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Tumor therapeutic agent improved through IL-12/CD62L fusion protein and preparation method and application thereof

A fusion protein and protein technology, applied in the field of medicine, can solve the problems of IL-12 deregulation, toxic and side effects, and insignificant toxic and side effects.

Active Publication Date: 2016-01-06
杨晶
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although IL-12 has a significant antitumor effect, it is greatly limited due to the systemic toxicity caused by its systemic administration.
Before the present invention, the clinical application of IL-12 was greatly limited due to the systemic toxic side effects induced by it, and the accidental death of two patients caused by systemic administration in early clinical trials.
[0005] In order to avoid the toxic and side effects of systemic administration and achieve the purpose of clinical application, clinical researchers have used local tumor injection, transient expression and multi-point injection in tumor clinical trials, but because the therapeutic effect of IL-12 is the same as The dose of reinfused IL-12 is directly related, and the above-mentioned local clinical scheme has not achieved significant anti-tumor effect
[0006] In order to solve this problem, the inventors once tried to maximize the anti-tumor effect by genetically modifying anti-tumor T cells to continuously secrete IL-12, but due to unknown reasons (one possible reason is the toxic side effects of IL-12 ) T cells genetically modified by the continuous secretion of IL-12 can not be effectively expanded in vitro, and cause a large number of T cell apoptosis
[0007] In order to solve this problem, the present inventors have also achieved the problem of effective expansion of T cells in vitro through the gene modification scheme of specifically regulating the expression of IL-12 through TCR activation, and also achieved a certain tumor-suppressing effect, but at the same time there is IL-12 -12 Toxic and side effects caused by deregulation cannot be effectively applied to clinical applications
[0008] In summary, there is still a lack of IL-12-based antitumor drugs with high efficiency and no obvious side effects in this field.

Method used

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  • Tumor therapeutic agent improved through IL-12/CD62L fusion protein and preparation method and application thereof
  • Tumor therapeutic agent improved through IL-12/CD62L fusion protein and preparation method and application thereof
  • Tumor therapeutic agent improved through IL-12/CD62L fusion protein and preparation method and application thereof

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preparation example Construction

[0174] Preparation of fusion protein expressed by lentiviral expression system

[0175] Culture 293T cells. One day before transfection, after adjusting the cell density with DMEM medium containing 10% fetal bovine serum, inoculate 25×10 cells per 15 cm cell culture dish. 6 293T cells at 37°C, 5% CO 2 Cultivate in an incubator, and use for transfection when the cell density grows to 80%-90% after 16h-24h. On the day of transfection, the medium was replaced with a complete medium (DMEM+10% FBS) without antibiotics (P / S). The lentiviral backbones of LVV-MSCV-MART-1TCR, CD62L, hscIL-12L and hscIL-12 / CD62L fusion protein were co-transfected into 293T cells with the other three packaging plasmids, using commercially available calcium phosphate as the medium. After culturing for 6 hours, discard the medium, wash with PBS 3 times and replace with 20 ml of fresh complete medium (DMEM+10%FBS+P / S). Collect the culture supernatant 30-72 hours after transfection, centrifuge at 6000rpm ...

Embodiment 1

[0183] Construction of fusion genes

[0184] The fusion gene was synthesized by Invitrogen Company, and the length and sequence of the fusion gene were confirmed by 1% agarose electrophoresis and sequencing.

[0185] The structure of the obtained fusion gene is shown in SEQ ID NO.:1, and the amino acid sequence of the encoded fusion protein is shown in SEQ ID NO.:2.

Embodiment 2

[0187] Construction of lentiviral expression vector

[0188] Adopt the method described in "Construction of Lentiviral Vector and Gene Modification of T Cells" in the general method: the humanized 293T cells cultured in low-generation DMEM (containing 10% FBS) medium in vitro are counted and transferred to 15CM In the petri dish, the bottom of the petri dish was treated with poly-D-Lysine, and 20X10 6 Cells, on the next day, add a mixed solution of DNA mixed solution and Lipofectamine in each transfection culture dish: the composition of the mixed solution is as follows, get 2mlOptimumI and add pLenti-MSCV (22.5ug), pMD2.G (7.5ug), gag / pol (15ug), pRev (10ug), mix well; at the same time take 2ml OptimumI and add Lipofectamine160ul (Invitrogen), mix well. Mix the two suspensions, incubate at room temperature for 5 minutes, and then drop evenly into the Petri dish. After 48-72 hours, harvest the supernatant containing the genetic engineering vector, centrifuge at 2000g to remo...

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Abstract

The invention provides an efficient and low-toxin tumor therapeutic agent and a preparation method and application thereof, and particularly provides IL-12 / CD62L fusion protein. Anti-tumor T cells are modified through slow viruses containing IL-12 / CD62L fusion genes, and by means of specific cleavage and release induced by activation of CD62L tumor antigen dependence, release of cell membrane surface IL-12 activated through tumor antigens is achieved. A is shown through experiments, fixed-point and timed release of IL-12 can be achieved locally on tumors attacked by the T cells, so that the immune microenvironment of T cell-tumor tissue is changed, and accordingly the anti-tumor immune effect is the maximum. Besides, the toxic and side effects are remarkably reduced.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a tumor therapeutic agent transformed by IL-12 / CD62L fusion protein and its preparation method and application. The invention provides a kind of IL-12 / CD62L gene-modified T cells with enhanced anti-tumor effect. Background technique [0002] IL-12 is a very important immune stimulating factor. IL-12 is a covalently linked heterodimeric cytokine composed of p35 and p40 subunits that is secreted by activated immune cells in vivo. IL-12 is an important regulatory factor of cellular immunity, and plays a role through NK cells and CTL in anti-infection immunity and immunity of malignant tumors. [0003] Individuals who are congenitally deficient or nonresponsive to IL-12 have profoundly deficient immunity against infection. A series of preclinical experimental models have confirmed that the intervention of IL-12 can significantly prolong the survival of tumor-bearing animal models. [0004...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N15/861C12N15/86C12N5/10C12P21/00A61K38/20A61K47/48A61P35/00
CPCA61K38/20C07K19/00C12N5/10C12N15/62C12N15/86C12N15/861C12N15/867C12P21/00Y02A50/30
Inventor 杨世成
Owner 杨晶
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