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.

Abstract 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, p<0.05) and T cell expansion (4.6E5 average radiance veh-treated vs 1.9E6 Rim-treated). When targeting PSCA+ HPAC cancer cells, PSCA DS CAR-T cells proliferated in a Rim dose-dependent manner and tumor control was maintained even after cessation of Rim treatment. In a second pancreatic tumor model, SU8686, Rim treatment induced long-term tumor control (>80 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<0.005). In vivo assessment of the iRC9 switch was performed via eGFPluciferase (eGFPfluc)-labeled PSCA dual-switch CAR-T cells in NSG mice. Temsirolimus (Tem; a Rap prodrug) treatment efficiently eliminated PSCA DS CAR-T cells within 6 hours (6.1E4 average radiance veh-treated vs 2.1E4 0.4mg/kg Tem-treated, p<0.0001). 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.

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.

Abstract 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

Background Improvement of the efficacy and safety of chimeric antigen receptor (CAR)-T immunotherapies requires controlled activation and termination of the T cells when transfused into patients. Here we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to their cognate ligands. T cell costimulation is controlled by the homodimerizer rimiducid that triggers signaling cascades downstream of MyD88 and CD40 via an engineered protein termed iMC. A rapamycin-controlled pro-apoptotic switch (iRC9) that induces dimerization of caspase-9 mitigates possible CAR-T cell toxicity. iRC9 is a chimeric protein comprisingan FKBP-rapamycin binding (FRB) domain in tandem with FKBP12 and caspase-9. This design permits rapamycin, a heterodimerizing ligand, to function as a homodimerizer. When combined with a first generation CD123-specific CAR, these molecular switches allow for controlled, robust expansion of engineered T cells to control acute myelogenous leukemia (AML) in vitro and in vivo combined with a rapid and efficient safety mechanism to block excessive cytokine release. Methods & Results T cells were activated and co-transduced with pSFG-iMC.2A.CD123CAR.ζ and pSFG-iRC9.2A.ΔCD19 vectors to generate Dual-switch (DS) CAR-T cells. Combined transduction of iRC9 and iMC-CAR vectors produced CD123-directed CAR-T cells that eliminated CD123+ THP1 and MOLM13 AML cells, but not CD123- HPAC tumor cells, in a co-culture assay. Cytokine secretion and target cell killing were dependent on the dose of rimiducid (EC50 Deployment of the off-switch induced fast (½ Vmax ~ 8 hours) and efficient T cell elimination of in a caspase-3 activation assay with real-time monitoring by Incucyte microscopy as well as Annexin V detection by flow cytometry (DS CAR-T = 77.6% versus untransduced = 2.2% Annexin V+ when treated with 1 nM rapamycin). In vivo assessment of the suicide switch was performed with eGFP Fluc -labeled CD123 DS CAR-T cells in NSG mice. Rapamycin, but not rimiducid, treatment efficiently eliminated DS CAR-T cells within 24 hours in NSG mice, which is similar to the clinically validated rimiducid-regulated iC9 switch. Importantly, the off-switch was insensitive to high rimiducid concentration, demonstrating that the on-switch regulator does not crosstalk with the safety switch. Summary Dual switch CAR-T, a novel platform comprising a first-generation CAR combined with regulated activation 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. Disclosures Bayle: Bellicum Pharmaceuticals: Employment, Equity Ownership. Duong: Bellicum Pharmaceuticals: Employment, Equity Ownership. Lu: Bellicum Pharmaceuticals: Employment. Morschl: Bellicum Pharmaceuticals: Employment, Equity Ownership. Collinson-Pautz: Bellicum Pharmaceuticals: Employment, Equity Ownership. Sharp: Bellicum Pharmaceuticals: Employment, Equity Ownership. Szymanski: Bellicum Pharmaceuticals: Employment, Equity Ownership. Brandt: Bellicum Pharmaceuticals: Employment, Equity Ownership. Slawin: Bellicum Pharmaceuticals: Consultancy, Equity Ownership. Toler: Bellicum Pharmaceuticals: Employment, Equity Ownership. Yvon: Bellicum Pharmaceuticals: Equity Ownership. Foster: Bellicum Pharmaceuticals: Employment, Other: stockholders . Spencer: Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders .

