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Chimeric antigen receptor targeting CD123 and double-target chimeric antigen receptor containing chimeric antigen receptor targeting CD123

A chimeric antigen receptor, targeting technology, applied in the field of biomedicine, can solve the problems of easy recurrence, ineffectiveness, poor curative effect, etc.

Active Publication Date: 2021-06-18
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention aims at the disadvantages of poor curative effect, easy relapse and ineffectiveness of leukemia treated with CD123 chimeric antigen in the prior art, and provides a CD123-targeted chimeric antigen receptor and its application

Method used

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  • Chimeric antigen receptor targeting CD123 and double-target chimeric antigen receptor containing chimeric antigen receptor targeting CD123
  • Chimeric antigen receptor targeting CD123 and double-target chimeric antigen receptor containing chimeric antigen receptor targeting CD123
  • Chimeric antigen receptor targeting CD123 and double-target chimeric antigen receptor containing chimeric antigen receptor targeting CD123

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Example 1: Construction of Chimeric Antigen Receptor CD123 scFv-CD8α-4-1BB-CD3ζ Vector 1. Using Nhe I and Bstb I endonucleases to digest the CD8α-4-1BB-CD3ζ fragment constructed by the inventor earlier The plasmid was used to obtain the CD8α-4-1BB-CD3ζ fragment, the amino acid sequence of which is shown in SEQ ID NO.18. The plasmid containing the CD8α-4-1BB-CD3ζ fragment can be prepared by any suitable method in the prior art, for example, the method in the patent No. ZL201510233748.0.

[0140] 2. Ligate the 6E11 CD123 scFv, 12H7 CD123 scFv, and 13C3 CD123 scFv fragments obtained by whole gene synthesis with the destination vector, and construct the 6E11-CD123 scFv-CD8α-4-1BB-CD3ζ CAR (6E11123CAR), 12H7-CD123 scFv -CD8α-4-1BB-CD3ζCAR (12H7 123CAR), 13C3-CD123 scFv-CD8α-4-1BB-CD3ζCAR (13C3 123CAR) target vector was identified by endonuclease Nhe I and Not I, the results are as follows figure 1 As shown in A, the enzyme digestion results show that the positive clones co...

Embodiment 2

[0141] Example 2: Preparation of chimeric antigen receptor CD123 scFv-CD8α-4-1BB-CD3ζ lentivirus modified T cells

[0142] 1. Using EndoFree Plasmid Maxi Plasmid Extraction Kit (QIAGEN Company) to extract CD123 scFv-CD8α-4-1BB-CD3ζ expression plasmid and packaging plasmid psPAX2, pMD.2G. The three plasmids were transfected with PEI transfection reagent (polyscience company) at a ratio of 4:3:1 (see the instructions of PEI transfection reagent for specific methods). Replace the fresh culture medium 12 hours after transfection, collect the virus supernatant 24 hours and 48 hours later, centrifuge at 4°C, 3000rpm for 15 minutes, filter through a 0.45μm filter, and use 50000g, 4°C, 1.5 hours after ultracentrifugation Concentrate 10 times, then transfer to -80°C for storage.

[0143] 2. Preparation of T cells: Take 10 ml of fresh healthy human peripheral blood, and use RosetteSep T cell enrichment Cocktail (Stemcell Company) and Ficoll-Paque PLUS (GE Healthcare Company) to extract...

Embodiment 3

[0146] Example 3: Killing effect of chimeric antigen receptor CD123 scFv-CD8α-4-1BB-CD3ζ lentivirus modified T cells on leukemia cells

[0147] 1. Expression level of CD123 in hematological tumor cell lines:

[0148] Both MV4-11 and K562 cell lines were purchased from ATCC, USA. After cultured separately, draw 5×10 5 The cell suspension was washed twice with PBS, labeled with APC anti-human CD123 monoclonal antibody (Biolegend Company), and labeled with APC-isotype as a control group, and incubated on ice for 30 minutes. The expression levels of CD123 in various cell lines were detected by flow cytometry, and the results were as follows: Figure 7 As shown, MV4-11 is CD123 positive cells, K562 cells are CD123 negative cells.

[0149] 2. Flow cytometry detection of residual tumor cells after co-culture of CAR-modified T cells with MV4-11 and K562 cell lines:

[0150] Divide the above cells into 2×10 5 Cells / well were inoculated into 24-well culture plates, and 2.5×10 4 (E...

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Abstract

The invention discloses a nucleic acid molecule for coding a chimeric antigen receptor targeting CD123. The chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular signal transduction region; the extracellular region coded by the chimeric antigen receptor comprises a CD123 binding structural domain; the CD123 binding structural domain is an anti-CD123 single-chain antibody variable region fragment; and the anti-CD123 single-chain antibody variable region fragment is an amino acid sequence shown as SEQ ID NO.13 or a sequence with 90%-99% identity with the amino acid sequence shown as SEQ ID NO.13, an amino acid sequence shown as SEQ ID NO.14 or a sequence with 90%-99% identity with the amino acid sequence shown as SEQ ID NO.14, or an amino acid sequence shown as SEQ ID NO.15 or a sequence with 90%-99% identity with the amino acid sequence shown as SEQ ID NO.15. According to the nucleic acid molecule for coding the chimeric antigen receptor targeting the CD123, the chimeric antigen receptor can be used for treating CD123+ and / or CD33+ hematologic tumors, so that the off-target effect is effectively prevented, the treatment effect is better, and escape is not prone to occurring.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a CD123-targeting chimeric antigen receptor and a CD123-targeting chimeric antigen receptor containing a double-target chimeric antigen receptor. Background technique [0002] Chimeric antigen receptor (CAR)-modified T cells, as an immunotherapeutic strategy, have received extensive attention and application in tumor treatment. The structure of CAR generally consists of four parts: an extracellular targeting linker region (usually a single-chain antibody with antigen recognition function), a hinge region, a transmembrane region, and an intracellular signal transduction region. Currently, according to the number of co-stimulatory molecules added to the intracellular signal transduction region, CARs are divided into one generation (no co-stimulatory molecule), second generation (with one co-stimulatory molecule) and third-generation (with two co-stimulatory molecules). Current...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2866C07K14/7051C12N15/86C12N5/0636A61K39/001116A61P35/00A61P35/02C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/804
Inventor 王建祥王敏熊冬生王珍珍卢杨饶青徐颖茜邢海燕
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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