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Whole-cell vaccine for inhibiting or preventing melanoma, and preparation method for whole-cell vaccine

A melanoma cell, whole cell vaccine technology, applied in the direction of tumor/cancer cells, genetically modified cells, biochemical equipment and methods, etc. The effect of improving the level and improving the immune killing ability

Active Publication Date: 2020-01-17
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The vaccine can improve the overall survival rate of patients by about 4 months, but due to its high cost, it has no competitive advantage compared with other drugs
The main reason for its poor effect is that tumor-associated antigens are also expressed in normal cells, and immune cells in the body have developed immune tolerance to this type of self-antigen after positive and negative selection, which cannot be effectively recognized and presented. Such antigens

Method used

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  • Whole-cell vaccine for inhibiting or preventing melanoma, and preparation method for whole-cell vaccine
  • Whole-cell vaccine for inhibiting or preventing melanoma, and preparation method for whole-cell vaccine
  • Whole-cell vaccine for inhibiting or preventing melanoma, and preparation method for whole-cell vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment one: see figure 1 , Take the bone marrow cells of C57 / B6J mice and add GM-CSF (10ng / ml) and IL4 (10ng / ml) in vitro, and culture until the 7th day; the tumor cells are first treated with zVAD (20uM) for 2 hours and then treated with TNF-a (100ng / ml) treatment for 24 hours, heated in a 65-degree water bath for 5 minutes; co-cultured 1.25 million DCs with 0.25 million B16 in a 24-well plate, and detected the activated markers CD86, CD80, and CD40 on the surface of DC cells after 24 hours. The control group was DC without co-culture treatment after induction of maturation; DC treated with CPG after 7 days of maturation; DC co-cultured with 50gy irradiated tumor cells; DC, compare the statistical graphs obtained; this graph proves that zVAD and TNF-A treatment of necroptotic tumor cells and co-culture of DCs can significantly improve the activity of DCs presenting tumor antigens.

[0025] Treat B16 cells with zVAD and TNF-A to cause necroptosis; when the necropto...

Embodiment 2

[0026] Embodiment two: see figure 2, using the same method as in Example 1 to treat tumor cells to cause necroptosis, compare normal melanocytes and knock out the MLH-1 gene on the melanoma cells to inactivate DNA damage repair enzymes, and produce a large number of neoantigens Tumor cells were co-cultured with DCs. Before co-culture with DC cells, the treated tumor cells were heated in a 65°C water bath for 5 minutes to determine the complete inactivation of tumor cells. Then culture 1 million DC cells and 0.25 million treated neoantigen tumor cells in a 24-well plate, and detect the expression of markers on the surface of DC cells after 24 hours; the diagram shows that the MLH-1 gene on tumor cells is knocked out, after Attached figure 1 Co-cultivation with DC cells after treatment by the method can significantly improve the activation degree of DC. Moreover, tumor cells expressing knockout MLH-1 but expressing neoantigens have a better ability to increase the degree of ...

Embodiment 3

[0027] Embodiment three: see image 3 , the vaccine obtained by co-culturing common tumor cells and DCs described in Example 2 and the vaccine obtained by co-culturing neoantigen cells and DCs were resuspended to 200ul in PBS and injected into mice. One week later, the mouse spleen cells were taken, and the spleen cells were stimulated with the tumor cell lysates of the corresponding components of the two vaccines, and 6 million spleen cells were stimulated with 1 million tumor lysates in a 24-well plate, and cultured in an incubator with 1640 as the medium for 4 Hours later, the ratio of intracellular secreted IFN-g of CD8+T cells was detected. The obtained value is the proportion of specific CD8+ T cells, and the figure shows that the vaccine obtained by co-cultivating neoantigen cells treated with necroptosis and DC is better than the vaccine obtained by co-culturing DC and ordinary tumor cells.

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Abstract

The invention relates to a whole-cell vaccine for inhibiting or preventing melanoma, and a preparation method for the whole-cell vaccine, in particular to a preparation method for a tumour vaccine which has a function of improving an immune response and is co-cultured by a new antigen cell and a dendritic cell, and application. The vaccine is characterized in that an MLH-1 gene with a DNA (deoxyribonucleic acid) repairing function is knocked out in a melanoma cell, so that a tumour cell generates mutation in a cell replication process so as to express a non-autologous antigen easy in immune cell recognition. After the mutated tumour cell is processed, the mutated tumour cell and a DC (Dendritic cell) are co-cultured, and therefore, costimulatory proteins expressed on the surface of the DCare obviously improved. The whole-cell vaccine prepared by the method obviously improves the immune response in the body of a mouse, in-vivo IFN-G (interferon-G) and TNF-A (Tumour necrosis factor-a) is improved than a non-injected vaccine group, and therefore, the whole-cell vaccine performs an important function of treating the tumor.

Description

technical field [0001] The invention relates to a whole cell vaccine for inhibiting or preventing melanoma and a preparation method thereof. Background technique [0002] Tumor has gradually become one of the main factors that endanger the life and health of the public. Because of its rapid progress, high degree of malignancy and low cure rate, it has become a hot spot for human exploration. The current treatment options for tumors mainly include surgical resection, chemotherapy, and radiation therapy. At present, the three methods are easy to lead to tumor invasion, metastasis and recurrence, so there is an urgent need to develop new methods to treat tumors clinically. [0003] Immunotherapy is to activate the body's own immunity, let immune cells actively exert their effectiveness, and lyse tumor cells through direct or indirect contact with tumor cells, thereby delaying tumor progression and inhibiting tumor recurrence and metastasis. At present, new types of immunother...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P35/00C12N5/0784C12N5/10
CPCA61K39/0011A61P35/00C12N5/0693C12N5/0639C07K14/47A61K2039/5154C12N2502/00C12N2501/25C12N2501/2304C12N2501/22C12N2510/00Y02A50/30
Inventor 杨翼铭冷启彬陈宪洋华来庆
Owner SHANGHAI UNIV
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