Human VEGFR-1 (Vascular Endothelial Growth Factor Receptor-1) targeting genetically engineered lymphocyte as well as preparation method and application thereof

A VEGFR-1, host cell technology, applied in the field of chimeric antigen receptors and lymphocytes, can solve the problems of increasing difficulty in clinical application, and achieve the effect of inhibiting tumor formation and good application prospects.

Active Publication Date: 2012-07-18
SICHUAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This makes the clinical application of TIL more difficult. 1

Method used

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  • Human VEGFR-1 (Vascular Endothelial Growth Factor Receptor-1) targeting genetically engineered lymphocyte as well as preparation method and application thereof
  • Human VEGFR-1 (Vascular Endothelial Growth Factor Receptor-1) targeting genetically engineered lymphocyte as well as preparation method and application thereof
  • Human VEGFR-1 (Vascular Endothelial Growth Factor Receptor-1) targeting genetically engineered lymphocyte as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Obtaining ScFv targeting hVEGFR-1

[0030] ScFv of hVEGFR-1 obtained by ribosome display

[0031] 1) BALB / c mice were immunized three times with eukaryotic expression of the extracellular domain of hVEGFR-1.

[0032] 2) Taking the splenocytes of the immunized mice, lysing the cells, and extracting the mRNA-ribosome complex.

[0033] 3) Incubate the extracted mRNA-ribosome complexes with the solid-phased extracellular segment of hVEGFR-1, and wash away unbound complexes.

[0034] 4) The mRNA is eluted from the antigen-ribosome-mRNA complex with an elution buffer.

[0035] 5) Using the RT-PCR method, the light chain and heavy chain variable regions with high affinity antibody sequences are obtained by using the light chain primers and heavy chain primers of the antibody sequences respectively.

[0036] 6) Linking the heavy chain and light chain with (G4S)3linker to obtain ScFv that recognizes hVEGFR-1.

[0037] 2. ScFv affinity screening

[0038] 1) The lig...

Embodiment 2

[0042] Example 2 Acquisition of full-length CAR gene targeting VEGFR-1 and construction of recombinant plasmid vector

[0043] Experimental route: 1. Synthesize primers to obtain ScFV-V-IgG1-Fc, CD4-TM-CD3-z fusion fragments by overlapping PCR method. Wherein, the signal peptide sequence is included in the upstream primer.

[0044] 2. Obtain the full-length CAR gene by overlapping PCR.

[0045] The specific scheme of the experiment is as follows:

[0046] a) Use primers F1 and R1 to amplify ScFv-V, use F2 and R2 to amplify IgG1-Fc, and finally use the two PCR products as templates, F1 and R2 as primers to amplify the target fragment ScFv-V-IgG1-Fc . Similarly, first use primers F3 and R3 to amplify CD4-TM, F4 and R4 to amplify CD3-z, and then use F3 and R4 to amplify to obtain CD4-TM-CD3-z. Among them, CD4-TM and CD3-z are amplified from human peripheral blood mononuclear cells by RT-PCR.

[0047] b) Using ScFv-V-IgG1-Fc and CD4-TM-CD3-z as templates, and F1 and R4 as pri...

Embodiment 3

[0069] Example 3 Lymphocyte transfection, expression detection and in vitro activity detection

[0070] 1. Electrotransfection of lymphocytes and detection of target gene expression.

[0071]After the lymphocytes were isolated from human peripheral blood by density gradient centrifugation, CD3 antibody (mouse anti-human CD3 monoclonal antibody, purchased from Wuhan Institute of Biological Products) (1ug / ml) and IL-2 (300IU / ml) Stimulate lymphocytes to obtain more than 95% CD3 (leukocyte antigen differentiation group molecule 3) positive T lymphocytes, which is a classic scheme for the expansion of lymphocytes widely used. Two days later, T lymphocytes were collected for transfection with pmax-CAR plasmid. After transfecting lymphocytes with Nucleofector (Lonza, Sweden, Lonza Company) or Multiporator (eppendorf, Eppendorf, Germany) using an electroporation instrument, culture lymphocytes for 16 hours, collect transfected lymphocytes, and extract total cell protein Perform we...

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Abstract

The invention relates to the field of gene engineering, and particularly relates to an anti-VEGFR-1 (Vascular Endothelial Growth Factor Receptor-1) mosaic type antigen receptor and lymphocyte capable of expressing the antigen receptor, and aims at providing a new and effective choice for the technical field of antitumor. The technical scheme for solving the technical problems is as follows: a single-chain antibody ScFv is provided, the single-chain antibody can recognize VEGFR-1, the single-chain antibody is prepared into the mosaic type antigen receptor which has a structure of (ScFv-V)-(IgG1-Fc)-(CD4-TM)-(CD3-z), the encoding gene of the antigen receptor is transformed into a plasmid vector, and the plasmid vector is transfected to lymphocyte, so that the lymphocyte has antitumor action, thereby providing the new and effective choice for the field.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an anti-VEGFR-1 chimeric antigen receptor and lymphocytes capable of expressing the antigen receptor. Background technique [0002] In the process of cellular immunity, T lymphocytes play a major role. Cellular immunity mediated by T cells is mainly through T cell receptor (T cell receptor, TCR) specific recognition of antigenic peptides displayed by the major histocompatibility complex (MHC) on the cell surface, thereby activating the intracellular signal of T cells , to specifically kill the target cells. This plays a vital role in timely removal of diseased cells in the body and prevention of tumors. [0003] Tumors can evade the surveillance of the immune system through various ways to produce immune escape, so that tumor cells can survive in the body and tumors occur. However, in tumor patients, there are still a small number of immune cells that can recognize and kill ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C07K19/00C12N15/62C12N15/63C12N5/10A61K35/26A61P35/00
CPCA61K39/0011A61K2039/5156A61K2039/5158C07K14/7051C07K2319/03
Inventor 魏于全王永生李炯王玮
Owner SICHUAN UNIV
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