Activation of a conditional safety switch has the potential to reverse serious toxicities arising from the administration of engineered cellular therapies, including chimeric antigen receptor (CAR) T cells. The functionally inert, non-immunogenic cell surface marker derived from human epidermal growth factor receptor (EGFRt) is a promising safety switch that has been used in multiple clinical constructs and can be targeted by cetuximab, a clinically available monoclonal antibody. However, this approach requires high and persistent cell surface expression of EGFRt to ensure that antibody-mediated depletion of engineered cells is rapid and complete. Here we show that incorporating a short juxtamembrane sequence into the EGFRt polypeptide enhances its expression on the surface of T cells and their susceptibility to antibody-dependent cellular cytotoxicity (ADCC). Incorporating this optimized variant (EGFRopt) into bicistronic and tricistronic CAR designs results in more rapid in vivo elimination of CAR T cells and robust termination of their effector activity compared to EGFRt. These studies establish EGFRopt as a superior safety switch for the development of next-generation cell-based therapeutics.

Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS-/--pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic β cells. Our results suggest that supplementation with exogenous thymidine affects stem cell proliferation, but not the function of stem cell-derived cells. After differentiation, postmitotic cells do not require thymidine in vitro or in vivo, as shown by the production of functional human insulin in mice up to 5 months after implantation of stem cell-derived pancreatic tissue.

Chimeric antigen receptor T cell (CAR-T) therapy is an innovative immunotherapeutic approach that utilizes genetically modified T-cells to eliminate cancer cells using the specificity of a monoclonal antibody (mAb) coupled to the potent cytotoxicity of the T-lymphocyte. CAR-T therapy has yielded significant improvements in relapsed/refractory B-cell malignancies. Given these successes, CAR-T has quickly spread to other hematologic malignancies and is being increasingly explored in solid tumors. From early clinical applications to present day, CAR-T cell therapy has been accompanied by significant toxicities, namely cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and on-target off-tumor (OTOT) effects. While medical management has improved for CRS and ICANS, the ongoing threat of refractory symptoms and unanticipated idiosyncratic toxicities highlights the need for more powerful safety measures. This is particularly poignant as CAR T-cell therapy continues to expand into the solid tumor space, where the risk of unpredictable toxicities remains high. We will review CAR-T as an immunotherapeutic approach including emergence of unique toxicities throughout development. We will discuss known and novel strategies to mitigate these toxicities; additional safety challenges in the treatment of solid tumors, and how the inducible Caspase 9 \"safety switch\" provides an ideal platform for continued exploration.

Gene edited and engineered cell-based therapies are a promising approach for treating a variety of disorders, including cancer. However, the ability of engineered cells to persist for prolonged periods along with possible toxicity raises concerns over the safety of these approaches. Although a number of different one-dimensional suicide systems have been incorporated into therapeutic cell types, the incorporation of a two-layered suicide system that allows controlled killing of therapeutic cells at different time points is needed. In this study, we engineered a variety of therapeutic cells to express two different kill switches, RapaCasp9 and HSV-TK and utilized Rapamycin and Ganciclovir respectively to activate these kill switches. We show that the function of both RapaCasp9 and HSV-TK molecules is preserved and can be activated to induce apoptosis detected early (24 h) and late (48 h) post-activation respectively, with no toxicity. In vivo, we show the eradication of a majority of cells after treatment in subcutaneous and orthotopic models. Furthermore, we demonstrate how both suicide switches work independently and can be activated sequentially for an improved killing, thus ensuring a failsafe mechanism in case the activation of a single one of them is not sufficient to eliminate the cells. Our findings highlight the reliability of the double suicide system, effective on a variety of cells with different biological characteristics, independent of their anatomic presence.

Gene-modified cellular therapies carry inherent risks of severe and potentially fatal adverse events, including the expansion of alloreactive cells or malignant transformation due to insertional mutagenesis. Strategies to mitigate uncontrolled proliferation of gene-modified cells include co-transfection of a suicide gene, such as the inducible caspase 9 safety switch (ΔiC9). However, the activation of the ΔiC9 fails to completely eliminate all gene-modified cells. Therefore, we tested a two suicide gene system used independently or together, with the goal of complete cell elimination. The first approach combined the ΔiC9 with an inducible caspase 8, ΔiC8, which lacks the endogenous prodomain. The rationale was to use a second caspase with an alternative and complementary mechanism of action. Jurkat cells co-transduced to co-express the ΔiC8, activatable by a BB homodimerizer, and the ΔiC9 activatable by the rapamycin analog sirolimus were used in a model to estimate the degree of inducible cell elimination. We found that both agents could activate each caspase independently, with enhanced elimination with superior reduction in cell regrowth of gene-modified cells when both systems were activated simultaneously. A second approach was employed in parallel, combining the ΔiC9 with the RQR8 compact suicide gene. RQR8 incorporates a CD20 mimotope, targeted by the anti-CD20 monoclonal antibody rituxan, and the QBend10, a ΔCD34 selectable marker. Likewise, enhanced cell elimination with superior reduction in cell regrowth was observed when both systems were activated together. A dose-titration effect was also noted utilizing the BB homodimerizer, whereas sirolimus remained very potent at minimal concentrations. Further in vivo studies are needed to validate these novel combination systems, which may play a role in future cancer therapies or regenerative medicine.

