Abstract A018: Functional, transcriptomic and genomic characterisation of CD19-directed CAR T-cells incorporating a TLR2 co-stimulatory domain

Abstract Second-generation anti-CD19 chimeric antigen receptor (CAR) T-cells incorporating CD28 (1928z) are now standard of care for treatment of refractory/relapsed large B-cell and mantle cell lymphomas. However, clinical use is limited by high toxicity rates, especially cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Modifying intracellular signalling domains can influence CAR T-cell toxicity risk. Although rare, reports of CAR T-cell-derived malignancies underscore the risks of gene-modified cell therapies and the need to assess transgene insertion sites in new products. Toll-like receptor 2 (TLR2) a pattern recognition receptor, is highly expressed by activated CD8+ human T-cells, and can serve as a T-cell costimulatory signal, enhancing, memory formation, and modifying T-cell cytokine profile. In support of a phase 1 clinical trial (ENABLE-1, NCT04049513) evaluating a new third-generation anti-CD19 CAR T-cell product incorporating CD28 and TLR2 co-stimulatory domains (1928T2z), we explored the functional impact of the TLR2 domain in vitro, and characterised gene insertion sites within the 1928T2z product. Incorporating TLR2-derived Toll/IL-1 receptor resistance domain (TIR) between CD28 and CD3ζ domains in the 1928T2z CAR provided comparable CAR T-cell efficacy while producing lower levels of IFN-γ and GM-CSF, cytokines linked to CRS and ICANS pathogenesis, than 1928z. In contrast, positioning the TLR2 domain at the cytoplasmic tail (1928zT2) did not lower IFN-γ and GM-CSF production. Inhibiting TLR2 TIR function using ortho-vanillin, or through targeted mutations abrogating MYD88 recruitment and/or dimerization, impaired CAR T-cell activity, indicating the TLR2 domain contributes to 1928T2z CAR function. Gene expression identified downregulation of IL-6 signalling and upregulation of an anti-inflammatory cytokine pathway in 1928T2z cells compared to comparator CAR T-cells. A phosphoprotein array revealed that a functional TLR2 domain was associated with in higher levels of phosphorylated MEK1/2 (involved in TLR signalling), GATA3 (Th2-linked transcription factor) and CREB (a transcription factor that limits inflammatory responses). Using long-read sequencing and bioinformatics, we characterised insertion sites in a clonal line and polyclonal human CAR T-cells. Results showed mostly intronic or intergenic sites, with less than 10% mapping to exons, aligning with known lentiviral transgene integration profiles. In summary, incorporating a TLR2 co-stimulatory domain into CD28-costimulated anti-CD19 CAR T-cells modulates cytokine profile in a position-dependent manner, with TLR2 placement between CD28 and CD3ζ reducing CRS-and ICANS-linked cytokines while preserving in vitro anti-tumour activity. These findings align with our phase 1 trial data suggesting that 1928T2z CAR T-cells are associated with low incidence of CRS and ICANS, while exhibiting similar clinical efficacy to commercial comparators. Citation Format: Nathaniel Dasyam, Yasmin Nouri, Joshua Halpin, Alexander McLellan, David Eccles, Simon Fink, Felix Schaefer-ruoff, Markus Templin, Qin Le, Peng Li, Ian Hermans, Rachel Perret, Robert Weinkove. Functional, transcriptomic and genomic characterisation of CD19-directed CAR T-cells incorporating a TLR2 co-stimulatory domain [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A018.