# Glypican 3-targeted chimeric antigen receptor T cells secreting TROP2-directed bispecific T cell engagers exhibit potent efficacy against lung squamous cell carcinoma

## TL;DR

A new CAR-T therapy targeting GPC3 and TROP2 shows strong effectiveness against lung squamous cell carcinoma in mice.

## Contribution

A novel dual-targeted CAR-T therapy combining GPC3 CAR-T and TROP2 BiTE is developed to overcome antigen heterogeneity in solid tumors.

## Key findings

- GPC3 CAR-T. TROP2 BiTE showed cytotoxicity against both GPC3+ and TROP2+ LUSC cells.
- The therapy exhibited faster killing and durable antitumor effects in heterogeneous LUSC cells.
- In mice, GPC3 CAR-T. TROP2 BiTE effectively eliminated tumors.

## Abstract

Chimeric antigen receptor T cell (CAR-T) therapy faces multiple challenges in solid tumors, especially the heterogeneity of tumor antigens. Glypican-3 (GPC3) and trophoblast cell-surface antigen 2 (TROP2) are highly expressed antigens in lung squamous cell carcinoma (LUSC) for development of dual-targeted therapy. The absence of GPC3 in any normal tissues of adults makes it an ideal target for CAR-T therapy. However, TROP2 is expressed in the epithelial cells of various normal tissues and thus is not acceptable for direct design of CAR-T therapy due to the high risk of “on-target off-tumor” effects. Here we developed a dual-targeted LUSC therapy featuring a GPC3-targeted CAR-T cell secreting TROP2-directed bispecific T cell engagers (GPC3 CAR-T. TROP2 BiTE), and verified the antitumor activity in vitro and in vivo, respectively.

Immunohistochemistry (IHC) was used to confirm the expression of GPC3 and TROP2 in LUSC and normal tissues. CAR-T cells were produced through lentiviral transduction of CAR genes. Real-time cytotoxicity assay (RTCA) was used to assess the cytotoxic effect of CAR-T cells on LUSC cells. Flow cytometry was utilized to examine the CAR-T cell phenotype, exhaustion and activation. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the release of cytokines. To evaluate the activity of CAR-T cells in vivo, tumor-bearing immunodeficient mice were given a single intravenous injection of CAR-T cells, and the tumor burden and CAR-T cell expansion were regularly monitored.

GPC3 was overexpressed in 70% of LUSC tissues, while negatively expressed in all normal tissues. Positive expression of TROP2 was observed in all LUSC tissues and also in many normal tissues. Compared with GPC3 CAR-T and TROP2 CAR-T, GPC3 CAR-T. TROP2 BiTE exhibited cytotoxicity to both GPC3+ and TROP2+ LUSC cells, and thereby showed faster killing and durable antitumor effect against LUSC cells with heterogenous expression of GPC3 and TROP2. In tumor-bearing mice, GPC3 CAR-T. TROP2 BiTE showed strong ability to eliminate tumors.

This study demonstrated that GPC3 CAR-T. TROP2 BiTE was a potent therapy for LUSC and provided a strategy for overcoming the antigen heterogeneity in solid tumors.