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Enhanced CAR-T cell targeting prostate cancer, and preparation method and drug thereof

An enhanced technology for prostate cancer, applied in the field of immunotherapy, can solve problems such as unsatisfactory treatment effects, achieve the effects of promoting proliferation and survival, reducing immunosuppressive effects, and enhancing lethality

Inactive Publication Date: 2019-12-13
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in solid cancers such as prostate cancer, the treatment effect is less than satisfactory

Method used

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  • Enhanced CAR-T cell targeting prostate cancer, and preparation method and drug thereof
  • Enhanced CAR-T cell targeting prostate cancer, and preparation method and drug thereof
  • Enhanced CAR-T cell targeting prostate cancer, and preparation method and drug thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Construct a plasmid vector containing an expression cassette for expressing the first receptor and the second receptor, and refer to the structure and positional relationship of each element on the expression cassette figure 1 , The original backbone of the plasmid vector is Carl June.

[0050] Wherein, the first receptor is a chimeric antigen receptor, and the chimeric antigen receptor includes the following elements: scFv+CD8hinge+CD8αTM+4-1BB+CD3ζ; its front end has a CD8α signal peptide.

[0051] Among them, the amino acid sequence and base sequence of each element are as follows:

[0052] The base sequence of the CD8α signal peptide is shown in SEQ ID NO.1.

[0053] The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.2.

[0054] The base sequence of scFv that specifically binds to PSMA is shown in SEQ ID NO.3.

[0055] The amino sequence of scFv is shown in SEQ ID NO.4.

[0056] The base sequence of CD8 hinge (hinge region) is shown in SEQ ID NO.5.

[0057]...

Embodiment 2

[0079] Construction of virus containing the above chimeric antigen receptor and T7R receptor

[0080] Methods as below:

[0081] The PSMA-T7R plasmid and the lentiviral packaging plasmid psPAX2 and the lentiviral envelope plasmid pMD2.G of Example 1 were transfected into 293T cells with the three-plasmid system using the transfection reagent PEI, and the culture supernatant was harvested and subjected to ultrafiltration and ultrafiltration. After isolation and concentration, a lentivirus expressing the first receptor and the second receptor is obtained.

[0082] The obtained virus was named PSMA-T7R virus.

Embodiment 3

[0084] Construction of T cells expressing chimeric antigen receptor and T7R receptor

[0085] T cells were separated from human blood using magnetic beads, activated by CD3 / CD28 complex, and infected with packaged lentivirus, and the expression level was detected by 48h flow cytometry. The specific process is as follows:

[0086] (1) First add the lymphocyte separation solution to a 50mL centrifuge tube, then slowly add the blood to the upper layer of the lymphocyte separation solution while keeping the interface clear (the volume ratio of lymphocyte separation solution to blood is 2:1), 800g Centrifuge for 25 minutes (during which the centrifuge is set to rise at 1 and drop at 0).

[0087] (2) After centrifugation, carefully aspirate the eukaryotic cell layer, add PBS and mix by pipetting gently, and centrifuge at 500g for 10 minutes.

[0088] (3) After resuspending the cells in PBS, count the cells. After counting, centrifuge at 300g for 10 minutes, discard the supernatant, and ad...

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Abstract

The invention discloses an enhanced CAR-T cell targeting prostate cancer, and a preparation method and a drug thereof, and relates to the technical field of immunotherapy. A first receptor and a second receptor are expressed on a cell membrane of the CAR-T cell. The first receptor is a chimeric antigen receptor, and the chimeric antigen receptor has an antigen binding domain for binding a prostatecancer antigen; the second receptor includes an extracellular TGF beta binding domain that specifically binds to TGF beta and an intracellular IL-7 activation signal transduction domain that persistently activates an IL-7 signaling pathway. The CAR-T cell provided by the invention can target prostate cancer, neutralize the inhibiting effect of TGF beta on the T cell in a tumor microenvironment and activate an IL-7-STAT 5 signaling pathway to promote proliferation and survival of the T cell. The CAR-T cell has enhanced anti-tumor activity and can be used for treating the prostate cancer.

Description

Technical field [0001] The present invention relates to the technical field of immunotherapy, in particular to an enhanced CAR-T cell targeting prostate cancer and a preparation method and medicine thereof. Background technique [0002] The adoptive cell therapy of chimeric antigen receptor (CAR) modified T cells (CAR-T) is currently the most promising immunotherapy method, especially for refractory leukemia and lymphoma, and has achieved great results. But for solid cancers such as prostate cancer, the therapeutic effect is not satisfactory. [0003] In view of this, the present invention is proposed. Summary of the invention [0004] The purpose of the present invention is to provide an enhanced CAR-T cell targeting prostate cancer and its preparation method and medicine. The CAR-T cell provided by the present invention can target prostate cancer, and has the characteristics of inhibiting the TGFβ signaling pathway while activating the IL-7 signaling pathway, and the CAR-T cell ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K39/00A61P35/00
CPCA61P35/00A61K39/001195C07K14/7051C07K14/71C07K14/7155C07K16/3069C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 张娜杜冰吴诗佳刘小红殷宏翔刘明耀
Owner EAST CHINA NORMAL UNIV
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