Neutralization of IFNγ improves the safety profile of CAR T-cells while maintaining unaffected efficacy against B-cell malignancies

Neutralization of IFNγ improves the safety profile of CAR T-cells while maintaining unaffected efficacy against B-cell malignancies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Neutralization of IFNγ improves the safety profile of CAR T-cells while maintaining unaffected efficacy against B-cell malignancies Simona Manni, Francesca Del Bufalo, Pietro Merli, Domenico Alessandro Silvestris, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1482837/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jun, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Chimeric antigen receptor (CAR) T-cell therapy represents a revolutionary approach to induce long-lasting remission in patients with B-cell malignancies not responding to conventional therapies. Nevertheless, possible severe side effects, including cytokine release syndrome (CRS), neurotoxicity and macrophage activation syndrome, whose management is still challenging, as well as lack of pathophysiological experimental models to investigate novel interventions, limit the widespread use of this therapy. In light of these considerations, we developed a comprehensive humanized mouse model to investigate the role of IFNγ neutralization, provided by the clinically approved monoclonal antibody, emapalumab, in controlling severe toxicity related to CAR T cells. We demonstrated that emapalumab reduces the pro-inflammatory environment in the animal model, allowing severe CRS control and preventing brain damage, characterized by multifocal hemorrhages. Furthermore, we proved that IFNγ inhibition does not affect the ability of CAR.CD19 T cells to eradicate CD19+ lymphoma cells, both in vitro and in vivo . Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Adoptive cellular immunotherapy with Chimeric Antigen Receptor (CAR) T cells represents a revolutionary approach for the treatment of B-cell lymphoproliferative disorders either relapsed or refractory to conventional therapies, including hematopoietic stem cell transplantation (HSCT). This innovative treatment, however, is characterized by peculiar toxicities. In particular, Cytokine Release Syndrome (CRS) and Immune effector Cell-Associated Neurotoxicity Syndrome (ICANS) have been largely reported in treated patients, with severity ranging from mild symptoms to life-threatening events or even death. 1 In addition, as a manifestation of severe CRS or as its pathophysiological consequence, the occurrence of haemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) has been reported in patients treated with CAR.CD19 2,3 or CAR.CD22 T-cells. 4 HLH/MAS are clinical syndromes characterized by a pathological hyperinflammation and uncontrolled macrophage activation, with symptoms largely overlapping those of CRS. Thus, CRS and HLH/MAS belong to a spectrum of systemic hyperinflammatory disorders. Notably, the recently published guidelines define this complication as a CRS/MAS overlap syndrome, typically characterized by persistent fever despite administration of the anti-IL6 receptor antibody, tocilizumab, organomegaly, cytopenias (± hemophagocytosis in the bone marrow), hyperferritinemia (> 10.000 ng/ml), liver dysfunction, coagulopathy (characterized, in particular, by hypofibrinogenemia) and hypertriglyceridemia. 5 CRS typically occurs within a few days after CAR T-cell infusion, often concomitantly with the maximal CAR T-cell expansion in vivo. 6 Notably, CRS incidence has been reported to range between 30% and 100%, with 10–30% of cases being either severe or life-threatening. 7 , 8 ICANS, a neurological complication characterized by irritability, dizziness, disorientation in mild forms, until global severe encephalopathy in the most severe cases, is reported to occur in 20–67% of lymphoma patients, 9 and in 29–72% of patients with B-cell precursor acute lymphoblastic leukemia (Bcp-ALL). 10 It can occur concurrently with CRS, slightly after CRS or following its resolution; approximately 10% of patients develop a form of “delayed ICAN” occurring > 3weeks after infusion. 11 , 12 Although the pathophysiology of ICANS remains less understood than that of CRS, both toxicities are characterized by a systemic inflammatory cascade with massive production of cytokines following the binding of CAR T-cell receptor to its target antigen. Upon activation of CAR T-cells, there is release of high levels of IFNγ, whose ability of inducing the activation of other immune cell subsets, mostly macrophages, is well-known. 13 This inflammatory loop, involving activated CAR T-cells, lysed cancer cells and macrophages, results into the massive production of additional cytokines, including IL-6, TNF-α, and IL-10, together with several catecholamines, 14 exponentially amplifying the inflammatory response. This process, on one hand, increases the anti-leukemia activity of CAR T-cells, 14 but on the other, can evolve into an uncontrolled, harmful condition. While low-grade CRS is usually easily managed using symptomatic measures and supportive care and, if needed, tocilizumab, high-grade CRS requires the administration of steroids after tocilizumab to control the inflammatory cascade before occurrence of organ failures. 6 Unfortunately, it has been reported that the prolonged use of high-dose steroids affects the anti-tumor activity of CAR Tcells, impairing long-term outcome of patients. 15 , 16 Moreover, the use of tocilizumab has some limitations. In particular, several groups found that an early intervention with tocilizumab largely mitigate the risk of sCRS, but does not affect neither most cases of fulminant refractory HLH/MAS, 17 nor treatment of ICANS. 18 This inefficacy on ICANS may be related to the different pathophysiology between CRS and ICANS, as well as to the poor penetration of tocilizumab across the blood-brain barrier (BBB). Indeed, it has been demonstrated that prophylactic use of tocilizumab decreases the incidence of sCRS, but increases the occurrence of severe ICANS, 6 , 11 likely owing to the higher circulating levels of IL-6 induced by the receptor blockade that, in turns, results into higher IL6 levels in the central nervous system (CNS). 19 – 21 In view of these findings, the most recent guidelines recommend the use of corticosteroid therapy for grade ≥ 2 ICANS. 5 As already mentioned, high levels of IFNγ have been found in patients with CRS, supporting the hypothesis that this cytokine plays a pivotal role of in driving the inflammatory process. 15 Notably, pericytes exposed to IFNγ contribute to the activation of endothelial cells and to the increase of BBB permeability, a dysfunction that appears to be a relevant finding in neurotoxicity: after occurrence of BBB disruption, IFNγ and TNF-α can activate the immune effector microglia cells, whose cytokines, released upon activation, induce neuro-inflammation and brain damage. 22 In view of all these considerations, we sought to evaluate whether inhibition of the IFNγ axis through emapalumab, a human monoclonal antibody directed against human IFNγ already approved by the FDA since November 2018 for treatment of refractory, relapsed primary HLH (pHLH), 23 can offer a therapeutic opportunity to manage these complications. Our results indicate that emapalumab is able to induce a significant reduction of CRS, HLH/MAS and brain damage, without impairing the anti-leukemia activity of CAR T-cells against CD19-positive B-cell malignancies. Results Neutralization of IFNγ does not impair CAR.CD19 T-cell anti-leukemic capacity in vitro In order to obtain preliminary evidence of the clinical suitability of emapalumab to manage CAR T-cell toxicities, we first evaluated whether neutralization of the IFNγ axis affects the cytotoxic activity CAR T cells against CD19 + tumor cells. For this purpose, peripheral blood mononuclear cells (PBMCs) derived from healthy donor (HD) were genetically modified with the 4-1BB-based CAR.CD19 construct used clinically at the Bambino Gesù Children Hospital of Rome (NCT03373071). In details, the anti-lymphoma activity of CAR.CD19 T-cells was tested in long-term co-culture assays with CD19 + lymphoma Daudi (Fig. 1 A) and Raji (Supplemental Fig. 1A) cell lines, either in the absence or in the presence of the anti-IFNγ monoclonal antibody emapalumab. Concentration of emapalumab was in the range of 0.2–100 µg/mL. This dose range corresponds to the concentration of drug observed in plasma of individual healthy subjects receiving emapalumab (study NI-0501-03). 