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A kind of chimeric antigen receptor car of cik cell and its application

A cell and antigen technology, applied to receptors/cell surface antigens/cell surface determinants, for targeting specific cell fusion, application, etc., can solve problems such as death, unknown CIK, weakened anti-tumor function, etc., to achieve The effect of reducing storm and improving safety

Active Publication Date: 2022-08-02
济南赛尔生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These CARs have achieved certain effects in T cells, but whether they are suitable for CIK is still unknown
Andreas et al. found that the CAR-modified CD3 of scFv-Fc-CD28-CD3ζ-OX40 + CD56 + cells, although able to secrete more interferons, resulted in more CD56 + Cell activation-induced cell death (AICD), which attenuates its antitumor function in vivo

Method used

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  • A kind of chimeric antigen receptor car of cik cell and its application
  • A kind of chimeric antigen receptor car of cik cell and its application
  • A kind of chimeric antigen receptor car of cik cell and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Packaging and purification of lentivirus

[0052] Will figure 2 The lentiviral vector pELPS-19-BB-z (named CAR-CK, the structure is SP-scFv-tag-CD8α-4-1BB-CD3ζ, i.e. CD19BBζ located downstream of the EF1α promoter) is shown. The CAR in the patent 2017108531905) was replaced with 7 different new CAR structures (CAR-1 to CAR-7) shown in Table 1, and 7 different recombinant lentiviral vectors were obtained, among which, figure 1 Shown is a recombinant lentiviral vector containing CAR-7 (structure SP-scFv-tag-DAP12-DNAM-1(CD226)-CD3ζ, CD19-12D-ζ located downstream of the EF1α promoter).

[0053] The above-mentioned eight different recombinant lentiviral vectors were separately packaged and purified according to the following methods to obtain eight different lentiviruses:

[0054] 1. Take a 10cm petri dish as an example, inoculate HEK293T cells in DMEM medium containing 10% fetal bovine serum; when they grow to a confluence of 70-80%, start packaging the virus...

Embodiment 2

[0066] Example 2. Acquisition of CAR-CIK cells

[0067] First, the acquisition of CIK cells

[0068] 1. Mononuclear cells were collected from peripheral blood, umbilical cord blood, and bone marrow by density gradient centrifugation;

[0069] 2. Press 2×10 6 / ml, cells were seeded in X-VIVO 15 medium containing 1000U / ml IFN-γ;

[0070] 3. After 24h, add OKT-3 and IL-2 to make the concentration reach 50-100ng / ml and 300-1000U / ml respectively;

[0071] 4. After every 2-3 days, half-change the medium and add X-VIVO 15 medium containing 300-500U / ml IL-2;

[0072] 5. On days 3-7, after the subpopulation of CIK cells reaches the doubling growth stage, cell transfection is started.

[0073] 2. Lentiviruses loaded with CAR gene were transfected into subpopulations of CIK cells

[0074] 1. Change the medium and adjust the cell density to 0.1-1×10 6 / ml;

[0075] 2. According to MOI=5-10, add eight different lentiviruses obtained in Example 1, and resuspend the supernatant;

[0...

Embodiment 3

[0083] Example 3. Killing of Raji cell lines by different CAR-CIK cells

[0084] The eight kinds of CAR-CIK cells obtained in Example 2 were respectively mixed and cultured with target cells-Raji cells (human lymphoma cells) at 1:1, 1:5, 1:25 (effect-target ratio E:T) for 24h, CIK cells without lentivirus transfection were set as the control group, and the luciferase quantitative killing efficiency evaluation method was used to detect the inhibitory and killing ability of CAR-CIK on target cells; among them, the results of CAR-7-CIK are shown in image 3 ; When E:T is 1:1, 1:5, the new CAR-CIK represented by CAR-7-CIK and traditional CAR-CIK have no significant difference in killing target cells, CAR-1-CIK, CAR-CIK The results of 2-CIK, CAR-3-CIK, CAR-4-CIK, CAR-5-CIK, CAR-6-CIK were not significantly different from those of CAR-7-CIK.

[0085] The cell supernatant of the 1:1 group E:T was collected, and the release levels of cytokines such as IFN-γ and IL-6 were detected by ...

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Abstract

The present application discloses a CIK cell chimeric antigen receptor CAR and its application. The CAR includes: an extracellular antigen binding domain, a hinge and transmembrane domain, and an intracellular signaling domain that bind to a specific target antigen; the intracellular signaling domain includes a costimulatory signaling domain, The costimulatory signaling domain contains intracellular segments of the following proteins: DAP12 and CD226. Experiments have shown that DAP12 or DAP10 are used as the hinge and transmembrane domain and part of the costimulatory signaling domain of CAR, and together with DNAM‑1 (CD226) or 2B4, they form a costimulatory signaling domain, and use these costimulatory signals. Molecules and CD3ζ to activate immune cells especially CD3 + CD56 + The related signaling pathways in CIK can inhibit and kill malignant tumor cells, which can significantly reduce cytokine storm and improve the safety of therapeutic products.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the technical field of immune cell therapy for malignant tumors, in particular to a chimeric antigen receptor CAR and its application. The CAR is especially suitable for CIK cells. Background technique [0002] Chimeric Antigen Receptors (CAR, Chimeric Antigen Receptors) are generally composed of an extracellular segment, a transmembrane region and an intracellular segment, which can specifically recognize tumor-associated antigens on the surface of target cells, and promote their location through the mechanism of receptor-activation signaling. The effector cells, such as T cells, NK cells, NKT cells, etc. activate, proliferate and kill the target cells. [0003] The structural design of CAR molecules has been refined over many generations. First-generation CARs contain single-chain variable fragments (scFv) that recognize tumor cell surface antigens, hinged and transmembrane structur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/13C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/2803C07K14/7051C12N5/0636C12N15/86A61K39/0011A61P35/00C07K2319/02C07K2319/03C07K2319/33C07K2317/622C12N2510/00C12N2740/15043
Inventor 罗昀宋珂慧郭栋刑晓
Owner 济南赛尔生物科技股份有限公司
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