In recent years, immunotherapy, which uses the body's own immune system to fight tumors, has expanded more possibilities for the development of tumor treatment. Compared with surgery, radiotherapy, chemotherapy and other treatment methods, immunotherapy has relatively few side effects and is also effective in some cases where other treatments do not work. However, because current immunotherapy mainly includes immune checkpoint monoclonal antibody therapy, adoptive immune cell therapy, human recombinant cytokines (non-specific immunotherapy), oncolytic virus therapy and tumor vaccine therapy, these therapies are still in clinical use. There are still various limitations in application and it is not foolproof. Tumor cells still have the possibility of escaping the "inspection" of immune cells. Therefore, there is an urgent need to develop new types of tumor immunotherapy.
Recently, the Yale University research team published the results of a new immunotherapy for the treatment of tumors in the journal Nature Immunology. The results presented in this article titled "Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity" show that Multiplex Activation of Endogenous Genes Immunotherapy (MAEGI) based on the CRISPR activation (CRISPRa) platform as Immunotherapy) can target specific types of cancer cells and activate related gene expression. By precisely locating, labeling and amplifying signals, the immune system can attack the labeled cancer cells, effectively killing or eliminating many types of tumors in mice. This treatment modifies the tumor microenvironment and is characterized by enhanced T cell infiltration and anti-tumor immunity. Multiplexed endogenous gene activation is a versatile, highly scalable strategy to elicit effective immune responses against tumors, distinct from all cancer therapies. This research is expected to help the human immune system recognize, attack and kill cancer cells.
First, researchers used adeno-associated viruses to deliver the CRISPRa library into tumors and precisely target the mutated genes of the tumors. Research shows that triple-negative breast cancer (TNBC) E0771? cells are transfected with CRISPRa lentiviral vector, allowing the cells to simultaneously express dCas9-VP64 and MS2-p65-HSF1, and then use the promoter of the phosphoglycerate kinase PGK gene to activate the OVA protein The E0071-OVA stably transfected cell line was obtained by expression, and was cultured with the isolated CD8+ T cells activated in vivo by OVA to evaluate the effectiveness of this tumor immunotherapy. Tumor cells with low expression of TAAs (tumor-associated antigens) can highly express TAAs on the cell membrane surface after being activated by CRISPRa, and are recognized and killed by CD8+? T cells of the immune system.
Based on the above-mentioned model research model, the researchers expanded MAEGI therapy to mice and used the AAV-CRISPR system to deliver the sgRNA library into the TNBC tumors of C57BL/6J mice. The results found that Compared with the PBS-treated group and the empty plasmid group, the tumor growth of mice introduced into AAV-g-MAEGI was significantly inhibited. At the same time, ELISA experiments also proved that mice in the AAV-g-MAEGI group had more IFNγ production and Greater numbers of active CD8+ T cells. The above results also show that the anti-tumor immune activity in mice has been significantly enhanced through MAEGI therapy.
Finally, the researchers used single-cell sequencing technology to further verify the composition of the activated immune cell population at the cellular level, and found that after C57BL/6J breast cancer mice were treated with different doses of AAV-MAEGI, Mice in the high-dose group had a higher proportion of CD4+ and CD8+ active cells, showing that MAEGI therapy can eliminate tumors by enhancing immune responses.
In summary, researchers have proven that direct activation of endogenous mutated genes through CRISPRa can amplify the "non-self" signals of tumor cells, thereby inducing powerful anti-tumor adaptive immunity.
Various forms of MAEGI, including AAV-g-MAEGI and AAV-p-MAEGI, as well as any future derivatives of endogenous gene activation therapies, provide an orthogonal modality for tumor immunotherapy as single agents. Or used in conjunction with other treatment modalities. Before entering Phase I clinical trials, the future clinical translation of MAEGI also requires the elimination of overexpressed potentially harmful genes, optimization of ingredients and design, evaluation of toxicity in animal models, and exploratory research on the development of finished drugs.
