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Sotillo, E.

Publications and source records attributed to Sotillo, E..

16 recordsLinked to original sources
bioRxiv2026

Targeted cancer ablation via programmable sensing of amplified oncogenic RNA

Selectively eliminating cancer cells based on their oncogenic drivers holds great promise but remains challenging for targeted cancer therapy. Here, we present ORCA (Oncogenic RNA-triggered Cancer Ablation), a programmable and modular platform that couples RADAR-based detection of oncogenic RNA with conditional expression of therapeutic payloads in cancer cells. We identify RADAR sensors targeting the transcripts of endogenously amplified oncogenes, including MYC, FGFR2 and wild-type KRAS in colorectal and gastric cancer cells. Focusing on colorectal cancer (CRC) driven by extrachromosomal DNA-amplified MYC, we demonstrate targeted ablation of high-MYC cancer cells using two orthogonal strategies: anti-CD19 CAR-T therapy and enzyme-prodrug therapy. In vivo delivery of ORCA using adeno-associated virus (AAV) suppresses tumor growth in a subcutaneous xenograft model and prolongs survival in a CRC liver metastasis model. ORCA establishes oncogenic RNAs as actionable inputs for targeted cancer therapy via programmable RNA sensing, thus offering a strategy to target tumors through the transcripts of otherwise difficult-to-drug oncogenes such as MYC.

synthetic biology↗
bioRxiv2026

Cell Cycle Sensing Shapes Human T Cell Fate and Exhaustion Programs

Cell cycle (CC) dynamics are reflected in diverse T cell processes such as TCR activation, expansion, contraction, differentiation, senescence, anergy, and exhaustion; linking CC behaviors to functional and dysfunctional T cell states in development and disease. Progression through CC checkpoints is also tightly linked to cell fate decisions across development. Yet, much remains unknown about the connection between CC sensing and T cell differentiation programs. To disentangle the relationship across T cell state, time-since-activation, receptor signaling, division, and CC, we leverage high-throughput single-cell mass cytometry for parallel measurement of these diverse biological states. By modulating CC progression and receptor signaling with inhibitors as well as tonic signaling Chimeric Antigen Receptor (CAR) models of T cell exhaustion, we reveal that earlier G1/S CC programs crosstalk with receptor signaling to control T cell fate, and that exhaustion programs are downstream to aberrant, S-G2 phase CC arrest signatures in tonic CAR signaling in vitro, in situ, and in vivo across human cancers in association with CD8 T-lymphocyte dysfunction.

immunology↗
bioRxiv2026

Dynamic, single-cell monitoring of CAR T cell identity and activation with Raman spectroscopy

Chimeric antigen receptor (CAR) T cell therapies have reshaped treatment for cancers and immune-mediated diseases, yet their safety and efficacy depend on both the proliferation of engineered cells and their dynamic functional state -- features that remain challenging to monitor in real-time clinical settings. Current methods require targeted labels, extensive processing, and provide only static snapshots of cell identity and activation. Here, we introduce a surface-enhanced Raman spectroscopy (SERS) and machine learning (ML) approach that enables single-cell identification of engineered CAR T cells without molecularly-targeted labels and time-resolved, semi-continuous monitoring of their functional activation state through a single physical readout spanning donor-derived cells and patient blood. From intrinsic vibrational signatures of live cells, we detect spectral differences resulting from engineered receptor expression in donor-derived CD19- and GD2-targeted CAR T cells (nine and five donors, respectively) with 81-85% donor-level accuracy, and resolve dynamic antigen-specific activation trajectories with temporal precision. Applying this same SERS-ML method to longitudinal samples from a four-patient CD19-CAR T therapy cohort, we classify patient peripheral blood mononuclear cells across pre-and post-infusion timepoints with a mean patient-level accuracy of 84%, and show that isolated CAR-positive T cells are distinguishable from both CAR-negative T cells and background populations with average 86-88% accuracies. These capabilities stem from biochemical signatures consistent with processes such as receptor expression, tonic signalling, and immune synapse formation, demonstrating a single method that reports both cellular identity and activation state with biochemical specificity across cell engineering and clinical monitoring contexts. Our results extend CAR T cell monitoring beyond static phenotyping and support the potential of SERS-ML analysis for rapid, point-of-care assessment of engineered immune cells across the therapeutic lifecycle.