24 The presence of emapalumab in the culture did not cause any significant modification of the cytotoxicity exerted by CAR.CD19 T-cells toward CD19 + lymphoma cells (Effector:Target, E:T ratio of 1:1) in vitro , as compared to untreated conditions, at any of the tested concentrations (Fig. 1 A for Daudi model and Supplemental Fig. 1A for Raji model). The evaluation of cytokines in the supernatant of the co-culture confirmed that IFNγ was completely neutralized at the doses used (Fig. 1 B and Supplemental Fig. 1B; p < 0.05 for the tested conditions), whereas no significant differences were observed for Granzyme B, IL2 and TNFα. In order to confirm these results in a more stressed condition, which could reproduce more reliably the clinical situation, the same experiment of co-culture with lymphoma cells was also performed at suboptimal conditions of decreased E:T ratio (from 1:1 to 1:80), to further evaluate whether the IFNγ neutralization could impact the cytotoxic activity of CAR.CD19 T-cells. In these experiments, emapalumab was added to the co-cultures at the highest dose of 100 µg/mL. As shown in Figs. 1 C and Supplemental Fig. 1C, the anti-lymphoma activity of CAR.CD19 T-cells was superimposable to the control condition, in which emapalumab was not added to the co-culture, at all the ratios tested. Furthermore, to test if the IFNγ neutralization interferes with the kinetics of target killing in a short (few hours) and long-time frame (up to 64 hours), the anti-lymphoma activity of CAR.CD19 T-cells against Daudi tumor cells was analyzed through a real-time, quantitative, live-cell imaging system that allows the visualization and quantification of live GFP + cells over time. As shown in Fig. 1 D, CAR.CD19 T cells exposed to emapalumab (dotted black line) exhibit a cytotoxicity profile superimposable to that of the untreated CAR.CD19 T-cells (black line). Moreover, we did not find differences in the proliferation index of CAR.CD19 T-cells induced by the 72-hour exposure to Daudi cells either in the absence (Fig. 1 E, blue area) or in the presence of 100µg/ml emapalumab (Fig. 1 E, purple area). Lastly, we analyzed CAR T-cell cytotoxicity at a very early time-point by measuring the bioluminescence of Raji cells, genetically modified to express firefly luciferase (FF-Luc), after 6 hrs of co-culture with CAR.CD19 T-cells, at the E:T ratio 1:1. As shown in Supplemental Fig. 1D, we did not observe any significant decrease of elimination index in the presence of Emapalumab. These data were further corroborated by the evaluation, by flow-cytometry, of the markers of apoptosis (Annexin V and 7-AAD) on Raji cells, after 6hrs of coculture with CAR.CD19 T-cells at a 1:1 E:T ratio. As shown in Supplemental Fig. 1E, the IFNγ neutralization did not impact significantly on the percentage of early apoptotic Raji cells. Several studies have reported that CAR T cells incorporating 4-1BB costimulatory domains have a significant differential signature and kinetics of lymphoma elimination as compared to CAR T-cells characterized by the presence of the CD28 costimulatory domain. 25 We thus investigated whether the antitumor activity of CAR.CD19.CD28 T-cells could be affected by the presence of emapalumab. As shown in Fig. 2 A, CAR.CD19.CD28 T-cells were co-cultured with Daudi tumor cells at suboptimal conditions of decreased E:T ratio (from 1:1 to 1:2,5), showing that, also in this case, the anti-tumor activity of CAR.CD19 T-cells exposed to emapalumab at the highest dose of 100 µg/mL was superimposable to that of CAR.CD19 T-cells in the absence of the drug. Moreover, the evaluation of cytokines in the supernatant collected after 24 hours of co-culture confirmed that IFNγ was completely neutralized (Fig. 2 B), whereas no significant differences were observed for Granzyme B, IL2 and TNFα (Fig. 2 B). Furthermore, the killing activity of CAR.CD19 T-cells against Daudi cells analyzed through the real-time imaging system (Fig. 2 C), as well as the proliferation assay (Fig. 2 D), reveal that IFNγ neutralization does not affect the functionality of CD28-based CAR.CD19 T-cells in either a short or long-time frame experimental setting. Emapalumab does not impair CAR.CD19 T-cell activation signaling In order to investigate if IFNγ neutralization could have an impact on the CAR T-cell gene signature, we analyzed the expression of 780 genes associated to some essential pathways of CAR-T biology, including phenotype, cell types, exhaustion, metabolic fitness, TCR diversity, toxicity, activation and persistence, in activated CAR.CD19 T-cells, with or without exposure to emapalumab. In details, un-transduced (NT) and CAR.CD19 T-cells were activated with 0.5 µg/ml recombinant human CD19 Fc Chimera Protein for 16 hours before the analysis of the gene signature. Overall, 90 genes resulted to be differentially regulated in the CAR.CD19 T-cell samples as compared to the control NT T-cells (46 up-regulated and 44 down-regulated genes), all involved in activation pathways of T-cells (Supplemental Fig. 2A and 2B, Supplemental Table 1). Upon addition of emapalumab (100µg/ml) to activated CAR.CD19 T-cells, we observed a differential expression of 14 genes (Fig. 3 A and Fig. 3 B). Among the 14 deregulated genes, the only upregulated is ITGAM, a gene associated to persistence and T-cell migration signaling (Supplemental Table 2). All the other 13 down-regulated genes, as expected, are involved in Interferon signaling pathways, or have already been reported as genes associated to toxicity (gene signature has been evaluated by bioinformatics analysis trough PubMed database, Supplemental Table 2), and the majority of them characterized by a linked signaling (Fig. 3 C). Moreover, seven out of the 14 genes deregulated by IFNγ neutralization, were specifically upregulated by the CAR.CD19 stimulation (Fig. 3 D and 3 E). Lastly, to confirm the data on gene expression with the phenotypic characterization of CAR.CD19 T-cells, the CAR.CD19 T-cell activation phenotype was tested upon challenge with lymphoma cells (E:T ratio of 1:1), in the presence or absence of emapalumab. As shown in Fig. 4 , we corroborated the gene expression data by monitoring the expression of several activation markers, including CD25, CD40L, HLA-DR, CD28, CD38, CD44 and CD69, showing no significant modulation when CAR.CD19 T-cells are stimulated by the lymphoma DAUDI cells in the presence of INFγ neutralization. IFNγ neutralization preserves CD19.CAR T-cell anti-lymphoma activity in a murine in vivo model We then tested the impact of emapalumab on the anti-lymphoma activity of CAR.CD19 T cells in the in vivo setting, in two different treatment settings. First, we considered an animal model in which CAR.CD19 T cells were infused when the tumor burden was low, mimicking most of clinical applications of CAR T-cells in Bcp-ALL patients. In this setting, clinical practice has shown that the onset of toxicity is delayed and both the incidence and severity of toxicity are limited. 26 In light of these considerations, we administered emapalumab 7 days after CAR T-cell infusion (Fig. 5 A shows the experimental design of the in vivo model). Mice were intravenously ( i.v. ) engrafted with Daudi FF-LUC cells on day − 2 and tumor burden was then monitored by in vivo imaging system (IVIS). After tumor engraftment, mice received i.v. control NT or 10X10 6 CAR.CD19 T-cells, followed by the intraperitoneal ( i.p. ) administration of either control vehicle or 100 mg/kg emapalumab on Days 7, 11 and 15, corresponding to the doses used for the non-clinical in vivo pharmacology/toxicology studies of emapalumab. [ https://www.fda.gov/drugs/fda-approves-emapalumab-hemophagocytic-lymphohistiocytosis ]. Notably, tumor was still largely detectable at the first day of emapalumab administration (Day 7). As shown in Fig. 5 B and 5 C, CAR.CD19 T cells in the presence of emapalumab exerted a significant lymphoma control (Fig. 5 B, mice from #13 to #16; Fig. 5 C-D, bioluminescence average value at day 28 = 5.87E + 05 ± 8.17E + 04), which did not differ from that of mice not exposed to emapalumab (Fig. 5 B, mice from #9 to #12; Fig. 5 C, bioluminescence average value at day 28 = 6.18E + 05 ± 9.59E + 04; p = 0.58). In the second animal model, we wanted to mimic the clinical situation of a high tumor burden, often associated with acute toxicity in patients treated with CAR.CD19 T-cells. 26 In this case, the administration of emapalumab was considered in a schedule of preventive treatment. In particular, mice were i.v. engrafted with Daudi FF-LUC cells on day − 8 to allow a tumor burden to grow up to 10 7 level of bioluminescence (sec/cm2/sr) before the i.v. infusion of either NT or CAR.CD19 T-cells (10x10 6 cells per mouse) at day 0 (Fig. 5 E shows the experimental design of the second in vivo model). Emapalumab (100 mg/kg) was administered i.p. on Days 0, 3 and 6. Also in this second animal model, we were able to provide evidence that CAR.CD19 T-cells exerted a significant lymphoma control in the presence of IFNγ neutralization (Fig. 5 F, mice #13 to #16; Fig. 5 G, bioluminescence values –dotted black lines- for each animal over time; Fig. 5 H, bioluminescence average value at day 15 = 5.67E + 07; p = 0.0002 vs the cohort of mice receiving NT T-cells), which did not differ from that of untreated counterpart (Fig. 5 F, mice #9 to #12; Fig. 5 G, bioluminescence values – black lines- for each animal over time; Fig. 5 H, bioluminescence average value at day 15 = 2.18E + 07; p = 0.47). As shown in Supplementary Fig. 3A, in this latter setting, we have also monitored the IFNγ neutralization that persists until the end of the in vivo experiment, without affecting the in vivo CAR.CD19 T-cell expansion (Supplementary Figs. 3B, 3C, 3D and 3E). This in vivo study was also performed using lower doses of CD19.CAR T-cells to investigate whether emapalumab administration affects CAR T-cell activity and proliferation in a more stringent and challenging setting characterized by a low CAR T-cell dose. For this reason, after tumor engraftment, mice received i.v. infusion of 1X10 6 or 0.1X10 6 CAR.CD19 T-cells, followed by the i.p. administration of 100 mg/kg of emapalumab or control vehicle on days 0, 3 and 6 (experimental setting in Fig. 5 E, control NT conditions showed in Fig. 5 F). We did not observe any difference in the bioluminescence between emapalumab-treated or untreated mice for both CAR T-cell doses we used (Supplementary Figs. 4A and 4B), with, in particular, a significant control of the lymphoma tumor being only observed in the cohort of mice receiving 1X10 6 CAR T-cells and Emapalumab (Supplementary Figs. 4A and 4B). Development of a simplified humanized murine model effectively reproducing CRS and brain damage hGM-CSF/hIL3 NOG mice engrafted with human CD34 + hematopoietic stem cells (HSCs) were used in order to establish an animal model recapitulating the human CRS and brain damage. hGM-CSF/hIL3 NOG