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Chimeric antigen receptor targeting CD22 and CD19 and application thereof

A chimeric antigen receptor, CD22 technology, applied in the field of biomedicine, can solve problems such as poor killing effect

Active Publication Date: 2021-03-05
JUVENTAS CELL THERAPY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, it has been found that if the antigen-binding part targeting CD19 and CD22 is inserted into the vector by simple connection, the killing effect after expression is not good, so it is necessary to find a specific connection method of the CD19 and CD22 antigen-binding part to Improve the effect of killing tumor cells

Method used

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  • Chimeric antigen receptor targeting CD22 and CD19 and application thereof
  • Chimeric antigen receptor targeting CD22 and CD19 and application thereof
  • Chimeric antigen receptor targeting CD22 and CD19 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Construction of dual-target chimeric antigen receptor vector

[0087] 1. Using BamH I and EcoR I endonucleases to digest the plasmid containing the CD8α-4-1BB-CD3ζ fragment constructed by the inventor in the early stage to obtain the CD8α-4-1BB-CD3ζ fragment, the amino acid sequence of which is as shown in SEQ ID NO.5 shown. The plasmid containing the CD8α-4-1BB-CD3ζ fragment can be prepared by any suitable method in the prior art, for example, the patent No. ZL201510233748.0.

[0088] 2. The synthesized CD 22scFv-CD 19scFv fragment, CD 19scFv-CD22scFv, CD19V L -CD22V L -CD22V H -CD19V H and CD22V L -CD19V L -CD19V H -CD22V H The fragments were respectively connected to the target vector, and the constructed CD22scFv-CD19scFv-CD8α-4-1BB-CD3ζCAR (22-19CAR), CD19scFv-CD22scFv-CD8α-4-1BB-CD3ζCAR (19-22CAR), CD19V L -CD22V L -CD22V H -CD19V H -CD8α-4-1BB-CD3ζ(19╳22CAR-T) and CD22V L -CD19V L -CD19V H -CD22V H -CD8α-4-1BB-CD3ζ(22╳19CAR-T) destinati...

Embodiment 2

[0089] Example 2: Preparation of dual-target chimeric antigen receptor lentiviral modified T cells

[0090] 1. Use EndoFree Plasmid Maxi Plasmid Extraction Kit (QIAGEN Company) to extract 22-19CAR, 19-22-CAR, 19╳22CAR and 22╳19CAR expression plasmids and packaging plasmids PRSV-Rev, pMDlg-PRRE, pMD.2G respectively . Each CAR plasmid and packaging plasmid (four plasmids) were transfected with PEI transfection reagent (polyscience company) at a ratio of 12.2:4.11:8.75:3.5 (see the instruction manual 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.

[0091] 2. Preparation of T cells: Take 10 ml of fresh healthy human peripheral blood, and use RosetteSep T cell enrichment...

experiment example 1

[0094] Experimental example 1: Chimeric antigen receptor 22-19CAR, 19-22-CAR, 19╳22CAR and 22╳19CAR lentiviral modification of T cells to kill leukemia cells

[0095] 1. CD22 expression level in hematological tumor cell lines:

[0096] Both Namalwa and MV4-11 cell lines were purchased from ATCC in the United States. MV4-11-CD19 and MV4-11-CD22 are monoclonal cell lines screened after MV4-11 cell lines were infected with CD19 and CD22, respectively. After cultured separately, draw 5×10 5 The cell suspension was washed twice with PBS, labeled with PE anti-human CD19 monoclonal antibody and APC anti-human CD22 monoclonal antibody (Biolegend Company), and labeled with PE-isotype and APC-isotype as the control group, and incubated on ice for 30 minutes. The expression levels of CD19 and CD22 in various cell lines were detected by flow cytometry, and the results were as follows: Figure 4 shown. Among them, A is the positive expression rate of CD19 target antigen molecules; B is ...

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PUM

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Abstract

The invention discloses a nucleic acid molecule encoding a chimeric antigen receptor targeting CD22 and CD19. The chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular signal transduction region, wherein the extracellular region encoded by the chimeric antigen receptor comprises a CD22 and CD19 binding domain, and the CD22 and CD19 binding domainis composed of an antibody single-chain variable region fragment of CD22 and an antibody single-chain variable region fragment of CD19; and the antibody single-chain variable region fragment of CD22 and the antibody single-chain variable region fragment of CD19 are arranged in the sequence of an amino acid sequence shown in SEQ ID No. 9, an amino acid sequence shown in SEQ ID No. 10, an amino acidsequence shown in SEQ ID No. 11 or an amino acid sequence shown in SEQ ID No. 12. The chimeric antigen receptor disclosed by the invention can be used for treating CD19 <+> and CD22 <+> B cell bloodtumors and combined treatment with CD19 CAR-T cells or CD22 CAR-T cells.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to chimeric antigen receptors targeting CD22 and CD19 and applications thereof. 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). The second-generation CAR is currently the most widely used. [0003] B-cel...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00A61K35/17A61P35/00A61P35/02
CPCC07K16/2803C07K14/7051A61K35/17A61P35/00A61P35/02C07K2319/02C07K2319/03
Inventor 王建祥王敏张瑜徐颖茜饶青廖晓龙
Owner JUVENTAS CELL THERAPY LTD
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