A composite protein of chimeric antigen receptor fused with inducible apoptosis enzyme

A technology of chimeric antigen receptors and complex proteins, which is applied in the fields of genetic engineering and oncology, can solve the problems of patients' body injury, cell off-target effects, etc., and achieve the effect of avoiding the storm of inflammatory factors

Active Publication Date: 2021-01-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main obstacle to the application of CAR-T therapy lies in the possible off-target effects of cells when specifically recognizing specific tumor cells, and the damage to the patient's body due to the possible inflammatory cytokine storm induced by the reinfusion of a large number of activated T lymphocytes

Method used

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  • A composite protein of chimeric antigen receptor fused with inducible apoptosis enzyme
  • A composite protein of chimeric antigen receptor fused with inducible apoptosis enzyme
  • A composite protein of chimeric antigen receptor fused with inducible apoptosis enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of a plasmid with a specific receptor protein that binds to the Her2 receptor, simultaneously strengthens the activation of immune T lymphocytes, and can regulate apoptosis

[0046] 1.1 Primer design: After finding the Her2 single-chain fragment antibody from the gene library, the activation fragment (CD8α chain, CD137 transmembrane segment, CD3ζ intracellular segment), and the sequence of the induction fragment (the conserved segment of Fkbp12 and caspase-9), use Primer software designed relevant primers and connecting primers respectively. The entire field sequence of the above-mentioned fragments can be searched and obtained from Gene Bank.

[0047] 1.1.1. Her2-ScFv primers:

[0048] Forward: NcoI is an endonuclease

[0049] 5'-GGGCCATGGCCCAGGTGCAGCTGTTGCAGTCTGGGGCAGAG-3' (SEQ ID NO: 3)

[0050] Reverse: NotI is an endonuclease

[0051] 5'-TTGCGGCCGCTCCGGAATTCACCTAGGACGGTCAGCTTGGTCCC-3' (SEQ ID NO: 4)

[0052] 1.1.2. Primers for activating...

Embodiment 2

[0114] Example 2 Small amount extraction of pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9 plasmid

[0115] 2.1 Bacteria collection: Collect bacteria by centrifugation at 12000g×1min. (Generally, 3-4ml bacterial liquid is used for extracting one portion);

[0116] 2.2 Discard the supernatant;

[0117] 2.3 Add 250u l ice-bath Buffer S1 (containing RNase A), vortex and oscillate to fully suspend the bacteria;

[0118] 2.4 Add 250ul Buffer S2, mix gently 6 times (this process does not exceed 5min); add 350ul Buffer S3, mix gently 6 times;

[0119] 2.5 Centrifuge at 12000g×10min;

[0120] 2.6 Take the supernatant, pass it through the DNA preparation tube, 12000g×1min, and discard the filtrate;

[0121] 2.7 Add 700ul Buffer W1 to the DNA preparation tube, 12000g×1min, discard the filtrate;

[0122] 2.8 Add 500ul BufferW2 to the DNA preparation tube, 12000g×1min, discard the filtrate;

[0123] 2.9 Centrifuge again at 12000g×1min;

[0124] 2.10 plus 40ulddH 2 Put O (or EB buffer...

Embodiment 3

[0126] Example 3 Identification of Chimeric Receptor Fusion-Induced Apoptosis Complex Protein by Enzyme Digestion Plasmid pMSCVpuro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9

[0127] The pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9 plasmid was analyzed with the sequence analysis software BioEdit, and the position of each restriction site was correctly located.

[0128] The restriction site of EcoRI (G^AATTC) is at 4537; the restriction site of XhoI (C^TCGAG) is at 1417; Dicer will cut from the 3 bases before and after the restriction site, therefore, the cut band size will be 6 bases more than the actual insertion sequence), and the position is accurate, such as figure 1 shown.

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Abstract

The invention discloses chimeric antigen receptor and induced apoptosis enzyme co-fused compound protein, a coding sequence thereof and modified T lymphocyte containing the compound protein. The invention also relates to application of the above compound protein and the modified T lymphocyte in prevention and treatment of breast cancer. The constructed specific chimeric antigen receptor and induced apoptosis enzyme co-fused compound protein has functions of binding to a Her2 receptor, enhancing activated state of T lymphocyte and regulating cell apoptosis. The modified T lymphocyte can efficiently kill Her-2 positive breast cancer cells and can induce modified T lymphocyte apoptosis under the action of the drug AP1903 to terminate the expanding killing effect as a safety guarantee.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and oncology, especially a composite protein of chimeric antigen receptor fused with inducible apoptosis enzyme and its construction method. The present invention also relates to the use of the above composite protein in the preparation of drugs for treating breast cancer use. Background technique [0002] The incidence of breast cancer in my country is showing an increasing trend year by year, especially the cases of advanced metastatic breast cancer are increasing. At present, the treatment of breast cancer is still a comprehensive treatment based on surgery. Since the concept of "breast cancer is a systemic disease" was put forward, more and more attention has been paid to systemic treatment. However, breast cancer that has metastasized throughout the body is still an intractable disease. These cases are often insensitive to multiple chemotherapy and radiotherapy regimens, and vari...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/395A61K38/43A61K35/17A61P35/00
CPCA61K35/17A61K38/00C07K14/7051C07K14/70517C07K14/70596C07K16/2863C12N5/0636C12N9/641C12Y304/22062
Inventor 宋尔卫姚燕丹张明霞
Owner SUN YAT SEN UNIV
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