RNA疫苗,CAR-T细胞 z-bo 2020-09-30 313

　　Genetically modified T cells (CAR-T) expressing chimeric antigen receptors (CAR) for adoptive T cell therapy have achieved clinical success in B cell malignancies. However, in patients with solid tumors, CAR-T cell therapy faces challenges and the effect is not very good. Important diseases are a limited number of cancer-specific high expression and low tumor/targeted toxicity (extra-tumor/targeted toxicity) cell surface targets, which can effectively eradicate tumors.

　　Scientists recently reported the expression of cludin6 (CLDN6), which is related to cancer. CLDN6 is a 4-transmembrane protein involved in the formation of tight junctions. In a new study, researchers from BioNTech (BioNTech) analyzed a set of human and mouse tissues to assess whether CLDN6 can be used as a target for CAR-T cell therapy. That expression. The analysis results showed that in humans, the transcript levels of CLDN6 in fetal stomach, lung, and kidney tissues are higher, but not in normal adult tissue samples. In addition, consistent with previous levels, CLDN6 transcription levels are generally elevated in various human cancers, including testicular cancer, ovarian cancer, cervical adenocarcinoma, and lung adenocarcinoma. In mice, CLDN6 is widely expressed in fetal organs, but it is not expressed in most adult mouse organs because it is down-regulated before delivery. Strictly speaking, this indicates that CLDN6 is a carcinoembryonic cell surface antigen and has an ideal expression profile suitable for CAR-T cell targeting.

　　These researchers designed CLDN6-CAR using the 4-1BB costimulatory domain (the CAR that recognizes and binds to CLDN6). For this receptor domain, they designed a single-chain variable fragment (scFv) with high specificity and binding affinity for CLDN6 in the nanomolar range. They found that after genetic modification, CLDN6-CAR-expressing T cells (CLDN6-CAR-T cells) can sensitively recognize and kill CLDN6-negative human lung cancer cells COLO699N transfected with CLDN6NA. In addition, CLDN6-CAR-T cells can only kill COLO699N cells transfected with CLDN6NA, but they can be killed by COLO699N cells transfected with CLDN3, CLDN4 or CLDN9NA, the latter having higher amino acid homology with CLDN6. You cannot kill the cell. When CLDN6-CAR-T cells were co-cultured with CLDN6-positive human tumor cell lines, the secretion of interferon gamma (IFN gamma) and the up-regulation of T cell activation markers were observed, but tumor cells that were CLDN6-negative were observed. This does not happen when co-cultivating with it. CLDN6-CAR-T cells can also effectively eliminate CLDN6-positive PA-1 ovarian cancer spheroids. CRISPR/Cas9 knocking out CLDN6 can completely disable the recognition of PA-1 ovarian cancer spheres by CLDN6-CAR-T cells. This further confirms the high efficiency and target specificity of CLDN6-CAR-T cells. Next, these researchers studied the anti-tumor activity of CLDN6-CAR-T cells in NSG mice xenotransplanted subcutaneously with human tumor cell lines. These mice received a single dose of human CLDN6-CAR-T cell or control cell transplantation. Compared with control mice with rapidly progressing disease, 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 can be detected in the treated mice. In order to evaluate the wide application of this method, these researchers chose CLDN 18.2, which is relatively far from CLDN 6. CLDN18.2 is expressed in various tumors with high medical needs (such as gastroesophageal cancer and pancreatic cancer). In humans and mice, its expression in normal tissues is limited to the tight junctions of differentiated cells in the gastric mucosa. They replaced CLDN6 specific scFv with CLDN18.2 specific scFv to construct CAR-T cells (called CLDN18.2-CAR-T) expressing CLDN18.2-CAR and CLDN18.2-CAR.

