Application of chimeric antigen receptor taking CD99 as target in combination with anti-tumor drugs

An anti-tumor drug and antigen technology, applied in the field of medicine and biology, can solve problems such as single, off-target, and unsatisfactory treatment effects, and achieve the effects of improving survival rate, improving tumor killing efficiency, and scientifically evaluating tumor killing effect

Active Publication Date: 2020-11-20
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To overcome the problems of off-target caused by high tumor heterogeneity and unsatisfactory treatment effect due to singleness of target in the process of CAR-T cell therapy

Method used

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  • Application of chimeric antigen receptor taking CD99 as target in combination with anti-tumor drugs
  • Application of chimeric antigen receptor taking CD99 as target in combination with anti-tumor drugs
  • Application of chimeric antigen receptor taking CD99 as target in combination with anti-tumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of PTK881-EF1α-C1 and PTK881-EF1α-C2

[0032] 1. Artificially synthesize the fragments shown in SEQ ID NO.2 and SEQ ID NO.4 respectively to form SP-C1 and SP-C2.

[0033] 2. Using the human cDNA library as a template, design primers for PCR to amplify fragments CD8 hinge, CD28 transmembrane region, CD28 intracellular domain, 4-1BB, and CD3ζ respectively, and obtain StrepⅡ fragments by primer complementation. Using Overlap PCR technology, SP-C1, SP-C2 and fragments of Strep tag II, CD8 hinge, CD28 transmembrane region, CD28 intracellular domain, 4-1BB, and CD3ζ were sequentially amplified and connected into EcoR with restriction sites. The C1-CAR and C2-CAR of I and BamH I, the structural diagrams of C1-CAR and C2-CAR are as follows figure 1 shown;

[0034] Among them, SP-C1 and SP-C2 are single-chain antibody ScFv capable of recognizing CD99 on the surface of tumor cells. Single-chain antibody ScFv: the amino acid sequences of SP-C1 and SP-C2 ...

Embodiment 2

[0038] Example 2, preparation and sequencing of plasmids

[0039] 1. Preparation of plasmid

[0040] Inoculate DH5α strains containing plasmids PTK881-EF1α-C1 and PTK881-EF1α-C2 into 250 mL of LB culture medium containing 100 μg / mL ampicillin, and culture overnight at 37°C and 220 rpm. The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0041] Take out Buffers P1 from the Endo Free 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 make the E. coli pellet completely in Buffers P1 dispersion.

[0042] 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.

[0043] Add 120mL Buffers P3 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to the maximum speed of...

Embodiment 3

[0056] Example 3, Preparation of Lenti3-C1-CAR, Lenti3-C2-CAR Lentiviral Vectors and Detection of Live Droplets

[0057] 1. Preparation of lentiviral vector

[0058] Insert 130.0~140.0×10 in the multilayer cell culture flask (Hyperflask) 6 Number of 293T cells (Takara), a total of 560 mL DMEM complete medium (50 mL fetal bovine serum, 5 mL Antibiotic-Antimycotic (100×)), at 37 °C with 5% CO 2 Cultivate in the incubator for 24h. Mix the shuttle plasmid PTK881-EF1α-C1 / PTK881-EF1α-C2 with the packaging plasmid in the following ratio, PTK881-EF1α-C1 / PTK881-EF1α-C2: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid = 12:10:5:6 , to get 320 μg of mixed plasmids, add 15 mL of DMEM complete medium. At the same time, 960 μg of PEI was added to 15 mL of DMEM complete medium, and the DMEM complete medium mixed with PEI was slowly added to the DMEM complete medium mixed with plasmid, and equilibrated at room temperature for 10 minutes. Mix the above 30mL mixed solution with 530mL DMEM complete me...

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Abstract

The invention provides a combined medication method for treating tumors and an application of the combined medication method. The combined medication method combines chimeric antigen receptor immune cell therapy and chemotherapy and has a stronger anti-tumor effect than single chemotherapy or single immune cell therapy. Meanwhile, the dosage of chemotherapeutic drugs can be reduced, and toxic andside effects generated by the chemotherapeutic drugs are reduced. The problems that off-target is caused by high heterogeneity of tumors in a process of treating the tumors with CAR-T cells, and the treatment effect is not ideal due to singleness of a target are solved.

Description

technical field [0001] The invention relates to the field of medicine and biology, and specifically refers to the application of a chimeric antigen receptor (CAR) combined with anti-tumor drugs targeting CD99 to treat broad-spectrum tumors. Background technique [0002] In early 2019, data released by the American Cancer Society showed that there were 18.1 million new cases of cancer and 9.6 million deaths worldwide in 2018. The incidence and death rate of cancer in the Chinese population ranked first in the world, and showed a rapid growth trend. Scientists around the world are also working on the research of various anticancer therapies. Immunotherapy, as the third revolution in anticancer therapy after chemotherapy and targeted therapy, has become a new generation of tumor treatment. In 2013, American "Science" magazine selected tumor immunotherapy as the biggest scientific breakthrough of the year, and in 2015, combined tumor immunotherapy was listed as one of the four m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K33/243A61K31/337A61P35/00A61P35/02C12N15/867C12N15/62C12N5/10
CPCA61K39/001111A61K33/243A61K31/337A61P35/00A61P35/02C12N15/86C12N5/0636C07K16/2896C07K14/7051C07K14/70517C07K14/70521C07K14/70578A61K2039/804A61K2039/812C12N2800/107C12N2740/15043C12N2510/00C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74A61K2300/00
Inventor 张同存顾潮江祝海川周经姣周勇史江舟
Owner WUHAN BIO RAID BIOTECH CO LTD
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