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Human IL1RAP (IL-1 receptor accessory protein) specific CAR (chimeric antigen receptor) and application thereof

A specific, co-stimulatory molecule technology, applied in the biological field, can solve the problems of reducing anti-tumor ability and low T cell activation rate, and achieve the effect of increasing T cell activation rate and enhancing killing ability

Active Publication Date: 2017-06-13
苏州艾凯利元生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology uses CD28 molecule as CAR co-stimulatory molecule, and the activation rate of T cells after transfection of T cells with IL1RAP-specific CAR virus prepared by this method is not high, thus reducing its anti-tumor ability in vivo

Method used

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  • Human IL1RAP (IL-1 receptor accessory protein) specific CAR (chimeric antigen receptor) and application thereof
  • Human IL1RAP (IL-1 receptor accessory protein) specific CAR (chimeric antigen receptor) and application thereof
  • Human IL1RAP (IL-1 receptor accessory protein) specific CAR (chimeric antigen receptor) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 V H and V L Gene cloning and identification

[0033] V H Gene refers to the variable region gene of the antibody heavy chain, V L Gene refers to the variable region gene of the antibody light chain, V H Gene and V L After the genes are connected by Linker, scFv is formed. V H and V L Genetically Encoded Antibody V H and V L The fragment is the key site for specific recognition and binding to the antigen, so the antibody V that specifically recognizes human IL1RAP protein was cloned and identified H and V L Genes are the first step.

[0034] Adopt Trizol (purchased from Invitrogen) reagent method to extract 10D8A7 hybridoma cell total RNA, carry out agarose gel electrophoresis detection to the total RNA of extraction, the result is as follows figure 1 Shown in B.

[0035] The first strand of cDNA was synthesized by reverse transcription with reverse transcriptase SuperScriptTMⅢ (purchased from Invitrogen), and stored at -20°C for future use.

[00...

Embodiment 2

[0039] Example 2 Construction of anti-human IL1RAP scFv

[0040] pMD18-10D8A7V H and pMD18-10D8A7V L The sequence is used as a template to amplify V H and V L Gene, amplified by overlap extension PCR and ligated as anti-human IL1RAP scFv, named 10D8A7scFv. The primer sequence is SEQ ID NO: 6-9.

[0041] Reaction conditions: Denaturation at 94°C for 1 rnin, annealing at 58°C for 1 rnin, extension at 72°C for 1.5 min, a total of 30 cycles, and finally 72°C for 10 min.

[0042] The total RNA of anti-human IL1RAP hybridoma cells was extracted and identified by agarose gel electrophoresis. The upper two bands are bright, clear and sharp, and the brightness of the first band (28S) is about twice the brightness of the second band (18S), and the lowermost band (5S) is the lightest, in line with Characterization of total RNA. The VH and VL gene fragments of two anti-human IL1RAP antibodies were amplified from total RNA of hybridoma cell lines. The results of agarose gel electro...

Embodiment 3

[0043] Example 3 Construction and Identification of Anti-human IL1RAP CAR Stable Expression Plasmid

[0044] Refer to the research of Uherek C et al. to design the gene sequence of CAR and synthesize ICD, including CD8α transmembrane segment-CD137 intracellular segment-TCRζ chain, such as figure 2 As shown in middle A, CAR is composed of anti-human IL1RAP antibody scFv, CD8α transmembrane segment, CD137 intracellular segment and TCRζ chain, and BamHI and SalI restriction enzyme sites are respectively introduced at both ends of the sequence, and BamHI site Then introduce hCD8αSignal peptide, and then introduce Stop codon before the SalI site.

[0045] Ligate the CAR into the LV-Lac vector, and the ligation product pattern is as follows figure 2 Shown in B. Transform the ligation product into competent cells DH5α, spread LB homogeneous medium (containing 150 μg / mL ampicillin) on a plate and culture at 37°C for 16 hours, pick a single clone colony in LB medium (containing 150...

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Abstract

The invention discloses a human IL1RAP (IL-1 receptor accessory protein) specific CAR (chimeric antigen receptor) and application thereof. The CAR consists of an IL1RAP specific single-chain antibody scFV, a costimulatory molecule and a T cell activated CD3zeta chain, wherein the IL1RAP specific single-chain antibody scFV comprises variable zone gene segments of a heavy chain and a light chain of an IL1RAP specific single-chain antibody McAb; the costimulatory molecule is CD137. Compared with the prior art, the human IL1RAP specific CAR has the advantages that (1) by introducing the CD137 cooperative costimulatory molecule, the activating rate of T cell transfected by an IL1RAP specific CAR virus is improved, and the ability of killing tumor cells is improved; (2) the function of killing leukemia cells is realized, the long-life antigen specific memory T cell can be formed in a human body, and the new leukemia cell is timely cleared.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a chimeric receptor, in particular to a human IL1RAP-specific CAR and its application. Background technique [0002] Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease that occurs on pluripotent hematopoietic stem cells, accounting for about 20% of leukemia. (q34; q11) is characterized by the formation of the Philadelphia chromosome (Ph). This translocation forms a new fusion gene BCR-ABL, and the fusion protein p210 or p190 encoded by this gene has activated tyrosine kinase activity, which leads to abnormal clonal proliferation of myeloid hematopoiesis, which is the root cause of the development of CML. It is also an important target for the treatment of CML. Tyrosine kinase inhibitors (TKIs), represented by imatinib, inhibit cell proliferation and induce apoptosis by inhibiting BCR-ABL autophosphorylation and substrate phosphorylation, and have now become ...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K39/00A61P35/02
CPCA61K39/0011C07K14/7051C07K14/70596C07K16/2866C07K2317/56C07K2317/622
Inventor 赵恺徐开林尹玲玲袁姝姝陈翀
Owner 苏州艾凯利元生物科技有限公司
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