Targeted resuscitation (NH) attenuates the development of acute trauma coagulopathy and systemic inflammation with improved tissue perfusion and reduced metabolic acidosis in a model of complex injury. This emphasizes the challenge of choosing a resuscitation strategy for trauma patients where the needs of tissue perfusion must be balanced against the risk of rebleeding during resuscitation.

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Chimeric antigen receptor (CAR) T cell therapy is routinely used to treat patients with refractory hematologic malignancies. However, a significant proportion of patients experience suboptimal CAR T cell cytotoxicity and persistence that can permit tumor cell escape and disease relapse. Here we show that a prototype pro-lymphoid growth factor is able to enhance CAR T cell efficacy. We demonstrate that a long-acting form of recombinant human interleukin-7 (IL-7) fused with hybrid Fc (rhIL-7-hyFc) promotes proliferation, persistence and cytotoxicity of human CAR T cells in xenogeneic mouse models, and murine CAR T cells in syngeneic mouse models, resulting in long-term tumor-free survival. Thus, rhIL-7-hyFc represents a tunable clinic-ready adjuvant for improving suboptimal CAR T cell activity.

Antibiotic efficacy is greatly enhanced the earlier it is administered following infection with a bacterial pathogen. However, in a clinical setting antibiotic treatment usually commences following the onset of symptoms, which in some cases (e.g., biothreat agents) may be too late. In a BALB/c murine intranasal model of infection for Francisella tularensis SCHU S4 infection, we demonstrate during a time course experiment that proinflammatory cytokines and the damage-associated molecular pattern HMGB1 were not significantly elevated above naive levels in tissue or sera until 72 h postinfection. HMGB1 was identified as a potential therapeutic target that could extend the window of opportunity for the treatment of tularemia with antibiotics. Antibodies to HMGB1 were administered in conjunction with a delayed/suboptimal levofloxacin treatment of F. tularensis. We found in the intranasal model of infection that treatment with anti-HMGB1 antibody, compared to an isotype IgY control antibody, conferred a significant survival benefit and decreased bacterial loads in the spleen and liver but not the lung (primary loci of infection) 4 days into infection. We also observed an increase in the production of gamma interferon in all tested organs. These data demonstrate that treatment with anti-HMGB1 antibody is beneficial in enhancing the effectiveness of current antibiotics in treating tularemia. Strategies of this type, involving antibiotics in combination with immunomodulatory drugs, are likely to be essential for the development of a postexposure therapeutic for intracellular pathogens.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds the receptors in the APC/T cell synapse and causes increased proliferation of T cells and a cytokine storm syndrome in vivo. Exposure to the toxin can be lethal and cause significant pathology in humans. The lack of effective therapies for SEB exposure remains an area of concern, particularly in scenarios of acute mass casualties. We hypothesized that blockade of the T cell costimulatory signal by the CTLA4-Ig synthetic protein (abatacept) could prevent SEB-dependent pathology. In this article, we demonstrate mice treated with a single dose of abatacept 8 h post SEB exposure had reduced pathology compared with control SEB-exposed mice. SEB-exposed mice showed significant reductions in body weight between days 4 and 9, whereas mice exposed to SEB and also treated with abatacept showed no weight loss for the duration of the study, suggesting therapeutic mitigation of SEB-induced morbidity. Histopathology and magnetic resonance imaging demonstrated that SEB mediated lung damage and edema, which were absent after treatment with abatacept. Analysis of plasma and lung tissues from SEB-exposed mice treated with abatacept demonstrated significantly lower levels of IL-6 and IFN-γ (p < 0.0001), which is likely to have resulted in less pathology. In addition, exposure of human and mouse PBMCs to SEB in vitro showed a significant reduction in levels of IL-2 (p < 0.0001) after treatment with abatacept, indicating that T cell proliferation is the main target for intervention. Our findings demonstrate that abatacept is a robust and potentially credible drug to prevent toxic effects from SEB exposure.

