Biology subjects

Pennati, A.

Publications and source records attributed to Pennati, A..

7 recordsLinked to original sources

bioRxiv2026

An autologous cell-based therapeutic vaccine expressing IL6/1 fusokine drives robust anti-tumor response against ovarian cancer.

BackgroundCytokines are immunomodulatory proteins that play central roles in regulating immune responses and represent attractive targets for cancer therapy. However, as single agents, cytokines have shown limited clinical benefit due to systemic toxicities and a short in vivo half-life. Our group has focused on engineering fusion cytokines (fusokines) that couple two cytokines into a single biologic to reprogram immune cell responses by enforcing non-canonical receptor engagement and signaling. A chimeric IL-6/IL-1{beta} fusokine was engineered to test the hypothesis that enforced co-engagement of IL-6 and IL-1{beta} signaling pathways would confer a gain-of-function phenotype in T cells and promote robust anti-tumor immunity. Here, we describe the immunomodulatory properties of IL6/1 fusokine and a method to deliver this fusokine to produce inhibition of ovarian tumor growth in a pre-clinical mouse model. MethodsLentiviral vectors encoding murine or human IL6/1 were designed using Vector Builder and expressed in either HEK293, CHO or ID8-F3 (p53-/-) cells depending on the downstream experiment to be conducted. IL6/1 expression was validated by ELISA and flow cytometry. Effects of human IL6/1 (hIL6/1) on T cell function (proliferation, memory phenotype, activation induced apoptosis) were monitored by flow cytometry. For in vivo studies, ID8-F3 murine ovarian cancer cells expressing mouse IL6/1 (mIL6/1) were administered intraperitoneally (I.P.) as a cell-based therapy to C57BL/6 female mice bearing established ID8-F3 luciferase tumors. Tumor progression was monitored by bioluminescence (BLI) imaging, and overall survival was evaluated. ResultshIL6/1 significantly enhanced T cell survival and selectively promoted activation and expansion of CD45RO memory T cells. mIL6/1 expressing ID8-F3 cells (ID8IL6/1) demonstrated stable transduction and sustained cytokine secretion. In vivo, ID8IL6/1 cell therapy significantly reduced tumor growth and improved overall survival compared to control groups, with 2 of 8 mice achieving complete tumor clearance. ConclusionThese findings indicate that IL6/1 fusokine enhances T cell survival and proliferation while promoting memory responses. Engineered cancer cells (ID8-F3) expressing mIL6/1 fusokine induced a strong anti-tumor response when delivered as a therapeutic vaccine in ovarian cancer mouse model. What is already known on this topicO_LIFusokines are a class of bifunctional proteins designed to achieve synergistic immune modulation. Previous studies in our lab have shown fusokine exhibit gain-of-function immunomodulating activity. Individually, IL-6 and IL-1{beta} are recognized for their roles in promoting T-cell proliferation and effector function. However, the potential for a fused IL-6/1 fusokine to reprogram the immune system and elicit a superior anti-tumor response in vivo in ovarian cancer model is not yet studied. C_LI What this study addsO_LIThis study develops a novel fusion cytokine (fusokine), combining IL-6 and IL-1{beta}, and demonstrate robust activation of T cells. In a preclinical ovarian cancer model, engineered cancer cells expressing IL6/1 used as a therapeutic vaccine showed significant tumor reduction and improved overall survival. C_LI How this study might affect research, practice or policyO_LIThis study demonstrates that in comparison to individual cytokines, fusokines have greater potential to activate T cell function and when delivered as a cell therapy, achieve clear therapeutic efficacy in an ovarian cancer model. Further translational and clinical studies may enable the development of novel and more effective fusokine cell therapy approaches for patients with ovarian cancer. C_LI

immunology↗

bioRxiv2026

A CD40-Targeted IL-21 Fusokine Enables Rapid Generation of Human IL-10⁺Granzyme B⁺ Regulatory B Cells