　　CLDN18.2-CAR-T cells show similar functional properties to CLDN6-CAR-T cells. The colonization and persistence of transplanted CAR-T cells in the body are the key to its clinical efficacy. However, in the case of solid tumors, CAR-T cells are difficult to reach tumor cells, and in the immunosuppressive tumor microenvironment, CAR-T cells lack growth signals when they contact target cells. Therefore, CAR-T cells are derived from T cells. cause. This percentage is rapidly declining. In recent years, these researchers have used liposomes (RNA encoding liposomal antigens, RNA-LPX) for intravenous injections, which carry RNA encoding antigens to produce tumor-associated T in the natural cell bank of cancer patients. cell. I am excited. This nano-scale vaccine delivers antigens to antigen-presenting cells (APCs) in the spleen, lymph nodes and bone marrow, and at the same time initiates a type I IFN-driven immune activation program that relies on high image receptor (TLR). Promote antigen-specific T. The cells are activated and proliferate strongly. These researchers conducted a series of experiments to verify whether this improved method can be used as an RNA vaccine to enhance CAR-T cells (RNA vaccines that expand CAR-T cells, called CARVac). First, they tested whether CLDN6 appeared on the surface of dendritic cells and stimulated CLDN6-CAR-T cells in vitro. They tested the dose-dependent expression of CLDN6 on the surface of dendritic cells treated with various doses of RNA-LPX encoding CLDN6 (CLDN6-LPX for short). The expression of CLDN6 obtained on the surface of dendritic cells induces the activation, secretion and proliferation of cytokines of CLDN6-CAR-T cells co-cultured with dendritic cells in a dose-dependent manner. After intravenous injection of CLDN6-LPX into BALB/c mice, CLDN6 expression can be detected on the surface of splenic dendritic cells and macrophages, but not on the surface of lymphocytes. This confirms that CLDN6 is unique in the body. Delivery to antigen-presenting cells (such as dendritic cells and macrophages). As the antigen-presenting cells matured, strong activation of natural killer cells (NK cells), B cells and T cells was detected in the spleen and lymph nodes of mice injected with CLDN6-LPX intravenously. Next, C57BL/6 mice were transplanted with CLDN6-CAR-T cells labeled with a cell proliferation dye and inoculated with CLDN6-LPX or control RNA-LPX. Compared with mice vaccinated with the control RNA-LPX vaccine, the spleen and lymph nodes of all major body parts of the mice vaccinated with the CLDN6-LPX vaccine had a certain proportion of CLDN6-CAR-T cell proliferation. It shows a significant increase. This indicates that the CLDN6 antigen is in the lymphatic compartment. Use various function expressions. In order to evaluate the performance of the CARVac strategy in vivo, these researchers allowed CLDN6-CAR-T cell transplantation in mice receiving whole body irradiation. They found that a single dose of CLDN6-LPX may cause a large number of circulating CLDN6-CAR-T cells to proliferate. This increase is related to the dose level of CLDN6-LPX. 3-4 days after CLDN6-LPX inoculation, the proportion of CLDN6-CAR-T cells reached a peak and then decreased. In another experiment, starting with 1,000 CLDN6-CAR-T cells and CLDN6-CAR- per mouse, multiple groups of mice were injected with different dose levels of CLDN6-CAR-T cells. .. Immediately after T cell transplantation, inoculate or not inoculate CLDN6-LPX. In mice not vaccinated with CLDN6-LPX, the colonization of the main CLDN6-CAR-T cells is linear with the number of injected cells, and remains stable or slow over time. And drop. It is worth noting that CLDN6-CAR-T cells are proliferated in mice vaccinated with CLDN6-LPX, and this proliferation is independent of the initial cell dose injected. The proliferation of 1000 CLDN6-CAR-T cells mediated by CLDN6-LPX resulted in a detectable level of CLDN6-CAR-T cells in peripheral blood. Almost all transplanted CLDN6-CAR-T cell populations undergo CLDN6-LPX-mediated activation and proliferation. Compared with CLDN6-CAR-T cells in mice not inoculated with CLDN6-LPX, CLDN6-CAR-T cells in mice inoculated with CLDN6-LPX were higher when co-cultured with CLDN6-positive tumor cells. A checkpoint was created. IFNγ levels showed significantly higher antigen-dependent cytolytic activity. In addition, mice receiving low-dose CLDN6-CAR-T cell transplantation also benefited from repeated CLDN6-LPX vaccination. In mice that received high-dose CLDN6-CAR-T cell transplantation, after reaching a high level, the proliferation of CLDN6-CAR-T cells in the body remained stable. To evaluate the repeat vaccination pair of CLDN6-LPX

　　"Influence on the long-term persistence of CLDN6-CAR-T cells, CLDN6-LPX is inoculated to mice undergoing CLDN6-CAR-T cell transplantation every week. Then add two longer doses. No treatment interval-4 and 4.5 weeks-CLDN6-LPX vaccination. The initial CLDN6-LPX vaccination will cause the rapid proliferation of CLDN6-CAR-T cells to more than double digits, and the subsequent weekly vaccination will maintain a high level of CLDN6-CAR-T cells, resulting in increased peripheral blood lymphocytes. 15%.

　　Given that the most prominent and most serious adverse event of CAR-T cell therapy is cytokine release syndrome (CRS), these researchers combined CLDN6-CAR-T cells with CARVac strategies. When we did this, we might have investigated the increase in systemic cytokine release. They analyzed the serum IFNγ, IL6 and NFα levels of mice transplanted with CLDN6-CAR-T cells after CLDN6-LPX. Except for the mild and transient increase in IFNγ in the early stages, no significant increase in the tested pro-inflammatory cytokines was observed. In addition, the spleens of mice that received single or repeated CLDN6-LPX vaccination had no obvious pathological changes; at different times after the mice received CLDN6-LPX repeated vaccination. The composition of their spleen CD11c + dendritic cells and F4/80 + macrophages showed a temporary decrease, but the number of T cells, B cells, and NK cells did not change. Finally, mice with cancer received a CLDN6-LPX or control RNA-LPX vaccination after mouse CLDN6-CAR-T cell transplantation. Without CLDN6-LPX, the control of tumors by CLDN6-CAR-T cells is incomplete and will only slow down the growth of tumors. In contrast, among the 10 mice that received the combination of CLDN6-CAR-T cells and CLDN6-LPX, 6 showed complete rejection of larger tumors, and the median survival time was significantly higher. When CLDN18.2-CAR-T cells and CLDN18.2-LPX are used in combination to treat tumor-bearing mice, similar treatment results can be obtained. Next, in mouse models with CLDN6-positive OV90 xenograft tumors, repeated CLDN6-LPX vaccination may lead to the specific proliferation of human CLDN6-CAR-T cells transplanted into these mice. I confirmed it. Effective tumor control is associated with a high proportion of CLDN6-CAR-T cells in the peripheral blood, and CLDN6-LPX vaccination can cause CLDN6-CAR-T cells to proliferate more effectively in the body. We have confirmed that it will bring good sustainability. In a mouse xenograft tumor model, the combination of CLDN18.2-CAR-T cells and CLDN18.2-LPX can achieve similar therapeutic effects. These findings indicate that CARVac can be used to improve the anti-tumor effect of CAR-ΔT cells. This provides a new strategy for using CAR-T cells to treat refractory solid tumors. However, these results were obtained in preclinical models, and whether they are the same as humans needs further research to verify.