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Preparation method of cell for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and application

A technology of chimeric antigen receptor and PD-L1, which is applied in the field of medicine and biology, can solve problems such as failure to achieve success, achieve the effect of prolonging the action time, alleviating immune tolerance, and improving tumor killing effect

Active Publication Date: 2020-11-17
UNIVERSITY OF MACAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Chimeric antigen receptor (CAR) T cell therapy has achieved great success in hematological malignancies, but not in solid tumors. Overcoming the immune resistance of solid tumors to CAR T cell therapy has become an urgent need. solved problem

Method used

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  • Preparation method of cell for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and application
  • Preparation method of cell for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and application
  • Preparation method of cell for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] In this example, a series of anti-CD22 vectors were constructed to co-express CAR and PD-L1 blocking protein. The anti-CD22 CAR constructs used in the present invention are second-generation CARs, all of which contain a CD8α leader sequence, an anti-CD22-specific scFv, a CD8α hinge, and a TM (transmembrane domain). The intracellular domain contains the 4-1BB co-stimulatory domain and the CD3ζ signaling domain, known as CD22AB.

[0068] To identify the efficiency of immune checkpoint molecules in CAR T cells, we constructed vectors co-expressing CAR and PD-1 extracellular domains with different transmembrane regions (CD4, CD8, and CD28). Each vector was designated 22ABPDR 4, 22ABPDR 8 and 22ABPDR28, respectively. Inserted gene and plasmid construction reference figure 1 shown.

[0069] The vector includes the first nucleic acid encoding a chimeric antigen receptor consisting of a single-chain antibody of an antibody against CD22 (Anti-CD22), a CD8 transmembrane domain...

Embodiment 2

[0104] In this example, T cells co-expressing CAR and PDR with CD28 were respectively constructed. Inserted gene and plasmid construction reference image 3 shown. Four kinds of plasmid vectors ZS, PDR, CD22AB and CD22ABPDR were constructed by conventional technical means in the field. Among them, the vector ZS is a control vector containing nucleic acids encoding IRES and ZsGreen1; the vector PDR is a control vector containing nucleic acids encoding PD-1 EM and CD28 TM; the vector CD22AB is a control vector containing nucleic acids encoding Anti-CD22, CD8 TM, 41-BB, CD3ζ, IRES and ZsGreen1 nucleic acid control vector; and the vector CD22ABPDR is the same as the vector CD22ABPDR 28 ZS in Example 1.

[0105] The above four plasmid vectors were transfected into human peripheral blood lymphocytes to construct CAR T cells.

[0106] The transfected human peripheral blood lymphocytes were cultured and harvested for FACS detection and Q-PCR identification.

[0107] The preparatio...

Embodiment 3

[0113] This example verifies the effect of the constructed CAR T cells.

[0114] (1) Construct a cell line expressing PD-L1.

[0115] To characterize the function of PDRs expressed on CAR T cells, tumor cells need to express PD-L1. Because the tumor cell lines of Raji, K562, Daudi, and BV173 do not express endogenous PD-L1, the inventors used lentivirus to construct PD-L1-expressing tumor cell lines on the basis of Raji, K562, Daudi, and BV173. Tumor cell lines Raji-PD-L1, K562-PD-L1, Daudi-PD-L1 and BV173-PD-L1. Through flow cytometry analysis, it was determined that all constructed cell lines could stably and highly express PD-L1 (refer to Figure 5 shown).

[0116] (2) Expression detection of key activation markers.

[0117] The ratios of CD4 and CD8 and other activated cell markers expressed on human PMBC transfected by the four plasmid vectors ZS, PDR, 971 and 971PDR in Example 2 were analyzed by flow cytometry.

[0118] Depend on Figure 6A It can be seen that the ...

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Abstract

The invention discloses a preparation method of a cell for expressing an anti-CD22 chimeric antigen receptor and a PD-L1 blocking protein, an expression vector and application, and relates to the technical field of medical biology. A first nucleic acid encoding the anti-CD22 chimeric antigen receptor and a second nucleic acid encoding the PD-L1 blocking protein are inserted into the expression vector. By utilizing a CD22 target and a PD-L1 target and targeting an antibody or a fragment thereof of CD22, the T cell can efficiently and specifically target a tumor cell for expressing an antigen CD22; and the PD-L1 blocking protein can compete with endogenously expressed PD-1 to be combined with PD-L1 on a target cell, so that a PD-1 / PD-L1 signal path is blocked, the action time of the T cell is prolonged, the tumor killing effect is improved, and the problem of immune tolerance of solid tumors to CAR T cell therapy is relieved.

Description

technical field [0001] The invention relates to the field of medical and biological technology, in particular to a preparation method, expression vector and application of cells expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein. Background technique [0002] In the field of tumor treatment, chimeric antigen receptor T cell therapy has attracted more and more attention. Chimeric antigen receptor T cells (CAR T cells) are T cells that have been genetically engineered to have chimeric antigen receptors for use in immunotherapy. [0003] Chimeric antigen receptor (CAR) is an artificially constructed transmembrane molecule encoded by a fusion gene, which can specifically target T cells to antigens on the surface of cancer cells to eliminate target cancer cells. A CAR consists of an extracellular domain (eg, a single-chain antibody, scFv of an antibody), a transmembrane domain, and an intracellular domain. The scFv of the extracellular domain is responsib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/62C12N15/12C12N5/10A61K39/00A61P35/00A61P35/02
CPCC12N15/86C07K16/2803C07K14/70578C07K14/70517C07K14/70514C07K14/70521C07K14/7051C07K14/70532C12N5/0636A61K39/001113A61K39/001111A61P35/00A61P35/02C12N2740/15043C12N2800/107C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2510/00A61K2039/5156A61K2039/804
Inventor 赵琦刘婕
Owner UNIVERSITY OF MACAU
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