206 Background: PSCA is a cell surface antigen that is overexpressed in a majority of metastatic prostate, transitional cell and pancreatic carcinomas. We describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), activated by a small molecule, rimiducid, to enhance survival, proliferation and anti-tumor activity of CAR-T cells targeting PSCA. Methods: T cells were transduced with a retrovirus encoding tandem rimiducid-binding domains,cloned in-frame with MyD88 and CD40 signaling elements and first generation CARs (CAR.ζ) targeting PSCA (SFG-iMC-2A-PSCA.ζ). iMC activation was assessed with and without rimiducid treatment of T cells. Coactivation via iMC and CAR was tested in coculture assays with or without rimiducid using various PSCA+tumor cells, e.g. Capan-1 and HPAC pancreatic adenocarcinoma. Efficacy of iMC-modified CAR-T cells in vivo was assessed using an NSG mouse tumor model. Results: T cells transduced with iMC-PSCA.ζ produced cytokines (e.g., IFN-γ and IL-6) in response to rimiducid; however, IL-2 was only produced when both iMC and CAR were activated simultaneously by rimiducid and tumor antigen. Treatment of NSG mice bearing large (> 200 mm3) HPAC tumors with a single i.v. dose of 1x107 iMC-PSCA.ζ cells resulted in complete tumor elimination in 10/10 mice including both rimiducid-treated and untreated animals, compared to mice receiving non-transduced T cells (p = 0.0003). Weekly rimiducid administration dramatically increased CAR-T cell numbers, resulting in a 23-fold expansion of iMC-PSCA.ζ-modified T cells in the spleen compared to mice not receiving rimiducid four weeks after infusion (p = 0.02). In a dose-titration study, rimiducid administration was required for tumor control, and led to 565- and 948-fold T cell expansion at the tumor site, respectively, when lower numbers (1.25x106 or 6.25x105, respectively) of iMC-PSCA.ζ-modified T cells were given. Conclusions: GoCAR-T cells targeting PSCA, which contain an inducible MyD88/CD40 activation switch, may be an effective adoptive cell therapy for patients with pancreatic, prostate, bladder, and other cancers that overexpress PSCA.

Introduction: Adoptive transfer of T cells, genetically engineered to express chimeric antigen receptors (CARs) containing costimulatory domains, such as CD28 or 4-1BB, has yielded impressive clinical results in some blood cancers, but severe toxicities have been observed due to unchecked T cell activation. In contrast, CAR-T cells have demonstrated limited clinical efficacy, associated with poor engraftment, survival and proliferation of adoptively transferred cells when used to target a variety of solid tumors. Thus, technologies that can regulate T cell activation and proliferation in vivo should both mitigate toxicities and maximize anti-tumor efficacy, expanding their clinical utility to a wider range of indications. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), activated by a small molecule chemical inducer of dimerization, rimiducid, to enhance survival and drive T cell proliferation.

Introduction: Efficacy of chimeric antigen receptor (CAR)-modified T cells is dependent on their in vivo survival and expansion following infusion. The addition of accessory molecules (e.g., costimulatory and cytokine genes) may improve CAR-T proliferation and potency, but may also increase toxicity of these next generation CAR-T cell therapies, suggesting that the incorporation of a built in "safety switch" would balance safety and efficacy in a single, controllable therapy. Here, we demonstrate that cytosolic coexpression of a MyD88/CD40-derived fusion protein dramatically enhances CAR-T activation, cytokine production, and proliferation in vivo, resulting in improved antitumor efficacy. Importantly, CAR-T cell numbers, elevated cytokine levels, and observed CAR-T-related toxicity could be controlled by titratable rimiducid administration to reduce or eliminate CAR-T cells by activating the inducible caspase-9 (iC9) suicide gene.

