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    Home > Medical News > Medical World News > Science found that RNA vaccine makes claudin car T cells more effective against solid tumors

    Science found that RNA vaccine makes claudin car T cells more effective against solid tumors

    • Last Update: 2020-01-21
    • Source: Internet
    • Author: User
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    Adoptive T cell therapy with car-t cells expressing car after gene transformation has achieved clinical success in B-cell malignant tumors However, in patients with solid tumors, car-t cell therapy has encountered challenges and is not so effective A key obstacle is the limited number of cell surface targets that allow high cancer specific expression and low risk of off-tumor / on-target toxicity of tumor eradication Recently, scientists have reported the cancer-related expression of CLDN6 CLDN6 is a quadruple membrane protein involved in tight junction In a new study, to assess whether CLDN6 can be used as a target for car-t cell therapy, researchers from biopharmaceutical New Technologies Corporation (bintech) in Germany analyzed its expression in a complete set of human and mouse tissues The results showed that the level of CLDN6 transcripts was higher in human fetal stomach, lung and kidney tissues, but could not be detected in normal adult tissue samples In addition, as before, CLDN6 transcript levels are generally higher in a variety of human cancers, such as testicular, ovarian, cervical, and lung adenocarcinoma In mice, CLDN6 is widely expressed in fetal organs, but it is down regulated before delivery, which results in its lack of expression in most organs of adult mice This shows that CLDN6 is a kind of carcinoembryonic cell surface antigen in a strict sense, and has an ideal expression profile suitable for car-t cell targeting The related research results were recently published in the Journal of science The title of the paper is "an RNA vaccine drives expansion and efficiency of claudin car T cells against solid tumors" The researchers designed a cldn6-car with a 4-1BB costimulatory domain (that is, a car that recognizes and combines CLDN6) In view of this receptor domain, they designed a single chain variable region fragment (scFv), which has high specificity and binding affinity for CLDN6 in the range of nanomoles They found that the transformed cldn6-car-t cells (cldn6-car-t cells) were highly sensitive to recognize and kill the CLDN6 negative human lung cancer cell line colo699n transfected with CLDN6 RNA In addition, cldn6-car-t cells can only kill colo699n cells transfected with CLDN6 RNA, but not those transfected with cldn3, cldn4 or cldn9 RNA which have high amino acid homology with CLDN6 When cldn6-car-t cells were co cultured with CLDN6 positive human tumor cell lines, they observed the up regulation of IFN γ secretion and T cell activation markers, but this did not happen when co cultured with CLDN6 negative tumor cells Cldn6-car-t cells were also able to effectively remove the globules of CLDN6 positive PA-1 ovarian cancer The identification of PA-1 ovarian cancer globules by knockout CLDN6 with CRISPR / cas9 could be completely abolished, which further confirmed the high efficacy and target specificity of cldn6-car-t cells Next, the researchers studied the antitumor activity of cldn6-car-t cells in NSG mice transplanted with human tumor cell lines The mice were transplanted with a single dose of human cldn6-car-t cells or control cells Compared with the control group, all mice treated with cldn6-car-t cells experienced complete tumor regression within 2 weeks Within 25 days after injection, circulating cldn6-car-t cells could still be detected in the treated mice In order to evaluate the applicability of this method, the researchers chose cldn18.2, which has a distant relationship with CLDN6 Cldn18.2 is expressed in a variety of tumors with high medical needs, such as gastroesophageal cancer and pancreatic cancer In human and mice, its expression in normal tissues is limited to the tight junction of differentiated cells in gastric mucosa By replacing ScFv specific to CLDN6 with ScFv specific to cldn18.2-car, they constructed cldn18.2-car and car-t cells expressing cldn18.2-car (cldn18.2-car-t for short) Cldn18.2-car-t cells showed similar functional characteristics to cldn6-car-t cells The key to the clinical efficacy of the transplanted car-t cells lies in their colonization and persistence in vivo However, as for solid tumors, car-t cells are difficult to contact with their tumor cells, and in the immunosuppressive tumor microenvironment, car-t cells lack the proliferation signal when contacting the target cells, so the proportion of car-t cells decreased rapidly In recent years, these researchers stimulated tumor related T cells in cancer patients' natural cell bank by intravenous injection of liposomal antigen encoding RNA (RNA LPX) The nanoparticles deliver antigens to APCs in the spleen, lymph nodes and bone marrow, and initiate a toll like receptor (TLR) - dependent type I IFN driven immune activation program to promote antigen-specific T cell activation and strong proliferation In order to verify whether the improved method can play a role as car-t cell amplifying RNA vaccine (carvac), these researchers carried out a series of experiments First, they tested whether CLDN6 was shown on the surface of dendritic cells to stimulate cldn6-car-t cells in vitro They detected the dose-dependent expression of CLDN6 on the surface of dendritic cells treated with different doses of rna-lpx encoding CLDN6 The resulting expression of CLDN6 on the surface of dendritic cells induces the activation, cytokine secretion and proliferation of cldn6-car-t cells co cultured with dendritic cells in a dose-dependent manner After intravenous injection of cldn6-lpx to