mice, indeed, stably develop extensive human myeloid and lymphoid cell lineages which are present in peripheral blood, bone marrow, thymus, spleen and non-lymphoid tissue including lung and liver. Engraftment of human hematopoietic CD45 + cells in the peripheral blood (Supplementary Fig. 5A), whose distribution was characterized by the presence of B cells, T cells and CD14 + cells (Supplementary Fig. 5B), was confirmed before proceeding with the experimental plan. Notably, in order to provide a non-proliferating tumor substrate, aimed exclusively to activate CAR T-cells and to avoid developing uncontrolled tumor burden, thus confounding the CRS symptoms and impacting on mouse overall survival, humanized hGM-CSF/hIL3 NOG mice were engrafted with irradiated CD19 + Daudi (irrDaudi) FF-LUC cell lines by i.v. injection (Fig. 6 A). After the infusion of Daudi cells, mice received i.v. infusion of CAR.CD19 T-cells (10x10 6 /mouse), generated from healthy donors (HDs). CRS signs, including general suffering, as well as circulating pro-inflammatory cytokines, were monitored over the course of 15 days. We observed that humanized mice infused with irrDaudi and CAR T-cells showed a significant increase of pro-inflammatory cytokines, including IFNγ (Fig. 6 B), IL6 (Fig. 6 C), TNF-α (Supplemental Fig. 6B), the INFγ-driven chemokine CXCL9 (Fig. 6 D), CXCL10 (Fig. 6 E) as well as IL-1β (Supplemental Fig. 6A), although this last detected to a low level of 3.96 pg/ml ± 2.8 pg/ml. Notably, this CRS model was characterized by a severe toxicity, all mice dying in the first 7 days after CAR.CD19 T-cell infusion, without the potentially confounding signs of sufferance due to lymphoma cell expansion, since the mice were engrafted with irradiated not-proliferating lymphoma cells (Fig. 6 F). In addition, considering the severity of the generated model, an ad-hoc experiment was designed to evaluate the brain damage occurrence in this murine model of acute CAR T-cell toxicity (Fig. 7 A), by sacrificing mice 3 days after CAR T-cell infusion. In this setting, the three doses of emapalumab were administered daily (Fig. 7 A). In mice given both irrDaudi and CAR.CD19 T-cells, we observed a significant increase in the occurrence of hemorrhagic areas in the central nervous system of mice developing CRS, as compared to control mice infused only with irrDaudi (Fig. 7 B, 7 C p = 0.035). To study the relevance of our experimental model for the occurrence of HLH/MAS, we analyzed bone marrow samples obtained from mouse tibiae. We observed a marked “starry sky” appearance, representing phagocytes that have engulfed apoptotic cells (Fig. 7 D, middle panels). HLH/MAS histological findings was never observed in control mice infused only with irrDaudi (Fig. 7 D, top panels). Emapalumab is highly effective in controlling acute and fatal toxicity related to CAR.CD19 T-cells The humanized model was instrumental to study the ability of emapalumab to control CAR.CD19 T-cell toxicity. As shown in Fig. 6 B, emapalumab infusion was associated with a significant reduction in the serum levels of the INFγ-driven chemokine CXCL9 (Fig. 6 D; p = 0.007 at Day + 3 and p = 0.01 at Day + 4), CXCL10 (Fig. 6 E; p = p = 0.002 at Day + 3 and p = 0.005 at Day + 4) and IL6 (Fig. 6 C; p = 0.009 at Day + 3 and p = 0.01 at Day + 4). Most importantly, mice engrafted with irrDaudi and treated with the concomitant infusion of CAR.CD19 T-cells and emapalumab showed an overall survival of 100%, even extending the follow-up to 15 days (Fig. 6 F, p = 0.02 vs control untreated mice). Moreover, in the animals receiving CAR T-cells and emapalumab, we observed a significant reduction of the hemorrhagic areas in brains compared to mice not given emapalumab, their histology being similar to that observed in control animals that did not receive CAR T-cells (Figs. 7 B and 7 C, p = 0.045). To deeply evaluate the impact of IFNγ neutralization in reducing brain damage in mice developing CRS, brain tissue sections were analyzed for the expression of 770 human genes involved in the cellular stress and injury response, glial regulatory pathways, inflammation and peripheral immune invasion, glial cell homeostasis and activation, and neurotransmission. As shown in Fig. 8 A and Supplementary Table 3, 32 genes were significantly downregulated in emapalumab-treated mice compared to their untreated counterparts. Notably, these genes are characterized by a linked signaling (Fig. 8 B), significantly associated to biological pathways relevant for our setting, namely oxidative stress, IFNγ signaling, chemokine- and cytokine-mediated inflammation (Fig. 8 C). Lastly, histopathologic evaluation of tibia bone marrow from mice infused with CAR.CD19 T-cells and treated with emapalumab showed a significant reduction in phagocytic cells or mitoses (Fig. 7 D, bottom panels) compared to mice not given emapalumab which develop severe CRS. Discussion As CAR T-cell field is exponentially growing, scientists are seeking for novel strategies to mitigate, cure or even prevent the toxicities associated with treatment, therefore increasing the therapeutic window of the approach. However, the lack of comprehensive animal models able to recapitulate human toxicities represents a relevant obstacle. A humanized model of CRS was previously published, 27 but the complexity of the approach employed has limited its large-scale application to further investigate CAR T-cell toxicity/pathophysiology and therapeutic interventions. In order to study CAR T-cell-related complications, we developed a humanized mouse model that reproduces the most relevant human acute toxicities observed in patients after CAR T-cell infusion, namely CRS, ICANS and HLH/MAS. After humanization, mice were engrafted with a high tumor burden and received a standard dose of CAR T-cells. Thanks to the sub-lethal tumor cell irradiation, we were able to discern signs of CAR T-cell-derived toxicity, minimizing those related to lymphoma engraftment and progression. The developed model is able to recapitulate severe CAR T-cell-related acute toxicities, and all mice died in the first 7 days following CAR T-cell infusion. Moreover, to the best of our knowledge, this is the first animal model in which a clear CAR T cell-induced brain damage has been reported. In particular, after infusing a high number of tumor cells and high dose of CAR T-cells, mice developed multifocal brain hemorrhages, which are well-known manifestation of loss of cerebral vascular integrity with evidence of endothelial activation. Although brain hemorrhages are not formally included in the definition of ICANS, endothelial activation and multifocal vascular disruption are commonly found in the brain of patients developing fatal neurotoxicity after CAR T-cell infusion. 12 Indeed, it has been documented that patients with severe ICANS (grade ≥ 3) show evidence of endothelial activation, disseminated intravascular coagulation, capillary leak, and increased BBB permeability, leading to increased risk of intracranial bleeding. 12 , 28 The permeable BBB fails to protect the cerebrospinal fluid (CSF) from accumulation of systemic cytokines, including IFNγ, which induces brain vascular pericyte stress and secretion of endothelium-activating cytokines, ultimately promoting multifocal brain bleeding. 29 In our animal model recapitulating the CAR-T cell-related toxicities, we decided to investigate the role of INFγ neutralization. We focused our study on the use of emapalumab, because this humanized monoclonal antibody, targeting both free and receptor-bound INFγ, is already clinically available and has shown a very promising toxicity profile in children with primary HLH relapsing, refractory or intolerant to conventional therapies, therefore representing an easily clinically translatable approach. 23 We documented the emapalumab ability to neutralize high concentration of IFNγ produced by CAR T-cells upon the engagement with tumor cells; we also showed that this neutralization does not affect the anti-tumor activity of CAR.CD19 T-cells. This finding was first obtained in vitro , through the functional study of CAR.CD19 T-cells cultured with lymphoma cells in the presence of high concentration of emapalumab, in standard co-culture conditions (E:T ratio of 1:1) and then confirmed with very low E:T ratio. Even in this unfavourable condition, the presence of emapalumab did not affect CAR T-cell activity. Proliferation assay and analysis of cytotoxicity at shorter timepoints confirm that CAR T-cell efficacy is preserved in presence of emapalumab. It is noteworthy that these findings were obtained independently from the costimulatory domain included in the second-generation CAR construct targeting CD19, namely either 4.1BB or CD28, the latter being reported to be associated with the occurrence of more severe acute toxicities than those triggered by 4-1BB CAR.CD19 T-cells. 