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Construct, transgenic lymphocyte, preparation method and use thereof

A technology of lymphocytes and constructs, applied in the field of biomedicine

Active Publication Date: 2019-09-27
BEIJING MARINO BIOTECH PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on the research status of the above-mentioned immune co-stimulatory molecules, the application of immune co-stimulatory molecules in the anti-tumor response of T cells needs to be further developed

Method used

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  • Construct, transgenic lymphocyte, preparation method and use thereof
  • Construct, transgenic lymphocyte, preparation method and use thereof
  • Construct, transgenic lymphocyte, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Cell lines and basic experimental techniques used in the embodiments of the present invention are as follows:

[0072] cell line

[0073] The following cell lines were used in the examples: Raji and Daudi (CD19+ Burkitt lymphoma cell line), Jurkat cells (CD19-T lymphoma cell line), and K562 (NK cell target cells). The above cells are all from ATCC (American Cell Bank), and cultured in RPMI-1640 medium (purchased from Gibco-BRL, San Francisco, CA, USA).

[0074] Transduction of peripheral blood lymphocytes

[0075] Retrovirus pMIP-CD19 infects peripheral blood lymphocytes, making peripheral blood lymphocytes stably express anti-CD19 chimeric antigen receptor and enhanced green fluorescent protein. Retroviruses were packaged and amplified by transfecting the Phoenix packaging cell line. The specific process was as follows: 2.5 micrograms of env (pVSV-G; Clontech, BD Biosciences) and 3.5 micrograms of retroviral plasmids were vortexed and mixed to form The DNA mixture wa...

Embodiment 24-1B

[0083] Example 24- The activation of 1BB enhances the anti-tumor effect of antigen-specific peripheral blood lymphocytes

[0084] In this example, the inventors examined the effect of activating 4-1BB on peripheral blood lymphocytes. In an embodiment, the inventor injects 5×10 5 B16-OVA mouse tumor cells. After 4 days, mice were injected intravenously with 2 × 10 6 activated OT-1 lymphocytes (taken from the spleen of OT1 transgenic mice and treated with 0.1 μg / ml SIINFEKL peptide for 48 hours in vitro to activate OT-1 lymphocytes) (spleen isolated cells of OT1 transgenic mice expressed T cell receptor body, and specifically recognize the SIINFEKL peptide and be activated) and intraperitoneally inject 150 μg of rat-derived anti-mouse-CD137 antibody (binding to 4-1BB and activating 4-1BB) (cloning site is 3H3) (purchased from BioXCell) . The mice in the experimental control group were injected intravenously with 2×10 6 Activated OT-1 lymphocytes or intraperitoneal injection...

Embodiment 3

[0086] Example 3 Construction of vectors co-expressing 4-1BB or OX40 and anti-CD19 chimeric antigen receptor

[0087] In this example, the inventors cloned the sequence encoding the single-chain antibody against human CD19, the ζ-chain sequence of the CD28 intracellular segment and the T cell receptor combination onto the pMIG retroviral vector. During the cloning process, the selected Restriction enzyme digestion is XbaI and NotI double enzyme digestion, and NotI and XhoI double enzyme digestion, through enzyme digestion, ligation, screening and amplification of the target plasmid, pMIG-CD19 chimeric antigen receptor retroviral plasmid (RV- CD19CAR). The sequence containing IRES and human 4-1BB or OX40 was cloned into the pMIG-CD19 chimeric antigen receptor retroviral plasmid, or the sequence containing the encoding linker peptide and human 4-1BB or OX40 was cloned into pMIG-CD19 chimeric antigen receptor retroviral plasmid Co-antigen receptor retroviral plasmids, or with di...

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Abstract

The invention proposes a construct, a transgenic lymphocyte and a preparation method and use thereof. The transgenic lymphocyte expresses a functional immune co-stimulatory molecule and a chimeric antigen receptor (CAR). The construct includes a first A nucleic acid molecule, the first nucleic acid molecule encoding a functional immune costimulatory molecule; and a second nucleic acid molecule encoding a chimeric antigen receptor. The transgenic lymphocytes proposed by the present invention have greatly improved viability in tumor patients and have stronger directional killing effect on tumor cells.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically, the present invention relates to a T lymphocyte, a retrovirus, a transgenic lymphocyte, a construct, a method for preparing a transgenic lymphocyte, and a method for treating Compositions for treating cancer and a method of increasing lymphocyte activity. Background technique [0002] Adoptive T cell therapy is the activation, expansion, and subsequent application of ex vivo T cells. Some T cells migrate to tumor sites and directly lyse tumor cells. Appropriately manipulated T cells in vitro have the ability to elicit significant therapeutic effects in ex vivo therapy. [0003] However, adoptive T cell therapy using tumor infiltrating lymphocytes still needs to be improved. [0004] Immune co-stimulation is required to activate effective T cell antitumor responses. Immune co-stimulatory molecules can be divided into two important families: B7 / CD28 family and tumor necrosis fac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867A61K48/00A61K35/17A61P35/00
CPCA61K38/00C12N5/0636C12N5/0638C12N2510/00C12N2740/10043C07K14/7051C07K2317/622C07K2319/00C07K2319/03C07K14/70521C07K14/70578A61P35/00A61K39/4644A61K39/4631A61K2239/31A61K39/4611A61K39/464412A61K2239/38A61K2239/57
Inventor 严勇朝苑锡铜朱益林陈思毅
Owner BEIJING MARINO BIOTECH PTY LTD
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