bioengineering↗
bioRxiv2025

OVEREXPRESSION OF TUMORPROTECTIVE CLUSTERIN PREVENTS EXHAUSTION OF TRANSGENIC T CELLS

1T cell therapies, such as chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) transgenic (tg) T cells, are a promising approach in the treatment of solid malignancies but are limited by T cell exhaustion caused by chronic antigen stimulation. Clusterin (CLU) is a chaperone protein known to protect both normal and malignant cells, from metabolic stress and reactive oxygen species (ROS). In this study, we investigated whether overexpression (OE) of CLU in TCRtg T cells and CAR-T cells respectively can reduce exhaustion induced by chronic antigen stimulation and enhance T cell functionality against Ewing sarcoma (EwS). Among other cytoprotective genes, we found that CLU was significantly downregulated in dysfunctional tumor infiltrating lymphocytes. Therefore, we engineered EwS-directed TCRtg T cells targeting a Chondromodulin-1 (Chm1)-derived peptide and GD2 CAR-T cells to overexpress CLU. We show here that CLU is downregulated in T cells following activation by tumor cells. CLU-overexpressing T cells exhibit decreased expression of exhaustion markers (PD1, LAG3) and reduced apoptosis after repetitive stimulation. These cells demonstrated improved infiltration into tumor spheroids and maintained functionality under hypoxic conditions. In vivo, CLU-overexpressing T cells showed enhanced persistence and a trend towards reduced tumor growth. Mechanistically, proteomic analysis suggested that reduced ribosomal activity might delay T cell exhaustion, implicating metabolic reprogramming. In conclusion, CLU OE in tg T cells enhances their persistence and functionality by mitigating exhaustion, possibly through modulation of ribosomal activity and metabolic pathways. This strategy holds potential for improving adoptive T cell therapies against solid tumors.

immunology↗
bioRxiv2025

A continuous landscape of signaling encodes a corresponding landscape of CAR T cell phenotype

Cytokine signaling is critical to the function of natural immune cells and engineered immune cell therapies such as chimeric antigen receptor (CAR) T cells. It remains unclear how the limited set of signal transducers and activators of transcription (STATs) and other proteins activated by these cytokine receptors can encode the observed diversity of immune cell phenotypes. To understand how signaling downstream of cytokines control immune cell phenotype, we sought to map the structure of Janus kinase (JAK)/STAT signaling domains to cell signaling and resulting CAR T cell function. We recombined 14 signaling motifs to construct a library of [~]30,000 constitutively active synthetic cytokine receptors (SCRs) with intracellular domains composed of novel signaling motif combinations that activate different signaling cascades. We experimentally tested [~]450 SCRs which generated a range of CAR T cell memory, cytotoxicity, and proliferation. SCRs with pSTAT1 and 3 signaling generated effector memory CAR T cells, while SCRs with strong pSTAT5 generated effector CAR T cells with potent anti-tumor activity. To map the structure-signaling-phenotype landscape we trained models to predict signaling and CAR T cell phenotype that result from varied motif combinations. From neural network predictions we identified features, including strong STAT5 and Shc signaling, that promote unsafe autonomous CAR T cell proliferation. Models also revealed a trade-off between memory and cytotoxicity, with a Pareto front encoded by a continuous change in signaling. These results demonstrate that recombination of a limited set of signaling motifs creates a continuous spectrum of signaling that encodes a corresponding spectrum of cell phenotype. This work synthetically expands the combinatorial space of JAK/STAT signaling and provides a foundation for rational design of CAR T cells with improved cytotoxicity, memory, and safety profiles. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/658149v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@cc1498org.highwire.dtl.DTLVardef@5c2864org.highwire.dtl.DTLVardef@5fc8b3org.highwire.dtl.DTLVardef@719f8f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗
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↗
bioRxiv2023

Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin like growth factor 2 (IGF2). After showing initial efficacy with wild type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially-selective targeted protein degradation. Significance StatementBetter therapeutic windows can be achieved by targeting therapeutics to their desired sites of action. For protein therapeutics, this might be achieved by engineering cell therapies that home to a tissue of interest and secrete the biologic drug locally. Here, we demonstrate that human primary T cells can be engineered to produce genetically encoded lysosome targeting chimeras (GELYTACs). These GELYTACs mediate the degradation of extracellular proteins associated with cancer progression. Thus, cells engineered to produce GELYTACs represent a potential new class of cancer therapeutics.

synthetic biology↗
bioRxiv2023

Engineered CD47 protects T cells for enhanced antitumor immunity

Adoptively transferred T cells and agents designed to block the CD47/SIRP axis are promising antitumor therapeutics, which activate distinct arms of the immune system. We administered anti-CD47 (CD47) with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which blunted therapeutic benefit. CD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRP and provided a "dont-eat-me" signal that was not blocked by CD47 antibodies. TCR or CAR T cells expressing 47E were resistant to clearance by macrophages following CD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy resulted in synergistic enhancement in antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests markedly enhanced potency against solid tumors.

