Prostate cancer-targeting CAR-T cell drug capable of secreting IL-23 antibody

A technology of prostate cancer and cells, applied in the field of immunocytology, can solve problems such as poor prognosis, side effects of bone health, unmet medical needs of treatment, etc., and achieve the effect of enhancing activity and enhancing curative effect

Active 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

Although this therapy has a certain curative effect, in addition to side effects on cardiovascular, sexual function, metabolism, psychology and bone health, the remission of most patients can only be maintained for 1 to 4 years, and then the patients are deprived of androgen. Resistance to therapy, allowing cancer cells to develop into the more aggressive form of castration-resistant prostate cancer (CRPC), treatment of these patients remains an unmet medical need because of the poor prognosis for CRPC patients

Method used

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  • Prostate cancer-targeting CAR-T cell drug capable of secreting IL-23 antibody
  • Prostate cancer-targeting CAR-T cell drug capable of secreting IL-23 antibody
  • Prostate cancer-targeting CAR-T cell drug capable of secreting IL-23 antibody

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Experimental program
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Effect test

Embodiment 1

[0068] Construction of a plasmid vector containing an expression cassette expressing a chimeric antigen receptor and a single-chain antibody specifically binding to IL-23, and the structure and positional relationship of each element on the expression cassette for reference figure 1 , plasmid vector backbone reference figure 2 .

[0069] Specific steps are as follows:

[0070] The first expression cassette (PSMA-CAR) was synthesized by GenScript to express the chimeric antigen receptor targeting prostate-specific membrane antigen. The first expression cassette (PSMA-CAR) includes: CD8α signal peptide, PSMA single-chain antibody heavy chain variable region , Linker1, PSMA scFv light chain, CD8 hinge region, CD8α transmembrane domain, intracellular costimulatory element of 4-1BB and intracellular domain of CD3ζ ( figure 1 In A), the above sequences are connected sequentially, and the Kozac sequence and the corresponding enzyme cutting site are introduced at the front end. Th...

Embodiment 2

[0112] (1) Construction of viruses expressing chimeric antigen receptors and anti-IL-23 scFv

[0113] The method is as follows: use Escherichia coli to amplify the above-mentioned IL-23ab-CAR plasmid and the lentiviral packaging helper plasmid psPAX2 (see Figure 8 ) and pMD2.G (see Figure 9 ), after the plasmid was extracted, agarose gel electrophoresis and sequencing were performed to identify the correctness of the plasmid. Select the 293T cells in good condition and the first generation as the lentivirus packaging cells, and use the transfection reagent PEI to transfect the 293T cells with the above three plasmids. The transfection is completed in a 10cm culture dish with a total system of 10mL, and the cell transfection mixture in each dish should be prepared into a 1mL system with serum-free DMEM, so that psPAX2 plasmid: pMD2.G plasmid: IL-23ab-CAR plasmid: PEI=5μg: 3 μg: 5 μg: 72 μl, mix the transfection mixture at room temperature, let it stand for 20 minutes, then ...

Embodiment 3

[0120] (1) Construction of T cells expressing chimeric antigen receptors and anti-IL-23 scFv

[0121] Methods as below:

[0122] Use lymphatic separation fluid to separate PBMC from human blood, and then use CD4 and CD8 magnetic bead sorting method to separate T cells. After being activated by CD3 / CD28 complex for 48 hours, the packaged PSMA-CAR and IL-23ab- CAR virus was infected by centrifugation at MOI=10 for 2h, and replaced with fresh medium (XVIVO+10%FBS+IL-2) after 24h. The two kinds of CART cells were named PSMA-CART cells and IL-23ab-CART cells respectively.

[0123] After 48 hours of infection, the CAR expression levels of the above two CARTs were detected according to the same method of titer detection.

[0124] see results Figure 4 It was shown that the CAR positive rates of IL-23ab-CART and PSMA-CART were 83% and 96%, respectively.

[0125] (2) Expression detection of IL-23-scFv:

[0126] For IL-23ab-CART, it is not only necessary to detect the expression of ...

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Abstract

The invention discloses a prostate cancer-targeting CAR-T cell drug capable of secreting an IL-23 antibody, and relates to the technical field of immunocytology. The prostate cancer-targeting CAR-T cell contains a nucleic acid molecule having a first nucleic acid sequence encoding a chimeric antigen receptor that binds to a prostate-specific membrane antigen and a second nucleic acid sequence encoding antibodies that specifically bind to IL-23 or functional fragments of the antibodies. The CAR-T cell or a drug containing the cell can target the prostate-specific membrane antigen of the prostate cancer and also can secrete antibodies that specifically bind to IL-23 or functional fragments of the antibodies. The secreted antibodies or functional fragments of the antibodies take effect in a paracrine mode to reverse castration resistance of prostate cancer. The activity of the CAR-T cells for resisting prostate cancer is enhanced, and the prostate cancer cell killing capacity is improved.

Description

technical field [0001] The invention relates to the technical field of immunocytology, in particular to a CAR-T cell drug targeting prostate cancer that secretes IL-23 antibody. Background technique [0002] Prostate cancer is one of the most common tumors of the male genitourinary system. The global incidence rate accounts for the second among male malignant tumors, and the global mortality rate accounts for the fifth among malignant tumors. It is the most common cancer and the second leading cause of death in the United States. Data show that in the past 10 years, my country's prostate cancer has shown a high incidence trend, and large cities have become "hardest hit areas". Based on the characteristics of an aging population base and age structure, along with the increase in the incidence of prostate cancer, the incidence of prostate cancer in China The rate of increase is worrying. [0003] Traditional prostate cancer treatments include radical prostatectomy, radiothera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62A61P39/00A61P35/00
CPCA61K2039/5156A61P35/00A61K39/00114A61K39/001195A61K2039/884C07K14/7051C07K14/70517C07K14/70578C07K16/244C07K16/3069C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/42C07K2319/74C12N5/0636C12N2510/00
Inventor 张娜杜冰刘小红吴诗佳殷宏翔刘明耀
Owner EAST CHINA NORMAL UNIV
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