Biology subjects

Thut, H.

Publications and source records attributed to Thut, H..

3 recordsLinked to original sources

bioRxiv2025

Longitudinal profiling of tumor-reactive T cells during TIL therapy in metastatic melanoma

Adoptive cell therapy (ACT) with expanded autologous tumor-infiltrating lymphocytes (TILs) can induce durable responses in metastatic melanoma, yet many patients relapse. We profiled tumor-reactive T cell dynamics during TIL therapy using single-cell RNA and TCR sequencing from seven patients to elucidate underlying reasons. We found that tumor-reactive T cells preferentially expanded early during ex vivo TIL culture, transitioning from exhausted to reinvigorated effector states. Particularly, CD8+ exhausted T cells (Tex) and CD4+ follicular helper T cell (Tfh), but not CD4+ Tex, were efficiently reinvigorated. Further, we resolved the heterogeneity of tumor-reactive CD8+ and CD4+ subsets, defining unique signatures for their identification during TIL expansion. In addition, non-responders (NRs) exhibit increased levels of Type 17 T cells in TIL products, suggesting a potential association with resistance to therapy. After transfer, tumor-reactive clones rapidly extravasated and established a stem-like reservoir. However, in NRs, CD4+ regulatory T cells (Tregs) expanded de novo and tumor-reactive CD8+ T cells reacquired exhaustion markers, limiting their functionality. By contrast, responders (Rs) retained a pool of less differentiated, stem-like cells. Collectively, these data provide a comprehensive analysis of T cell fates during TIL-ACT providing the basis for new approaches to enhance therapeutic strategies.

immunology↗

bioRxiv2025

In vivo screening of TCR-based chimeric antigen receptors for improved anti-tumor functionality

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in hematological malignancies, but its success in solid tumors remains limited. Conventional CAR-T designs do not integrate into the T-cell receptor (TCR) and rely on non-natural CD3{zeta} signaling. They thus often suffer from tonic signaling, rapid T-cell exhaustion, and antigen escape due to reduced sensitivity. To address these limitations, we explored alternative CAR architectures that take into account the evolutionary optimized TCR signaling machinery. Specifically, we employed T-cell receptor fusion constructs (TRuCs), where a single-chain variable fragment (scFv) targeting the Sialyl-Thomson-Nouveau antigen (STn) is attached to the CD3{varepsilon} subunit. We then systematically screened a library of costimulatory domains and their combinations, fused to the TRuC, using a novel in vitro and in vivo screening approach. This screen identified a potent TRuC variant incorporating both CD28 and CD27 costimulatory domains. This optimized construct exhibited increased in vitro and in vivo proliferation and enhanced 4-1BB and IFN{gamma} expression upon repeated antigen stimulation. It further showed improved antitumor efficacy compared to conventional second-generation CAR-T cells in a mouse melanoma model. Moreover, we validated the adaptability of this approach by targeting B7H3 in a sarcoma model. The TRuC with costimulatory domains again outperformed other CAR-variants. Our findings highlight the potential of incorporating combined CD28 and CD27 costimulatory domains into TCR-based CAR architectures to overcome limitations associated with conventional CAR-T therapy and improve efficacy against solid tumors.

bioengineering↗

bioRxiv2024

Targeting cancer-associated glycosylation for adoptive T cell therapy of gastro-intestinal and gynecological cancers

CAR-T cell therapy has provided a significant improvement for patients with chemotherapy-resistant B cell malignancies. However, CAR-T cell treatment of patients with solid cancers has been more difficult, in part because of the heterogeneous expression of tumor-specific cell surface antigens. Here, we describe the generation of a fully human CAR targeting altered glycosylation in secretory epithelial cancers. The expression of the target antigen - the truncated, sialylated O-glycan sialyl-Thomsen-Nouveau antigen (STn) - was studied with a highly STn-specific antibody across various different tumor tissues. Strong expression was found in a high proportion of gastro-intestinal cancers including pancreatic cancers and in gynecological cancers, in particular ovarian and endometrial tumors. T cells expressing anti-STn CAR were tested in vitro and in vivo. Anti-STn CAR-T cells showed activity in mouse models as well as in assays with primary ovarian cancer samples. No significant toxicity was observed in mouse models, although some intraluminal expression of STn was found in gastro-intestinal mouse tissue. Taken together, this fully human anti-STn CAR construct shows promising activity in preclinical tumor models supporting its further evaluation in early clinical trials.

cancer biology↗