Biology subjects

Labanieh, L.

Publications and source records attributed to Labanieh, L..

7 recordsLinked to original sources

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

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

Delivery of CAR-T Cells in a Transient Injectable Stimulatory Hydrogel Niche Improves Treatment of Solid Tumors

Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers such as B cell malignancies, but traditional approaches to ACT are poorly effective in treating the multifarious solid tumors observed clinically. Locoregional cell delivery methods have shown promising results in treating solid tumors compared to standard intravenous delivery methods, but the approaches that have been described to date have several critical drawbacks ranging from complex manufacturing and poor modularity to challenging adminstration. In this work, we develop a simple-to-implement self-assembled and injectable hydrogel material for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improves treatment of solid tumors. We evaluate a range of hydrogel formulations to optimize the creation of a transient inflammatory niche that affords sustained exposure of CAR-T cells and cytokines. This facile approach yields increased CAR-T cell expansion, induces a more tumor-reactive CAR-T phenotype, and improves efficacy in treating solid tumors in mice.

bioengineering↗

bioRxiv2018

Live imaging of Aiptasia larvae, a model system for studying coral bleaching, using a simple microfluidic device

Coral reefs, and their associated diverse ecosystems, are of enormous ecological importance. In recent years, coral health has been severely impacted by environmental stressors brought on by human activity and climate change, threatening the extinction of several major reef ecosystems. Reef damage is mediated by a process called coral bleaching where corals, sea anemones, and other cnidarians lose their photosynthetic algal symbionts (genus Symbiodinium) upon stress induction, resulting in drastically decreased host energy harvest and, ultimately, coral death. The mechanism by which this critical cnidarian-algal symbiosis is lost remains poorly understood. Here, we report Traptasia, a simple microfluidic device with multiple traps designed to isolate and image individual live larvae of Aiptasia, a sea anemone model organism, and their algal symbionts over extended time courses. Aiptasia larvae are ~100 m in length, deformable, and highly motile, posing particular challenges for long-term imaging. Using a trap design optimized via fluid flow simulations and polymer bead loading tests, we trapped Aiptasia larvae containing algal symbionts and demonstrated stable imaging for >10 hours. We visualized algal migration within Aiptasia larvae and observed algal expulsion under an environmental stressor. To our knowledge, this device is the first to enable live imaging of cnidarian larvae and their algal symbionts and, in further implementation, could provide important insights into the cellular mechanisms of coral bleaching under different environmental stressors. The device is simple to use, requires minimal external equipment and no specialized training to operate, and can easily be adapted to study a variety of large, motile organisms.

ecology↗

bioRxiv2017

Engineering Cell Sensing and Responses Using a GPCR-Coupled CRISPR-Cas System

G-protein coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes, and detects a wide array of physiological cues in the human body. We describe a new molecular device that couples CRISPR-Cas9 programmed genome regulation to natural and synthetic extracellular signals via GPCRs. The design of our synthetic device, named CRISPR ChaCha, displays superior performance over an architecture proposed by the previously reported Tango system. Using a parsimonious mathematical model and gene-reporter assays, we find that CRISPR ChaCha can recruit and activate multiple Cas9 molecules for each GPCR molecule. We also characterize key molecular features that modulate CRISPR ChaCha performance. We adopt the design to diverse GPCRs that sense synthetic and natural ligands including chemokines, mitogens, and fatty acids, and observe efficient conversion of signals to customizable genetic programs in mammalian cells, including regulation of endogenous genes. The new class of CRISPR-coupled GPCRs provides a robust and efficient platform for engineering cells with novel behaviors in response to the diverse GPCR ligand repertoire.

synthetic biology↗