25 Moreover, we have performed gene expression analysis to compare eight essential pathways related to CAR T-cell biology in either the presence or absence of IFNγ neutralization, showing that the only 14 genes (out of the 780 investigated) modulated in the presence of emapalumab were mainly genes associated to IFNγ signalling and toxicity pathways, as expected. This result is also corroborated by the flow-cytometry analysis of CAR.CD19 T-cell with respect to the expression of activation markers, which reveals an identical activation profile between CAR.CD19 T-cells exposed to emapalumab and the untreated counterpart. The preserved functionality of CAR T-cells in presence of emapalumab was confirmed also in vivo , in a lymphoma animal model, in the presence of high doses of emapalumab, administered with a schedule of 3 infusions, 4-day apart, concomitant with the onset of toxicity or concurrent with the infusion of CD19.CAR T-cells. No significant reduction of the activity of CAR.CD19 T-cell against Daudi cells was observed in mice infused with emapalumab. In addition, in the NSG mouse model, our data indicate that CAR T-cell persistence and expansion were unaffected by IFNγ neutralization. Encouraged by these data, we then tested the possibility to control toxicity of CAR T-cells by using emapalumab in a comprehensive humanized model. Notably, the administration of emapalumab induced a complete neutralization of IFNγ with significant reduction of other inflammatory cytokines and chemokines, including CXCL-9, a molecule shown to be highly correlated with HLH/MAS activity. In particular, we measured in mice blood, human IL-6, one of the most elevated cytokines during CRS, likely released by activated endothelial cells, as well as by activated macrophages stimulated by IFNγ, which represents the most relevant therapeutic target to manage CRS in humans. In patients, IL-6 elevation can cause capillary leakage, hypotension, activation of complement pathway and coagulation cascades, and myocardial dysfunction. 30 – 32 We clearly showed that the neutralization of IFNγ represents a valid approach to mitigate the elevation of this key component of the inflammatory cascade downstream of IFNγ itself. Indeed, we detected a significant reduction of circulating human IL6 levels in mice treated with emapalumab, as compared to untreated mice. Moreover, in our animal model of CRS, we measured circulating levels of two human chemokines, CXCL9 (also known as MIG) and CXCL10 (also known as IP-10), specifically induced by IFNγ and synthesized and secreted by histiocytes and dendritic cells, thus allowing quantification of IFNγ-inhibition. 33 Both chemokines have been found to be significantly increased in patients experiencing grade 4–5 versus grade 0–3 CRS. 34 In our model, we found a significant increase of CXCL9 and CXCL10 in mice developing CRS after CAR.CD19 infusion. Notably, IFNγ neutralization was able to inhibit their elevation. Most importantly, emapalumab administration was able to completely protect mice from acute CRS, as all mice treated with emapalumab survived behind the time-line of the experimental plan (15 days), while untreated mice died within 7 days after CAR.CD19 T-cell infusion. We also studied the brain damage in mice developing acute toxicity, and proved that emapalumab administration is able to significantly control the multifocal bleeding in CNS, which has been associated to high-grade neurotoxicity in patients treated with CAR T-cells, 12 , 28 as well as to downregulate the expression of key signalling molecules associated to IFNγ pathway and inflammation mediated by chemokines/cytokines. Altogether, these results provide the biological rationale for considering the use of emapalumab as pathogenetic treatment of ICANS. Our data are in line with a recent experimental study demonstrating that an anti-INFγ approach, performed using a no azide/low endotoxin (NA/LE)-produced mouse anti-human IFNγ monoclonal antibody, abrogates macrophage activation in an in vitro model of CRS. 35 However, in the study of Bailey and colleagues 35 , the impact of IFNγ neutralization on severe systemic CRS, BBB damage or secondary HLH was not investigated in vivo . The potential importance of INFγ neutralization in management of CAR T-cell toxicities is supported by a recently published case report of a patient treated with emapalumab for life-threatening grade 4 CRS and neurotoxicity refractory to tocilizumab. 36 Emapalumab (administered at the dosage of 1 mg/kg) was infused on day 9 after numerous interventions, including repeated doses of tocilizumab, corticosteroids and siltuximab. Although it is not possible to attribute definitive causality to any particular intervention, patient fully recovered from CRS at day + 18, and one week later from neurotoxicity. Notably, this patient maintained clinical remission and sustained B-cell aplasia for 12 months after CAR T-cell infusion, this observation corroborating our experimental observation that INFγ neutralization does not interfere with CAR T-cell efficacy. In summary, in a simplified but comprehensive humanized model of CRS that overcomes the experimental limitations of previously reported CRS models, our study provides, for the first time, consistent evidence that emapalumab is a promising drug able to contain the CAR T-cell inflammatory cascade associated to high-grade CRS, MAS/HLH and brain injury, while sparing anti-tumor activity. Methods Cell cultures CD19 positive human Burkitt's lymphoma cell lines Daudi and Raji (ATCC, USA), genetically modified with firefly luciferase (FF-Luc), were cultured in RPMI 1640 medium (EuroClone, Italy) supplemented with 10% heat-inactivated foetal bovine serum (EuroClone), 2mM l-glutamine (GIBCO, USA), 25 IU/mL of penicillin, and 25 mg/mL of streptomycin (EuroClone), in a humidified atmosphere containing 5% CO2 at 37°C. All cell lines were authenticated by PCR-single-locus-technology (Promega, USA. PowerPlex 21 PCR) performed in "BMR Genomics s.r.l." (Italy), and routinely checked for mycoplasma (Venor®GeM Advance, MB Minerva biolabs, UK) and surface marker expression. CAR.CD19 T-cells generation PBMC derived from buffy coats of HDs were isolated, activated, transduced and expanded as previously described. 37 HDs signed a written informed consent, in accordance with rules set-up by the Institutional Review Board (IRB) of Bambino Gesù Children Hospital, IRCCS, Rome, Italy (OPBG; Approval of Ethical Committee N°969/2015 prot.N°669LB, and N°1422/2017 prot.N°810). The retroviral CAR.CD19 construct includes the anti-human CD19-scFv derived from FMC63 clone, in frame with 16aa sequence of the human CD34 antigen, the CD8 stalk domain, the CD8 transmembrane domain and the 4.1BB and CD3ζ cytoplasmic domains. 37 The retroviral CD28 based-CAR.CD19 construct includes the anti-human CD19-scFv derived from FMC63 clone, in frame with 16aa sequence of the human CD34 antigen, the CD8 stalk domain, the CD8 transmembrane domain and the CD28 and CD3ζ cytoplasmic domains. Phenotypic analysis. Expression of cell surface molecules was determined by flow-cytometry using standard methodology. The following monoclonal antibodies (mAbs) were used: CD3, CD4, CD8, CD14, CD19, CD25, CD28, CD34 (for CAR detection), CD38, CD40L, CD44, CD45, CD69 and HLA-DR (BD Bioscience). Samples were acquired with a BD LSRFortessa X-20 and analysed using the FACSDiva software (BD Biosciences). For each sample, we analysed a minimum of 20,000 events. Cytokine analysis IFNγ, Granzyme B, IL-2, TNF-α, IL-6, IL-1β, CXCL9/MIG and CXCL10/IP-10 levels found either after 24h co-culture assays or in peripheral blood of mice were analyzed by the enzyme-linked lectin assay Ella Automated Immunoassay System (Bio-Techne, California, USA). Cytotoxicity Assays The IncuCyte S5 Live Cell Assay System (Sartorius, Michigan, MI, USA) was used for kinetic monitoring of CAR T-cell cytotoxicity against tumor in presence or absence of emapalumab. Effector T-cells (NT or CAR.CD19) and FF-LUC Daudi tumor cells were plated at 0.25 x10 6 cells/well on 48-well plates at 1:1 E:T ratio in presence or absence of 100µg/ml emapalumab and incubated for 64 hours at 37°C in a humidified atmosphere (5% CO2). Growth curves were generated by the algorithm in the “2021C” software (Schrödinger; New York, NY, USA) evaluating GFP values ​​expressed by Daudi tumor cells from data points acquired during imaging at 2-hour intervals. All samples were plated in triplicate. Short-term (6 hours) CAR.CD19 T-cell cytotoxic assay was performed with a luciferase-based test. Briefly, effector T-cells (NT or CAR.CD19) and FF-LUC-expressing Raji tumor cells were plated, at 0.5 x10 6 cells/well, on 24-well plates, at 1:1 E:T ratio, either in the presence or in the absence of 100µg/ml emapalumab, and incubated at 37°C in a humidified atmosphere (5% CO2). After 6 hours, 15µg/ml of luciferin (Xenolight D-luciferin; Perkin Elmer) was added to the media and bioluminescence (BLI) was quantified after 10 min of incubation at 37°C in a humidified atmosphere (5% CO2) on an ENSPIRE Multimode plate reader (Perkin Elmer). Apoptosis Assay Apoptosis of Raji target cells was evaluated by flow-cytometry-based assay. In particular, effector T-cells (NT or CAR.CD19) and Raji tumor cells were plated at 0.5 x10 6 cells/well, on 24-well plates, at 1:1 E:T ratio, either in the presence or in the absence of 100µg/ml emapalumab, and incubated at 37°C in a humidified atmosphere (5% CO2). After 6 hours, cells were labeled with Annexin V BUV-395 (BD Bioscences)/ 7-Amino-Actinomycin D (7-AAD; BD Bioscences) according to the manufacturer's instructions and evaluated by flow-cytometry, as specified in the phenotypic analysis. Cell proliferation assay Cell proliferation assay was performed by labeling cells with the CellTraceTM Cell Proliferation Kit (Far Red, Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, CAR.CD19 T-cells were washed with PBS and resuspended at 10 6 cells/mL in working dye solution (1µM in 1xPBS) for 20 minutes at 37°C. Thereafter, labeled cells were resuspended in five volumes of cell culture medium, centrifuged, and resuspended in culture media to be co-cultured with Daudi tumor cells (at 1:1 E:T ratio) in presence or absence of 100µg/ml emapalumab for up to three days. Nanostring analysis RNA was isolated from samples using the RNeasy mini kit (Qiagen) according to supplier’s instructions. Total RNA was quantified with NanoDrop ND-100. We analyzed the expression of 780 genes (including 10 reference genes) related to components of CAR-T biology using the nCounter CAR-T Characterization Panel™ (XT