Preparation and application of engineered immune cells

An immune cell and molecular technology, applied in the field of the preparation of engineered immune cells, can solve problems such as the inability to achieve universal CART cells and the removal of CART cells

Active Publication Date: 2020-10-23
NANJING BIOHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, knocking out TCR alone cannot meet the requirements of universal CART cells, because there are still HLA molecules expressed on the surface of CART cells, and there is a risk of being cleared after being recognized by recipient immune system T cells
[0005] In clinical treatment, alemtuzumab is usually used to clear T cells in patients to prevent rejection, but this also leads to the risk of being cleared of reinfused CART cells

Method used

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  • Preparation and application of engineered immune cells
  • Preparation and application of engineered immune cells
  • Preparation and application of engineered immune cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Preparation of sgRNA

[0039] The sgRNA targeting the exons of the CD52 gene is designed, and its target sequence on the gene is unique. Mix the forward and reverse sequences of the synthesized sgRNA oligonucleotides in equal proportions, react under the action of PNK enzyme at 37°C for 30 minutes, then react at 95°C for 5 minutes to inactivate the PNK enzyme, and slowly anneal to room temperature to obtain Double-stranded DNA product of sgRNA.

[0040] The DNA product obtained above was cloned into a T7U2 vector, and the correct insertion of the target sequence was confirmed by sequencing.

[0041] Then, the above expression vector was digested with EcoRI enzyme, and the linearized vector was obtained after purification and recovery. Then, using the linearized vector as a template, sgRNA was prepared with the MEGAscript® T7 High YieldTranscription Kit (Invitrogen, Cat. -01) for purification to obtain purified sgRNA.

Embodiment 2

[0042] Example 2. Knockout of the CD52 gene in T cells

[0043] The T cells used in the examples of the present invention are primary human T cells isolated from healthy donors by Ficoll-Paque™ PREMIUM (GE Healthcare) using leukapheresis.

[0044] T cells were stimulated with DynaBeads CD3 / CD28 CTSTM (Gibco, Cat. No. 40203D) and incubated at 37°C and 5% CO 2 cultured for 3 days. Then, using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX), 10ug Cas9 protein and 10ug sgRNA were electrotransfected into activated T cells at 400V and 0.7ms to obtain CD52 knockout T cells. Immediately after electrotransfection, T cells were placed in 1 mL of pre-warmed medium and cultured in the presence of IL-2 (300 IU / mL) at 37°C and 5% CO2. After 3 days, use PE Mouse Anti-Human CD52 (BD Pharmingen, catalog number 562945) antibody to detect the expression of CD52 by flow cytometry, so as to obtain the knockout efficiency of CD52. T cells not transfected with Cas9 protein and sgRNA ...

Embodiment 3

[0053] Example 3. Preparation of UCART cells knocked out of TCRα and CD52

[0054] 1. Preparation of CD19-22 CART cells

[0055] The following coding sequences were synthesized and cloned into pGEM-T Easy vector (Promega, Cat. No. A1360): CD8α signal peptide (SEQ ID NO: 31), anti-CD19 scFv (SEQ ID NO: 25), anti-CD22 scFv (SEQ ID NO: 29), CD8α hinge region (SEQ ID NO: 39), CD8α transmembrane region (SEQ ID NO: 33), 4-1BB costimulatory domain (SEQ ID NO: 35), CD3ζ intracellular signaling domain (SEQ ID NO: 38), and the correct insertion of the target sequence was confirmed by sequencing.

[0056] Add 3mL Opti-MEM (Gibco, Cat. Cat. No. 12260) and the envelope vector pMD2.G (Addgene, Cat. No. 12259). Then, add 120uL X-treme GENE HP DNA Transfection Reagent (Roche, Cat. No. 06366236001), mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid / vector / transfection reagent mixture dropwise to the culture flask of 293T cells middle. Viruses were coll...

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Abstract

The invention relates to a preparation and an application for an engineered immune cell with a CD52 gene knocked out. Specifically, an sgRNA molecule is selected and can target the CD52 gene with highspecificity, so the efficiency of gene editing is improved; and preparation of universal CAR cells is facilitated.

Description

technical field [0001] The invention relates to the technical field of biomedicine. More specifically, it relates to the preparation and application of an engineered immune cell in which the CD52 gene has been knocked out. Background technique [0002] Chimeric antigen receptor (CAR) cells, especially CART cells, are currently one of the most promising tumor immunotherapies. A specific chimeric antigen receptor that recognizes and binds to tumor cell surface antigens, thereby targeting and killing tumor cells. CART has achieved remarkable efficacy in the treatment of leukemia and lymphoma. On August 30, 2017, the U.S. FDA approved the official launch of the CART cell therapy Kymriah for the treatment of relapsed or refractory children and adolescents with B-cell acute lymphoblastic leukemia (rALL). Acute lymphoblastic leukemia accounts for about 25 percent of cancer diagnoses in children under the age of 15 and is the most common childhood cancer in the United States. In...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N9/22C12N5/10A61K35/17A61K35/15A61P35/00A61P31/00A61P37/02
CPCA61K35/15A61K35/17A61P31/00A61P35/00A61P37/02C12N5/0636C12N9/22C12N15/113C12N15/85C12N15/907C12N2310/10C12N2510/00
Inventor 周亚丽任江涛贺小宏王延宾韩露
Owner NANJING BIOHENG BIOTECH CO LTD
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