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Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I(−), MIC-A(+)/B(+) K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Natural killer (NK) cells expressing chimeric antigen receptors (CARs) are a promising anticancer immunotherapy, leveraging both innate NK cell antitumor activity and target-specific cytotoxicity. Inducible MyD88/CD40 (iMC) is a potent, rimiducid-regulated protein switch that has been deployed previously as a T-cell activator to enhance proliferation and persistence of CAR-modified T cells. In this study, iMC was extended to CAR-NK cells to enhance their growth and augment cytotoxicity against tumor cells. iMC-activated NK cells substantially increased cytokine and chemokine secretion and displayed higher levels of perforin and granzyme B degranulation. In addition, iMC activation could be coupled with ectopic interleukin-15 (IL-15) to further enhance NK cell proliferation. When coexpressed with a target-specific CAR (CD123 or BCMA), this IL-15/iMC system showed further augmented antitumor activity through enhanced CAR-NK cell expansion and cytolytic activity. To protect against potential toxicity from engineered NK cells, an orthogonal rapamycin-regulated Caspase-9 (iRC9) was included in a 4-gene, dual-switch platform. After infusion of dual-switch NK cells, pharmacologic iRC9 dimerization led to rapid elimination of a majority of expanded transduced NK cells. Thus, CAR-NK cells utilizing dual molecular switches provide an innovative and effective approach to cancer immunotherapy with controlled specificity, efficacy, and safety.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns. Keywords: CAR-T, dimerizer, cell therapy, costimulation switch, apoptosis, rapamycin, rimiducid, iMC, iRC9, safety switch

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting “low” cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Background: Natural Killer (NK) cells possess potent innate anti-tumor cytotoxicity that can be augmented and focused by engineering with chimeric antigen receptors (CARs). Because NK cells do not express T cell receptors that can direct alloreactivity, they have potential as an off-the-shelf (OTS) cell therapy for the treatment of cancer. We recently demonstrated that a drug-inducible co-activation molecule (inducible MyD88/CD40; iMC) synergizes with transgenic IL-15 to boost CAR-NK cell proliferation, survival and anti-tumor cytotoxic effects (Blood Adv.4:1950 [2020]). Here, we describe the pre-clinical development of an OTS iMC/IL-15-enhanced CAR-NK cell platform targeting B cell maturation antigen (BCMA) for the treatment of multiple myeloma. Methods: NK cells were isolated from peripheral blood mononuclear cells by CD56+ selection, activated with IL-15 and microparticles conjugated with IL-21 and 4-1BB ligand. Activated NK cells were transduced with retrovirus encoding an optimized iMC and IL-15-expressing BCMA CAR construct (iMC-BCMA.z-IL15) where iMC signaling could be activated by exposure to rimiducid (Rim), a small molecule dimerizing ligand. Anti-tumor cytotoxicity and cytokine production was assessed using co-culture assays with control or modified CAR-NK cells against BCMA-expressing myeloma cells (NCIH929, RPMI8226, MM1S, U266 and NALM-6-BCMA). Additional experiments were performed with BCMA-edited cell lines (CRISPR/Cas9) to evaluate the innate cytotoxic potential of GoCAR-NK cells. In vivo anti-tumor efficacy and NK cell expansion was measured using immunodeficient NSG mice engrafted with 1.5 x 106 NCIH929-GFPffluc, MM1S-GFPffluc or THP1-GFPffluc cells followed by i.v. treatment with up to 1 x 107 BCMA GoCAR-NK cells. Tumor and NK cells were tracked via bioluminescence imaging. Results: Following IL-15 and IL-21/4-1BBL microparticle stimulation, NK cells were efficiently transduced (40-70%) and exhibited rapid ex vivo expansion (200-fold in 13 days). iMC-BCMA.CAR-IL15-modified NK cells exhibited potent cytotoxicity against BCMA+target cells compared with mock-transduced NK cells (MM1S, 58±4% versus 17±2%; Nalm-6-BCMA, 61±2% versus 19±6%) after 24 hours. Long-term (7 day) co-culture assays revealed the effect of iMC/IL-15 enhancement on NK cell potency, proliferation and cytokine production where iMC-BCMA.z-IL15-modified NK cells stimulated with Rim showed a >70% increase in tumor-specific killing compared to cells without iMC activation. Further, rimiducid-induced activation led to NK cell persistence and proliferation, 8.1±4.0-fold expansion compared to the start of the coculture. In comparison, there was an 80% reduction mock transduced NK cells or GoCAR-NK cells in cocultures without rimiducid. Induced-MC signaling also drove production of cytokines such as TNF-α, IFN-g (6.6X stimulation with 1 nM Rim relative to no drug), GMCSF, IP-10, and IL-13. In addition, activation of the iMC co-activation protein in combination with IL-15 secretion prevented NK cell exhaustion and led to retained functional activity of the modified GoCAR-NK cells for over 4-weeks in culture. In contrast, unmodified NK cells or modified GoCAR-NK cells without Rim exposure became functionally deficient. Of interest, a comparison of NK and T cells modified with the iMC/IL-15 BCMA CAR construct indicated that CAR-NK cells display more rapid target killing, which is further augmented by iMC-mediated cell signaling in the presence of Rim. Furthermore, GoCAR-NK cells were capable of lysis of BCMA-null target cells due to their innate anti-tumor activity. In vivo efficacy studies showed that neither iMC activation nor IL-15 secretion alone were sufficient to support CAR-NK cell engraftment in NSG mice but, in combination, they resulted in CAR-NK cell expansion and persistence. iMC/IL-15-enhanced BCMA GoCAR-NK cells proliferation was associated with improved control of tumor outgrowth in mice challenged with BCMA+ myeloma cells. Summary: These results indicate that the synergistic activity of iMC signaling combined with transgenic IL-15 production can enhance BCMA-specific CAR-NK cytotoxicity, cytokine production, long-term proliferation and persistence and may improve overall anti-tumor efficacy of a potential OTS cell therapy for the treatment of myeloma. Disclosures Wang: Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Duong:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Guerrero:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Mahendravada:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharp:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Brandt:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gagliardi:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Foster:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Bayle:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