CAR-T Code-Set Panel, Nanostring, Seattle, WA) and of 770 genes (including 10 reference genes) related to components of glial biology using the nCounter Human Glial Profiling panel (XT Human Glial Profiling Code-Set Panel, Nanostring, Seattle, WA). Total RNA was used as input and sample hybridization was performed according to the manufacturer's instructions. Sample detection and analysis were completed on an nCounter® Digital Analyzer. Raw data processing, quality control, and normalization were performed using the nSolver™ 4.0 analysis software (NanoString nCounter Technologies, Seattle, WA). Background subtraction from raw transcript counts was performed through negative input controls. Normalization to 10 housekeeping genes and differential expression analysis were completed using the Advanced Analysis software plugin (version 2.0.115). For differential expression analysis, a p-value of ≤ 0.05, were applied as cut-offs. Gene set enrichment was evaluated with the Enrichr ( https://maayanlab.cloud/Enrichr/ ) web tool. The basic interaction unit in STRING has been used to evaluate possible network between genes differentially expressed ( https://string-db.org/ ). In vivo CAR + lymphoma mouse model All procedures were performed in accordance with the Guidelines for Animal Care and Use of the National Institutes of Health (Ethical committee for animal experimentation Prot. N 088/2016-PR). To perform anti-tumor in vivo study, Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) female mice were purchased from Charles Rives Laboratories and maintained in the Plaisant animal facility in Castel Romano, Rome, Italy. Mice were i.v. engrafted with 0.25x10 6 FF-Luciferase positive Daudi cells at Day-2 or at Day-8, and treated with 10x10 6 un-transduced (NT) or CAR.CD19 T-cells/mouse at Day 0. At Day 7, 11, 15 (Fig. 5 A) or at Day 0, 3, 6 (Fig. 5 D) respectively, mice received 100mg/Kg Emapalumab. Tumor growth was monitored weekly by IVIS Imaging System, 38 after D-Luciferin (PerkinElmer, D-Luciferin potassium salt) intraperitoneal (i.p.) administration. Humanized murine model of CRS and brain injury after CAR.CD19 T-cell administration hGM-CSF/hIL3 NOG mice were purchased from Taconic Laboratories and maintained at the Plaisant animal facility in Castel Romano, Rome, Italy. Mice were engrafted with human umbilical cord blood-derived CD34 + hematopoietic stem cells (HSCs). Mice were aged 10 weeks post engraftment and quality checked for human leukocyte reconstitution by flow-cytometry. Only mice reaching ≥ 25% hCD45 + cells were used in the experiments. Humanized mice were infused with irradiated (30cGy) FF-Luciferase positive Daudi cells at Day-2 and treated either with 10x10 6 un-transduced (NT) or with CAR.CD19 T-cells/mouse either in the presence or absence of 100mg/Kg/day emapalumab, according to the treatment schedules. Mice were subject to blood bleeding at Day 3 and Day 4 for the monitoring of cytokines. For brain injury evaluation, 3 days after CAR.CD19 T-cell infusion, animals were sacrificed, and brains were fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) and paraffin embedded. The histopathologic haematoxylin and eosin staining was performed on the middle axial sections. 39 For HLH/MAS bone marrow analysis, tibiae of experimental mice were collected at sacrifice, and tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. Deparaffined sections were stained with hematoxylin and eosin (Thermo Fischer Scientific). Images were acquired on a ScanScope XT scanner and digitized to scalable images up to a 20× objective (Leica Biosystems). Statistical analysis Unless otherwise noted, data are summarized as mean ± standard deviation (SD). The Student’s t-test or Mann-Whitney test (two-sided) were used to determine statistically significant differences between samples, with a p-value < 0.05 indicating a significant difference. Mouse survival data were analyzed using Kaplan-Meier survival curves and the log-rank test was used to measure differences between groups. No valuable samples were excluded from the analyses. Neither randomization nor blinding were performed during in vivo studies. However, mice were matched based on the tumor signal for control and treatment groups before infusion of NT or CAR T-cells. To compare the growth of tumours over time, bioluminescence signal intensity was collected blindly. Bioluminescence signal intensity was log-transformed and, then, compared using a two-sample t-test. We estimated the sample size considering no significant variation within each group of data. The principle of using the smallest sample size possible was adopted in planning the animal experiments. We estimated the sample size in order to detect a difference in averages of 2 standard deviations at the 0.05 level of significance with an 80% power. Graphic representations and statistical analysis were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). Declarations Acknowledgments The experimental work was supported by grants awarded by Accelerator Award – Cancer Research UK/AIRC – INCAR project (F.L.), Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC)-Special Project 5×1000 no. 9962 (F.L.), AIRC IG 2018 id. 21724 (F.L.), MFAG 21979 (C. Quintarelli), id.26915-2021 AIRC Fellowships Call (S.M.), Ricerca Corrente (C. Q., B.D.A.), Ministero dell’Università e della Ricerca (Grant PRIN 2017 to F.L.); Italian Healthy Ministry project on CAR T RCR-2019-23669115 (Coordinator F.L.), GR-2016-02364546 (B.D.A), RF-2016-02364388 (F.L.), Independent Research grant AIFA (F.L.: 2016 call), “PNRR M4C2- Investment 1.4-CN00000041”– NextGenerationEU, PNC HLS-TA 2022 (F.L.). We are very grateful to SOBI for the collaboration in providing emapalumab used for the in vitro and in vivo experiments. Competing Interests F.L and P.M. on September 2022 participated into an advisory board on primary HLH organized by Sobi, receiving honoraria. All the other authors do not have any conflicting financial interests to disclose. SOBI has not supported the study, beside the provision of the emapalumab drug. Author Contributions C.Q., B.D.A., and F.L. designed experimental studies, supervised the project conduction, analysed the data and wrote the manuscript. 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Cancer Res 78 , 3337–3349, doi: 10.1158/0008-5472.CAN-17-3140 (2018). Supplementary Files SupplementalInformationGateStrategy.pdf NCOMMS2211266B01RS.pdf Reporting Summary SupplementaryFigures.pdf Cite Share Download PDF Status: Published Journal Publication published 09 Jun, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Angelis","email":"data:image/png;base64,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","orcid":"","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Biagio","middleName":"","lastName":"De Angelis","suffix":""},{"id":179985747,"identity":"02ca51a0-ab66-40b3-8a7f-95f4b51cc97e","order_by":23,"name":"Franco Locatelli","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Franco","middleName":"","lastName":"Locatelli","suffix":""},{"id":179985748,"identity":"ef46c96c-e456-4219-a3ff-6f50d1889e99","order_by":24,"name":"Concetta Quintarelli","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Concetta","middleName":"","lastName":"Quintarelli","suffix":""}],"badges":[],"createdAt":"2022-03-23 18:25:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1482837/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1482837/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-023-38723-y","type":"published","date":"2023-06-09T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":33628671,"identity":"55f35614-6e17-40f5-8019-3ef58c19f87b","added_by":"auto","created_at":"2023-03-01 15:02:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":258078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmapalumab does not affect CD19.CAR T-cell \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecytotoxicity against Daudi tumor cells. (a)\u003c/strong\u003e Effector T-cells (NT or CAR.CD19.4-1BB T-cells) and GFP+ Daudi tumor cells were plated at the 1:1 E:T ratio in presence or absence of emapalumab within the concentration range of 0.2-100μg/ml. Following 7 days of incubation, tumor cells and T cells were collected and assessed by fluorescence-activated cell-sorting (FACS) analysis to detect GFP+ residual tumor cells. Data are expressed in percentage, as mean ± SD of 3 independent experiments, with effector cells generated from 3 HDs.\u003cstrong\u003e (b)\u003c/strong\u003e Heatmaps displaying the IFNγ, Granzyme B, IL-2 and TNF-α levels that are secreted by effector T-cells (NT or CAR.CD19.4-1BB) after 24h of co-culture with Daudi cells in media containing emapalumab within the concentration range of 0.2-100μg/ml. Color scale represents cytokine levels as mean of 3 independent experiments with effector cells generated from 3 HDs expressed in percentage. Only IFNγ was differentially detected between the control condition and those in which emapalumab has been added to different concentrations (p\u0026lt;0.05)\u003cstrong\u003e (c)\u003c/strong\u003e Effector T-cells (NT or CAR.CD19.4-1BB) and GFP+ Daudi cells were plated at decreasing E:T ratios, from 1:1 to 1:80 either in the presence or in the absence of 100μg/ml emapalumab. At the end of the experiments, tumor cells and T-cells were collected and assessed by FACS analysis evaluating the residual GFP+ tumor cells. Data are expressed in percentage,\u003cstrong\u003e \u003c/strong\u003eas mean ± SD of 3 independent experiments with effector cells generated from 3 HDs \u003cstrong\u003e(d)\u003c/strong\u003e Real-time kinetics of Daudi elimination exerted by T-cells (NT or CAR.CD19.4-1BB) (1:1 E:T ratio) either in the presence or absence of 100μg/ml emapalumab. The indicated statistical significance refers to the end point of the experimental assay. \u003cstrong\u003e(e) \u003c/strong\u003eProliferation of CAR.CD19.4-1BB T-cells activated by Daudi cells and monitored for 3 days in absence (blue area) or in presence of 100μg/ml emapalumab (purple area) compared to CAR T-cells at time of cell plating (day 0, pink area). Exemplificative histograms, relative to one representative experiment, out of the three performed with three different CAR.CD19 T-cell donors (p value of the proportion of proliferating CAR.CD19 T-cells with vs without Emapalumab: \u0026gt;0.05). \u0026nbsp;Data are expressed as mean ± SD. ** p-value=\u0026lt;0.01, *** p-value=\u0026lt;0.001. The Student’s t-test was employed to calculate statistically significant differences between groups.