Engineering chimeric antigen receptor (CAR) T cells with novel costimulatory domains

Abstract

CD19⁺ B cell malignancies represent a substantial proportion of haematological cancers. While chimeric antigen receptor (CAR) T cell therapy has revolutionised treatment, durable efficacy remains limited by relapse, toxicity, and insufficient long-term persistence. These limitations are tightly linked to CAR design, particularly the intracellular costimulatory domain (CSD), which governs activation dynamics, differentiation, survival, and metabolic fitness.

In this study, CD19-specific CARs incorporating distinct CSDs were engineered to systematically define how alternative signalling architectures shape CAR T cell fate. Constructs included a CD3ζ-only first-generation CAR, a clinically established 4-1BB CAR, and novel CARs containing TNFR2 orDNAM-1 signalling domains. Early activation was assessed in Jurkat T cells, while primary human CAR T cells were evaluated under prolonged antigen stimulation for differentiation, exhaustion, and metabolic adaptation. In vivo function was examined using a Raji xenograft model and aCD34⁺ haematopoietic stem progenitor cell (HSPC)-derived humanised mouse model.

TNFR2- and 4-1BB-based CAR T cells exhibited enhanced activation compared with CD3ζ-only andDNAM-1 CARs. While 4-1BB CAR T cells demonstrated strong early functional responses, TNFR2CAR T cells showed features consistent with improved long-term persistence, reduced exhaustion, and sustained metabolic fitness under chronic antigen exposure. In vivo, TNFR2-based CAR T cells mediated more durable tumour control in the Raji xenograft model and, together with DNAM-1CARs, significantly improved survival compared to 4-1BB and CD3ζ-only CAR T cells. TNFR2 CAR T cells exhibited prolonged peripheral persistence and favourable memory differentiation. In humanised mice, CARs incorporating TNFR2, 4-1BB, or DNAM-1 induced sustained CD19⁺ B cell depletion, with 4-1BB exhibiting faster kinetics and DNAM-1 and TNFR2 supporting slower but durable activity.

Collectively, these findings establish CSD architecture as a central determinant of CAR T cell durability and demonstrate that incorporation of TNFR2 signalling is associated with enhanced metabolic resilience, reduced exhaustion, and sustained antitumour activity, highlighting TNFR2as a promising alternative costimulatory domain for next-generation CAR T cell therapies targeting relapsed or refractory CD19⁺ B cell malignancies.

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Identifiers

ORCID for Hatice Nurdan Aksoy Kilinc: orcid.org/0000-0002-4081-977X

ORCID for Ali Roghanian: orcid.org/0000-0003-1316-4218

ORCID for Aymen Al-Shamkhani: orcid.org/0000-0003-0727-4189

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