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Chimeric antigen receptor combined anti-tumor medicine composition and application thereof

An anti-tumor drug and tumor drug technology, applied in the field of biomedicine, can solve the problems of off-target, limited therapeutic effect, poor therapeutic effect, etc., and achieve the effect of improving tumor killing efficiency and significant effect.

Pending Publication Date: 2022-04-08
WUHAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the current PS targeted therapy has shown potential anti-tumor activity, there are problems in the treatment of tumors by CAR-T cells, such as off-target caused by high tumor heterogeneity and poor therapeutic effect due to the singleness of the target. The limitations of the therapy, which limit the therapeutic effect

Method used

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  • Chimeric antigen receptor combined anti-tumor medicine composition and application thereof
  • Chimeric antigen receptor combined anti-tumor medicine composition and application thereof
  • Chimeric antigen receptor combined anti-tumor medicine composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Anti-PS ScFv and protein selection targeting PS

[0031] In order to select a suitable protein structure that can target PS, we selected two ScFvs of two known antibodies that have entered clinical trials, named A1 (US_2016_0015826_A1) and A2 (US_2018_0289771_A1) respectively. For the comparison of the combined non-antibody protein sequences, the most common AnnexinA5 protein was selected and named A3. The amino acid sequence of A1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence of A2 is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4 shown; the amino acid sequence of A3 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6. The survey results showed that the three anti-PS scFvs or proteins all had high affinity and could be used for the subsequent construction of CAR-T cells.

Embodiment 2

[0032] The construction of embodiment 2 plasmid

[0033] 1. Fragments A1, A2, and A3 were artificially synthesized, and PS, StrepII+G4S-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ fragments were artificially synthesized.

[0034] 2. Using Overlap PCR amplification, PS, A1 / A2 / A3 and StrepII+G4S-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ were used as templates to obtain A1 with restriction sites EcoR I and BamH I -Structure diagram of CAR, A2-CAR, A3-CAR, A1-CAR, A2-CAR, A3-CAR figure 1 shown;

[0035]Wherein, the amino acid sequence of the signal peptide (PS) is shown in SEQ ID NO.7; the nucleotide sequence is shown in SEQ ID NO.8; the amino acid sequence of StrepII+G4S is shown in SEQ ID NO.9, and the nucleotide The sequence is shown in SEQ ID NO.10; the amino acid sequence of CD8 hinge is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12; the amino acid sequence of CD28TM is shown in SEQ ID NO.13, nucleoside The acid sequence is shown in SEQ ID NO.14; the amino acid seq...

Embodiment 3

[0039] Example 3 Preparation and sequencing of plasmids

[0040] 1. Preparation of plasmid

[0041] Inoculate Escherichia coli DH5α strains containing plasmids PTK881-EF1α-A1, PTK881-EF1α-A2, PTK881-EF1α-A3, respectively into 250mL LB culture medium containing 100μg / mL ampicillin, and culture overnight at 37°C and 220rpm . The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0042] Take out Buffers P1 from the EndoFree plasma mega kit (Qiagen), add 120mL pre-cooled Buffers P1 to the centrifuged E. coli pellet, cover the cap of the centrifuge bottle, shake the centrifuge bottle vigorously to completely disperse the E. coli pellet in Buffers P1 .

[0043] Add 120mL Buffers P2 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to 50rpm, mix thoroughly and place at room temperature for 5min.

[0044] Add 120mL Buffers P3 to the centrifuge bottle, put the cap on the roller mixer, slowly increase t...

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Abstract

The invention provides a chimeric antigen receptor combined anti-tumor medicine composition, a chimeric antigen receptor comprises a single-chain antibody ScFv capable of recognizing phosphatidylserine on the surface of a tumor cell, and an anti-tumor medicine is a chemotherapeutic medicine capable of increasing expression of phosphatidylserine on the surface of a tumor cell line or a primary tumor cell. The chimeric antigen receptor taking phosphatidylserine as a target spot is used for modifying immune cells, and the modified immune cells can be used for treating tumors with positive phosphatidylserine on the surface and particularly have a remarkable tumor killing effect on ewing sarcoma, acute lymphoma / leukemia, acute myelogenous leukemia and breast cancer.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a chimeric antigen receptor combined anti-tumor pharmaceutical composition and its application. Background technique [0002] Chimeric antigen receptor T cells (CAR-T-Cell), the principle is to combine the single-chain variable region (Scfv) of an antibody that can recognize a certain tumor antigen and the intracellular region of the CD3-ζ chain through genetic engineering. It is coupled to a chimeric protein in vitro, and is transfected into patient T cells cultured in vitro by gene transduction to express chimeric antibody receptor (CAR). After the patient's T cells are "reprogrammed", a large number of lethal CAR-T cells are generated, which can specifically target tumor cells. Compared with traditional immunotherapy, CAR-T has significant advantages such as more precise treatment, more precise targeting, wider tumor killing, and longer-lasting effects. [0003] Phosphat...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K45/06C07K19/00C12N5/10C12N15/867C12N15/861C12N15/864A61P35/00A61P35/02
Inventor 张同存刘家兴祝海川史江舟
Owner WUHAN UNIV OF SCI & TECH
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