Anti-cancer hero immune cells
American biologist George Daley once said: If the 20th century was the era of drug treatment, then the 21st century is the era of cell therapy. Based on the precise location and identification of cancer cell-related antigens, the body's immune system will begin to attack the marked cancer cells. Killer immune cells serve as the vanguard and constitute the core force of the anti-tumor immune system. Therefore, immune cells are the key to the anti-cancer battle. The real hero. A normal human body will produce hundreds of cancerous cells every day, but they are basically eliminated by immune cells before forming tumor lesions. Therefore, a high-quality immune system is the body’s own best doctor. Adoptive Cell Transfer Therapy (ACT) refers to isolating immune active cells from tumor patients, amplifying and functionally identifying them in vitro, and then infusing them back to the patient to directly kill the tumor or stimulate the body's immune response. The purpose of killing tumor cells. Adoptive immune cell therapy mainly includes TIL, LAK, CIK, DC, NK, TCR-T, CAR-T and other categories. Among them, CIK (cytokine-induced killer) is also called a killer cell induced by multiple cytokines. Because this type of cell expresses two membrane protein molecules, CD3+ and CD56+, it is also called NK cell-like T lymphocyte. It has both It has the advantages of powerful anti-tumor activity of T lymphocytes and non-MHC-restricted tumor killing of NK cells. Therefore, the application of CIK cells is considered to be the first choice for the new generation of anti-tumor adoptive cell immunotherapy.
Effector cells CD3+CD56+ cells in CIK cells are extremely rare in normal human peripheral blood, only 1% to 5%. After being cultured with multiple factors in vitro for 28 to 30 days, CD3+CD56+ cells increased rapidly. , the increase can reach more than 1000 times compared with before culture, and then infused back into the body to exert the corresponding functional activity. The activity of CIK mainly includes the following aspects:
(1) CIK cells proliferate quickly and are resistant to Tumor active cells can proliferate in large numbers, and cell activity is also greatly enhanced. (2) CIK cells have a mechanism to recognize tumors and have no toxic effects on normal cells. (3) It has a broad tumor-killing spectrum and can be used to treat leukemia, lymphoma, lung cancer, gastric cancer, intestinal cancer and other tumors. It is also sensitive to multi-drug-resistant tumor cells. (4) is a typical personalized biological treatment model. After infusion of such cells, it can also improve the body's immunity and produce specific antiviral effects, thus exerting a dual effect on tumor treatment. (5) Since CIK cells are activated autologous cells, they are very safe to use.
However, immune cells also vary in quality. As age increases, the body's immune cells will also undergo corresponding changes, and their cell viability and number will weaken with age. For example, when a person is 40-50 years old, the function (vitality and number) of immune cells is only 1/2 of what the body was at its peak. This increases the risk of human disease (such as tumors and other diseases) to a certain probability. To avoid this problem, cutting-edge biotechnology cell storage technology has emerged. This technology can store cells for a certain period of time to ensure that their functions and activities are not significantly affected by the passage of time. If high-quality immune cells from a young human body can be stored and extracted and used when needed, it will undoubtedly be of great help in fighting infections, fighting tumors, and improving one's own immunity.
Immune cell storage—your “healthy seeds”
Immune cell storage refers to the use of advanced biotechnology to separate and enrich a certain number of immune cells from human blood, and combine them The biophysical factors of cells preserve immune cells at a low temperature of -196°C, thereby maintaining the diversity and high activity potential of immune cells, keeping the cells in a dormant state, and then resuscitating and amplifying them when needed for precise Cell therapy, beauty and anti-aging and other fields.
Specifically, specific cell cryopreservation matrices are used. These matrices usually contain cryoprotectant DMSO or glycerol and serum components, and are cooled through a slow gradient through 4°C, -20°C, -80°C and finally reaching -196°C. , you can also use a commercial cell freezing box to place the cells directly into -80°C and then into liquid nitrogen. Cell recovery emphasizes speed, that is, cells in liquid nitrogen are directly placed in a water bath at 37°C. Rapid temperature rise reduces the damage caused by melting of ice crystals during cell recovery. Fast recovery through slow cryopreservation can minimize the damage to cells. degree of damage and maintain high cell activity.
The purpose of cell storage is for future use. Whether it is disease prevention, treatment or anti-aging, it has high requirements on the scientific research capabilities and storage capabilities of the institution that stores cells. Among related domestic companies, Jitao Health and the Chinese Academy of Sciences recently signed a strategic cooperation agreement on immune cell scientific research. With strong scientific research strength and professional market-oriented management, the cooperation between the two parties has formed a four-in-one full-process cell service chain in terms of cell storage, preparation and scientific research transformation. The frozen cell service of the Chinese Academy of Sciences Seed Bank has created a multi-point disaster preparedness safe for users of Jitao Biotech, which can store cells for life for users.