bioengineering↗
bioRxiv2023

Inosine Induces Stemness Features in CAR T cells and Enhances Potency

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing. Statement of SignificanceAdenosine is well known to inhibit T cell function and substantial effort has focused on inhibiting adenosine generation and signaling. Here, we show that exhausted T cells are suppressed by adenosine, which is only modestly impacted by inhibiting adenosine generation or signaling. In contrast, metabolism of adenosine to inosine augmented T cell function and culture of T cells with inosine induced multi-level reprogramming leading to stemness and improved anti-tumor potency. We demonstrate the feasibility of introducing inosine during GMP cell manufacturing as a novel strategy to generate enhanced CAR-T cells.

immunology↗
bioRxiv2022

Enhanced Effector Activity of Mediator Kinase Module Deficient CAR-T Cells

Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes limiting T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were MED12 and CCNC, components of the cyclin-dependent kinase (CDK) module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. MED12 or CCNC deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. Chemical inhibition of CDK8/19 kinase activity recapitulated some features of genetic loss of MED12, including increased T cell expansion. MED12 deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation at enhancers used by transcription factors playing a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in MED12 deficient T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK module as a target for enhancing the potency of anti-tumor T cell responses. One Sentence SummaryThe Mediator kinase module is a primary regulator of T cell differentiation, and genetic or small molecule-based inhibition of this module enhances effector T cell potency.

immunology↗
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↗
bioRxiv2021

Dynamic chromatin regulatory landscape of human CAR T cell exhaustion

Dysfunction in T cells limits the efficacy of cancer immunotherapy1-6. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion7. We found widespread, dynamic changes in chromatin accessibility and 3D chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mouse, we find that the regulatory networks of T cell exhaustion differ between the species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.

genomics↗
bioRxiv2021

Anti-GD2 antibody disrupts GD2:Siglec-7 interactions and synergizes with CD47 blockade to mediate tumor eradication

The disialoganglioside GD2 is consistently overexpressed in neuroblastoma and osteosarcoma, and is variably expressed in other sarcomas, gliomas, neuroendocrine tumors, and epithelial cancers. Anti-GD2 antibodies have improved the survival rates of patients with neuroblastoma only when administered as part of intense chemotherapy-based cytotoxic regimens, which are associated with debilitating late effects including hearing loss, growth retardation, and secondary leukemias. Despite broad expression of GD2 on osteosarcoma, anti-GD2 antibody has not mediated significant antitumor activity in that disease or any other GD2+ cancers. CD47 is a checkpoint molecule overexpressed on tumor cells that inhibits macrophage activity, and CD47 blockade has demonstrated promising clinical activity in early human trials. We investigated whether anti-CD47 antibody could enhance the efficacy of anti-GD2 antibody in neuroblastoma and other GD2+ malignancies. We demonstrate substantial synergy of these two agents, resulting in the recruitment of tumor associated macrophages (TAMs) to mediate robust and durable anti-tumor responses. The responses are driven by GD2-specific factors that reorient the balance of macrophage activity towards phagocytosis of tumor cells, including disruption of a newly described GD2:Siglec-7 axis. These results demonstrate the unique synergy of combining anti-GD2 with anti-CD47, which has the potential to significantly enhance outcomes for children with neuroblastoma and osteosarcoma and will soon be investigated in a first-in-human clinical trial.

immunology↗
bioRxiv2020

Transient "rest" induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells

T cell exhaustion limits immune responses against cancer and is a major cause of resistance to CAR-T cell therapeutics. Using a model wherein tonic CAR signaling induces hallmark features of exhaustion, we employed a drug-regulatable CAR to test the impact of transient cessation of receptor signaling (i.e. "rest") on the development and maintenance of exhaustion. Induction of rest in exhausting or already-exhausted CAR-T cells resulted in acquisition of a memory-like phenotype, improved anti-tumor functionality, and wholescale transcriptional and epigenetic reprogramming. Similar results were achieved with the Src kinase inhibitor dasatinib, which reversibly suppresses CAR signaling. The degree of functional reinvigoration was proportional to the duration of rest and was associated with expression of transcription factors TCF1 and LEF1. This work demonstrates that transient cessation of CAR-T cell signaling can enhance anti-tumor potency by preventing or reversing exhaustion and challenges the paradigm that exhaustion is an epigenetically fixed state.

immunology↗
bioRxiv2019

c-Jun Overexpressing CAR-T Cells are Exhaustion-Resistant and Mediate Enhanced Antitumor Activity

CAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmarks of exhaustion described in other settings. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs, and overexpression of numerous bZIP and IRF transcription factors that have been implicated in inhibitory activity. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in numerous in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.

immunology↗