Human mesenchymal stem cells (MSCs) are multipotent stem cells that have been intensively studied as therapeutic tools for a variety of disorders. To enhance the efficacy of MSCs, therapeutic genes are introduced using retroviral and lentiviral vectors. However, serious adverse events (SAEs) such as tumorigenesis can be induced by insertional mutagenesis. We generated lentiviral vectors encoding the wild-type herpes simplex virus thymidine kinase (HSV-TK) gene and a gene containing a point mutation that results in an alanine to histidine substitution at residue 168 (TK(A168H)) and transduced expression in MSCs (MSC-TK and MSC-TK(A168H)). Transduction of lentiviral vectors encoding the TK(A168H) mutant did not alter the proliferation capacity, mesodermal differentiation potential, or surface antigenicity of MSCs. The MSC-TK(A168H) cells were genetically stable, as shown by karyotyping. MSC-TK(A168H) responded to ganciclovir (GCV) with an half maximal inhibitory concentration (IC50) value 10-fold less than that of MSC-TK. Because MSC-TK(A168H) cells were found to be non-tumorigenic, a U87-TK(A168H) subcutaneous tumor was used as a SAE-like condition and we evaluated the effect of valganciclovir (vGCV), an oral prodrug for GCV. U87-TK(A168H) tumors were more efficiently ablated by 200 mg/kg vGCV than U87-TK tumors. These results indicate that MSC-TK(A168H) cells appear to be pre-clinically safe for therapeutic use. We propose that genetic modification with HSV-TK(A168H) makes allogeneic MSC-based ex vivo therapy safer by eliminating transplanted cells during SAEs such as uncontrolled cell proliferation.

BACKGROUND: Cell therapy as a promising therapeutic modality to treat cancer has been intensively studied for decades. However, the clinical trials have indicated that patients under T cell therapy may develop severe cytokine release syndrome resulting in hospitalization or even death. Furthermore, genetic modifications to promote proliferation and persistence of T cells could result in high numbers of long-lived engineered cells in patients after treatment.
METHODS: We incorporated the pro-apoptotic truncated BH3 interacting-domain death agonist (tBID) with the mutant ecDHFR destabilizing domain to form a novel recombinant protein as the major component of an engineered tBID-based safety switch system, which would be unstable and quickly degraded in the absence of trimethoprim (TMP) but, upon TMP treatment, should become stabilized and allow tBID to induce cell death experimentally.
RESULTS: The novel tBID-based safety switch could be regulated through a small molecule inducer, TMP, to control undesired toxicity or ablate the engineered cells as needed. We systematically compared and assessed several tBID-based safety switch constructs with the clinically validated safety switches, including human herpes simplex virus thymidine kinase (HSV-TK) and inducible Caspase 9 (iCasp9). With optimization, we were able to achieve significant killing potency in vitro in Jurkat or human primary T cells.
CONCLUSIONS: We demonstrated that our engineered tBID-based safety switch was able to eliminate up to ~90% of transduced human primary T cells within 72 h after activation, providing an alternative switch system to manage safety concerns for cell therapy.

Immune cell-based therapies can induce potent antitumor effects but are also often associated with severe toxicities. We previously developed a PD-1-based small molecule-regulated reversible T cell activation switch to control the activity of cellular immunotherapy products. This chemically regulated and SH2-delivered-inhibitory tail (CRASH-IT) switch relies on the noncovalent interaction of switch SH2 domains with phosphorylated ITAM motifs in either chimeric antigen receptors or T cell receptors. After this interaction, the immunoreceptor tyrosine-based inhibition motif/switch motif (ITIM/ITSM) containing PD-1 domain present in the CRASH-IT switch induces robust inhibition of T cell signaling, and CRASH-IT-mediated suppression of T cell activity can be reversed by small molecule-induced switch proteolysis. With the aim to develop improved second-generation switch systems, we here analyze the possibility space of both the immune cell receptor docking and inhibitory signaling domains that allow control over T cell activity. Importantly, these analyses demonstrate that the inhibitory domains that most potently suppress antigen receptor signaling in primary human T cells are not derived from inhibitory receptors, such as PD-1 and BTLA, that are endogenously expressed in T cells, but include ITIM/ITSM containing inhibitory domains derived from receptors present in myeloid cells. In addition, we demonstrate that physical proximity of the inhibitory domain to the antigen receptor is crucial to efficiently suppress T cell activation, as only switch designs that employ SH2 domains directly interacting with ITAM motifs in antigen receptors efficiently and reversibly inhibit T cell functionality. These data demonstrate the flexible and interchangeable nature of immune cell signaling domains, and inform the design of a synthetic proximity-based switch system with a superior dynamic range.

Induced pluripotent stem cell (iPSC)-derived cell products hold great promise as a potential cell source in personalized medicine. As concerns about the potential risk of graft-related severe adverse events, such as tumor formation from residual pluripotent cells, currently restrict their applicability, we established an optimized tool for therapeutic intervention that allows drug-controlled, specific and selective ablation of either iPSCs or the whole graft through genetic safety switches. To identify the best working system, different tools for genetic iPSC modification, promoters to express safety switches and different safety switches were combined. Suicide effects were slightly stronger when the suicide gene was delivered through lentiviral (LV) vectors compared to integration into the AAVS1 locus through TALEN technology. An optimized HSV-thymidine kinase and the inducible Caspase 9 both mediated drug-induced, efficient in vitro elimination of transgene-positive iPSCs. Choice of promoter allowed selective elimination of distinct populations within the graft: the hOct4 short response element restricted transgene expression to iPSCs, while the CAGs promoter ubiquitously drove expression in iPSCs and their progeny. Remarkably, both safety switches were able to prevent in vivo teratoma development and even effectively eliminated established teratomas formed by LV CAGs-transgenic iPSCs. These optimized tools to increase safety provide an important step towards clinical application of iPSC-derived transplants.

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