OBJECTIVE:In the everyday life, stress is deemed as something unfavorable that may enhance the risk for the development or worsen a disease. However, in its nature, stress is adaptive reaction of the body. Its main characteristic is the activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Previously, we have shown that activation of the HPA axis plays a gastroprotective role during acute stress. The aim of our study was to clarify the effects of chronic stress and chronically elevated basal corticosterone levels on the gastric ulceration and cardiovascular vulnerability in rats.METHODS:Male Wistar rats were repeatedly restrained 60 min daily for 14 days and examined on day 15th. The gastric ulceration was induced by a s.c. injection of indomethacin (35 mg/kg). The cardiovascular vulnerability was examined in urethane-anaesthetized rats in an experimental angina pectoris model (epinephrine, 10 µg/kg, 30 s later phentolamine, 15 mg/kg, both i.v.).RESULTS:We confirmed the development of chronic stress consequences by changes in several somatic parameters (body weight decrease, thymus involution, adrenal gland hypertrophy), and elevated resting corticosterone levels. However, the gastroprotective effect of chronic stress was not manifested and there was no aggravation of indomethacin-induced gastric ulceration, either. In the experimental angina pectoris model, previous chronic stress did not have any profound effect on the blood pressure, heart rate, and electrocardiogram changes.CONCLUSIONS:In contrast to the general view on the harmfulness nature of the stress, we were unable to find a harmful effect of chronic stress on the internal diseases (gastric ulceration and angina pectoris). However, its protective effect was also missing among present experimental conditions.

Activation of complement is one of the earliest immune responses to exogenous threats, resulting in various cleavage products including anaphylatoxin C3a. In addition to its contribution to host defense, C3a has been shown to mediate Th2 responses in animal models of asthma. However, the role of C3a on pulmonary Th17 responses during allergic inflammation remains unclear. Here, we show that mice deficient in C3a receptor (C3aR) exhibited (i) higher percentages of endogenous IL-17-producing CD4+ T cells in the lungs, (ii) higher amounts of IL-17 in the bronchoalveolar lavage fluid, and (iii) more neutrophils in the lungs than wild-type mice when challenged with intranasal allergens. Moreover, adoptive transfer experiments showed that the frequencies of antigen-specific IL-17-producing CD4+ T cells were significantly higher in the lungs and bronchial lymph nodes of C3aR-deficient recipients than those of wild-types recipients. Bone-marrow reconstitution study indicated that C3aR-deficiency on hematopoietic cells was required for the increased Th17 responses. Furthermore, C3aR-deficient mice exhibited increased percentages of Foxp3+ regulatory T cells; however, depletion of these cells minimally affected the induction of antigen-specific Th17 cell population in the lungs. Neutralization of IL-17 significantly reduced the number of neutrophils in bronchoalveolar lavage fluid of C3aR-deficient mice. Our findings demonstrate that C3a signals negatively regulate antigen-specific Th17 responses during allergic lung inflammation and the size of Foxp3+ regulatory T cell population in the periphery.

Adenosine is an extracellular signaling molecule that is generated in response to cell injury where it orchestrates tissue protection and repair. Whereas adenosine is best known for promoting anti-inflammatory activities during acute injury responses, prolonged elevations can enhance destructive tissue remodeling processes associated with chronic disease states. The generation of adenosine and the subsequent activation of the adenosine 2B receptor (A(2B)R) is an important processes in the regulation of both acute and chronic lung disease. The goal of this study was to examine the contribution of the A(2B)R in models of bleomycin-induced lung injury that exhibit varying degrees of acute and chronic injury. Intratracheal bleomycin exposure results in substantial acute lung injury followed by progressive fibrosis. In this model, genetic removal of the A(2B)R resulted in enhanced loss of barrier function and increased pulmonary inflammation, with few differences in indexes of pulmonary fibrosis. These results support an anti-inflammatory role for this receptor in this model of acute lung injury. In contrast, systemic exposure of mice to bleomycin resulted in modest acute lung injury together with progressive pulmonary fibrosis. In this model, the effects of A(2B)R removal on acute lung injury were negligible; however, there were substantial reductions in pulmonary fibrosis, supporting a profibrotic role for this receptor. A(2B)R-dependent regulation of IL-6 production was identified as a potential mechanism involved in the diminished pulmonary fibrosis seen in A(2B)R knockout mice exposed to i.p. bleomycin. These studies highlight the distinct roles of A(2B)R signaling during acute and chronic stages of lung injury.