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/c29c2d40ad3e52cc0e860be3.png"},{"id":33627909,"identity":"2d21813e-eb6f-41f6-8fa7-9958d3d58580","added_by":"auto","created_at":"2023-03-01 14:54:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258759,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmapalumab does not affect CD28-based CAR.CD19 T-cell \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecytotoxicity. (a)\u003c/strong\u003e Effector T-cells (NT or CAR.CD19.CD28) and GFP+ Daudi cells were plated at decreasing E:T ratios, from 1:1 to 1:2.5 either in the presence or absence of 100μg/ml emapalumab. At the end of the experiments, tumor cells and T-cells were collected and assessed by FACS analysis evaluating the residual GFP+ tumor cells. Data are expressed in percentage, as mean ± SD of 3 independent experiments, with effector cells generated from 3 HDs\u003cstrong\u003e (b)\u003c/strong\u003e Heatmaps displaying the IFNγ, Granzyme B, IL-2 and TNF-α levels that are secreted by effector T-cells (NT or CD28-based CAR.CD19) after 24h of co-culture with Daudi cells in media containing emapalumab within the concentration range of 0.2-100μg/ml. Color scale represents cytokine levels as mean of 3 independent experiments with effector cells generated from 3 HDs expressed in percentage. \u003cstrong\u003e(c)\u003c/strong\u003e Real-time kinetics of Daudi elimination exerted by T-cells (NT or CAR.CD19.CD28) (1:1 E:T ratio) either in the presence or absence of 100μg/ml emapalumab. The indicated statistical significance refers to the end point of the experimental assay. \u003cstrong\u003e(d) \u003c/strong\u003eProliferation of CAR.CD19.CD28 T-cells activated by Daudi cells and monitored for 3 days in absence (blu area) or in presence of 100μg/ml emapalumab (purple area) compared to CAR T-cells at time of cell plating (day 0, pink area). Exemplificative histograms, relative to one representative experiment, out of the three performed with three different CAR.CD19 T-cell donors (p value of the proportion of proliferating CAR.CD19 T-cells with vs without Emapalumab: \u0026gt;0.05). * p-value=\u0026lt;0.05, ** p-value=\u0026lt;0.01, *** p-value=\u0026lt;0.001. The Student’s t-test was employed to calculate statistically significant differences between groups.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/3571684f741669926178abe8.png"},{"id":33627899,"identity":"2e9650e8-05d2-4aa6-a688-155ce6154744","added_by":"auto","created_at":"2023-03-01 14:54:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":378310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFNγ neutralization does not impair CAR.CD19 T-cell activation signaling. (a) \u003c/strong\u003eVolcano plot of the 14 deregulated genes in CAR.CD19 T-cells activated with 0.5 μg/ml Recombinant Human CD19 Fc Chimera Protein for 16 hours in the absence or presence of 100μg/ml emapalumab. The log2 FC indicates the mean expression level for each gene. Genes with negative log2FC are downregulated in CAR.CD19 T-cells in presence of emapalumab. \u003cstrong\u003e(b)\u003c/strong\u003e Heatmap displaying the expression of the same genes in untreated NT T-cells, untreated CAR.CD19 T-cells and CAR.CD19 T-cells treated with 100μg/ml emapalumab. \u003cstrong\u003e(c) \u003c/strong\u003eSTRING tool has been applied to deconvolute the\u003cstrong\u003e \u003c/strong\u003epotential interaction network among the 14 genes deregulated in CAR.CD19 T-cells in presence of emapalumab. \u003cstrong\u003e(d) \u003c/strong\u003eVenn diagram that summarizes common genes deregulated both in Untreated CAR.CD19 compared to Untreated NT T-Cells and in CAR.CD19 cultured either in the presence or absence of emapalumab (Untreated CAR.CD19 T-Cells). \u003cstrong\u003e(e)\u003c/strong\u003e Heatmaps displaying the 7 genes specifically upregulated in activated CAR.CD19 T-cells, but deregulated in the presence of emapalumab. Data are obtained from three independent experiments, with effector cells generated from 3 HDs.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/48acc1cb3ee02ab95c3eb546.png"},{"id":33627900,"identity":"d60cea8b-a541-4d62-a75f-966e915ceca2","added_by":"auto","created_at":"2023-03-01 14:54:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFNγ neutralization does not affect the activation phenotypic profile of CD19.CAR T-cells. \u003c/strong\u003ePhenotypic analysis on\u003cstrong\u003e \u003c/strong\u003edifferent\u003cstrong\u003e \u003c/strong\u003eactivation markers expressed by T-cells (NT or CAR.CD19) after 16 hours of co-cultures with Daudi cells either in the presence or absence of 100μg/ml emapalumab. Data are expressed as mean ± SD and obtained by three independent experiments, with CAR.CD19 T cells generated by three different donors. The Student’s t-test was employed to evaluate the statistical relevance of the differences between groups. * p-value=\u0026lt;0.05, ** p-value=\u0026lt;0.01, *** p-value=\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/97758ff0ad0bb5d712f8329d.png"},{"id":33628849,"identity":"6db5dfbd-1e57-4a7d-82a1-e35d71270cc8","added_by":"auto","created_at":"2023-03-01 15:10:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":293575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmapalumab does not affect CD19.CAR T-cell anti-lymphoma activity in NSG mouse model. (a)\u003c/strong\u003e Schematic representation of the first experimental setting: mice (n=4 in each cohort) were infused with 0.25x10\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;FF-LUC Daudi cells/mouse at Day-2 and treated with 10x10\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;effector T (NT or CAR.CD19) cells/mouse at Day 0. At Day 7, 11, 15 mice received 100mg/Kg emapalumab and were evaluated for lymphoma eradication. \u003cstrong\u003e(b) \u003c/strong\u003eBioluminescence imaging of each treatment cohort. \u003cstrong\u003e(c) \u003c/strong\u003eMean ± SD of bioluminescence values of the four mice cohorts over the time, receiving only tumor (black dotted line), NT (grey line), CAR.CD19 T-cells in absence of emapalumab (black line) or CAR.CD19\u003csub\u003e \u003c/sub\u003eT-cells in presence of emapalumab (red line). \u003cstrong\u003e(d) \u003c/strong\u003eMean ± SD of bioluminescence values of the four mice cohorts at Day 28.\u003cstrong\u003e (e) \u003c/strong\u003eSchematic representation of the second experimental setting: mice (n=4 in each cohort) were infused with 0.25x10\u003csup\u003e6\u003c/sup\u003e FF-LUC Daudi cells/mouse at Day-8 and treated with 10x10\u003csup\u003e6\u003c/sup\u003e effector T (NT or CAR.CD19) cells/mouse at Day 0. At Day 0, 3, 6 mice received 100mg/Kg emapalumab and evaluated for lymphoma eradication. \u003cstrong\u003e(f) \u003c/strong\u003eBioluminescence imaging of each treatment cohort. \u003cstrong\u003e(g) \u003c/strong\u003eBioluminescence values of each mouse, receiving NT cells in absence of emapalumab (red line) or in presence of emapalumab (red dotted line) or CAR.CD19 T-cells in absence of emapalumab (black line) or CAR.CD19 T-cells in presence of emapalumab (black dotted line). \u003cstrong\u003e(h) \u003c/strong\u003eMean ± SD of bioluminescence values of the four mice cohorts at Day 15.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;* p-value=\u0026lt;0.05, *** p-value=\u0026lt;0.001. The Student’s t-test was employed to calculate statistically significant differences between groups.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/72973aac8d725ed3d89be95f.png"},{"id":33628851,"identity":"bff56947-8f36-4d70-bef0-b60078b6e396","added_by":"auto","created_at":"2023-03-01 15:10:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":137687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmapalumab displays therapeutic effect in humanized mice developing CRS. (a\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSchematic representation of the experimental design: hGM-CSF/hIL3 NOG mice were infused with irradiated FF-Luciferase positive sub-acutely irradiated Daudi cells (10x10\u003csup\u003e6\u003c/sup\u003e cells/mouse. n=10 total humanized mice) at Day-2 and treated with 10x10\u003csup\u003e6\u003c/sup\u003e CAR.CD19 T-cells/mouse in presence of either 100mg/Kg/day emapalumab (n=4) or vehicle (n=4). Two mice were considered as for negative control infused only with DAUDI cells. (\u003cstrong\u003eb\u003c/strong\u003e) IFNγ, (\u003cstrong\u003ec\u003c/strong\u003e) IL-6, (\u003cstrong\u003ed\u003c/strong\u003e) CXCL9/MIG and (\u003cstrong\u003ee\u003c/strong\u003e) CXCL10/IP-10 levels were measured on peripheral blood of mice at Day+3 and Day+4 after effector T-cell infusion. The Student’s t-test was employed to calculate statistically significant differences between groups. (\u003cstrong\u003ef\u003c/strong\u003e) Kaplan-Meier survival curve analysis of CAR.CD19 T-cell treated mice without emapalumab (black line) or with emapalumab (red line). * p-value=\u0026lt;0.05, ** p-value=\u0026lt;0.01, *** p-value=\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/6a40dce8d93585332ea21dd7.png"},{"id":33629300,"identity":"a6c4b9da-4cbf-4f0f-8877-a8f340231447","added_by":"auto","created_at":"2023-03-01 15:18:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":712108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmapalumab prevents brain injury and histological bone marrow signs of macrophage activating syndrome in humanized mice developing toxicity associated to CD19.CAR T-cells. (a)\u003c/strong\u003e Schematic representation of the experimental design: hGM-CSF/hIL3 NOG mice (n=12) were infused with irradiated FF-Luciferase positive Daudi cells at Day-2 and treated with 10x10\u003csup\u003e6\u003c/sup\u003e CAR.CD19 T-cells/mouse either in the presence of 100mg/Kg/day emapalumab (n=5) or of the vehicle (n=5), as reported in the cartoon. Two mice were considered as for negative control, being infused only with DAUDI cells.