Biology subjects

Bonini, C.

Publications and source records attributed to Bonini, C..

5 recordsLinked to original sources

bioRxiv2026

Engineered α-Synuclein-specific nanobody CAR iTregs restrain neuroinflammation and proteinopathy in Parkinson's disease mice

Parkinson's disease (PD) is characterized by progressive DAergic neurodegeneration and the accumulation of aggregated -Synuclein (Syn), which drives chronic neuroinflammation through sustained activation of innate and adaptive immune responses. Regulatory T cells (Tregs) exert potent immunosuppressive functions and have shown neuroprotective effects in preclinical PD models; however, clinical translation of polyclonal Treg therapies has been limited by poor tissue specificity and insufficient therapeutic efficacy. To overcome these limitations, we engineered induced human Tregs (iTregs) expressing chimeric antigen receptors (CARs) directed against pathological Syn aggregates. Among the CAR designs tested, only a nanobody-based construct incorporating NbSyn87 displayed selective antigen-dependent activation in response to Syn preformed fibrils (PFFs). Intriguingly, despite the ability of the parental NbSyn87 nanobody to bind both monomeric and aggregated Syn, incorporation into the CAR architecture conferred functional selectivity for aggregated conformers. This feature enabled discrimination between pathological extracellular aggregates and physiological monomeric Syn, providing an important safety advantage. To evaluate therapeutic activity in vivo, we established an immunodeficient mouse model of synucleinopathy permissive to human cell engraftment. iTregs preferentially accumulated within Syn-rich brain regions and, in the presence of astrocyte-derived human IL-2 with antigen-independent mechanism. Conversely, only CAR iTregs directed against Syn significantly reduced microglial and astrocytic activation, decreased pro-inflammatory cytokine expression, and attenuated Syn pathology. Collectively, these findings demonstrate that Syn-specific CAR iTregs can selectively exert potent local immunomodulatory effects, establishing a promising antigen-specific cellular immunotherapy platform for PD and other synucleinopathies.

neuroscience↗

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

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

WRAD core perturbation impairs DNA replication fidelity promoting immunoediting in pancreatic cancer

It is unclear how cells counteract the potentially harmful effects of uncoordinated DNA replication in the context of oncogenic stress. Here, we identify the WRAD (WDR5/RBBP5/ASH2L/DPY30) core as a modulator of DNA replication in pancreatic ductal adenocarcinoma (PDAC) models. Molecular analyses demonstrated that the WRAD core interacts with the replisome complex, with disruption of DPY30 resulting in DNA re-replication, DNA damage, and chromosomal instability (CIN) without affecting cancer cell proliferation. Consequently, in immunocompetent models, DPY30 loss induced T cell infiltration and immune-mediated clearance of highly proliferating cancer cells with complex karyotypes, thus improving anti-tumor efficacy upon anti-PD-1 treatment. In PDAC patients, DPY30 expression was associated with high tumor grade, worse prognosis, and limited response to immune checkpoint blockade. Together, our findings indicate that the WRAD core sustains genome stability and suggest that low intratumor DPY30 levels may identify PDAC patients who will benefit from immune checkpoint inhibitors.

cancer biology↗

bioRxiv2022

Therapy-induced senescence upregulates antigen presentation machinery and triggers anti-tumor immunity in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematological malignancy often curable only by using intensive chemotherapy. Nonetheless, resistance/early relapses are frequent, underscoring the need to investigate the molecular events occurring shortly after chemotherapy. Therapy-induced senescence (TIS) is a fail-safe tumor suppressive mechanism that may elicit immune-mediated responses contributing to senescent cell clearance. Yet, TIS functional role in AML eradication and immune surveillance early post-chemotherapy remains ill-defined. By combining transcriptional and cellular-based evaluation of senescence markers in AML patient samples, we found upregulation of senescence-associated genes and interferon gene categories with concomitant induction of HLA class I and class II molecules, pointing to a causal link between TIS and leukemia immunogenicity. Consistently, senescence-competent AML samples activated autologous CD4+ and CD8+ T cells and improved leukemia recognition by both T-cell subsets. Lastly, the anti-leukemic activity of Immune Checkpoint Blockades (ICBs) was enhanced upon senescence engagement in AML. Altogether, our results identify senescence as a potent immune-related anti-leukemic mechanism that may rapidly translate into innovative senescence-based strategies to prevent AML relapse. STATEMENT OF SIGNIFICANCEOur findings uncover a novel link between senescence induction and leukemia immune recognition by T cells via upregulation of antigen presentation machinery components, providing the basis for conceptually novel senescence-based targeted immunotherapeutic regimens for AML patients.

cancer biology↗