# Intein‐based modular chimeric antigen receptor platform for specific CD19/CD20 co‐targeting

## TL;DR

Researchers developed a new CAR platform called CARtein that targets two B-cell antigens, CD19 and CD20, to improve cancer therapy and reduce tumor escape.

## Contribution

The novel CARtein platform uses intein-based modular assembly to enable co-targeting of CD19 and CD20 with a universal signaling backbone.

## Key findings

- CARtein generates fully active CARs by covalently binding scFv-intein recognition partners to a universal signaling backbone.
- T cell activation was observed in Raji and K562 cells expressing CD19 and/or CD20, with NFAT and NFκB promoter activity and CD69 upregulation.
- The modular design supports flexible multi-antigen targeting to reduce relapse from antigen escape in CAR-T therapy.

## Abstract

Development of chimeric antigen receptor T‐cell therapy has revolutionized the treatment of B‐cell malignancies, although challenges such as antigen escape and tumor heterogeneity often decrease treatment success. Modular CARs targeting multiple antigens have been proposed as an interesting solution to address these challenges by reducing the likelihood of tumor cells evading treatment through the loss of a single antigen. In this study, we present a new modular CAR platform, termed CARtein, which takes advantage of intein interactions to jointly target CD19 and CD20 antigens. We demonstrate that the CARtein system, which features a universal CAR signaling backbone that covalently binds to specific scFv‐intein recognition partners, generates fully active CARs. Functionality was validated using Raji cells and K562 cells expressing CD19 and/or CD20, observing significant T cell activation through NFAT and NFκB promoter activity and CD69 upregulation. Overall, our study lays the foundation for the establishment of a new way to target multiple antigens through a universal and inert CAR backbone with highly specific activation.

CARtein is a modular CAR platform that uses split inteins to splice antigen‐recognition modules onto a universal signaling backbone, enabling precise, scarless assembly without re‐engineering signaling domains. Deployed here against CD19 and CD20 in B‐cell malignancies, the design supports flexible multi‐antigen targeting to boost T‐cell activation and reduce relapse driven by antigen escape, a common failure mode in CAR‐T therapy.