Biology subjects

Ruggiero, E.

Publications and source records attributed to Ruggiero, E..

5 recordsLinked to original sources

bioRxiv2026

Engineering antigen-driven co-stimulation and T helper cell activity into TCR-T cells with CD8-41BB fusion receptors enhances anti-tumor activity

1Adoptive cell therapy using tumor antigen-targeting T cell receptors (TCRs) offers a compelling approach to treat both hematological cancers and solid tumors due to broad antigen accessibility and the ability to target cancer-specific neoantigens. However, unlike clinically validated second generation CAR-T cells bearing built-in co-stimulatory signaling modules (i.e. 41BB or CD28), TCR-T cells receive little to no co-stimulation within most tumor microenvironments leading to attenuated cellular responses. Additionally, CD4+ TCR-T cells engineered to express HLA-Class I restricted TCRs possess minimal T-helper cell activity and thus do not effectively mobilize CD8+ TCR-T cells or host anti-tumor immune responses. To address these limitations, we used CRISPR-Cas9 to engineer TCR-T cells with targeted integration of chimeric CD8 constructs containing intracellular co-stimulatory domains. We found that expression of wild-type CD8{beta}, but not CD8, could promote CD4+ T cell activities in HLA-Class I restricted TCR-T cells. However, this was insufficient to drive durable anti-tumor responses in challenging tumor mouse models when using a high-affinity WT1-directed TCR. To address this, several CD8 co-stimulatory fusion constructs containing CD28 or 41BB intracellular domains were designed and screened, identifying two CD8-41BB based chimeras that substantially increased TCR-T cell activity relative to wild-type CD8{beta}. WT1-TCR-T cells co-expressing the CD8-41BB fusions demonstrated not only enhanced CD4+ activity including strong and polarized Th1-type cytokine secretion, but also enhanced the proliferation, cytokine release, and cytotoxicity of CD8+ CTLs. Remarkably, when combined with TGFBR2 gene disruption, WT1-TCR-T cells co-expressing CD8-41BB receptors were able to completely regress established cell line-derived ovarian tumors, showed robust in vivo expansion and persistence, and provided long-term protection from tumor rechallenge. Importantly, the specificity profile of the WT1-TCR including its HLA-A*02:01 restriction and WT1 peptide recognition motif was preserved upon expression of CD8-41BB. To simplify cell engineering processes for clinical applications, we configured a homology directed repair (HDR) cassette to allow for efficient CRISPR-Cas9-based insertion of both the TCR and CD8-41BB transgenes in the TRAC locus in a single step with >80% efficiency. Lastly, the enhanced activity conferred by CD8-41BB expression was validated with a second clinically relevant TCR targeting PRAME, suggesting this platform can be a universal approach for enhancing the therapeutic potential of TCR-based cell therapies.

synthetic biology↗

bioRxiv2025

HTLV-1 antisense transcription is promoted by increased SP1 binding at 3'-LTR G-Quadruplexes

The human T-cell lymphotropic virus type 1 (HTLV-1) is a highly oncogenic delta-retrovirus. It presents 5- and 3-long terminal repeats (LTR) that are enriched in putative G-quadruplex (G4)-forming sequences. G4s are non-canonical nucleic acid structures that regulate key biological processes in both human and viral genomes. We here investigated the presence and functional role of G4s within the HTLV-1 3-LTR, which governs the antisense transcription of the viral bZIP factor (HBZ), the main responsible for T-cell transformation. We identified seven highly conserved sequences that folded into two-layer G4s in both single- and double-stranded DNA in vitro. We demonstrated G4 folding in infected cells by chromatin immunoprecipitation and showed SP1 enrichment at the 3-LTR G4s. We showed that G4 stabilization with a ligand enhances antisense transcription by promoting recruitment of SP1. Our findings unveil a G4-mediated regulatory mechanism sustaining HTLV-1 antisense transcription and provide new insights into the complex interplay between the HTLV-1 genome and host cellular factors, contributing to our understanding of retroviral replication strategies to be exploited as new therapeutic targets.

