# c-JUN enhances CRISPR knockin anti-B7-H3 CAR T cell function in small cell lung cancer and thoracic SMARCA4-deficient undifferentiated tumors

## TL;DR

Researchers improved CAR T cell therapy for aggressive lung cancers by combining a new target (B7-H3) with a genetic modification (c-JUN) to overcome tumor resistance.

## Contribution

A novel CRISPR-based CAR T cell design combining B7-H3 targeting and c-JUN expression to enhance anti-tumor activity in SCLC and SMARCA4-deficient tumors.

## Key findings

- B7-H3 is a viable CAR T cell target in SCLC and thoracic SMARCA4-deficient undifferentiated tumors.
- c-JUN enhances CAR T cell function by inducing cytokines and overcoming TGF-β1 suppression.
- Non-viral CRISPR knockin of c-JUN+B7-H3 CAR is feasible at clinical scale.

## Abstract

Small cell lung cancer (SCLC), a highly lethal disease, limits T cell responses by downregulating major histocompatibility (MHC) class I molecules. Because chimeric antigen receptor (CAR) T cells are not MHC restricted, they may provide a powerful strategy against SCLC. However, few CAR targets for SCLC are known. Here, we show that B7-H3/CD276 is expressed in SCLC and thoracic SMARCA4-deficient undifferentiated tumors (UTs) that can clinicopathologically mimic SCLC. Thoracic SMARCA4-deficient UTs limit killing by B7-H3 CAR T cells via secretion of transforming growth factor β1 (TGF-β1). To overcome tumor-driven CAR T cell suppression, we knock in c-JUN alongside a B7-H3 CAR into the TRAC locus of primary human T cells utilizing CRISPR-Cas9. Non-viral c-JUN+B7-H3 CAR T cells show enhanced killing of both SCLC cells with low antigen density and thoracic SMARCA4-deficient UTs, providing a platform to address these highly aggressive entities. We also provide evidence that good manufacturing practice (GMP) clinical-scale manufacturing is feasible for c-JUN+B7-H3 CAR T cells.

•B7-H3 can be targeted in SCLC and thoracic SMARCA4-deficient UTs with CAR T cells•TGF-β1 drives resistance in thoracic SMARCA4-deficient UTs•c-JUN enhances killing of B7-H3 CAR T cells by inducing type I/II cytokines•CRISPR knockin of c-JUN+B7-H3 CAR (3.6 kb) is feasible at the clinical scale

B7-H3 can be targeted in SCLC and thoracic SMARCA4-deficient UTs with CAR T cells

TGF-β1 drives resistance in thoracic SMARCA4-deficient UTs

c-JUN enhances killing of B7-H3 CAR T cells by inducing type I/II cytokines

CRISPR knockin of c-JUN+B7-H3 CAR (3.6 kb) is feasible at the clinical scale

Balke-Want et al. show that B7-H3 is an actionable CAR target in SCLC and thoracic SMARCA4-deficient UTs. However, in thoracic SMARCA4-deficient UTs, TGF-β1 suppresses their activity. By engineering CRISPR knockin c-JUN+B7-H3 CAR T cells with combined expression of type I/II cytokines, the authors overcome TGF-β1-mediated suppression.