Chimeric antigen receptor taking CD99 as target spot and application of chimeric antigen receptor

A technology of receptors and antigens, applied in the field of medicine and biology, can solve the problems of off-target, limited treatment, broad-spectrum, singleness, etc., and achieve the effect of improving tumor killing efficiency and scientific evaluation of tumor killing effect

Pending Publication Date: 2022-05-17
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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 CD99 as target spot and application of chimeric antigen receptor
  • Chimeric antigen receptor taking CD99 as target spot and application of chimeric antigen receptor
  • Chimeric antigen receptor taking CD99 as target spot and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Affinity determination of scFv against CD99

[0037] After multiple rounds of screening in a large-capacity CD99 phage antibody library prepared with the extracellular domain of CD99 as an antigen, a scFv that can specifically recognize CD99 on the surface of tumor cells was obtained, which was named C6. Sequencing analysis of its sequence shows that the nucleotide sequence of C6 is shown in SEQ ID NO.2., and its amino acid sequence is SEQ ID NO.1.

[0038] In order to characterize the advantages and disadvantages of the ScFv obtained by the screening of the present application relative to the known anti-CD99 ScFv of the prior art and whether it is applicable to the construction of chimeric antigen receptors, four ScFvs known in the prior art were selected (CN 110590960 AC2 C3 ; sequences 47, 83 in WO 2019136419) to conduct research simultaneously. Next, the four ScFvs known in the prior art were named C2, C3, C4 and C5 respectively, and their amino acid seq...

Embodiment 2

[0042] Example 2: PTK881-EF1α-C6, PTK881-EF1α-C2, PTK881-EF1α-C3, PTK881-EF1α-C4, PTK881-EF1α-C5, PTK881-EF1α-C6-7x19, PTK881-EF1α-C2-7x19, Construction of PTK881-EF1α-C3-7x19, PTK881-EF1α-C4-7x19, PTK881-EF1α-C5-7x19 plasmids

[0043] 1. Artificially synthesize fragments C6, C2, C3, C4, and C5, respectively, and artificially synthesize fragments of SP and strepII-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ.

[0044] 2. Use Overlap PCR to amplify with SP, C6 / C2 / C3 / C4 / C5 and strepII-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ, or with SP, C6 / C2 / C3 / C4 / C5 and strepII-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ+F2A peptide+IL-7+F2A peptide+CCL19 as a template to obtain C6-CAR, C2-CAR, C2-CAR, C3-CAR, C4-CAR, C5-CAR, 6-7x19 CAR, C2-7x19 CAR, C3-7x19 CAR, C4-7x19 CAR, C5-7x19 CAR, C6-CAR, C2-CAR, C3-CAR, C4 Schematic diagram of the structure of -CAR and C5-CAR figure 1 Shown; Schematic diagram of the structure of C6-7x19CAR, C2-7x19 CAR, C3-7x19 CAR, C4-7x19 CAR, C5-7x19 CAR fragments figure 2 shown.

[0045] Wh...

Embodiment 3

[0049] Example 3, preparation and sequencing of plasmids

[0050] 1. Preparation of plasmid

[0051] Plasmids PTK881-EF1α-C6, PTK881-EF1α-C2, PTK881-EF1α-C3, PTK881-EF1α-C4, PTK881-EF1α-C5, PTK881-EF1α-C6-7x19, PTK881-EF1α-C2-7x19, PTK881 -EF1α-C3-7x19, PTK881-EF1α-C4-7x19, PTK881-EF1α-C5-7x19 Escherichia coli DH5α strains were inoculated into 250mL LB culture medium containing 100μg / mL ampicillin, cultured at 37°C and 220rpm overnight. The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0052] 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 .

[0053] 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 ...

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Abstract

The invention discloses an optimized chimeric antigen receptor with a CD99 receptor as a target spot and application of the optimized chimeric antigen receptor. The chimeric antigen receptor is sequentially spliced with 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 CD3 zeta chain from the N end to the C end, and the single-chain antibody ScFv can specifically recognize CD99 protein on the surface of a tumor cell. The chimeric antigen receptor taking the CD99 protein as the target spot is used for modifying immune cells so as to be used for treating surface CD99 positive tumors.

Description

[0001] The present invention claims the priority of the Chinese patent application CN202110485551.1, and the content described in the specification, drawings and claims of the priority document is incorporated into the specification of the present invention in its entirety and is regarded as a part of the original description of the specification of the present invention. The applicant further declares that the applicant has the right to amend the description and claims of the present invention based on the priority document. technical field [0002] The invention relates to the field of medicine and biology, in particular to a chimeric antigen receptor (CAR) targeting CD99 for treating broad-spectrum tumors and its application. Background technique [0003] 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. Scientists around the world are also working on the research o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K35/17A61P35/00G01N33/574
CPCC07K14/7051C07K16/18C12N15/85C12N5/0636A61K35/17A61P35/00G01N33/574C07K2319/02C07K2319/03C12N2800/107C12N2510/02C07K2317/622
Inventor 张同存祝海川史江舟
Owner WUHAN BIO RAID BIOTECH CO LTD
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