Biology subjects

Rotiroti, M. C.

Publications and source records attributed to Rotiroti, M. C..

5 recordsLinked to original sources

bioRxiv2026

Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

Chimeric Antigen Receptor (CAR) therapy for Acute Myeloid Leukemia (AML) is hindered by disease heterogeneity, antigen overlap with normal hematopoiesis, and the persistence of leukemic stem cells (LSCs). To overcome these barriers, we employed the IF-BETTER strategy to simultaneously target CD33 and the LSC-associated marker TIM-3 by pairing a second-generation CAR with a Cytokine-Costimulatory Receptor (CCR) in two dual CAR configurations (CD33.CAR/TIM-3.CCR and TIM-3.CAR/CD33.CCR). Both constructs displayed potent antigen-restricted cytotoxicity against AML cell lines and primary blasts, achieving leukemia clearance, while sparing normal immune and hematopoietic cells. Mechanistic studies revealed that the TIM-3.CAR single-chain fragment variable (scFv) recognizes a protein-proximal epitope whose interaction is selectively enhanced by AML-specific hyper-fucosylated and hyper-sialylated N-glycans. Fucosylation blockade reduced TIM-3.CAR avidity and cytotoxicity, confirming a glycosylation-modulated interaction. Integrating this glycosylation-tolerant TIM-3 scFv into a dual CAR framework enables selective targeting of AML cells, providing a rational strategy for safer and more effective AML-directed immunotherapy.

immunology↗

bioRxiv2025

Synthetic signaling platform uncovers and rewires cellular responses to PD-1 perturbation

Tyrosine phosphorylation motifs are central regulators of cell signaling, yet methods to selectively detect and reprogram these events have been lacking. Here we introduce Sphyder (Selective PHosphotYrosine DEtection and Rewiring), which enables precise detection of signaling at the resolution of individual phosphorylation motifs. Using Sphyder biosensors, we resolved phosphorylation dynamics and uncovered regulatory mechanisms of the checkpoint receptor PD-1 in living cells. Sphyder also provided a framework for reconstructing phosphosignaling pathways. With this approach, we redirected PD-1 signaling from immunosuppressive to immunoactivating outputs and engineered synthetic receptors that linked extracellular sensing to customized transcriptional programs. In addition, Sphyder biosensors revealed previously unrecognized mechanisms of the PD-1/VEGF bispecific antibody Ivonescimab, showing that it induces VEGF-dependent clustering, phosphorylation, and degradation of PD-1. These findings may underlie its promising clinical activity relative to conventional PD-1 blockade. Together, our study establishes a broadly applicable strategy for sensing and reprogramming cell signaling, while also providing mechanistic insights into a new class of immune checkpoint inhibitors of major clinical interest.

immunology↗

bioRxiv2024

IKAROS facilitates antigen escape in the face of CD19- and CD22-targeted therapies for B-cell acute lymphoblastic leukemia

Relapse due to antigen escape is a major cause of treatment failure for patients with B-cell malignancies following targeted immunotherapies, including CD19- and CD22-directed chimeric antigen receptor T (CAR T) cells. To identify tumor intrinsic factors associated with antigen loss, we performed single-cell analyses on 61 primary patient samples or patient-derived xenografts from patients with B-cell acute lymphoblastic leukemia (B-ALL) treated with CAR T cells. We identified that low levels of the transcription factor IKAROS in pro-B-like B-ALL cells before CAR T treatment are associated with antigen escape. We demonstrate that IKAROSlow B-ALL cells lose features of B cell identity and resemble progenitor cells based on their epigenetic and transcriptional state, resulting in the downregulation of B-cell immunotherapy antigens, including surface expression of CD19 and CD22. We find that modulation of CD19 and CD22 protein expression is IKAROS dose-dependent and reversible. Further, we demonstrate that IKAROSlow cells are resistant to CD19- and CD22-targeted therapies. Together, we describe a novel role for IKAROS in the regulation of B-cell immunotherapy targets and the risk of antigen escape relapse, identifying it as a potential prognostic target. HighlightsO_LIIKAROSlow pro-B-like B-ALL cells are associated with CD19neg relapse C_LIO_LIIKAROSlow B-ALL cells resemble progenitor cells and have lower B-cell commitment C_LIO_LIIKAROS modulates CD19 and CD22 surface expression in a dose-dependent and reversible manner C_LIO_LIIKAROSlow B-ALL cells are more resistant to CD19- and CD22-targeted therapies C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/621347v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@109f75eorg.highwire.dtl.DTLVardef@14f41fborg.highwire.dtl.DTLVardef@74e428org.highwire.dtl.DTLVardef@2f275c_HPS_FORMAT_FIGEXP M_FIG C_FIG Before immunotherapy, IKAROSlow pro-B-like B-ALL cells possess chromatin and gene expression states poised for loss of B-cell identity while maintaining expression of CD19 and CD22. Under immune pressure, IKAROShigh cells maintain their antigen expression, making them more susceptible to T cell-mediated killing. Conversely, IKAROSlow cells are more likely to downregulate their antigen expression, giving them a relative advantage to escape immunotherapies, resulting in antigen escape relapse.

cancer biology↗

bioRxiv2024

Immunotherapy-related cognitive impairment after CAR T cell therapy in mice

Persistent central nervous system (CNS) immune dysregulation and consequent dysfunction of multiple neural cell types is central to the neurobiological underpinnings of a cognitive impairment syndrome that can occur following traditional cancer therapies or certain infections. Immunotherapies have revolutionized cancer care for many tumor types, but the potential long-term cognitive sequelae are incompletely understood. Here, we demonstrate in mouse models that chimeric antigen receptor (CAR) T cell therapy for both CNS and non-CNS cancers can impair cognitive function and induce a persistent CNS immune response characterized by white matter microglial reactivity and elevated cerebrospinal fluid (CSF) cytokines and chemokines. Consequently, oligodendroglial homeostasis and hippocampal neurogenesis are disrupted. Microglial depletion rescues oligodendroglial deficits and cognitive performance in a behavioral test of attention and short-term memory function. Taken together, these findings illustrate similar mechanisms underlying immunotherapy-related cognitive impairment (IRCI) and cognitive impairment following traditional cancer therapies and other immune challenges.

neuroscience↗

bioRxiv2022

Coopting T cell proximal signaling molecules enables Boolean logic-gated CAR T cell control

Introductory paragraphWhile CAR T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumor toxicity has hampered their development for solid tumors because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean logic gating to CAR T cells to prevent on-target, off-tumor toxicity3-7; however, a truly safe and effective logic-gated CAR has remained elusive8. Here, we describe a novel approach to CAR engineering in which we replace traditional ITAM-containing CD3{zeta} domains with intracellular proximal T cell signaling molecules. We demonstrate that certain proximal signaling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumors in vivo while bypassing upstream signaling proteins such as CD3{zeta}. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for the propagation of T cell signaling. We leveraged the cooperative role of LAT and SLP-76 to engineer Logic-gated Intracellular NetworK (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and the prevention of on-target, off-tumor toxicity. LINK CAR will dramatically expand the number and types of molecules that can be targeted with CAR T cells, enabling the deployment of these powerful therapeutics for solid tumors and diverse diseases such as autoimmunity9 and fibrosis10. In addition, this work demonstrates that the internal signaling machinery of cells can be repurposed into surface receptors, a finding that could have broad implications for new avenues of cellular engineering.

synthetic biology↗