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Engineered immune cells and uses thereof

An immune cell, engineering technology, applied in animal cells, genetically modified cells, cells modified by introducing foreign genetic material, etc., can solve the problem that CAR cells cannot infiltrate tumor tissue.

Active Publication Date: 2020-10-30
NANJING BIOHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a common problem with CAR cell therapy at present, that is, the tumor microenvironment has an inhibitory effect on CAR cells, making it impossible for CAR cells to infiltrate tumor tissue.

Method used

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  • Engineered immune cells and uses thereof
  • Engineered immune cells and uses thereof
  • Engineered immune cells and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1 Construction of mouse pancreatic cancer cell line Panc02-mCD19

[0111] 1. Preparation of pLV-mCD19 plasmid

[0112] Using the total mouse spleen mRNA as a template, the total cDNA sequence of the mouse spleen was obtained by reverse transcription PCR, and then the nucleic acid sequence of mCD19 (SEQ ID NO: 41) was obtained by PCR. Then, the mCD19 sequence was cloned into the pGEM-T Easy vector (Promega, Cat. No. A1360) to obtain the pLV-mCD19 plasmid.

[0113] 2. lentiviral packaging

[0114] In a T175 culture flask, 30×10 6 293T cells were inoculated in 30ml of DMEM medium containing 10% fetal calf serum, and cultured overnight in a 37°C, 5% CO2 incubator.

[0115] Add 3ml Opti-MEM (Gibco, Cat. No. 31985-070), 34 μg pLV-mCD19 plasmid, 8.5 μg pMD2.G vector (Addgene, Cat. No. 12259) and 17 μg psPAX2 vector (Addgene, Cat. No. 12260) into a sterile tube. Then add 120 μl X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately,...

Embodiment 2

[0119] Example 2. Preparation of CAR-T cells

[0120] 1. Construction of Retroviral Plasmids

[0121] The DLL1 gene fragment (SEQ ID NO: 29) was artificially synthesized and recombined into the MSCV vector by Gibson to obtain the MSCV-DLL1 plasmid.

[0122] Artificially synthesized sequentially connected mCD19-scFv (SEQ ID NO: 13), CD8α hinge region (SEQ ID NO: 21), CD8α transmembrane region (SEQ ID NO: 15), 4-BB co-stimulatory domain (SEQ ID NO : 17) and the coding sequence fragment of CD3ζ intracellular domain (SEQ ID NO: 19), and XhoI / EcoRI restriction sites were added at both ends. The fragment was cloned into the MSCV vector to obtain the MSCV-mCD19-CAR plasmid.

[0123] Artificially synthesized sequentially connected mCD19-scFv (SEQ ID NO: 13), CD8a hinge region (SEQ ID NO: 21), CD8α transmembrane region (SEQ ID NO: 15), 4-BB co-stimulatory domain (SEQ ID NO : 17), CD3ζ intracellular domain (SEQ ID NO: 19), T2A (SEQ ID NO: 31) and Flt3L (SEQ ID NO: 27) coding sequence...

Embodiment 3

[0132] Example 3. Detecting the expression of CAR-T cells

[0133] 1. The expression level of CAR on the cell surface

[0134] Take out the 2 * 10 that embodiment 2 prepares 5 CAR-T cells were detected by flow cytometry using Goat Anti-Rat IgG (H&L) Biotin (BioVision, Cat. No. 6910-250) as the primary antibody and APC Streptavidin (BD Pharmingen, Cat. No. 554067) as the secondary antibody The expression level of CAR on the cells, the results are as follows figure 2 shown.

[0135] It can be seen that the CAR positive efficiencies in mCD19-CAR, mCD19-CAR+Flt3L, mCD19-CAR+DLL1, mCD19-CAR+Flt3L+DLL1 and mCD19-CAR+Flt3L+DLL1+XCL1 cells compared with control NT cells Both were greater than 50%, indicating that these cells can effectively express CAR.

[0136] 2. Expression level of DLL1

[0137] Take out the 2 * 10 that embodiment 2 prepares 5 For each CAR-T cell, PE Anti-DLL1 antibody (Biolegend, catalog number 128307) was used to detect the expression level of DLL1 in CAR-...

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Abstract

The present invention relates to an engineered immune cell, which is capable of expressing a chimeric antigen receptor, an exogenous Flt3L gene and a DLL1 gene, and optionally an XCL1 and / or XCL2 gene. The invention also provides the use of the engineered immune cell in the treatment of cancer, infection or autoimmune disease. Compared with a traditional CAR cell, the engineered immune cell has the advantage that the tumor killing activity is remarkably improved.

Description

technical field [0001] The invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered immune cell expressing a chimeric antigen receptor, exogenous Flt3L and DLL1 genes, and optionally XCL1 and / or XCL2 genes. Background technique [0002] Tumor immunotherapy mainly relies on autoimmunity to eliminate tumor cells by regulating the human immune system and tumor microenvironment. The immune system is a unified whole, and innate immunity also plays a very important role in tumor immunity. [0003] Some antigen-presenting cells, such as dendritic cells and macrophages, are the bridge between innate immunity and acquired immunity. Antigen-presenting cells can recognize tumor antigens and present them to the adaptive immune system, activate tumor-specific T cells, and then clear the tumor. Therefore, increasing the effect of the immune system on killing tumors by enhancing the antigen presentation process is an important re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867A61K39/00A61K48/00A61K38/19A61K38/17A61P35/00A61P31/00A61P33/00A61P37/00
CPCA61K38/1709A61K38/19A61K48/0025A61K48/005A61P31/00A61P33/00A61P35/00A61P37/00A61K39/001102C07K14/47C07K14/52C07K14/521C07K14/7051C07K16/2803C07K2317/622C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107Y02A50/30
Inventor 邢芸闫忠辉熊瑛浦容容任江涛贺小宏王延宾韩露
Owner NANJING BIOHENG BIOTECH CO LTD
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