Despite remarkable clinical efficacy, CAR-T therapy has been limited by life-threatening toxicities in over 30% of patients (Maude, NEJM 2014 and Davila, SciTransMed 2014). Toxicities primarily manifest as Cytokine Release Syndrome (CRS) characterized by an early phase with fever, hypotension, and elevations of cytokines including IFNγ, GM-CSF, TNF, IL-10, and IL-6. Using a protein kinase inhibitor library containing 644 independent compounds, we aimed to identify compounds that could block CRS-related cytokine production without inhibiting CAR-T function. We identified, duvelisib (kindly provided by Verastem Oncology, Needham, MA), a novel and selective dual PI3K-δ,γ inhibitor as a potent inhibitor of CRS in vitro and in vivo without attenuating CAR-T function. Duvelisib (Copiktra) is approved for the treatment of relapsed/refractory CLL after 2 prior therapies and follicular lymphoma after 2 prior systemic therapies; the latter gained accelerated approval status based on overall response rate and continued approval may be contingent on confirmatory trials. To assess the ability of duvelisib to inhibit CAR-T mediated CRS, we performed an in vitro CRS assay (Singh, Cytotherapy, 2017). CART19 (19-28BBζ, 25,000 cells), Ramos (CD19+, 50,000 cells), and immature dendritic cells (iDC, 2,500 cells) were co-cultured in a 96 well plate for 48hrs in the presence of varying concentrations of duvelisib (0.3nM-1000nM). Secreted IL-6, a surrogate marker of CRS, was determined using a human IL-6 ELISA (R&D Systems). Duvelisib reduced IL-6 levels in a dose-dependent manner with 30nM duvelisib reducing IL-6 secretion more than 10-fold (Fig 1a). To confirm that duvelisib did not inhibit CAR-T function, we performed in vitro killing assays, in which CAR-T efficacy was determined using BLI imaging of luciferase labeled CD19+ Ramos targets. At clinically relevant therapeutic doses (C max of 200 to 500nM) of duvelisib, there was no effect on CAR-T function in vitro. Treatment with 10nM duvelisib resulted in a statistically insignificant ~20% reduction of CART19 efficacy (p>0.05) (Fig 1b). Of interest, although selective inhibitors of either or PI3Kδ (GSK2292767) or PI3Kγ (IPI549) had modest effects on blocking CAR-T induced IL-6 production in this in vitro model, the effect of combining both inhibitors had a more dramatic effect similar to the dual PI3K-δ,γ inhibitor, duvelisib. Next we assessed the ability of duvelisib to block IL-6 secretion in vivo using a fully immunocompetent murine model of CRS. Six-week old BALB/c mice were injected with the mitogenic anti-CD3ε antibody, 145-2C11 (10 µg/mouse). Duvelisib (450µg/mouse) was administered daily intraperitoneally (I.P), with the first dose of duvelisib injected 24 hours prior to injection of 145-2C11. Plasma IL-6 levels were determined using a mouse IL-6 ELISA (R&D Systems). Injection of 145-2C11 acutely elevated plasma IL-6 >32 fold relative to non-treated controls (4hrs; Control 34.4 pg/mL versus vs. 145-2C11 1088±99.6 pg/mL). Duvelisib significantly reduced mean plasma IL-6 >54% at 4hrs (Vehicle 1088±99.6 pg/ml vs duvelisib 492±99.6 pg/ml, p≤ 0.01) and >78% at 24hrs (Vehicle 220±101 pg/ml vs. duvelisib 49.3±16.1, p≤ 0.01) (Fig 1c) consistent with the effect of duvelisib in our in vitro CAR-T-induced CRS model described above. These studies demonstrate that duvelisib can inhibit CAR-T induced IL-6 production from iDC while having no inhibitory effect on CAR-T. Experiments are currently in progress to further characterize PI3K-δ,γ inhibition in humanized mouse models of CRS and CAR-T efficacy. Our preclinical data suggest that dual PI3K-δ,γ inhibition with duvelisib may represent an attractive alternative to IL-6 receptor antagonists, such as tocilizumab, for the treatment of CAR-T-associated cytokine release syndrome in the clinic which warrants further clinical evaluation. Figure 1. Dose dependent effect of duvelisib on IL-6 secretion in co-culture of CART19, Ramosluc, and iDC after 48 hours (a) Dose dependent inhibition of CART19 