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Engineered immune cell and use thereof

An immune cell, engineering technology, applied in blood/immune system cells, animal cells, genetic engineering, etc., can solve the problem of limited recruitment ability of cDC1 dendritic cells

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

[0007] CCL-19 has the ability to recruit dendritic cells and endogenous T cells into tumors, but its ability to recruit cDC1 dendritic cells is limited. Therefore, a new immunotherapy approach that can effectively differentiate or recruit cDC1 dendritic cells is needed Cytomorphic cells to improve the efficiency of tumor antigen presentation, induce the body's adoptive immune response, and solve the problem of tumor heterogeneity, thereby improving the efficacy of CAR cell therapy

Method used

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  • Engineered immune cell and use thereof
  • Engineered immune cell and use thereof
  • Engineered immune cell and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1 Construction of mouse pancreatic cancer cell line Panc02-mCD19

[0125] 1. Preparation of pLV-mCD19 plasmid

[0126] 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 mouse mCD19 sequence containing XbaI and SalI restriction sites was obtained by PCR. Then, the mCD19 gene was recombined into the pLV-BHAm plasmid to obtain the pLV-BHAm-mCD19 plasmid.

[0127] 2. lentiviral packaging

[0128] In a T175 culture flask, 30×10 6 293T cells were inoculated in 30ml of DMEM medium containing 10% fetal bovine serum at a density of 1 cell / flask at 37°C, 5% CO 2 Incubate overnight in the incubator.

[0129] Add 3ml Opti-MEM (Gibco, catalog number 31985-070), 34 μg pLV-BHAm-mCD19 plasmid, 8.5 μg pMD2.G vector (Addgene, catalog number 12259) and 17 μg psPAX2 vector (Addgene, catalog number 12260) to a sterile tube ). Then add 120 μl X-treme GENE HP DNA transfection reag...

Embodiment 2

[0133] Example 2. Preparation of CAR-T cells

[0134] 1. Construction of Retroviral Plasmids

[0135] The coding sequence fragments of mCD19-scFv, mCD8a hinge region and transmembrane region, mouse 41bb intracellular domain and mouse CD3ζ intracellular domain were artificially synthesized sequentially, 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.

[0136] The coding sequence fragments of T2A and mouse IL-7 were artificially synthesized sequentially, and EcoRI / SalI restriction sites were added at both ends. The fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-IL-7 plasmid.

[0137] The coding sequence fragments of T2A and mouse XCL1 were artificially synthesized sequentially, and EcoRI / SalI restriction sites were added at both ends. The fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-XCL1 plasmid.

[0138] The coding ...

Embodiment 3

[0147] Example 3. Detecting the expression of CAR-T cells

[0148] 1. The expression level of CAR on the cell surface

[0149] Take out the 2 * 10 that embodiment 2 prepares 5 CAR-T cells, using Goat Anti-Rat IgG (H&L) Biotin (BioVision, Cat. No. 6910-250) as the primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) as the secondary antibody, and detecting CAR T cells by flow cytometry On the expression level of CAR, the results are as follows Figure 4 shown.

[0150] It can be seen that compared with the control, mCD19-CAR, mCD19-CAR-XCL1, mCD19-CAR-Flt3L, mCD19-CAR-IL-7-XCL1, mCD19-CAR-IL-7-CCL19 cells, mCD19-CAR- The CAR positive efficiencies in IL-7-Flt3L cells were all greater than 50%, indicating that these cells can effectively express CAR.

[0151] 2. Expression level of XCL1

[0152] The supernatant of the CAR-T cells prepared in Example 2 was collected, and according to the manufacturer's suggestion, the XCL1 secretion level in the cells was detec...

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Abstract

The present invention relates to an engineered immune cell expressing (i) a cell surface molecule that specifically recognizes a ligand, (ii) an exogenous interleukin, and (iii) an exogenous Flt3L, XCL2, and / or XCL1. The invention also provides the use of the engineered immune cell in the treatment of cancer, infection or autoimmune disease. Compared with the traditional engineered immune cell, the engineered immune cell has remarkably improved tumor killing activity.

Description

technical field [0001] The invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered immune cell expressing (i) cell surface molecules that specifically recognize ligands, (ii) exogenous interleukins, and (iii) exogenous Flt3L, XCL2 and / or XCL1 genes. More preferably, the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor. 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, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867A61K39/00A61K35/17A61P35/00A61P31/00A61P37/02
CPCC12N5/0636C07K16/2803C07K14/7051C12N15/86A61P35/00A61P31/00A61P37/02C12N2510/00C12N2740/15043C12N2800/107C07K2317/622C07K2319/03C07K2319/33C07K2319/74A61K39/46444A61K39/4631A61K2239/54A61K2239/38A61K39/464412A61K39/4611A61K39/464442C12N2740/16043C12N2740/13043C07K2317/70A61K48/00A61K39/4635A61K39/4637C07K14/54C07K14/52
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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