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Chimeric antigen receptor using CD99 as target site and application of chimeric antigen receptor

A technology of antigen and receptor, applied in the field of medicine and biology, to achieve the effect of improving tumor killing efficiency and scientific evaluation of tumor killing effect

Active Publication Date: 2019-12-20
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no effective way to treat tumors by targeting CD99

Method used

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  • Chimeric antigen receptor using CD99 as target site and application of chimeric antigen receptor
  • Chimeric antigen receptor using CD99 as target site and application of chimeric antigen receptor
  • Chimeric antigen receptor using CD99 as target site and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Affinity determination of scFv against CD99

[0040] Anti-CD99 scFv with strong specificity and high affinity was screened from a large-capacity CD99 phage antibody library prepared with the extracellular domain of CD99 as an antigen. After multiple rounds of screening, three scFvs that could specifically recognize CD99 on the surface of tumor cells were obtained, respectively. Named as C1, C2, and C3, and the sequencing analysis of their sequences shows that the nucleotide sequences of C1, C2, and C3 are shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively, and their amino acids The sequences are shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively.

[0041] In order to characterize the advantages and disadvantages of the ScFv obtained by screening in this application relative to the ScFv against CD99 known in the prior art and whether it is suitable for the construction of chimeric antigen receptors, two ScFvs known in the prio...

Embodiment 2

[0044] Example 2: PTK881-EF1α-C1, PTK881-EF1α-C2, PTK881-EF1α-C3, PTK881-EF1α-C4, PTK881-EF1α-C5, PTK881-EF1α-C1-7x19, PTK881-EF1α-C2-7x19, Construction of PTK881-EF1α-C3-7x19, PTK881-EF1α-C4-7x19, PTK881-EF1α-C5-7x19 plasmids

[0045] 1. Fragments C1, C2, C3, C4, and C5 were artificially synthesized, and SP, strepII-CD8hinge-CD28TM+ICD-4-1BB-CD3ζ fragments were artificially synthesized.

[0046] 2. Using Overlap PCR to amplify with SP, C1 / C2 / C3 / C4 / C5 and strepII-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ, or with SP, C1 / 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 C1-CAR, C2-CAR, C3-CAR, C4-CAR, C5-CAR, C1-7x19 CAR, C2-7x19 CAR, C3-7x19 CAR, C4-7x19 CAR, C5-7x19 CAR, C1-CAR, C2-CAR, C3-CAR, C4 Schematic diagram of the structure of -CAR and C5-CAR figure 1 shown; the structural schematic diagram of C1-7x19CAR, C2-7x19 CAR, C3-7x19 CAR, C4-7x19 CAR, C5-7x19 CAR fragments is shown in figure 2 shown.

[0047] Wherei...

Embodiment 3

[0051] Example 3, preparation and sequencing of plasmids

[0052] 1. Preparation of plasmid

[0053] Plasmids PTK881-EF1α-C1, PTK881-EF1α-C2, PTK881-EF1α-C3, PTK881-EF1α-C4, PTK881-EF1α-C5, PTK881-EF1α-C1-7x19, 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.

[0054] 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 .

[0055] 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 present invention discloses a chimeric antigen receptor using a CD99 receptor as a target site and a use of the chimeric antigen receptor. A splice peptide, a single-chain antibody ScFv, strepII,CD8 hinge, a CD28 transmembrane region, a CD28 intracellular structural domain, an intracellular co-stimulatory domain 4-1BB and a CD3-zeta chain are sequentially spliced from a N-terminus to a C-terminus, and more preferably, the splice signal peptide, the single-chain antibody ScFv, the strepII, the CD8 hinge, the CD28 transmembrane region, the CD28 intracellular structural domain, the intracellular co-stimulatory domain 4-1BB, the CD3-zeta chain, a F2A peptide, IL-7, a F2A peptide and CCL19 are sequentially spliced from the N-terminus to the C-terminus. The single chain antibody ScFv can specifically recognize the CD99 receptor on surfaces of tumor cells. The chimeric antigen receptor using the CD99 receptor as the target site is used to modify immune cells and the modified immune cellscan be used for treatment of surface CD99-positive tumors.

Description

technical field [0001] 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 [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 most noteworthy scientific advances. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10C12N15/62A61K39/00A61P35/00
CPCA61K39/0011A61K2039/5158A61P35/00C07K14/52C07K14/5418C07K14/7051C07K16/2896C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 张同存祝海川史江舟
Owner WUHAN BIO RAID BIOTECH CO LTD
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