\u003cstrong\u003e (b)\u003c/strong\u003e Hematoxylin and eosin staining on brain slides of a control mouse (only tumor; top panels), untreated mouse (tumor and CAR.CD19 T-cells in absence of emapalumab; middle panels) and treated mouse (tumor and CAR.CD19 T-cells in presence of emapalumab; bottom panels). \u003cstrong\u003e(c) \u003c/strong\u003eAverage of the haemorrhagic area in assial brain slide of control mouse (only tumor), untreated mouse (tumor and CAR.CD19 T-cells in the absence of emapalumab) and treated mouse (tumor and CAR.CD19 T-cells in the presence of emapalumab).\u0026nbsp; \u003cstrong\u003e(d)\u003c/strong\u003e The histopathologic haematoxylin and eosin staining was performed on bone marrow tibiae of experimental control mice engrafted only with lymphoma (#1 and #2), mice developing CRS after CAR T-cell infusion (#3 and #4), and mice treated with emapalumab (#5 and #6). * p-value=\u0026lt;0.05. The Student’s t-test was employed to calculate statistically significant differences between groups.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/d9129a94339459d276323e8e.png"},{"id":33629299,"identity":"46007257-7499-4f06-ba4e-7fc9f58cea3f","added_by":"auto","created_at":"2023-03-01 15:18:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":383915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression profile associated to glial signalling in humanized mice developing neurotoxicity and treated with emapalumab. (a) \u003c/strong\u003eVolcano plot of the 33 significantly deregulated genes (32 downregulated and one upregulated) in brain tissue sections of mice developing toxicity after CAR T-cell infusion treated with emapalumab or not. The log2 FC indicates the mean expression level for each gene. Genes with negative log2FC are downregulated in mice receiving emapalumab. \u003cstrong\u003e(b)\u003c/strong\u003e STRING tool has been applied to deconvolute the potential interaction network among the 32 genes downregulated in brain tissue sections of mice treated with emapalumab, compared to brain tissues from control mice that did not receive the drug. \u003cstrong\u003e(c)\u003c/strong\u003eGene enrichment analysis performed through the Enrichr web tool displays the pathways in which the 32 genes downregulated in emapalumab treated mice are involved.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/e7549ddd3d10fcafb9f385dd.png"},{"id":38323501,"identity":"3cb269c4-c0e0-4637-ba0e-2d0024312ac6","added_by":"auto","created_at":"2023-06-10 07:07:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3290956,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/cfd233e0-bef8-4722-b9d4-995736280759.pdf"},{"id":33627904,"identity":"21120d76-b7ce-44dc-95ae-db555003721e","added_by":"auto","created_at":"2023-03-01 14:54:21","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1267147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementalInformationGateStrategy.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/7f0913227b81cff3bc684d6a.pdf"},{"id":33627907,"identity":"4916387e-bef9-4044-bafb-43efaba4ead9","added_by":"auto","created_at":"2023-03-01 14:54:21","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4156708,"visible":true,"origin":"","legend":"\u003cp\u003eReporting Summary\u003c/p\u003e","description":"","filename":"NCOMMS2211266B01RS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/3e959a9df41fe9c0cfb66b03.pdf"},{"id":33627903,"identity":"700e6695-31f5-4d14-8244-fd3c08bd601e","added_by":"auto","created_at":"2023-03-01 14:54:21","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":585696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1482837/v1/77e72daf46da6957e6a3dfef.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eNeutralization of IFNγ improves the safety profile of CAR T-cells while maintaining unaffected efficacy against B-cell malignancies\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdoptive cellular immunotherapy with Chimeric Antigen Receptor (CAR) T cells represents a revolutionary approach for the treatment of B-cell lymphoproliferative disorders either relapsed or refractory to conventional therapies, including hematopoietic stem cell transplantation (HSCT). This innovative treatment, however, is characterized by peculiar toxicities. In particular, Cytokine Release Syndrome (CRS) and Immune effector Cell-Associated Neurotoxicity Syndrome (ICANS) have been largely reported in treated patients, with severity ranging from mild symptoms to life-threatening events or even death.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e In addition, as a manifestation of severe CRS or as its pathophysiological consequence, the occurrence of haemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) has been reported in patients treated with CAR.CD19\u003csup\u003e2,3\u003c/sup\u003e or CAR.CD22 T-cells.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e HLH/MAS are clinical syndromes characterized by a pathological hyperinflammation and uncontrolled macrophage activation, with symptoms largely overlapping those of CRS. Thus, CRS and HLH/MAS belong to a spectrum of systemic hyperinflammatory disorders. Notably, the recently published guidelines define this complication as a CRS/MAS overlap syndrome, typically characterized by persistent fever despite administration of the anti-IL6 receptor antibody, tocilizumab, organomegaly, cytopenias (\u0026plusmn;\u0026thinsp;hemophagocytosis in the bone marrow), hyperferritinemia (\u0026gt;\u0026thinsp;10.000 ng/ml), liver dysfunction, coagulopathy (characterized, in particular, by hypofibrinogenemia) and hypertriglyceridemia.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCRS typically occurs within a few days after CAR T-cell infusion, often concomitantly with the maximal CAR T-cell expansion \u003cem\u003ein vivo.\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e Notably, CRS incidence has been reported to range between 30% and 100%, with 10\u0026ndash;30% of cases being either severe or life-threatening.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e ICANS, a neurological complication characterized by irritability, dizziness, disorientation in mild forms, until global severe encephalopathy in the most severe cases, is reported to occur in 20\u0026ndash;67% of lymphoma patients,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and in 29\u0026ndash;72% of patients with B-cell precursor acute lymphoblastic leukemia (Bcp-ALL).\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e It can occur concurrently with CRS, slightly after CRS or following its resolution; approximately 10% of patients develop a form of \u0026ldquo;delayed ICAN\u0026rdquo; occurring\u0026thinsp;\u0026gt;\u0026thinsp;3weeks after infusion.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough the pathophysiology of ICANS remains less understood than that of CRS, both toxicities are characterized by a systemic inflammatory cascade with massive production of cytokines following the binding of CAR T-cell receptor to its target antigen. Upon activation of CAR T-cells, there is release of high levels of IFNγ, whose ability of inducing the activation of other immune cell subsets, mostly macrophages, is well-known.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e This inflammatory loop, involving activated CAR T-cells, lysed cancer cells and macrophages, results into the massive production of additional cytokines, including IL-6, TNF-α, and IL-10, together with several catecholamines,\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e exponentially amplifying the inflammatory response. This process, on one hand, increases the anti-leukemia activity of CAR T-cells,\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e but on the other, can evolve into an uncontrolled, harmful condition.\u003c/p\u003e \u003cp\u003eWhile low-grade CRS is usually easily managed using symptomatic measures and supportive care and, if needed, tocilizumab, high-grade CRS requires the administration of steroids after tocilizumab to control the inflammatory cascade before occurrence of organ failures.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Unfortunately, it has been reported that the prolonged use of high-dose steroids affects the anti-tumor activity of CAR Tcells, impairing long-term outcome of patients.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Moreover, the use of tocilizumab has some limitations. In particular, several groups found that an early intervention with tocilizumab largely mitigate the risk of sCRS, but does not affect neither most cases of fulminant refractory HLH/MAS,\u003csup\u003e17\u003c/sup\u003e nor treatment of ICANS.