Regulatory B cells (Bregs) have emerged as important modulators of immune homeostasis, complementing the classical roles of B cells in antibody production, antigen presentation, and provision of costimulatory signals and cytokines. Beyond promoting immunity, B cells can actively suppress inflammatory responses through specialized regulatory programs that limit effector T cell expansion and restrain pathogenic myeloid activation. These suppressive functions are mediated by mechanisms including secretion of IL-10, IL-35, and TGF-{beta}, expression of inhibitory ligands, and release of granzyme B. However, Bregs are rare and heterogeneous in humans, typically comprising less than 1-2% of circulating B cells, and defects in their frequency or function are associated with autoimmune and inflammatory diseases. The scarcity of endogenous Bregs has driven interest in their ex vivo generation as a cellular immunotherapy. Although several strategies efficiently induce regulatory B cells in murine systems, translation to human B cells has been limited by inefficiency, lack of scalability, or reliance on poorly defined culture conditions. Consequently, a robust and clinically compatible approach for generating human Bregs has remained elusive. With human peripheral blood B cells as starting materiel, we show that a CD40 targeted IL-21 gain of function fusion protein in combination with TLR9 activation induces a stable regulatory phenotype characterized by IL-10 and granzyme B expression in more than 95% of input B-cells. These induced Bregs display a coordinated transcriptional program distinct from conventional activation, significantly suppress activated human T cell proliferation and modulate inflammatory myeloid responses in vitro. In mice with human T-cell driven xenoGVHD, transfusion of T-cell donor matched Bregs significantly improves survival. These data support the feasibility of Breg manufacturing at scale for use a cellular pharmaceutical.

immunology↗

bioRxiv2025

Synthesis of mRNA lipid nanoparticles for engineering GD2 CAR T and CAR NK cells against neuroblastoma

AbstractEngineering T and natural killer (NK) cells with chimeric antigen receptors (CAR) creates effective adoptive cell transfer therapies for cancer treatment. However, using viral transduction as a primary genetic modification method adds regulatory burdens and is expensive to produce at scale. Delivering mRNA encoding CAR via lipid nanoparticles (LNPs) has been explored as a potent non-viral method to generate CAR immune cells. Still, it has not been optimized for CAR treatment of neuroblastoma to date. An LNP formulation to deliver mRNA encoding a GD2 CAR into human T and NK cells was designed and characterized by dynamic light scattering for size distribution, z-average diameter, polydispersity index, and {zeta} potential. Fluorescent reporter detection persisted for more than 1 week after mRNA LNP transfection, without affecting T or NK cell viability. The potency of GD2 CAR T cells with 79.9% reporter positivity and GD2 CAR NK cells with 26.6% reporter positivity was assessed in vitro against the GD2+ neuroblastoma cell line CHLA20. GD2 CAR T or CAR NK cells could effectively target and kill neuroblastoma cells in a dose-dependent fashion, and GD2 CAR T cells showed increased IFN{gamma} production. This study shows mRNA LNPs are a promising non-viral approach for generating GD2 CAR T and CAR NK cells, potentially offering a safer and more cost-effective alternative to current viral vector-based methods.

cancer biology↗

bioRxiv2025

Revised evolutionary relationships within Brachycera and the early origin of bicoid in flies

The specification of the anterior-posterior axis in the embryo is a crucial step in early insect development. Despite its importance, the underlying genetic and regulatory architecture controlling this process varies significantly between species. In cyclorrhaphan flies, such as Drosophila melanogaster, anterior determination is controlled by the transcription factor bicoid, which emerged through duplication of the ancestral Hox3 gene (called zen in insects). With new, high-quality genomic data we mine the genomes of 186 dipteran species, and find presence of bicoid in non-cyclorrhaphan flies, including in the bee flies (family Bombyliidae) and the stiletto flies (Therevidae). We confirm maternal expression and localisation of the non-cyclorrhaphan bicoid mRNA to the anterior region of the unfertilised oocyte in the dark edged bee fly, Bombylius major. To determine the timing and origin of bicoid, we address uncertainty in the dipteran phylogeny, uncovering a ladder-like topology in the branching orders of the early Brachycera lineages. This new species phylogeny suggests that bicoid emerged at the common ancestor of Bombyliidae, Asiloidea, and Eremoneura (collectively Heterodactyla), and was subsequently lost at least 16 times. These findings expand our understanding of the early developmental processes in flies and provide new insights into the backbone phylogeny of Diptera and the evolution of bicoid.