Background: Chimeric antigen receptor (CAR) T cell therapy targeting solid tumors is challenged by inhibitory signals in the tumor microenvironment that may lead to CAR-T exhaustion. Previously, we described a GoCAR platform that uses a rimiducid (Rim)-inducible cytoplasmic costimulatory switch, inducible MyD88/CD40 (iMC), to drive the proliferation, survival, cytokine release and anti-tumor efficacy of T cells expressing a 1st generation CAR. To further understand how iMC signaling improves CAR-T potency, we directly compared iMC-based costimulation to conventional costimulatory domains (i.e., CD28 and 4-1BB) against hematological and solid tumor models. Methods: PBMCs from four donors were activated with anti-CD3/CD28 and transduced with retrovirus encoding 1st generation (CAR.ζ), 2nd generation (CAR.CD28.ζ CAR.4-1BB.ζ or GoCAR (iMC-2A-CAR.ζ) components targeting HER2, CD19 or GD2. Cocultures were established at a 1:1 ratio with antigen-expressing targets and cultures serially passaged weekly against fresh targets for up to six weeks. For in vivo efficacy evaluations NSG mice were engrafted s.c. with 1 × 106 HER2+ OE19-GFPffLuc esophageal carcinoma cells and challenged on day 4 with 5 × 106 CAR-T cells marked with RLuc each monitored by IVIS imaging. Results: 1st generation CAR-T cells produced low levels of IL-2 and IFNγ after the first antigen stimulation and quickly expressed markers of exhaustion including PD-1. 2nd generation CAR-T cells proliferated in early passage but became phenotypically and functionally exhausted following a second stimulation. Conversely, GoCAR-T cells were capable of expanding and sustaining IL-2 production beyond five passages, but only when stimulated with Rim. Lower levels of PD-1, TIM-3, and LAG-3 expression were observed in Rim-treated GoCAR-T cells when compared to 1st gen, 2nd gen and GoCAR-T cells without iMC activation, and PD1 expression remained repressed for six weeks in Rim-treated GoCAR-T cells. In NSG mice engrafted with HER2+ OE19 tumors, Rim treatment drove robust iMC-HER2.ζ CAR-T cell expansion compared to HER2.ζ, HER2.BB.ζ and HER2.28.ζ CAR-T cells over 5 weeks (p < 0.05). iMC-HER2.ζ CAR-T cells significantly enhanced tumor killing with Rim activation relative to vehicle-control and HER2.ζ-expressing T cells days post T cell infusion (tumor radiance = 1.63 × 107 ± 1.7E7 vs 1.40 × 108 ± 5.27E7 vs 1.70 × 108 ± 8.65E7, respectively, p Conclusions: iMC activation via Rim infusion provides on-demand control of CAR-T cell signaling to resist CAR-T cell exhaustion, enhance persistence, produce cytokines, and induce antitumor toxicity against solid tumors. Citation Format: MyLinh T. Duong, Aruna Mahendravada, Mary E. Brandt, Kelly L. Sharp, Aaron E. Foster, J. Henri Bayle. Inducible MyD88/CD40 (iMC) enhances CAR-T cell expansion and persistence by overcoming T cell exhaustion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2194.