BALB / c mice, CLDN6 expression was detected on the surface of spleen dendritic cells and macrophages, but not on the surface of lymphocytes, which confirmed that CLDN6 was only delivered to antigen-presenting cells (such as dendritic cells and megaphagocytes) in vivo NK cells, B cells and T cells were strongly activated in spleen and lymph nodes of mice injected cldn6-lpx Then, C57BL / 6 mice were transplanted with cldn6-car-t cells labeled with cell proliferation dye and inoculated with cldn6-lpx or control rna-lpx Compared with the mice inoculated with control rna-lpx, the spleen and lymph nodes of all the main body parts of the mice inoculated with cldn6-lpx showed a significant increase in the proportion of proliferated cldn6-car-t cells, which indicated that CLDN6 antigen had a wide range of functional expression in the lymphoid compartment In order to assess the performance of carvac strategy in vivo, the researchers used cldn6-car-t cell transplantation in mice that received whole-body irradiation in advance They found that intravenous injection of a single dose of cldn6-lpx resulted in significant proliferation of circulating cldn6-car-t cells This proliferation was associated with cldn6-lpx dose levels The proportion of cldn6-car-t cells peaked 3 to 4 days after cldn6-lpx inoculation, and then decreased In another experiment, multiple groups of mice were injected with cldn6-car-t cells at different dose levels, at the beginning as low as 1000 cldn6-car-t cells were injected into each mouse, and then cldn6-lpx was inoculated or not inoculated shortly after cldn6-car-t cell transplantation In mice not inoculated with cldn6-lpx, the number of primary cldn6-car-t cells was linearly correlated with the number of injected cells, and remained stable or decreased slowly over time It should be noted that cldn6-car-t cells proliferated in mice inoculated with cldn6-lpx, which was not related to the initial dose of the injected cells The proliferation of 1000 cldn6-car-t cells mediated by cldn6-lpx results in the detectable level of cldn6-car-t cells in peripheral blood Almost all the transplanted cldn6-car-t cell populations experienced cldn6-lpx mediated activation and proliferation Compared with cldn6-car-t cells from mice not inoculated with cldn6-lpx, cldn6-car-t cells from mice inoculated with cldn6-lpx, once co cultured with CLDN6 positive tumor cells, will produce higher levels of IFN γ and show significantly higher antigen-dependent cytolytic activity In addition, mice receiving low-dose cldn6-car-t cell transplantation benefited from repeated inoculation of cldn6-lpx In mice receiving high dose of cldn6-car-t cells transplantation, the proliferation of cldn6-car-t cells in vivo remained stable after reaching a high level In order to evaluate the effect of repeated cldn6-lpx vaccination on the long-term persistence of cldn6-car-t cells, mice receiving cldn6-car-t cells transplantation were inoculated with cldn6-lpx three times a week, followed by two times of cldn6-lpx vaccination with longer treatment free interval - 4 weeks and 4.5 weeks The first cldn6-lpx vaccination resulted in the rapid proliferation of cldn6-car-t cells over two orders of magnitude, and the subsequent weekly vaccination maintained a high level of cldn6-car-t cells, resulting in their proportion in the total peripheral blood lymphocytes more than 15% Since the most prominent serious adverse event of car-t cell therapy is cytokine release syndrome (CRS), these researchers explored the possibility of increased systemic cytokine release when cldn6-car-t cells are used in combination with carvac strategy They analyzed the levels of IFN γ, IL6 and NF α in the serum of cldn6-car-t cells transplanted mice after cldn6-lpx inoculation In addition to the slight transient increase of IFN γ in the early stage, no significant increase of proinflammatory cytokines was observed In addition, the spleen of mice inoculated with cldn6-lpx single time or repeatedly did not show any obvious pathological changes in structure; at different time points after receiving cldn6-lpx repeated inoculation, the cell composition of their spleen decreased slightly in CD11c + dendritic cells and F4 / 80 + macrophage population, while in T cells, B cells and NK cell population, there was no significant pathological change There was no change in quantity Finally, in tumor bearing mice, they received a single inoculation of cldn6-lpx or control rna-lpx after receiving cldn6-car-t cell transplantation In the absence of cldn6-lpx, the control of cldn6-car-t cells on tumor is incomplete, and the growth of tumor is only delayed In contrast, of the 10 mice treated with cldn6-car-t cells and cldn6-lpx, 6 showed complete rejection of large tumors and had significantly higher median survival When the tumor bearing mice were treated with cldn18.2-car-t cells and cldn18.2-lpx, they also achieved similar results Then, in mice with xenografted tumors of CLDN6 positive ov90, they confirmed that repeated inoculation of cldn6-lpx resulted in specific proliferation of human cldn6-car-t cells transplanted into these mice Effective tumor control is related to the high proportion of cldn6-car-t cells in the peripheral blood, which proves that cldn6-lpx can cause cldn6-car-t cells to proliferate efficiently and persist better in vivo In mice xenotransplantation tumor model, cldn18.2-car-t cells and cldn18.2-lpx combined treatment can achieve similar results These findings suggest that carvac can be used to improve the antitumor effect of car-t cells This provides a new strategy for the use of car-t cells in the treatment of solid tumors that are difficult to treat However, these results are achieved in preclinical models, and whether they are the same in human body remains to be verified by further research ginseng
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