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Chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, nucleic acid molecule, immune effector cell and application thereof

A technology of immune effector cells and chimeric receptors, applied in the field of biomedicine or bioengineering, can solve the problems of poor efficacy and disadvantages of CAR therapy

Active Publication Date: 2020-04-10
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These soluble ligands can significantly inhibit the activity of traditionally designed NKG2D-CAR, thereby blocking the immune response to form immune escape, which is very unfavorable for clinical treatment of tumors, which is also an important reason why many CAR therapy clinical trials are not effective at present. reason

Method used

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  • Chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, nucleic acid molecule, immune effector cell and application thereof
  • Chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, nucleic acid molecule, immune effector cell and application thereof
  • Chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, nucleic acid molecule, immune effector cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Rational design and construction of chimeric receptors targeting human membrane-bound and soluble NKG2D ligands in this example:

[0064] The NKG2D-CAR fusion gene fragment was designed according to the following coding sequence: extracellular signal peptide, Flag tag, NKG2D ligand-binding domain based on NKG2D molecule, hinge region, transmembrane region, intracellular co-stimulatory signal domain and intracellular Signal transduction domain. figure 1 It is a structural schematic diagram and a vector schematic diagram of the chimeric receptor (NKG2D-CAR) targeting human membrane-bound and soluble NKG2D ligands constructed in Example 1 of the present invention. figure 1 a is a schematic diagram of the structure of NKG2D-CAR in the present invention, wherein the signal peptide part is preferably CD8α molecule, the transmembrane domain part is preferably CD28 molecule, and the signal transduction domain is CD28, CD137 and CD3ζ signal molecules as examples. figure 1 b ...

Embodiment 2

[0069] In this example, the synthesis of nucleic acid molecules encoding chimeric receptors targeting human membrane-bound and soluble NKG2D ligands and the construction of viral vectors (ie, the synthesis of NKG2D-CAR fusion genes and the construction of expression vectors):

[0070] Firstly, the nucleotide sequence (SEQ ID NO: 16), Nanjing GenScript Biotechnology Co., Ltd. provides gene synthesis technical services.

[0071] Cloning the synthetic fusion gene fragment into the pCDH-CMV-MCS-P2A-copGFP-T2A-Puro lentiviral vector, such as figure 1 shown. Restriction endonucleases Xba I and EcoR I were used to digest the lentiviral vector and the gene fragment (see Table 1), respectively, to obtain the linearized lentiviral vector and the NKG2D-CAR gene fragment after digestion, and T4DNA Ligase system, incubated at 16°C for 3h (see Table 2). Then transform Escherichia coli Stbl3 competent cells and smear the medium plate containing ampicillin, pick multiple clone colonies fo...

Embodiment 3

[0078] Packaging, concentration and titer determination of viral vector (ie NKG2D-CAR lentiviral expression vector) in this example:

[0079] (1) Packaging of NKG2D-CAR lentiviral expression vector

[0080] Take 8×10 6 HEK293T cells (purchased from ATCC, product number CRL-1573) were inoculated in 175cm 2 In a cell culture flask, culture overnight at 37°C. When the cell density reaches 75%-85%, replace 25 mL of fresh penicillin- and streptoxan containing 6% FBS (BI, product number 04-001-1ACS) 1 hour in advance. Mycin-based DMEM medium (Hyclone, product number SH30022.01), continued to be placed in the incubator for cultivation, and virus packaging was carried out after 1 hour.

[0081]Use calcium phosphate kit (Beiyuntian, article number is C0508) for packaging, packaging steps: the recombinant NKG2D-CAR expression plasmid (pCDH-NKG2D-CAR) extracted in Example 2, and helper plasmids pSPAX2, pMD2.G according to 4 : 3:1 ratio mix, add to CaCl 2 solution (provided by the kit...

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Abstract

The invention discloses a chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, a nucleic acid molecule and an immune effector cell and an application thereof, which belong to the technical field of biomedicine or bioengineering. According to the chimeric receptor targeting the targeting human membrane-bound and soluble NKG2D ligands, a ligand binding structural domain of a human natural NKG2D molecule is used for targeting the various NKG2D ligands; the tumor-associated surface antigen NKG2DLs is specifically recognized through the ligand or receptor, and an activation signal is transmitted into immune cells through intracellular signal transduction molecules, so that the killing activity of the immune cells is activated, and finally, tumor cells are removed. Based on an NKG2D-NKG2DLs protein complex structure and a receptor dimerization activation mechanism, a haplotype hinge region is used, so that the chimeric receptor can respond to NKG2DLs on the surface ofa tumor cell and soluble NKG2DLs falling into a tumor microenvironment at the same time, and the immunosuppression effect of the soluble NKG2DLs is converted into the immune activation effect again.

Description

technical field [0001] The invention relates to a chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, nucleic acid molecules, immune effector cells and applications thereof, belonging to the technical field of biomedicine or bioengineering. Background technique [0002] In recent years, tumor immune cell therapy technology represented by chimeric antigen receptor engineered T cells (CAR-T) has attracted much attention due to its excellent curative effect on malignant tumors. At present, CAR-T therapy targeting CD19 has achieved remarkable results in hematological malignancies, opening a new door of hope for humans to overcome malignant tumors (Nature Reviews Clinical Oncology, 2018, 15:31–46). However, the existing CAR-T technology has several defects, such as easy induction of cytokine storm (CRS), poor efficacy on solid tumors, etc. (International Reviews of Immunology, 2015, 34(2):154-187). The treatment of solid tumors is the real main battlefiel...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K39/00A61P35/00
CPCC07K14/7051C07K16/2851C12N5/0636C12N15/86A61K39/0011A61P35/00C07K2319/02C07K2319/03C07K2319/33C12N2510/00C12N2740/15043
Inventor 郭长江陈涵张会勇牛志远支灵通朱武凌
Owner XINXIANG MEDICAL UNIV
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