of the artificial chimeric antigen cell receptor in this example is described as follows:

[0083] The amino acid sequence of the chimeric antigen cell receptor is shown in SEQ ID NO.19.

[0084] The chimeric antigen cell receptor includes an antigen binding domain, and an α chain and a β chain respectively connected to the antigen binding domain, the antigen binding domain is a Fab fragment of an anti-CD19 protein antibody, and the anti-CD19 protein antibody has The characteristic of specific binding to the target protein CD19.

[0085]Wherein, the α chain is connected with the heavy chain constant region of the Fab fragment to form the first chain, and the first chain includes the heavy chain variable region of the Fab fragment (SEQ ID NO.12), the humanized heavy chain CH1 constant of the Fab fragment Region (SEQ ID NO.13), hinge region, transmembrane region and intracellular active reg...

Embodiment 2

[0090] Preparation of Chimeric Antigen Cell Receptor Lentivirus

[0091] (1) Take out HEK293T cells from the liquid nitrogen tank, and quickly put them into a 37°C water bath to thaw. In a biological safety cabinet, use a pipette to add the cell suspension in the tube to a 15mL centrifuge tube containing 5mL of medium, mix by inverting, and centrifuge at 250xg for 10 minutes at room temperature.

[0092] (2) After the centrifugation, add 5 mL of complete medium (DMEM, 10% FBS), gently blow the cell mass, then transfer the cell suspension to a 10 cm culture dish, supplement the complete medium (DMEM, 10% FBS) to Final volume 10 mL. Place the Petri dish at 37°C, 5% CO 2 Incubate overnight in an incubator.

[0093] (3) Preparation of liposome / DNA complex. Take 500uL PBS into a sterile centrifuge tube, add 22μg LV6-EF1a-CAFabR-puroGFP, 12μg Lenti-Gag, 9μg Lenti-Rev, 5μg pTetOff, 5μg pVSVG respectively, pipette up and down to mix well, then add 500μL 1μg / μL liposomes, immedia...

Embodiment 3

[0098] Preparation of Chimeric Antigen Cell Receptor T Cells

[0099] (1) Transfer the anticoagulated blood sample to a 15mL sterile centrifuge tube, tighten the cap, adjust the centrifugal force of the centrifuge to 800xg, set the centrifugal speed down to the lowest, and centrifuge the blood sample at room temperature for 20 minutes.

[0100] (2) After centrifugation, take out the centrifuge tube from the centrifuge, discard the upper light yellow serum layer with a disposable pipette; then add an equal volume of normal saline to the lower red peripheral blood cell layer, and tighten the cap of the centrifuge tube , and mix by gently inverting.

[0101] (3) Take out the lymphocyte separation solution, turn it upside down several times, and mix well.

[0102] (4) In a biological safety cabinet, first add 5 mL of lymphocyte separation solution to a 15 mL centrifuge tube with a disposable sterile pipette, and then use a pipette to slowly add the blood sample diluted with norma...

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Abstract

The invention discloses a chimeric antigen cell receptor and application thereof, and relates to the technical field of biology. The chimeric antigen cell receptor comprises an antigen binding structure domain, an alpha chain and a beta chain, wherein the alpha chain and the beta chain are connected with the antigen binding structure domain; the antigen binding structure domain is an antibody Fabfragment; the antibody has the characteristic combined with target protein specificity. Immune cells for expressing the chimeric antigen cell receptor can be combined with target proteins on target cells; activating signals are transmitted; immune cells are activated, so that the target cells for expressing the target proteins are killed; the process does not depend on MHC molecules for submissionof target proteins. The chimeric antigen cell receptor can be applied to the preparation of corresponding antitumor medicine, and is used in the fields of malignant cell clearing, autoimmune diseasetreatment and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chimeric antigen cell receptor and its application. Background technique [0002] The T cell antigen receptor (TCR) in mammals recognizes protein peptides bound to the major histocompatibility complex (MHC). These protein peptide-MHC complexes are usually displayed on the surface of different types of abnormal cell membranes. These sources The protein polypeptide-MHC complex derived from pathogens or the neoantigen peptide-MHC complex derived from tumors are molecular markers on abnormal cells in vivo. [0003] Natural T cell receptor (TCR) is a multi-subunit antigen-specific transmembrane complex that can mediate antigen-specific activation of T cells. TCR is composed of two different polypeptide chains (such as figure 1 Schematic), T cell receptor alpha chain and T cell receptor beta chain. The two chains have an N-terminal variable region (V region) and a C-terminal constant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K35/17A61P35/00A61P37/00
CPCA61P35/00A61P37/00A61K35/17C07K14/7051C07K2319/02C07K2319/03
Inventor 荆光军岳庆李成欧晓红
Owner 安诺柏德生物医药科技(苏州)有限公司
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