Background: Effective chimeric antigen receptor (CAR) T cell therapy against solid tumors must overcome a hostile, tumor microenvironment that includes tumor-associated macrophages (TAM). Pancreatic adenocarcinomas (PDAC) are commonly infiltrated with TAMs polarized to a tumor-promoting M2 phenotype rather than a T cell-stimulatory and tumor-inhibitory M1 phenotype. The GoCAR platform combines an inducible MyD88/CD40 (iMC) costimulation protein with a 1st generation CAR. Our previously published results demonstrated that iMC costimulation, activated by the small molecule dimerizer, rimiducid (Rim), enhanced CAR-T proliferation and anti-tumor efficacy. Here, we examined the extrinsic effects of iMC signaling on CAR-T cell immune-activating ligands, cytokine production and their ability to polarize M2 macrophage to an anti-tumor phenotype. Methods: Macrophages were prepared from peripheral blood monocytes of four or more random blood donors from the Gulf Coast Regional Blood Center (Houston, TX) and differentiated in vitro to an M2 phenotype with TGF-β and IL-10, or an M1 phenotype (as a positive control). GoCAR-T cells targeting prostate stem cell antigen (PSCA) were prepared by retroviral transduction from the autologous donors. To test the effects of iMC activation on macrophage polarization, contact-dependent or independent (i.e., separation of cell populations with transwell inserts) coculture assays were performed with and without activation of iMC with 1 nM Rim and/or surface-bound PSCA. Anti-tumor cytotoxicity was measured by coculture with PSCA+ Panc1-GFP cells. Results: CAR activation by PSCA antigen recognition or iMC activation with Rim decreased CD163, an M2 macrophage marker, and increased CD80, expressed on M1 macrophage. Full activation of the GoCAR-T cells with both Rim and the target antigen fully repolarized M2 macrophage to an M1 marked phenotype (CD163lowCD80high). This repolarization could be directed partially in the absence of cell-cell contact by diffusion of soluble factors through Transwell membranes. M2 macrophages repolarized by conditioning media from activated GoCAR-T cells also exhibited the functionality of M1 macrophages and acquired cytotoxicity against tumor cells. Furthermore, cultures of conditioned macrophages and limiting dilutions of GoCAR-T cells demonstrated a cooperative enhancement of the cytotoxicity of PSCA GoCAR-T cells toward Panc-1 targets. This cooperation was more effective for GoCAR-T cells than CD28- or 4-1BB-enhanced 2nd generation PSCA CAR-T cells. Conclusions: These results predict that GoCAR-T activation with Rim will convert TAMs within a solid tumor microenvironment from T cell inhibitors to tumor-caustic agents. Citation Format: Xiaohong Wang, Konrad Gabrusiewicz, David M. Spencer, Aaron E. Foster, J. Henri Bayle. Small molecule inducible MyD88/CD40 (iMC) in CAR-T cells can repolarize M2 macrophage to an anti-tumor M1 phenotype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2193.

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.