microbiology↗

bioRxiv2025

A Simple Generative Model for the Prediction of T-Cell Receptor - Peptide Binding in T-Cell Therapy for Cancer

ObjectiveTo develop a deep learning model capable of predicting epitope peptides recognized by specific CDR3 (Complementarity-Determining Region 3) sequences of T-cell receptors (TCRs) in the context of Major Histocompatibility Complex (MHC) molecules, addressing the challenges of incomplete datasets and the need for novel sequence generation in adoptive T-cell therapy for cancer. MethodsWe implemented a sequence to sequence generative model named "GRIP" (Generative Reconstruction of antIgen Peptides) using a Long Short-Term Memory (LSTM) network with attention mechanisms. The model was trained and validated on publicly available datasets, employing data balancing, label smoothing, and dynamic learning rate scheduling to enhance performance and generalization. Accuracy was assessed at the amino acid level. ResultsThe model achieved a training accuracy of 97% and a test accuracy of 85% for predicting epitope sequences at the amino acid level. Probabilistic sequence generation allowed GRIP to produce biologically plausible epitope sequences, even for unseen CDR3 inputs. Attention-based interpretability provided insights into the models focus on critical sequence elements. The model outperformed existing approaches in handling data imbalance and generalization to novel epitopes. ConclusionGRIP offers a novel solution to the TCR-epitope binding problem by generating potential epitope sequences instead of matching to known data, addressing a fundamental gap in existing models. This approach has significant implications for personalized immunotherapy, facilitating the design of targeted T-cell therapies for cancer.

bioinformatics↗

bioRxiv2024

The iMab antibody selectively binds to intramolecular and intermolecular i-motif structures

i-Motifs are quadruplex nucleic acid conformations that form in cytosine-rich regions. Because of their acidic pH dependence, iMs were thought to form only in vitro. The recent development of an iM-selective antibody, iMab, has allowed iM detection in cells, which revealed their presence at gene promoters and their cell cycle dependence. However, recently evidence emerged which seemed to suggest that iMab recognizes C-rich sequences regardless of their iM conformation. To further investigate the selectivity of iMab, we examined the binding of iMab to C-rich sequences, using a combination of pull-down and Western blot assays. Here we observe that the composition of buffers used during binding and washing steps strongly influences the selectivity of antibody binding. In addition, we demonstrate by NMR that several of the previously reported C-rich sequences, which were not expected to form iMs, actually form intermolecular iMs which are selectively recognized by iMab. Our results highlight the specificity of the iMab antibody, emphasize the importance of optimizing DNA concentrations, blocking and washing conditions, and confirm iMab selectivity not only for intramolecular iMs, but also for intermolecular iMs.

genetics↗

bioRxiv2021

Outer Membrane Vesicles from the gut microbiome contribute to tumor immunity by eliciting cross-reactive T cells

The gut microbiome plays a key role in cancer immunity. One proposed mechanism is through the elicitation of T cells, which incidentally recognize neo-epitopes arising from cancer mutations ("molecular mimicry (MM)" hypothesis). To support MM, Escherichia coli Nissle was engineered with the SIINFEKL epitope (OVA) and orally administered to C57BL/6 mice. The treatment elicited OVA-specific CD8+ T cells in the lamina propria and inhibited the growth of OVA-B16F10 tumors. Importantly, the administration of Outer Membrane Vesicles (OMVs) engineered with different T cell epitopes elicited epitope-specific T cells and inhibited tumor growth. Microbiome shotgun sequencing and TCR sequencing provided evidence that cross-reacting T cells were induced at the mucosal level and subsequently reached the tumor site. Overall, our data support the role of MM in tumor immunity, assign a new role to OMVs and pave the way to new probiotics/OMV-based anti-cancer immunotherapies.

microbiology↗