Adoptive T cell therapy has achieved clinical success in B cell malignant tumors. However, CAR-T cell therapy is challenged and less effective in patients with solid tumors. A key obstacle is the limited number of cell surface targets that allow the efficient eradication of tumors with cancer-specific high expression and low off-target toxicity risk.
-
Introduction of research
Recently, scientists have reported the cancer-related expression of claudin 6 (CLDN6). CLDN6 is a four-transmembrane protein involved in the formation of tight junction. In a new study, to assess whether CLDN6 can be used as a target for CAR-T cell therapy, the researchers analyzed its expression in a complete set of human and mouse tissues. In a new study, to assess whether CLDN6 can be used as a target for CAR-T cell therapy, the researchers analyzed its expression in a complete set of human and mouse tissues. In addition, consistent with previous studies, CLDN6 transcript levels are generally high in a variety of human cancers such as testicular, ovarian, cervical and lung cancers. In mice, CLDN6 is widely expressed in fetal organs, but down-regulated in prenatal, resulting in a lack of expression in most organs of adult mice. This indicates that CLDN6 is a strict carcinoembryonic cell surface antigen and has an ideal expression profile suitable for the targeting of CAR-T cells. The results of the study were recently published in the journal Science, and the paper is entitled “An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors”.
These researchers designed a CLDN6-CAR with a 4-1BB costimulatory domain (that is, CAR that recognizes and binds to CLDN6). Aiming at this receptor domain, they designed a single-stranded variable region fragment (scFv), which has high specificity and binding affinity for CLDN6 in the range of nano-moles. They found that genetically modified T cells expressing CLDN6-CAR could highly sensitively recognize and kill CLDN6 RNA-transfected CLDN6-negative human lung cancer cell line COLO699N. In addition, CLDN6-CAR-T cells could only kill COLO699N cells transfected with CLDN6 RNA, but could not kill CLDN3, CLDN4 or CLDN9 RNA transfected COLO699N cells which had high amino acid homology with CLDN6.
When CLDN6-CAR-T cells were co-cultured with CLDN6-positive human tumor cell lines, they observed an up-regulation of interferon-gamma (IFN-γ) secretion and T cell activation markers, but this did not occur when co-cultured with CLDN6-negative tumor cells. CLDN6-CAR-T cells can also efficiently remove CLDN6-positive PA-1 ovarian cancer globules. Knockout of CLDN6 by CRISPR/Cas9 can completely abolish the recognition of PA-1 ovarian cancer globules by CLDN6-CAR-T cells, which further confirms the high potency and target specificity of CLDN6-CAR-T cells.
Next, the researchers studied the anti-tumor activity of CLDN6-CAR-T cells in NSG mice subcutaneously xenotransplanted with human tumor cell lines. These mice were transplanted with a single dose of human CLDN6-CAR-T cells or control cells. Compared with the control group with rapid progression of the 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 could still be detected in treated mice.
In order to assess the wider applicability of this method, the researchers chose CLDN18.2, which is closely related to CLDN6. CLDN18.2 is expressed in a variety of tumors with high medical needs, such as gastroesophageal and pancreatic cancer. In humans and mice, its expression in normal tissues is limited to the tight junction of differentiated cells in the gastric mucosa. They constructed CLDN18.2-CAR and CLDN18.2-CAR-expressing CAR-T cells (referred to as CLDN18.2-CAR-T) by replacing CLDN6-specific scFv with CLDN18.2-specific scFv. The functional characteristics of CLDN18.2-CAR-T cells were similar to those of CLDN6-CAR-T cells.
The colonization and persistence of transplanted CAR-T cells in vivo is the key to their clinical efficacy. However, as far as solid tumor is concerned, it is difficult for CAR-T cells to contact its tumor cells, and in the immunosuppressive tumor microenvironment, CAR-T cells lack proliferation signals when they come into contact with target cells, so the proportion of CAR-T cells decreases rapidly.
In recent years, these researchers have stimulated tumor-associated T cells in cancer patients’ natural cell banks by intravenous injection of liposomes (liposomal antigen-encoding RNA, RNA-LPX) that carry antigens encoding RNA. The nanoparticle vaccine delivers antigens to antigen-presenting cells (APC), in spleen, lymph nodes and bone marrow and initiates type I IFN-driven immune activation procedures dependent on Toll-like receptor (TLR), thus promoting the activation and strong proliferation of antigen-specific T cells.
In order to verify whether this improved method can be used as a CAR-T cell enhanced RNA vaccine (CAR-T cell Amplifying RNA Vaccine, referred to as CARVac), these researchers carried out a series of experiments. First, they tested whether CLDN6 was displayed on the surface of dendritic cells to stimulate CLDN6-CAR-T cells in vitro. They detected dose-dependent expression of CLDN6 on the surface of dendritic cells treated with different doses of CLDN6-coding RNA-LPX (CLDN6-LPX). The resulting CLDN6 expression on the surface of dendritic cells induced 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 into BALB/c mice, CLDN6 expression could be detected on the surface of splenic dendritic cells and macrophages, but not on the surface of lymphocytes, which confirmed that CLDN6 uniquely delivered antigen-presenting cells (such as dendritic cells and macrophages) in vivo. Antigen-presenting cells experienced maturation, and natural killer cells (NK cells), B cells and T cells were strongly activated in the spleen and lymph nodes of mice injected with CLDN6-LPX.
