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Chimeric antigen receptor targeting human GPC3 and application thereof

A chimeric antigen receptor and targeting technology, applied in the direction of polypeptides containing positioning/targeting motifs, anti-animal/human immunoglobulins, hybrid peptides, etc., can solve the limitations of clinical applications, tumor cell recognition, Reduce the killing ability and other problems, and achieve the effect of reducing tumor recurrence rate, prolonging the survival period, and strong killing activity

Inactive Publication Date: 2020-04-24
NANJING BLUE SHIELD BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the application of CAR-T technology is still limited to hematological tumors such as leukemia, myeloma, and lymphoma, and it is rarely used in solid tumors such as liver cancer. In addition, in the complex tumor microenvironment, CAR-T cells have a negative effect on tumor cells. The ability to recognize and kill is greatly reduced, which limits its clinical application

Method used

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  • Chimeric antigen receptor targeting human GPC3 and application thereof
  • Chimeric antigen receptor targeting human GPC3 and application thereof
  • Chimeric antigen receptor targeting human GPC3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of chimeric antigen receptor targeting human GPC3 in the present invention.

[0031] (1) Construction of chimeric antigen receptor (CAR) lentiviral expression vector

[0032] Using the intracellular domain of CD137 and the immunoreceptor tyrosine activation motif (ITAM) region of CD3Zeta as the activation signal, fused with the single-chain antibody of GPC3 to construct a chimeric antigen receptor expression vector, and subcloned it into PLVX - In the EF1a vector (purchased from clontech), the combination sequence of each element in the constructed chimeric antigen receptor lentiviral expression vector is as follows figure 1 Shown:

[0033] The amino acid sequences of each element in the constructed chimeric antigen receptor are (synthesized by Nanjing GenScript Biotechnology Co., Ltd.):

[0034] Signal peptide: SEQ ID NO.4

[0035] Sequence of extracellular antigen binding region: SEQ NO.1 and SEQ NO.2

[0036] CD8a hinge region: SEQ ID NO.5 ...

Embodiment 2

[0067] Example 2 Lysis of target cells by CAR-T cells.

[0068] The specific experimental steps are as follows:

[0069] S1. Adjust the target cell state to the logarithmic growth phase, and it needs to be continuously passaged twice before the experiment;

[0070] S2. Digest the adherent target cells with trypsin and resuspend them in complete medium, adjust the cell density to 5*10 5 cells / mL, take a new 96-well plate, and inoculate target cells at 100 μL / well. To the unused wells around the 96-well plate, add 100 μL sterile water to each well to prevent water evaporation in the middle experimental well. Place the orifice plate in 5% CO 2 , Cultivate overnight in a 37°C incubator;

[0071] S3. Collect the above-prepared CAR-T cells by centrifugation, and resuspend them in serum-free 1640 medium; take out the 96-well plate from the incubator, completely suck out the medium in the wells, and gently wash the cells once with sterile PBS , and then according to the E / T ratio...

Embodiment 3

[0078] Example 3 Detection of secretion level of CAR-T cytokines.

[0079] The specific experimental steps are as follows:

[0080] S1. Adjust the target cell state to the logarithmic growth phase, and it needs to be continuously passaged twice before the experiment;

[0081] S2. Digest the adherent target cells with trypsin and resuspend them in complete medium, adjust the cell density to 5*10 5 cells / mL, take a new 96-well plate, and inoculate target cells at 100 μL / well. Add 100 μL of sterile water to the unused wells around the 96-well plate to prevent water evaporation in the middle experimental well; place the orifice plate in 5% CO 2 , Cultivate overnight in a 37°C incubator;

[0082] S3. Collect the above-prepared CAR-T cells by centrifugation, and resuspend them in serum-free 1640 medium; take out the 96-well plate from the incubator, completely suck out the medium in the wells, and gently wash the cells once with sterile PBS , and then according to the E / T ratio ...

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Abstract

The invention discloses a chimeric antigen receptor targeting human GPC3, the chimeric antigen receptor comprises an extracellular antigen binding region, a transmembrane region and an intracellular signal structural domain which are sequentially connected in series, the extracellular antigen binding region is combined with a GPC3 antigen, the extracellular antigen binding region comprises anti-GPC3 scFv used for combining the GPC3 antigen, wherein a heavy chain amino acid sequence is shown as SEQ ID NO. 1, and a light chain amino acid sequence is shown as SEQ ID NO. 2. Compared with the priorart, the chimeric antigen receptor is constructed by taking the human GPC3 as a target spot, and particularly, amino acids in ITAM2 and ITAM3 regions of CD3zeta are mutated, so that constructed T cells modified by the chimeric antigen receptor have stronger killing activity on tumor cells; moreover, more central memory T cells are provided, GPC3 positive tumor cells are killed in a targeted manner, the tumor recurrence rate after treatment is effectively reduced, and the life cycle of a patient is prolonged.

Description

technical field [0001] The invention relates to a chimeric antigen receptor targeting human GPC3 and an application thereof, belonging to the technical field of chimeric antigen receptors. Background technique [0002] Chimeric antigen receptor (CAR) is an artificial receptor that mimics the function of T cell receptor, which combines the recognition and binding specificity of antigen and antibody or ligand and receptor, and the killing of recognized tumor cells by effector T cells ability. CAR is sequentially connected by CD8a leader peptide, antigen recognition region (ligand or single-chain antibody or Fab fragment), transmembrane region, and a series of signal transduction regions of T cells (CD28, CD3, CD137 intracellular signaling domain) made. After T cells are modified, the CAR expressed on the surface first binds to the surface antigen of tumor cells through the antigen recognition region, and then transmits the activation signal into the cell through its signal t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62
CPCC07K16/18C07K2319/02C07K2319/03
Inventor 李俊郭志刚
Owner NANJING BLUE SHIELD BIOTECH CO LTD
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