Background Natural Killer (NK) lymphocytes possess innate anti-tumor activity that has the potential to be used as an allogeneic cell therapy due to reduced GvHD risk relative to αβ T cells. Despite their potential, adoptive NK cell immunotherapies have been limited by poor expansion in vivo. Using our previously developed Chimeric Antigen Receptor-T cell (CAR-T) strategy that relies on rimiducid-based dimerization of inducible MyD88/CD40 (iMC) to regulate T cell expansion and survival, we demonstrate that iMC can also be applied to NK cell growth and anti-tumor efficacy in vitro and in vivo. Moreover, a rapamycin-inducible Caspase-9 (iRC9) was used to provide an orthogonally regulated safety switch. Methods and Results CD56+ NK cells were isolated from peripheral blood of human donors, stimulated overnight with IL-15 then activated by seeding with K562 erythroleukemia target cells. NK cells were then transduced with γ-retrovirus encoding control iRC9-2A-ΔCD19, iRC9-2A-ΔCD19-2A-iMC (dual-switch NK) or iRC9-2A-IL-15-2A-ΔCD19-2A-iMC (dual-switch/IL-15 NK). ΔCD19 marked transduced cells in 50:50 cocultures with untransduced NK cells. NK cells containing only iRC9 grew at the same rate as untransduced cells, but iMC-expressing NK cells displayed enhanced growth that was further augmented by 1 nM rimiducid treatment. In cocultures with THP1 acute myeloid leukemia cells at increasing Target:Effector (T:E) ratios, presence (P < 0.001, two way ANOVA) and activation (P To study in vivo anti-tumor activity, immunodeficient NSG mice were engrafted with dual-switch NK cells with or without autocrine IL-15 expression in the presence or absence of THP-1 tumor targets. When tumor was present, unstimulated iMC with IL-15 or activation of iMC without IL-15 expression supported modest NK cell expansion, but rimiducid stimulation of iMC plus autocrine IL-15 showed enhanced NK expansion in vivo. Furthermore, in tumor-free animals only dual-switch/IL-15 NK cells with weekly rimiducid stimulation expanded and persisted in vivo (up to 7 weeks). Cotransduction of a first generation CD123-targeted CAR to produce dual-switch/IL-15 CD123CAR-NK cells led to rimiducid-dependent control of THP1 tumor outgrowth in vivo beyond 40 days. Conversely, temsirolimus-mediated activation of the iRC9 safety switch rapidly (< 24 hours) ablated dual-switch NK cells in vivo. Conclusions Inducible MyD88/CD40 is an activation switch that supports NK cell expansion, persistence and anti-tumor activity. When paired with autocrine IL-15 expression, this platform supports NK expansion and persistence in vivo, and AML tumoricidal activity that can be further activated by target-specific CAR expression. Moreover, the fast-acting, orthogonally regulated proapoptotic switch, iRC9, mitigates the risk of off-tumor targeting. Therefore, we describe a novel, regulated NK cell platform that solves many of the challenges of NK cell-based therapy and should be amenable to a readily translatable off-the-shelf cellular therapy for malignancies. Disclosures Wang: Bellicum Pharmaceuticals: Employment, Equity Ownership. Chang:Bellicum Pharmaceuticals: Employment, Equity Ownership. Jasinski:Bellicum Pharmaceuticals: Employment, Equity Ownership. Medina:Bellicum Pharmaceuticals: Employment, Equity Ownership. Zhang:Bellicum Pharmaceuticals: Employment, Equity Ownership. Foster:Bellicum: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Bayle:Bellicum Pharmaceuticals: Employment, Equity Ownership.

Background Despite impressive efficacy in liquid tumors, improved CAR-T efficacy and persistence appear necessary to control solid tumors, but this increased potency will likely increase the risk of toxicity. Here, we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to cognate ligands. Cell activation is controlled by rimiducid (Rim), which triggers signaling cascades downstream of MyD88 and CD40 via an engineered chimeric protein termed iMC. A rapamycin (Rap)-controlled apoptotic switch, iRC9, is co-expressed, which induces dimerization of caspase-9 active domains to mitigate possible toxicity from excessive CAR-T function. When combined with a first generation CAR in a single vector, these molecular switches allow for specific and efficient regulation of engineered T cells to eliminate HER2+ and PSCA+ cancer cells. Results Transduction of activated T cells using γ-retroviruses encoding the unified dual-switch (DS) HER2- or PSCA-CAR components (SFG-iRC9.2A-CAR.ζ.2A-iMC) yielded efficient transgene expression despite the large insert size (60.5±8.7% CAR+). Greater transduction efficiency was achieved with single-cell producer clones encoding the PSCA DS CAR vector (79.3±2.9% CAR+). When exposed to target antigen in a coculture assay with tumor cells, robust IL-2 and IFN- γ production by DS CAR-T cells was Rim-dependent. Tumor cell killing and T cell expansion were also enhanced by Rim stimulation. In an OE19 tumor-bearing mouse model, Rim stimulation of HER2 DS CAR-T cells significantly enhanced tumor killing (205.8±58.3 mm2 vehicle (veh)-treated vs 55.9±10.9 mm2 Rim-treated, p80 days). When necessary, deployment of the off-switch (iRC9) rapidly (½ Vmax ~ 8 hours) and efficiently eliminated DS CAR-T cells in a caspase-3 activation assay with real-time (IncuCyte) monitoring, as well as AnnV/PI detection by flow cytometry (3.2±1.0% AnnV+/PI+ veh-treated vs 80.0±5.2% 10nM Rap-treated, p Summary Dual-switch CAR-T, a novel platform comprising a CAR combined with regulated costimulation and apoptotic signaling elements, effectively control solid tumor growth and T cell expansion and elimination. This technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells. Citation Format: MyLinh Duong, Eva Morschl, Aruna Mahendravada, Matthew Collinson-Pautz, Mary Brandt, Ming Zhang, Aaron Foster, J. Henri Bayle, David Spencer. A unified dual-switch CAR vector to target solid cancer with controllable "on" and "off" states [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-141.

