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Engineered immune cells and application thereof

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

Inactive Publication Date: 2020-12-11
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 application thereof
  • Engineered immune cells and application thereof
  • Engineered immune cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Construction of mouse pancreatic cancer cell line Panc02-mCD19

[0127] 1. Preparation of pLV-mCD19 plasmid

[0128] 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 pLVX vector (PPL, Cat No.: PPL00157-4a) to obtain the pLV-mCD19 plasmid.

[0129] 2. lentiviral packaging

[0130] 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.

[0131] 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 (R...

Embodiment 2

[0135] Example 2. Preparation of CAR-T cells

[0136] 1. Construction of Retroviral Plasmids

[0137]Artificially synthesized CD19-scFv (SEQ ID NO: 26), CD8a hinge region and transmembrane region (SEQ ID NO: 35 and 29), 41bb intracellular domain (SEQ ID NO: 31) and CD3ζ intracellular domain ( SEQ ID NO: 33) coding sequence fragment, and add XhoI / EcoRI restriction sites at both ends. The fragment was cloned into the MSCV vector to obtain the MSCV-mCD19-CAR plasmid.

[0138] The coding sequence fragments of T2A (SEQ ID NO: 37) and IL-7 (SEQ ID NO: 43) 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.

[0139] The coding sequence fragments of T2A (SEQ ID NO: 37) and CCL4 (SEQ ID NO: 83) were artificially synthesized sequentially, and EcoRI / SalI restriction sites were added at both ends. The fragment was cloned into the MSCV-mCD1...

Embodiment 3

[0148] Example 3. Detecting the expression of CAR-T cells

[0149] 1. The expression level of CAR on the cell surface

[0150] 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 2 shown.

[0151] It can be seen that compared with the control, CAR positive in mCD19-CAR cells, mCD19-CAR+CCL4 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+IL7+CCL4 cells, and mCD19-CAR+IL7+CCL5 cells The efficiencies are all about 60%, indicating that these cells can effectively express CAR.

[0152] 2. IL-7 expression level

[0153] The supernatant of the CAR-T cells prepared in Example 2 was collected, and according to the manufacturer's suggestion, the IL-7 secretion level in the cells was detected with the Mou...

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Abstract

The invention relates to an engineered immune cell, and the engineered immune cell expresses (i) a chimeric receptor, and (ii) exogenous CCL3, CCL4 and / or CCL5. The invention further provides application of the engineered immune cell to treatment of cancers, infections or autoimmune diseases. Compared with a traditional engineered immune cell, the engineered immune cell provided by the invention has the 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) a chimeric receptor, and (ii) exogenous CCL3, CCL4 and / or CCL5 genes. More preferably, said chimeric receptor is a chimeric antigen receptor or T cell 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, and then clear the tumor. Therefore, increasing the effect of the immune system on killing tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/12C12N15/24C12N15/62C12N15/867A61K39/00A61P35/00A61P31/00A61P33/00
CPCC12N5/0636C07K14/523C07K14/5418C07K16/2803C07K14/7051C12N15/86A61P35/00A61P31/00A61P37/02A61P33/00C12N2510/00C07K2317/622C07K2319/03C07K2319/33C12N2740/10043C12N2800/107A61K39/4631A61K2239/54A61K39/464412A61K39/4611A61K2239/31A61K2239/13Y02A50/30A61K48/005C12N2740/16043C12N2740/13043A61K2039/505A61K39/4635A61K39/464442A61K39/4622A61K39/46444A61K2239/21A61K2239/22C07K14/52C07K2319/02C12N2501/51C12N2501/515
Inventor 邢芸任江涛贺小宏王延宾韩露
Owner NANJING BIOHENG BIOTECH CO LTD
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