Chimeric antigen receptor targeting ADGRE2 and application thereof

A chimeric antigen receptor and targeting technology, which can be used to target specific cell fusion, polypeptides containing positioning/targeting motifs, applications, etc., can solve problems such as low expression

Active Publication Date: 2022-03-01
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study found that ADGRE2 was highly expressed in AML cells and was detectable in 93% of patients; however, the protein was only very low expressed in the intestine, ovary and spleen

Method used

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  • Chimeric antigen receptor targeting ADGRE2 and application thereof
  • Chimeric antigen receptor targeting ADGRE2 and application thereof
  • Chimeric antigen receptor targeting ADGRE2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0050] The preparation method of CAR-T cells in accordance with an embodiment of the present invention, comprising the step of collecting peripheral blood mononuclear cells; T cells such as CD3 + T cells are selected from the above peripheral blood mononuclear cells; T cells are activated The T cells containing the above nucleic acids were used, and then amplification culture was then carried out.

[0051] In a specific example, retronectin can also be used to improve the infection efficiency of Virus on T cells during infection.

[0052] In a specific example, the preparation method comprises the steps of:

[0053] Peripheral blood mononuclear cells were selected from CD3 + T cells with CD3, transferred to GT-T551 H3 medium to carry out cultures, and the proportion of bearing ratio of 1: 1 was added to the CD3-CD28 stimulating magnetic beads for stimulation. 48h after viral infection;

[0054] The retronectin is packaged in a hole plate, placed in a 37 ° C cell incubator for 2 h;...

Embodiment 1

[0056] Example 1 vector construction

[0057] In this embodiment, a single-chain antibody (SCFV) using anti-human ADGRE2 antibodies is used as an antigen binding domain, combined with signal peptide, hinge region, transmembrane region, a common thorns, and CD3Z intracellular domain, and constructs a chimeric antigen receptor of target ADGRE2. , Schematic figure 1 Indicated. After obtaining the ADGRE2-CAR amino acid sequence, all genetically synthesis of Guangzhou Aiji Biotechnology Co., Ltd. and constructed to a slow viral expression vector.

[0058] ADGRE2-Car protein sequence (482AA):

[0059] / / mdmrvpaqllgllllwlrgarc DIQMTQSPSSLSSSLGGKVTITCRASQDINKFISWYQHRPGKGP Rllihyastlqpgipsrfsgsgsgrdysfsisnlepediatyphyclqydnlwtfggggtkleirggggggggggggggggggg SevqlqqsgpelvkpgasvrmsckasgyTFTDynmywvkqslgkslewigyiyPntcgatynqkfkgkatltvnks SSTAFMELSLTSEDSAVYCGRGGPFAYWGQGTLVTVSATTPAPRPTPAPTISQPLSLRPEACRPAAGGAV HtrgldfacdiyiwaplagtcgvllslvitlyckrgrkkkllyifkqpfMrpvqttqeedgcscrfpeeeeggcel Rvkfsr...

Embodiment 2

[0062] Example 2 Virus Packaging

[0063] Plasmid transfection: Plasmid, PEI, OPTI-MEM medium was placed at room temperature for 5 min; Take OPTI-MEM436 μL in 1.5 MLEP tube, then add 64 μl of PEI to mix, stand for 5 min at room temperature; press 6: 9: 9: 16 PLP1, PLP2, PLP-VSVG, the purpose gene expression plasmid, and OPTI-MEM to 500 μL were added to stand for 5 min at room temperature; the mixed PEI-OPTI-MEM solution was added to the plain-containing OPTI-MEM, and the room temperature was allowed to stand for 20 min; The 1 ml DNA / PEI mixture slowly drops into HEK 293T, gently mixed, incubated at 37 ° C incubation, replace fresh medium after 16 to 18 hours, add 5% CO 2 The 37 ° C incubator continued to incubate.

[0064] Virus collection and concentration: Plasmid was transfected with 48 h, collected by superprint, centrifuged at 4 ° C for 20 min, remove cell debris, and filtered with a 0.45 μm filter to obtain supernatants; 500000 centrifugation 2H, removing the supernatant, ...

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Abstract

The invention relates to an ADGRE2-targeting chimeric antigen receptor and application thereof. The ADGRE2-targeting chimeric antigen receptor comprises an ADGRE2-targeting single-chain antibody, a hinge region, a transmembrane structural domain, a costimulatory structural domain and a signal transduction structural domain which are sequentially connected from an amino terminal to a carboxyl terminal, the amino acid sequences of a light chain variable region and a heavy chain variable region of the ADGRE2-targeting single-chain antibody are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2. An ADGRE2-targeting chimeric antigen receptor T cell constructed by using the chimeric antigen receptor can specifically kill ADGRE2 positive tumor cells, so that an effective cell treatment choice is provided for treating ADGRE2 positive acute myelogenous leukemia.

Description

Technical field [0001] The present invention relates to the field of molecular biological techniques, and in particular, to a chimeric antigen receptor targeting ADGRE2 and its application thereof. Background technique [0002] Acute myeloid leukemia (AML) is the most common leukemia in adults, which is the second common leukemia in children. The standard chemotherapy induction program has not substantial changes in the past few decades, and most patients have responded to chemotherapy at the beginning, and they have reached a completely alleviated state (CR), but only some of the people can survive for a long time because chemotherapy Resistance leads to recurrence of diseases. Studies have shown that patients who have never achieved disease relief or recurrence within six months of CR will have a response to any treatment. [0003] In recent years, chimeric antigen receptor T cells (CAR T) therapy showed very good results in tumor therapy, especially CAR T cell therapy for CD19...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/02
CPCC07K16/2896C07K14/7051C12N15/86C12N5/0636A61K39/001129A61P35/02C07K2317/622C07K2317/56C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156
Inventor 李光超罗敏周兆
Owner GUANGZHOU BIO GENE TECH CO LTD
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