Compared with the mice inoculated with RNA-LPX, the spleen and lymph nodes of all major body parts of mice inoculated with CLDN6-LPX showed a significant increase in the proportion of proliferated CLDN6-CAR-T cells, indicating that CLDN6 antigen is widely expressed in the lymphatic compartment.
To assess the performance of the CARVac strategy in vivo, the researchers transplanted CLDN6-CAR-T cells into mice that had received whole-body irradiation in advance. They found that intravenous injection of a single dose of CLDN6-LPX could lead to significant proliferation of circulating CLDN6-CAR-T cells. This proliferation is related to the dose level of CLDN6-LPX. The proportion of CLDN6-CAR-T cells peaked 3 to 4 days after CLDN6-LPX inoculation, and then decreased.
-
Other support
In another experiment, multiple groups of mice were injected with different doses of CLDN6-CAR-T cells, initially as low as 1000 CLDN6-CAR-T cells per mouse, followed by CLDN6-LPX or no CLDN6-LPX shortly after CLDN6-CAR-T cell transplantation. In mice not vaccinated with CLDN6-LPX, the colonization of major CLDN6-CAR-T cells was linearly related to the number of cells injected and remained stable or decreased slowly over time. It is worth noting that CLDN6-CAR-T cells proliferated in mice inoculated with CLDN6-LPX, which was independent of the initial dose of cells injected. The proliferation of 1000 CLDN6-CAR-T cells mediated by CLDN6-LPX leads to the presence of detectable 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 from non-CLDN6-LPX-inoculated mice, CLDN6-CAR-T cells from CLDN6-LPX-inoculated mice produced higher levels of IFN γ and significantly higher antigen-dependent cytolytic activity when co-cultured with CLDN6-positive tumor cells. In addition, mice that received low-dose CLDN6-CAR-T cell transplantation benefited from repeated CLDN6-LPX vaccinations. In mice transplanted with high-dose CLDN6-CAR-T cells, the proliferation of CLDN6-CAR-T cells remained stable after reaching a high level in vivo.
To evaluate the effect of repeated CLDN6-LPX inoculation on the long-term persistence of CLDN6-CAR-T cells, mice that received CLDN6-CAR-T cell transplantation were inoculated with three weekly doses of CLDN6-LPX, followed by two CLDN6-LPX inoculations with longer untreated intervals (treatment-free interval)-4 weeks and 4. 5 weeks. The first CLDN6-LPX inoculation led to the rapid proliferation of CLDN6-CAR-T cells by more than two orders of magnitude, followed by weekly inoculation to maintain a high level of CLDN6-CAR-T cells, resulting in more than 15% of total peripheral blood lymphocytes.
Given that 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 were used in combination with CARVac strategies. They analyzed serum levels of IFN γ, IL6 and NF α in mice that received CLDN6-CAR-T cell transplantation after inoculation with CLDN6-LPX. Except for the slight transient increase of IFN γ in the early stage, no significant increase in proinflammatory cytokines was observed. In addition, the spleen of mice inoculated with single or repeated CLDN6-LPX did not show any obvious pathological changes; at different time points after repeated inoculation with CLDN6-LPX, the composition of spleen cells decreased slightly in CD11c+ dendritic cells and F4/80+ macrophages, while there was no quantitative change in T cells, B cells and NK cells.
Finally, in tumor-bearing mice, after receiving mouse CLDN6-CAR-T cell transplantation, they were inoculated with a single dose of CLDN6-LPX or control RNA-LPX. In the absence of CLDN6-LPX inoculation, the control of CLDN6-CAR-T cells on the tumor is incomplete, and the tumor growth is only delayed. In contrast, of the 10 mice treated with CLDN6-CAR-T cells combined with CLDN6-LPX, 6 showed complete rejection of larger tumors and had a significantly longer median survival. When tumor-bearing mice were treated with CLDN18.2-CAR-T cells and CLDN18.2-LPX, they achieved similar results.
Then, in a mouse model with CLDN6-positive OV90 xenografts, they confirmed that repeated inoculation of CLDN6-LPX could lead to specific proliferation of human CLDN6-CAR-T cells transplanted into these mice. Effective tumor control is associated with a higher proportion of CLDN6-CAR-T cells in peripheral blood, thus confirming that inoculation of CLDN6-LPX can lead to efficient proliferation and better persistence of CLDN6-CAR-T cells in vivo. In the mouse xenograft tumor model, the combination of CLDN18.2-CAR-T cells and CLDN18.2-LPX can achieve similar results.
These findings suggest that CARVac can be used to improve the anti-tumor effect of CAR-T cells. This provides a new strategy for using CAR-T cells to treat refractory solid tumors. However, these results are achieved in preclinical models, and whether they are the same in humans remains to be verified by further research.