Anti-CD1a CAR-T cells(The Affiliated Hospital of Xuzhou Medical University)

One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues.

EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC.

EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3ζ endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice.

These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.

Gastric cancer is a deadly malignancy and is a prognostically unfavorable entity with restricted therapeutic strategies available. Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein widely expressed in bladder, prostate, and pancreatic cancers. Existing studies have thoroughly recognized the availability of utilizing anti-PSCA CAR-T cells in the treatment of metastatic prostate cancer and non-small-cell lung cancer. However, no previous study has investigated the feasibility of using anti-PSCA CAR-T cells to treat gastric cancer, irrespective of the proven expression of PSCA on the gastric cancer cell surface.

We determined the expression of PSCA in several primary tumor tissues and constructed third-generation anti-PSCA CAR-T cells. We then incubated anti-PSCA CAR-T cells and GFP-T cells with target tumor cell lines at E:T ratios of 2:1, 1:1, 1:2, and 1:4 to evaluate the therapeutic efficacy of anti-PSCA CAR-T cells in vitro. We also assayed canonical T cell activation markers after coculturing anti-PSCA CAR-T cells with target cell lines by flow cytometry. The detection of a functional cytokine profile was carried out via enzyme-linked immunosorbent assays. We then evaluated the antitumor activity of anti-PSCA CAR-T cells in vivo by establishing two different xenograft GC mouse models.

Anti-PSCA CAR-T cells exhibited upregulated activation markers and increased cytokine production profiles related to T cell cytotoxicity in an antigen-dependent manner. Moreover, anti-PSCA CAR-T cells exhibited robust anti-tumor cytotoxicity in vitro. Importantly, we demonstrated that anti-PSCA CAR-T cells delivered by peritumoral injection successfully stunted tumor progression in vivo. However, intravenous administration of anti-PSCA CAR-T cells failed to reveal any therapeutic improvements.

Our findings corroborated the feasibility of anti-PSCA CAR-T cells and their efficacy against gastric cancer, implicating the potential of applying anti-PSCA CAR-T cells to treat GC patients in the clinic.