mediated cytotoxicity using duvelisib (b) Duvelisib effectively lowers IL-6 plasma level in an immunocompetent BALB/cJ mouse model of CRS (c). (** p≤ 0.01 and ns p>0.05). Figure 1 Disclosures Cooper: Wugen: Consultancy, Current equity holder in private company, Patents & Royalties. Pachter:Verastem Therapeutics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Introduction: Despite remarkable clinical efficacy, CAR-T therapy has been limited by life threatening toxicities in over 30% of patients.1, 2 Toxicities primarily manifest as cytokine release syndrome (CRS) characterized by an early phase with fever, hypotension and elevations of cytokines including IFNγ, GM-CSF, TNF, IL-10, and IL-6 and a later phase associated with life-threatening or life-ending neurologic events. We hypothesized that reversible inhibition of CAR mediated signaling will enable controllable regulation of CAR-T cell activity in vivo and mitigate CAR-T mediated toxicities. There are specific protein kinases such as the SRC kinases, LCK, and ZAP-70 that are known to be involved in various cellular signaling pathways, especially T cell receptor mediated signaling and may also be appropriate targets for modulating (both enhancing and inhibiting) CAR-T function in a rapid and reversible fashion in vivo. Our hypothesis is that small molecule inhibitors of TCR signaling and downstream pathways could be identified using specific high throughput screens. Methods: To identify novel inhibitors of CAR-T cell proliferation, we developed a high throughput kinase inhibitor screen to identify compounds that reversibly inhibit CAR-T function. T cells containing a third generation CAR targeting CD19 cells (CAR19) and CD19+ tumor cells (Ramos cells expressing both GFP and luciferase) were incubated at an effector to target ratio of 1:1 in 96 well plates in the presence of 1µM of each inhibitor. After 24 hours, tumor cell death induced by CAR-T was measured using bioluminescence (BLI) imaging. Small molecules that inhibited CAR-T proliferation and cytotoxicity were determined by assessing the BLI signal in each well. Results: A protein kinase inhibitor library (Selleckchem, Texas) containing 644 independent compounds was tested (Figure 1). Of the 644 kinase inhibitors tested, 32 were found to be potent inhibitors of CART19 cell activation and cytotoxic killing of CD19+ target tumor cells, reducing anti-tumor viability in 24 hours by >50% compared to vehicle control. Compounds such as Nintedanib (C3), Dasatinib (C9), and Saracatinib (C12), all SRC kinase inhibitors, were able to inhibit cell killing by 99%, 84%, and 76% respectively. Next we assessed the reversibility of CAR-T cell mediated killing upon removal of inhibitors from the cultures. Reinitiation of potent, anti-tumor activity was observed within 24 hours after inhibitor removal, confirming reversible nature of CAR-T cell inhibition by the three most potent compounds. Conclusions: Recent publications (Weber et al Blood Adv, 2018, Westermann et al. Sci Transl Med, 2019) have also shown that dasatinib can reversibly suppresses CAR-T cell cytotoxicity, cytokine secretion, and proliferation in vitro and in vivo. 3, 4 Here we confirm the reports of others regarding dasatinib and that show for the first time that reversible inhibition of CAR-T activity by kinase inhibitors is not limited solely to dasatanib, but is observed with other small molecules targeting many different kinases. This work further demonstrates the potential applications of tyrosine kinase inhibitors as a safety switch to modulate CAR-T cell toxicity. 1. Maude, NEJM 2014 2. Davila, SciTransMed 2014 3. Mestermann SciTransMed 2019 4. Weber. Blood Adv 2019 Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents & Royalties. DiPersio:WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Magenta Therapeutics: Equity Ownership; Celgene: Consultancy; Karyopharm Therapeutics: Consultancy; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; Amphivena Therapeutics: Consultancy, Research Funding.