Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. ...Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.
The immunostimulatory intracellular domains (ICDs) of chimaeric antigen receptors (CARs) are essential for converting antigen recognition into antitumoural function. Although there are many possible ...combinations of ICDs, almost all current CARs rely on combinations of CD3𝛇, CD28 and 4-1BB. Here we show that a barcoded library of 700,000 unique CD19-specific CARs with diverse ICDs cloned into lentiviral vectors and transduced into Jurkat T cells can be screened at high throughput via cell sorting and next-generation sequencing to optimize CAR signalling for antitumoural functions. By using this screening approach, we identified CARs with new ICD combinations that, compared with clinically available CARs, endowed human primary T cells with comparable tumour control in mice and with improved proliferation, persistence, exhaustion and cytotoxicity after tumour rechallenge in vitro. The screening strategy can be adapted to other disease models, cell types and selection conditions, and could be used to improve adoptive cell therapies and to expand their utility to new disease indications.
Abstract
Despite the success of HER2 targeted therapies in HER2+ breast and gastric cancer, additional therapies are needed to address treatment-resistant metastatic disease. Adoptive immune cell ...therapy is a promising therapeutic modality given the remarkable clinical responses seen with autologous chimeric antigen receptor (CAR) T cells in hematological malignancies. However, success of cell therapy in solid tumors has been more limited. Three major impediments to the success of adoptive cell therapies in solid tumors are the heterogeneity of antigen expression, the immunosuppressive tumor microenvironment (TME), and the inherent challenges of manufacturing autologous cells and consequent variability of these cell products. Engineered, off-the-shelf, allogeneic Natural Killer (NK) cells provide a solution to these challenges. We describe here CAT-179, a novel engineered CAR-NK cell therapeutic for HER2+ solid tumors. CAT-179 cells express three transgenes: a HER2-directed CAR to effectively eliminate tumor cells, a Transforming Growth Factor (TGF) β dominant negative receptor (DNR) for resistance to TGFβ -mediated immunosuppression in the TME, and Interleukin 15 (IL15) cytokine to enhance NK cell persistence and activity for durable response. High efficiency engineering of the large (~3.7Kb) cargo containing CAR, IL15, and DNR in CAT-179 is enabled by the non-viral TC Buster™ Transposon System. Transposon engineering of CAT-179 results in high and stable expression of CAR (45% CAR at day 7 post gene delivery) without the need for post-engineering selection. CAT-179 demonstrates both CAR-dependent and innate NK receptor-dependent tumor cell killing in vitro, reducing the likelihood of tumor escape through antigen loss. CAT-179 effectively kills in vitro both high HER2-expressing SKOV3 cells as well as lower HER2-expressing HT-29 cells. CAT-179 also demonstrates resistance to TGFβ mediated immunosuppression, as evidenced by 75% reduction in TGFβ -induced phosphorylation of SMAD2 as well as prevention of TGFβ induced downregulation of NK cell activating receptors and restoration of NK cell cytotoxic activity. These data suggest CAT-179 cells will be protected from TGFβ -mediated immune suppression in the TME. Finally, the addition of IL15 in CAT-179 significantly enhances persistence for at least fourteen days in vitro without the need for exogenous cytokines. Moreover, CAT-179 administration to NSG mice showed expansion and persistence of the transferred cell product. CAT-179 addresses key hurdles to allogeneic cell therapy for solid tumors and is a promising new therapeutic approach for HER2 expressing breast, gastric and other tumors.
Citation Format: Celeste Richardson, Finola Moore, Andres Alvarez, Alexia Barandiaran, Luke Barron, Eugene Choi, Tucker Ezell, Charlotte Franco, Bashar Hamza, Jennifer Johnson, Annie Khamhoung, Taeyoon Kyung, Marilyn Marques, Dominic Picarella, Jared Sewell, Alex Storer, Meghan Walsh, Vipin Suri. Allogeneic Natural Killer cells engineered to express HER2 CAR, Interleukin 15 and TGF beta dominant negative receptor effectively control HER2+ tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 555.
Optogenetic approaches for controlling Ca
channels provide powerful means for modulating diverse Ca
-specific biological events in space and time. However, blue light-responsive photoreceptors are, ...in principle, considered inadequate for deep tissue stimulation unless accompanied by optic fiber insertion. Here, we present an ultra-light-sensitive optogenetic Ca
modulator, named monSTIM1 encompassing engineered cryptochrome2 for manipulating Ca
signaling in the brain of awake mice through non-invasive light delivery. Activation of monSTIM1 in either excitatory neurons or astrocytes of mice brain is able to induce Ca
-dependent gene expression without any mechanical damage in the brain. Furthermore, we demonstrate that non-invasive Ca
modulation in neurons can be sufficiently and effectively translated into changes in behavioral phenotypes of awake mice.
Calcium (Ca super(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. ...However, study of Ca super(2+) responses has been hampered by technological limitations of existing Ca super(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca super(2+) levels through activation of Ca super(2+)-selective endogenous Ca super(2+) release-activated Ca super(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca super(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca super(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca super(2+) signals.
Calcium (Ca.sup.2+) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis (1). ...However, study of Ca.sup.2+ responses has been hampered by technological limitations of existing Ca.sup.2+-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca.sup.2+ levels through activation of Ca.sup.2+-selective endogenous Ca.sup.2+ release-activated Ca.sup.2+ (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor (2,3) and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca.sup.2+ levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca.sup.2+-associated processes and facilitate screening for drug candidates that antagonize Ca.sup.2+ signals.
Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, ...study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.
PDF
