Signaling role of viral protein motif and its application in CAR T cell therapy

Abstract

Abstract CD19-targeted Chimeric Antigen Receptor (CAR) T cell therapy is a leading treatment for B-cell malignancies, achieving complete remission rates of ~50% in pediatric non-Hodgkin's lymphoma. However, relapse remains a major barrier, driven by immune evasion, T-cell exhaustion and/or dysfunction, and limited persistence. These challenges highlight the need for innovative CAR vector designs to improve durability and efficacy. One strategy is to enhance recruitment of the T cell kinase LCK to the immune synapse, thereby strengthening proximal signaling (Feucht et al., 2019; Hartl et al., 2020; James, 2018). This compensates for the absence of co-receptor–MHC interactions in native T cell receptor (TCR) signaling, which position LCK near the immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3ζ. Optimizing LCK-mediated ITAM phosphorylation may yield next-generation CARs with cytotoxic potency approaching that of the native TCR. We engineered an optimized CAR incorporating a 37-amino-acid segment (TIP) of the Herpesvirus saimiri tyrosine-protein kinase–interacting protein that binds LCK with high specificity and modulates its activity. Incorporation of the TIP co-stimulation motif into the CAR-BB-CD3ζ domain produced several notable effects: The TIP motif altered early CAR T cell activation, with antigen-independent recruitment of LCK and other proximal signaling mediators to the CAR–CD3ζ complex, indicating ligand-independent priming of signaling. Despite elevated basal phosphorylation, cytokine secretion was absent and downstream transcription remained inactive, thereby preserving antigen specificity while accelerating early activation kinetics. Ex vivo expansion revealed a consistent enrichment of CD8⁺ T cells in TIP-CAR cultures (72% vs 35% in BBζ-CAR, p<0.0001), accompanied by higher Ki67 (46% vs 28%, p<0.05) and CD38 (85% vs 64%, p<0.01) expression in CD8+ subset across eight donors. Phenotyping also showed a skew toward a CCR7⁻CD45RA⁺ effector phenotype within the CD8⁺ compartment (59% vs 41%, p<0.01), suggesting distinct TIP-driven memory imprinting patterns across T cell lineages. Integrated transcriptomic profiling and flow cytometric phenotyping demonstrated that TIP co-stimulation reprograms T cell transcriptional landscape relative to BBζ-CARs, with upregulation of key TCR signaling genes (TCR variants, ZAP70, LAT) and the transcription factor TCF7, while showing no changes in exhaustion-associated transcripts, including PD1, LAG3 and TIGIT. These findings suggest that TIP co-stimulation reinforces T cell activation programs while restraining terminal differentiation and exhaustion. The functional performance of TIP-CAR T cells, evaluated through standard in vitro assays, demonstrated that TIP-CAR T cells showed a statistical increase in target-cell killing (p<0.0001) with robust proliferation comparable to that of BBζ-CAR T cells. To assess in vivo efficacy, we conducted a xenograft study using NSG mice engrafted with luciferase-Raji lymphoma cells. Mice (n = 6–7/group) received TIP-CAR T cells, BBζ-CAR T cells, or no treatment, using cells manufactured from two healthy donors. Bioluminescent imaging revealed a statistically significant reduction of tumor burden in TIP-CAR–treated mice compared to BBζ-CAR (p < 0.05), with rapid and sustained tumor clearance. Survival analysis showed significantly prolonged overall survival in the TIP-CAR-treated group (log-rank p < 0.001); mice treated with BBζ-CAR T cells succumbed to progressive disease, while TIP-CAR treated mice survived long-term. Notably, upon rechallenge with Raji cells several weeks after initial clearance, TIP-CAR-treated mice resisted tumor re-engraftment, indicating durable antitumor immunity and memory formation. Flow cytometric analysis of peripheral blood and bone marrow of rechallenged mice confirmed enhanced persistence and expansion of TIP-CAR T cells, enriched for CD8⁺effector-memory phenotypes. These findings demonstrate that the TIP co-stimulation motif enhances in vivo CAR T cell function by improving synapse formation, sustaining cytotoxic competence, and enabling long-term immune surveillance. Altogether, this study introduces a modular, virus-derived co-stimulatory motif that rewires early CAR signaling and programs durable, high-functioning T cell responses. TIP co-stimulation motif offers a broadly translatable strategy to enhance CAR T cell efficacy, persistence, and memory in hematologic malignancies.