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Chimeric antigen receptor taking IFNGR1 as target spot and application of chimeric antigen receptor

An antigen-receptor technology, applied in the field of medicine and biology, can solve the problems of off-target, singleness, and broad-spectrum limitation of treatment, and achieve the effect of improving tumor killing efficiency and remarkable effect

Pending Publication Date: 2022-06-03
WUHAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Despite the continuous success in the field of CAR-T treatment of tumors, CAR-T cells still face problems such as off-targets caused by high tumor heterogeneity and singleness of targets in the treatment of tumors.
The high heterogeneity of tumor cells directly leads to the limitations of CAR-T therapy in the treatment process, and the singleness of the target limits the broad-spectrum of treatment

Method used

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  • Chimeric antigen receptor taking IFNGR1 as target spot and application of chimeric antigen receptor
  • Chimeric antigen receptor taking IFNGR1 as target spot and application of chimeric antigen receptor
  • Chimeric antigen receptor taking IFNGR1 as target spot and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Affinity determination of scFv against IFNGR1

[0034] Anti-IFNGR1 scFvs with strong specificity and high affinity were screened from the large-capacity IFNGR1 phage antibody library prepared with the extracellular domain of IFNGR1 as antigen. Named as B1, B2 and B3, the sequence analysis of their sequences shows that the nucleotide sequences of B1, B2 and B3 are shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, respectively, and their amino acids are shown in The sequences are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, respectively.

[0035] In order to determine the affinity of the above scFv to the antigen IFNGR1, the binding kinetics of the soluble scFv and the extracellular domain of IFNGR1 was further analyzed by surface plasmon resonance analysis, and the Kd values ​​of purified B1, B2 and B3 were calculated. The steps are briefly described as follows:

[0036] According to the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, a...

Embodiment 2

[0037] Example 2: Construction of PTK881-EF1α-B1, PTK881-EF1α-B2, PTK881-EF1α-B3, PTK881-EF1α-B1-7x19, PTK881-EF1α-B2-7x19, PTK881-EF1α-B3-7x19 Plasmids

[0038] 1. Artificially synthesize fragments B1, B2, B3, and artificially synthesize SP, strepII-CD8hinge-CD28TM+ICD-4-1BB-CD3ζ fragment.

[0039] 2. Use Overlap PCR to amplify with SP, B1 / B2 / B3 and strepII-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ, or with SP, B1 / B2 / B3 and strepII-CD8 hinge-CD28TM+ICD -4-1BB-CD3ζ+F2A peptide+IL-7+F2A peptide+CCL19 as template to obtain B1-CAR, B2-CAR, B3-CAR, B1-7x19 CAR with restriction sites EcoR I and BamH I , B2-7x19 CAR, B3-7x19 CAR, the schematic diagram of the structure of B1-CAR, B2-CAR, B3-CAR figure 1 shown; the structural schematic diagrams of B1-7x19 CAR, B2-7x19 CAR, B3-7x19 CAR fragments are shown in figure 2 shown.

[0040] The amino acid sequence of signal peptide (SP) is shown in SEQ ID NO.7, the amino acid sequence of strepII is shown in SEQ ID NO.9, the amino acid sequence of CD...

Embodiment 3

[0044] Example 3. Preparation and sequencing of plasmids

[0045] 1. Plasmid preparation

[0046] Escherichia coli DH5α strains containing plasmids PTK881-EF1α-B1, PTK881-EF1α-B2, PTK881-EF1α-B3, PTK881-EF1α-B1-7x19, PTK881-EF1α-B2-7x19, PTK881-EF1α-B3-7x19 They were inoculated into 250 mL of LB medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C and 220 rpm. The culture medium was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0047] Take out Buffers P1 in the EndoFree plasmid mega kit (Qiagen), add 120 mL of pre-cooled Buffers P1 to the E. coli pellet obtained by centrifugation, cover the centrifuge bottle, and shake the centrifuge bottle vigorously to completely disperse the E. coli pellet in Buffers P1 .

[0048] Add 120 mL of Buffers P2 to the centrifuge bottle, cover the bottle and place it on a roller mixer, slowly increase the speed to 50 rpm, mix thoroughly and place at room temperature for 5 minutes.

[0049] Add 12...

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Abstract

The invention discloses a chimeric antigen receptor taking an IFNGR1 receptor as a target spot and application of the chimeric antigen receptor, a signal peptide, a single-chain antibody ScFv, strepII, CD8hinge, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular costimulatory domain 4-1BB and a CD3zeta chain are sequentially spliced from an N end to a C end, and more preferably, the signal peptide, the single-chain antibody ScFv, strepII, CD8hinge, CD28 transmembrane region, CD28 intracellular domain, the intracellular costimulatory domain 4-1BB, the CD3zeta chain, F2A peptide, IL-7, F2A peptide and CCL19 are sequentially spliced from the N end to the C end. The single-chain antibody ScFv can specifically recognize an IFNGR1 receptor on the surface of a tumor cell. The chimeric antigen receptor taking the IFNGR1 receptor as the target spot is used for modifying immune cells, and the modified immune cells can be used for treating surface IFNGR1 positive tumors.

Description

technical field [0001] The invention relates to the field of medicine and biology, in particular to a chimeric antigen receptor (CAR) that uses IFNGR1 as a target to treat broad-spectrum tumors and its application. Background technique [0002] At the beginning of 2019, data released by the American Cancer Society showed that in 2018, there were 18.1 million new cases of cancer and 9.6 million deaths in the world. The incidence and mortality 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 various anti-cancer therapies. Immunotherapy, as the third revolution in anti-cancer therapy after chemotherapy and targeted therapy, has become a new generation of tumor treatment methods. In 2013, the American "Science" magazine selected tumor immunotherapy as the biggest scientific breakthrough of the year, and in 2015, tumor immunotherapy was listed as one of the four most noteworthy scientif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N15/62C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2866C07K14/7051C07K14/5418C07K14/521C12N15/86C12N5/0636A61K39/00112A61P35/00A61P35/02C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N2740/15043C12N2800/107C12N2510/00A61K2039/5158
Inventor 张同存冷颖奇史江舟
Owner WUHAN UNIV OF SCI & TECH
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