SummaryDeveloping myocardial cells respond to signals from the endocardial layer to form a network of trabeculae that characterize the ventricles of the vertebrate heart. Abnormal myocardial trabeculation results in specific cardiomyopathies in humans and yet trabecular development is poorly understood. We show that trabeculation requires Brg1, a chromatin remodeling protein, to repress ADAMTS1 expression in the endocardium that overlies the developing trabeculae. Repression of ADAMTS1, a secreted matrix metalloproteinase, allows the establishment of an extracellular environment in the cardiac jelly that supports trabecular growth. Later during embryogenesis, ADAMTS1 expression initiates in the endocardium to degrade the cardiac jelly and prevent excessive trabeculation. Thus, the composition of cardiac jelly essential for myocardial morphogenesis is dynamically controlled by ADAMTS1 and its chromatin-based transcriptional regulation. Modification of the intervening microenvironment provides a mechanism by which chromatin regulation within one tissue layer coordinates the morphogenesis of an adjacent layer.

We have developed a general method of making conditional alleles that allows the rapid and reversible regulation of specific proteins. A mouse line was produced in which proteins encoded by the endogenous glycogen synthase kinase-3 beta (GSK-3beta) gene are fused to an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3beta, producing a severe loss-of-function allele. In the presence of C20-MaRap, a highly specific, nontoxic, cell-permeable small molecule, GSK-3betaFRB* binds to the ubiquitously expressed FKBP12 protein. This interaction stabilizes GSK-3betaFRB* and restores both protein levels and activity. C20-MaRap-mediated stabilization is rapidly reversed by the addition of an FKBP12 binding competitor molecule. This technology may be applied to a wide range of FRB*-tagged mouse genes while retaining their native transcriptional control. Inducible stabilization could be valuable for many developmental and physiological studies and for drug target validation.

The bifunctional protein DCoH (Dimerizing Cofactor for HNF1) acts as an enzyme in intermediary metabolism and as a binding partner of the HNF1 family of transcriptional activators. HNF1 proteins direct the expression of a variety of genes in the liver, kidney, pancreas, and gut and are critical to the regulation of glucose homeostasis. Mutations of the HNF1α gene underlie maturity onset diabetes of the young (MODY3) in humans. DCoH acts as a cofactor for HNF1 that stabilizes the dimeric HNF1 complex. DCoH also catalyzes the recycling of tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases. To examine the roles of DCoH, a targeted deletion allele of the murine DCoH gene was created. Mice lacking DCoH are viable and fertile but display hyperphenylalaninemia and a predisposition to cataract formation. Surprisingly, HNF1 function in DCoH null mice is only slightly impaired, and mice are mildly glucose-intolerant in contrast to HNF1α null mice, which are diabetic. DCoH function as it pertains to HNF1 activity appears to be partially complemented by a newly identified homolog, DCoH2.

Background: While chimeric antigen receptor (CAR)-T immunotherapies have shown remarkable efficacy against leukemias and lymphomas, improved CAR-T efficacy and persistence are needed to overcome solid tumors, without compromising safety. Here, we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to their cognate ligands. Cell activation is controlled by the homodimerizer rimiducid that triggers signaling cascades downstream of MyD88 and CD40 via an engineered chimeric protein termed iMC. A rapamycin-controlled pro-apoptotic switch, iRC9, is co-expressed, which induces dimerization of the caspase-9 domain to mitigate possible toxicity from excessive CAR-T function. When combined with a first generation CAR, these molecular switches allow for specific and efficient regulation of engineered T cells. Methods & Results: T cells were activated and co-transduced with the HER2 GoCAR (SFG-iMC.2A-CAR.ζ) and RapaCIDe (SFG-iRC9.2A-ΔCD19) vectors to generate “Dual-switch GoCAR-T” cells. Combined transduction of RapaCIDe and HER2 GoCAR vectors into T cells did not adversely affect the antitumor efficacy of the GoCAR-T cells, which eliminated OE19 esophageal tumor cells in a 7-day coculture assay at a 1:20 effector to target ratio (3.9±4.3% OE19-eGFPFluc cells remained in GoCAR-modified cultures vs. 1.1±0.1% for the dual-switch GoCAR), and promoted T cell expansion (53.4±9.4% CAR+ for GoCAR vs. 44.6±13.2% for the dual-switch). When challenged in an OE19 tumor-bearing mouse model, rimiducid stimulation of the dual-switch GoCAR-T cells enhanced tumor killing and T cell expansion. Deployment of the off-switch induced fast (½ Vmax ~ 8 hours) and efficient elimination of T cells (Dual-switch GoCAR-T = 94.1% AnnV+/PI+ vs. GoCAR-T = 5.1%) in a caspase-3 activation assay with real-time (IncuCyte) monitoring as well as AnnV/PI detection by flow cytometry. In vivo assessment of the RapaCIDe switch was performed via eGFPluciferase (eGFPfluc)-labeled RapaCIDe-T cells in NSG mice. Rapamycin, but not rimiducid, treatment efficiently eliminated RapaCIDe-T cells within 24 hours, which is similar to the rate observed by the clinically validated rimiducid-regulated CaspaCIDe switch. Summary: Dual-switch GoCAR-T, a novel platform comprising a first-generation CAR combined with regulated costimulation and apoptotic signaling elements, effectively controlled tumor growth and T cell expansion and elimination in vitro and in vivo. This dual-switch technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells. Citation Format: MyLinh T. Duong, Matthew R. Collinson-Pautz, Eva Morschl, Mary E. Brandt, Ming Zhang, Kevin W. Slawin, Aaron E. Foster, J. Henri Bayle, David M. Spencer. Dual-switchGoCAR-T cells: small molecule-regulated “GO” and “STOP” switches to target solid cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-184. doi:10.1158/1538-7445.AM2017-LB-184