Pharmacologic evidence suggests that activation of A(2B) adenosine receptors results in proinflammatory effects relevant to the progression of asthma, a chronic lung disease associated with elevated interstitial adenosine concentrations in the lung. This concept has been challenged by the finding that genetic removal of An receptors leads to exaggerated responses in models of acute inflammation. Therefore, the goal of our study was to determine the effects of A(2B) receptor gene ablation in the context of ovalbumin-induced chronic pulmonary inflammation. We found that repetitive airway allergen challenge induced a significant increase in adenosine levels in fluid recovered by bronchoalveolar lavage. Genetic ablation of A(2B) receptors significantly attenuated allergen-induced chronic pulmonary inflammation, as evidenced by a reduction in the number of bronchoalveolar lavage eosinophils and in peribronchial eosinophilic infiltration. The most striking difference in the pulmonary inflammation induced in A(2B) receptor knockout (A(2B)KO) and wildtype mice was the lack of allergen-induced IL-4 release in the airways of A(2B)KO animals, in line with a significant reduction in IL-4 protein and mRNA levels in lung tissue. In addition, attenuation of allergen-induced transforming growth factor-beta release in airways of A(2B)KO mice correlated with reduced airway smooth muscle and goblet cell hyperplasia/hypertrophy. In conclusion, genetic removal of A(2B) adenosine receptors in mice leads to inhibition of allergen-induced chronic pulmonary inflammation and airway remodeling. These findings are in agreement with previous pharmacologic studies suggesting a deleterious role for A(2B) receptor signaling in chronic lung inflammation.

Extracellular adenosine is produced in a coordinated manner from cells following cellular challenge or tissue injury. Once produced, it serves as an autocrine- and paracrine-signaling molecule through its interactions with seven-membrane-spanning G-protein-coupled adenosine receptors. These signaling pathways have widespread physiological and pathophysiological functions. Immune cells express adenosine receptors and respond to adenosine or adenosine agonists in diverse manners. Extensive in vitro and in vivo studies have identified potent anti-inflammatory functions for all of the adenosine receptors on many different inflammatory cells and in various inflammatory disease processes. In addition, specific proinflammatory functions have also been ascribed to adenosine receptor activation. The potent effects of adenosine signaling on the regulation of inflammation suggest that targeting specific adenosine receptor activation or inactivation using selective agonists and antagonists could have important therapeutic implications in numerous diseases. This review is designed to summarize the current status of adenosine receptor signaling in various inflammatory cells and in models of inflammation, with an emphasis on the advancement of adenosine-based therapeutics to treat inflammatory disorders.

Adenosine is a signaling nucleoside that is generated in response to cellular injury and orchestrates the balance between tissue protection and the progression to pathological tissue remodeling. Adenosine deaminase (ADA)-deficient mice develop progressive airway inflammation and remodeling in association with adenosine elevations, suggesting that adenosine can promote features of chronic lung disease. Furthermore, pharmacological studies in ADA-deficient mice demonstrate that A2BR antagonism can attenuate features of chronic lung disease, implicating this receptor in the progression of chronic lung disease. This study examines the contribution of A2BR signaling in this model by generating ADA/A2BR double-knockout mice. Our hypothesis was that genetic removal of the A2BR from ADA-deficient mice would lead to diminished pulmonary inflammation and damage. Unexpectedly, ADA/A2BR double-knockout mice exhibited enhanced pulmonary inflammation and airway destruction. Marked loss of pulmonary barrier function and excessive airway neutrophilia are thought to contribute to the enhanced tissue damage observed. These findings support an important protective role for A2BR signaling during acute stages of lung disease.