evolutionary biology↗

bioRxiv2024

STRESS GRANULE-RELATED GENES DURING EMBRYOGENESIS OF AN INVERTEBRATE CHORDATE

Control of global protein synthesis through the assembly of stress granules (SGs) represents a strategy adopted by eukaryotic cells to face various stress conditions. TIAR, TTP and G3BP are key components of SGs, allowing the regulation of mRNA stability and thus controlling not only stress responses but also cell proliferation and differentiation. In the present work we aimed to investigate the role of tiar, ttp and g3bp during embryogenesis of the solitary ascidian Ciona robusta, both in physiological and stress conditions. We carried out CRISPR/Cas9 knockout to evaluate the effects on normal embryonic development, and gene reporter assays to study time and tissue specificity of gene expression, together with whole mount ISH and qRT-PCR. To induce acute stress conditions, we used iron and cadmium as "essential" and "non-essential" metals, respectively. Our results highlight, for the first time, the importance of tiar, ttp and g3bp in the control of development of mesendodermal tissue derivatives during embryogenesis of an invertebrate chordate. HIGHLIGHTS- Mesenchyme, nervous cells, and endoderm are the embryonic regions mainly transcribing cr-tiarx1, cr-ttp and cr-g3bp2 during metal stress conditions. - Only cr-ttp transcription is regulated at mid gastrula stage. - The highest transcription levels are reached in hatching larvae after iron exposure. - cr-tiarx1, cr-ttp and cr-g3bp2 knockouts resulted in impaired embryogenesis.

developmental biology↗

bioRxiv2021

Independent mechanisms underlie the protective effect of dietary polyunsaturated fatty acid supplementation and G&alphaz deficiency on the early type 1 diabetes phenotype of Non-obese diabetic (NOD) mice

Non-obese diabetic (NOD) mice deficient in Gz alpha subunit (Gz) are protected from developing hyperglycemia, even with early islet insulitis similar to wild-type mice. Similarly, wild-type (WT) NOD mice are protected from glucose intolerance when fed a diet enriched in eicosapentaneoic acid (EPA). In the beta-cell, Prostaglandin EP3 receptor (EP3), whose primary endogenous ligand is the arachidonic acid (AA) metabolite, prostaglandin E2, is specifically coupled to Gz. In this work, we tested whether dietary EPA supplementation, thereby reducing systemic PGE2 levels, would complement Gz loss in the NOD mouse model. WT and Gz-null NOD mice were fed an AA-enriched diet, EPA-enriched diet, or control diet upon weaning. After 12 weeks of diet feeding, glucose tolerance tests were performed and pancreatic islets and whole pancreas collected for ex vivo analyses, with the longer-term effect of an EPA-enriched diet on splenic T-cell populations quantified via flow cytometry. Our results reveal a polyunsaturated fatty acid-enriched diet, whether AA or EPA, improves wild-type NOD glucose tolerance by the same magnitude as Gz loss, but through almost completely different physiological and cellular mechanisms. Our results shed critical light on future research into novel pharmacological and dietary adjuvant therapies for T1D.

physiology↗

bioRxiv2020

Hmx gene conservation identifies the evolutionary origin of vertebrate cranial ganglia

The evolutionary origin of vertebrates included innovations in sensory processing associated with the acquisition of a predatory lifestyle1. Vertebrates perceive external stimuli through sensory systems serviced by cranial sensory ganglia (CSG) which develop from cranial placodes; however understanding the evolutionary origin of placodes and CSGs is hampered by the gulf between living lineages and difficulty in assigning homology between cell types and structures. Here we use the Hmx gene family to address this question. We show Hmx is a constitutive component of vertebrate CSG development and that Hmx in the tunicate Ciona is able to drive the differentiation program of Bipolar Tail Neurons (BTNs), cells previously thought neural crest homologs2,3. Using Ciona and lamprey transgenesis we demonstrate that a unique, tandemly duplicated enhancer pair regulated Hmx in the stem-vertebrate lineage. Strikingly, we also show robust vertebrate Hmx enhancer function in Ciona, demonstrating that deep conservation of the upstream regulatory network spans the evolutionary origin of vertebrates. These experiments demonstrate regulatory and functional conservation between Ciona and vertebrate Hmx, and confirm BTNs as CSG homologs. Our analysis also identifies derived evolutionary changes, including a genetic basis for secondary simplicity in Ciona and unique regulatory complexity in vertebrates.

evolutionary biology↗