T cells engineered to express the α and β chains of antigen-specific T cell receptors (TCRs) have shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor persistence of gene-modified T cells. Additionally, severe toxicities, including patient deaths, have occurred upon infusion of large numbers of TCR-modified T cells. To enhance T cell persistence while providing a safeguard against life-threatening toxicity, we developed a dual-switch αβ TCR platform that uses a rapamycin (Rap)-induced caspase-9 (iRC9) together with a rimiducid (Rim)-controlled activation switch, inducible MyD88/CD40 (iMC). The αβ TCR sequence derived from an HLA-A2-restricted, PRAME-specific T cell clone was synthesized and placed in-frame with iMC, comprising signaling domains from MyD88 and CD40 fused to tandem Rim-binding mutant FKBP12v36 domains to generate the iMC-PRAME TCR. Caspase-9 was fused to FRB and wild-type FKBP12 domains and cloned in-frame with a selectable marker, truncated CD19 (ΔCD19), to generate iRC9-ΔCD19 retrovirus. All modules were separated by 2A polypeptide, bond-skipping sequences. Activated human T cells were dual-transduced with iMC-PRAME TCR and iRC9-ΔCD19 viruses and subsequently enriched for CD19 expression using magnetic selection. iMC and iRC9 were activated by exposing transduced T cells to 10 nM Rim or Rap, respectively. Proliferation, cytokine production and cytotoxicity of TCR-modified T cells were assessed in co-culture assays with U266 (myeloma) and THP-1 (AML) cells in the presence or absence of inducible ligands. T cells transduced with iMC-PRAME TCR and iRC9-ΔCD19 showed efficient and stable expression for TCR and ΔCD19 post-CD19 selection (82±9% CD3+Vβ1+, 96±2% CD3+CD19+). In coculture assays, dual-switch PRAME TCR-modified T cells demonstrated specific lysis of HLA-A2+PRAME+ THP-1 and U266 tumor cells compared to an irrelevant TCR (CMVpp65-specific)-modified T cells with or without iMC activation. However, Rim exposure induced a 42-fold induction of IL-2 (9±0.3 versus 385±180 pg/ml IL-2) and resulted in 13-fold greater expansion of TCR-modified T cells. The expression of iRC9 did not interfere with TCR function, nor with the synergy between TCR and iMC activation. Furthermore, exposure to Rap triggered rapid apoptosis of dual-switch TCR-modified T cells (72±5% Annexin-V+ with Rap versus 14±4% without drug), indicating that the suicide switch is also functional. iMC is a potent “Go” switch that utilizes rimiducid to provide costimulation to TCR-engineered T cells. In addition, iRC9 is a novel rapamycin-inducible suicide switch that can eliminate T cells in case of severe toxicity. This iMC-enhanced iRC9-incorporating TCR is the first reported prototype of a dual switch TCR-engineered T cell therapy to increase efficacy, durability and safety of adoptive T cell therapies. Citation Format: Tsvetelina Pentcheva-Hoang, Tania Rodriguez, David Torres, Ana Korngold, Jeannette Crisostomo, MyLinh T. Duong, Matthew Collinson-Pautz, J. Henri Bayle, Mirjam HM Heemskerk, J.H. Frederik Falkenburg, Annemarie Moseley, Kevin M. Slawin, David Spencer, Aaron E. Foster. Dual-switch TCR: A two-ligand system to control PRAME TCR-modified T cell proliferation and death using inducible MyD88/CD40 and caspase-9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2017-3745

Rapamycin at high doses (2-10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 μg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca(2+) influx to enhance refilling of sarcoplasmic reticulum Ca(2+) stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.

Introduction: Promising clinical results with CD19-specific chimeric antigen receptor (CAR)-directed T cells for the treatment of B cell leukemia and lymphoma suggest that CARs may be effective in other hematological malignancies, such as acute myeloid leukemia (AML). CD123/IL-3Rα is an attractive CAR-T cell target due to its high expression on both AML blasts and leukemic stem cells (AML-LSCs). However, the antigen is also expressed at lower levels on normal stem cell progenitors presenting a major toxicity concern should CD123-specific CAR-T cells show long-term persistence. Here, we describe a CAR platform, "GoCAR-T", which uses a proliferation-deficient, first generation, CD123-specific CAR together with a ligand (rimiducid (Rim))-dependent costimulatory switch (inducible MyD88/CD40 (iMC)) to provide physician-controlled eradication of CD123+ tumor cells and regulate long-term CAR-T cell engraftment. Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a bicistronic retrovirus encoding tandem Rim-binding domains (FKBP12v36),cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and first generation CAR targeting CD123 (SFG-iMC-CD123.ζ). The effects ofiMC costimulation on CD123-targeted CARs were assessed in coculture assays with CD123+, EGFPluciferase (EGFPluc)-modified leukemic cell lines (KG1, THP-1 and MOLM-13) with and without Rim using the IncuCyte live cell imaging system. IL-2 production was examined by ELISA from coculture supernatants. In vivo efficacy of iMC-CD123.ζ-modified T cells was assessed using an immune-deficient NSG tumor xenograft model. One million EGFPluc-expressing CD123+ THP-1 tumor cells were injected i.v. into the animals, followed by a single i.v. injection on day 7 with 2.5x106 non-transduced or iMC-CD123.ζ-modified T cells. Groups receiving CAR-T cells subsequently received i.p. injections of Rim (1 mg/kg) or vehicle only on days 0 and 15 post-T cell injection. Animals were evaluated for THP-1-EGFPluc tumor burden and weight change on a weekly basis using IVIS bioluminescent imaging (BLI) and for T cell persistence by flow cytometry and qPCR at day 30 post-T cell injection. Results: SFG-iMC-CD123.ζ efficiently transduced activated T cells (66±8%) and showed antigen-specific cytolytic function against CD123+ leukemic cell lines. However, in coculture assays both CAR antigen recognition and Rim-dependent iMC costimulation were required for IL-2 production (285±41 versus 2,541±255 pg/ml for control and 1 nM Rim, respectively), robust CAR-T cell proliferation (87-fold increase with Rim stimulation) and enhanced tumor cell killing. In NSG mice engrafted with CD123+ THP-1-EGFPluc tumor cells, only animals were treated with iMC-CD123.ζ-modified T cells and 1 mg/kg Rim on days 0 and 15 post-T cell injection controlled tumor growth, showing a 2-log (average radiance: NT = 3.0x108; CAR + Vehicle = 2.4x108; CAR + Rim=8.4x105 p/sec/cm2/sr) reduction in tumor burden compared to mice receiving CAR-T cells and vehicle only (Figure 1). Approximately two weeks after the second Rim injection (day 30), CAR-T cells were infrequent ( Summary: GoCAR-T, a platform comprising a ligand-dependent activation switch and a proliferation-deficient first generation CAR, efficiently eradicates CD123+ leukemic cells when costimulation is provided by systemic rimiducid administration. Deprivation of iMC costimulation results in reduction of CAR-T levels, providing a user-controlled system for managing persistence and safety of CD123-specific CAR-T cells. Disclosures Foster: Bellicum Pharmaceuticals: Employment. Duong:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Bayle:Bellicum Pharmaceuticals: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.

Background: While chimeric antigen receptor (CAR)-T immunotherapies are remarkably effective against a subset of leukemias and lymphomas, three current hurdles for broad deployment include lack of regulation once administered to the patient, modest efficacy against solid tumors, and the necessity to make separate GMP vectors for each tumor target. Methods: We developed two methods that utilize chemical induction of protein dimerization (CID) to regulate the activity of engineered T cells containing a CAR of broad utility. “Uni-iC9CAR” combines Bellicum's caspase-9-based, rimiducid-inducible safety switch, CaspaCIDe, with a first generation CD16/FCGR3A–CAR. Antigen receptor specificity relies on the interaction of the Fc-binding domain of CD16 with various tumor-targeted antibodies. In the “Uni-GoCAR” strategy, signaling domains from MyD88 and CD40 are fused to two copies of FKBPv36 to generate iMC, which is co-expressed with the CD16-based CAR. In each strategy, the same dimerizer, rimiducid, binds to FKBPv36 with sub-nanomolar affinity to cause the activation of signaling molecules with distinct functions and outcomes. Lastly, we generated the “Uni-CIDeCAR” vector that combines the iCaspase-9 and CD16-CAR activities from Uni-iC9CAR with augmented ligand-independent MyD88/CD40 costimulation to generate a potent universal CAR with a rapid and effective suicide gene, activated by the normally bio-inert ligand, rimiducid. Results: Both Uni-GoCAR and Uni-iC9CAR constructs demonstrated rapid, effective and durable, rituximab-dependent antitumor activity when expressed in human T cells and mixed with Raji B cells as early as 7 days after T cell co-culture at a 1:1 ratio. Additionally, rimiducid induced robust cytokine production, including IL-2 and IL-6, and proliferation in T cells transduced with the Uni-GoCAR vector, which expresses the iMC activation switch. In contrast, co-expression of iC9 in the Uni-iC9CAR vector demonstrated robust rimiducid-dependent T cell apoptosis, thus providing a valuable safety mechanism for clinical applications. Finally, in the enhanced, but regulated, Uni-CIDeCAR vector, iC9 maintains safety in a CD16-CAR that is functionally enhanced by rimiducid-independent, basal MC activity. Conclusion: We report an improved “universal” CAR-T technology that employs a CD16-based CAR (described by Kudo et al (14) Cancer Res) coupled with Bellicum's costimulatory and safety switches to effectively target tumor cells while providing a broad clinical safety net. Citation Format: MyLinh T. Duong, Matthew R. Collinson-Pautz, Aaron E. Foster, J. Henri Bayle, David M. Spencer. Uni-CIDeCAR-T cells: MyD88/CD40-enhanced, Ab-directed CAR incorporating the CaspaCIDe® safety switch. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A057.

We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell, 2003, 12, 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Delta G) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilized by up to 6 kcal mol(-1) relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Delta G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Delta G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.

Histone acetyltransferases and deacetylases are involved in the regulation of gene transcription. Recently, tumor suppressor protein p53 has been shown to be a target for transcriptional coactivators that have histone acetyltransferase activity, suggesting acetylation is also involved in the regulation of cell proliferation and tumorigenesis.

The delicate leaflets that make up vertebrate heart valves are essential for our moment-to-moment existence. Abnormalities of valve formation are the most common serious human congenital defect. Despite their importance, relatively little is known about valve development. We show that the initiation of heart valve morphogenesis in mice requires calcineurin/NFAT to repress VEGF expression in the myocardium underlying the site of prospective valve formation. This repression of VEGF at E9 is essential for endocardial cells to transform into mesenchymal cells. Later, at E11, a second wave of calcineurin/NFAT signaling is required in the endocardium, adjacent to the earlier myocardial site of NFAT action, to direct valvular elongation and refinement. Thus, NFAT signaling functions sequentially from myocardium to endocardium within a valvular morphogenetic field to initiate and perpetuate embryonic valve formation. This mechanism also operates in zebrafish, indicating a conserved role for calcineurin/NFAT signaling in vertebrate heart valve morphogenesis.

SummaryControlling protein dimerization with small molecules has broad application to the study of protein function. Rapamycin has two binding surfaces: one that binds to FKBP12 and the other to the Frb domain of mTor/FRAP, directing their dimerization. Rapamycin is a potent cell growth inhibitor, but chemical modification of the surface contacting Frb alleviates this effect. Productive interactions with Frb-fused proteins can be restored by mutation of Frb to accommodate the rapamycin analog (a rapalog). We have quantitatively assessed the interaction between rapalogs functionalized at C16 and C20 and a panel of Frb mutants. Several drug-Frb mutant combinations have different and nonoverlapping specificities. These Frb-rapalog partners permit the selective control of different Frb fusion proteins without crossreaction. The orthogonal control of multiple target proteins broadens the capabilities of chemical induction of dimerization to regulate biologic processes.