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e This inefficacy on ICANS may be related to the different pathophysiology between CRS and ICANS, as well as to the poor penetration of tocilizumab across the blood-brain barrier (BBB). Indeed, it has been demonstrated that prophylactic use of tocilizumab decreases the incidence of sCRS, but increases the occurrence of severe ICANS,\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e likely owing to the higher circulating levels of IL-6 induced by the receptor blockade that, in turns, results into higher IL6 levels in the central nervous system (CNS).\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e In view of these findings, the most recent guidelines recommend the use of corticosteroid therapy for grade\u0026thinsp;\u0026ge;\u0026thinsp;2 ICANS.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAs already mentioned, high levels of IFNγ have been found in patients with CRS, supporting the hypothesis that this cytokine plays a pivotal role of in driving the inflammatory process.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Notably, pericytes exposed to IFNγ contribute to the activation of endothelial cells and to the increase of BBB permeability, a dysfunction that appears to be a relevant finding in neurotoxicity: after occurrence of BBB disruption, IFNγ and TNF-α can activate the immune effector microglia cells, whose cytokines, released upon activation, induce neuro-inflammation and brain damage.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn view of all these considerations, we sought to evaluate whether inhibition of the IFNγ axis through emapalumab, a human monoclonal antibody directed against human IFNγ already approved by the FDA since November 2018 for treatment of refractory, relapsed primary HLH (pHLH),\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e can offer a therapeutic opportunity to manage these complications. Our results indicate that emapalumab is able to induce a significant reduction of CRS, HLH/MAS and brain damage, without impairing the anti-leukemia activity of CAR T-cells against CD19-positive B-cell malignancies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eNeutralization of IFNγ does not impair CAR.CD19 T-cell anti-leukemic capacity\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIn order to obtain preliminary evidence of the clinical suitability of emapalumab to manage CAR T-cell toxicities, we first evaluated whether neutralization of the IFNγ axis affects the cytotoxic activity CAR T cells against CD19\u0026thinsp;+\u0026thinsp;tumor cells. For this purpose, peripheral blood mononuclear cells (PBMCs) derived from healthy donor (HD) were genetically modified with the 4-1BB-based CAR.CD19 construct used clinically at the Bambino Ges\u0026ugrave; Children Hospital of Rome (NCT03373071). In details, the anti-lymphoma activity of CAR.CD19 T-cells was tested in long-term co-culture assays with CD19\u003csup\u003e+\u003c/sup\u003e lymphoma Daudi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and Raji (Supplemental Fig.\u0026nbsp;1A) cell lines, either in the absence or in the presence of the anti-IFNγ monoclonal antibody emapalumab. Concentration of emapalumab was in the range of 0.2\u0026ndash;100 \u0026micro;g/mL. This dose range corresponds to the concentration of drug observed in plasma of individual healthy subjects receiving emapalumab (study NI-0501-03).\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The presence of emapalumab in the culture did not cause any significant modification of the cytotoxicity exerted by CAR.CD19 T-cells toward CD19\u0026thinsp;+\u0026thinsp;lymphoma cells (Effector:Target, E:T ratio of 1:1) \u003cem\u003ein vitro\u003c/em\u003e, as compared to untreated conditions, at any of the tested concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA for Daudi model and Supplemental Fig.\u0026nbsp;1A for Raji model). The evaluation of cytokines in the supernatant of the co-culture confirmed that IFNγ was completely neutralized at the doses used (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Supplemental Fig.\u0026nbsp;1B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for the tested conditions), whereas no significant differences were observed for Granzyme B, IL2 and TNFα. In order to confirm these results in a more stressed condition, which could reproduce more reliably the clinical situation, the same experiment of co-culture with lymphoma cells was also performed at suboptimal conditions of decreased E:T ratio (from 1:1 to 1:80), to further evaluate whether the IFNγ neutralization could impact the cytotoxic activity of CAR.CD19 T-cells. In these experiments, emapalumab was added to the co-cultures at the highest dose of 100 \u0026micro;g/mL. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Supplemental Fig.\u0026nbsp;1C, the anti-lymphoma activity of CAR.CD19 T-cells was superimposable to the control condition, in which emapalumab was not added to the co-culture, at all the ratios tested.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, to test if the IFNγ neutralization interferes with the kinetics of target killing in a short (few hours) and long-time frame (up to 64 hours), the anti-lymphoma activity of CAR.CD19 T-cells against Daudi tumor cells was analyzed through a real-time, quantitative, live-cell imaging system that allows the visualization and quantification of live GFP\u0026thinsp;+\u0026thinsp;cells over time. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, CAR.CD19 T cells exposed to emapalumab (dotted black line) exhibit a cytotoxicity profile superimposable to that of the untreated CAR.CD19 T-cells (black line). Moreover, we did not find differences in the proliferation index of CAR.CD19 T-cells induced by the 72-hour exposure to Daudi cells either in the absence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, blue area) or in the presence of 100\u0026micro;g/ml emapalumab (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, purple area). Lastly, we analyzed CAR T-cell cytotoxicity at a very early time-point by measuring the bioluminescence of Raji cells, genetically modified to express firefly luciferase (FF-Luc), after 6 hrs of co-culture with CAR.CD19 T-cells, at the E:T ratio 1:1. As shown in Supplemental Fig.\u0026nbsp;1D, we did not observe any significant decrease of elimination index in the presence of Emapalumab. These data were further corroborated by the evaluation, by flow-cytometry, of the markers of apoptosis (Annexin V and 7-AAD) on Raji cells, after 6hrs of coculture with CAR.CD19 T-cells at a 1:1 E:T ratio. As shown in Supplemental Fig.\u0026nbsp;1E, the IFNγ neutralization did not impact significantly on the percentage of early apoptotic Raji cells.\u003c/p\u003e \u003cp\u003eSeveral studies have reported that CAR T cells incorporating 4-1BB costimulatory domains have a significant differential signature and kinetics of lymphoma elimination as compared to CAR T-cells characterized by the presence of the CD28 costimulatory domain.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e We thus investigated whether the antitumor activity of CAR.CD19.CD28 T-cells could be affected by the presence of emapalumab. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, CAR.CD19.CD28 T-cells were co-cultured with Daudi tumor cells at suboptimal conditions of decreased E:T ratio (from 1:1 to 1:2,5), showing that, also in this case, the anti-tumor activity of CAR.CD19 T-cells exposed to emapalumab at the highest dose of 100 \u0026micro;g/mL was superimposable to that of CAR.CD19 T-cells in the absence of the drug.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the evaluation of cytokines in the supernatant collected after 24 hours of co-culture confirmed that IFNγ was completely neutralized (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), whereas no significant differences were observed for Granzyme B, IL2 and TNFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, the killing activity of CAR.CD19 T-cells against Daudi cells analyzed through the real-time imaging system (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), as well as the proliferation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), reveal that IFNγ neutralization does not affect the functionality of CD28-based CAR.CD19 T-cells in either a short or long-time frame experimental setting.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEmapalumab does not impair CAR.CD19 T-cell activation signaling\u003c/h2\u003e \u003cp\u003eIn order to investigate if IFNγ neutralization could have an impact on the CAR T-cell gene signature, we analyzed the expression of 780 genes associated to some essential pathways of CAR-T biology, including phenotype, cell types, exhaustion, metabolic fitness, TCR diversity, toxicity, activation and persistence, in activated CAR.CD19 T-cells, with or without exposure to emapalumab. In details, un-transduced (NT) and CAR.CD19 T-cells were activated with 0.5 \u0026micro;g/ml recombinant human CD19 Fc Chimera Protein for 16 hours before the analysis of the gene signature. Overall, 90 genes resulted to be differentially regulated in the CAR.CD19 T-cell samples as compared to the control NT T-cells (46 up-regulated and 44 down-regulated genes), all involved in activation pathways of T-cells (Supplemental Fig.\u0026nbsp;2A and 2B, Supplemental Table\u0026nbsp;1). Upon addition of emapalumab (100\u0026micro;g/ml) to activated CAR.CD19 T-cells, we observed a differential expression of 14 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Among the 14 deregulated genes, the only upregulated is ITGAM, a gene associated to persistence and T-cell migration signaling (Supplemental Table\u0026nbsp;2). All the other 13 down-regulated genes, as expected, are involved in Interferon signaling pathways, or have already been reported as genes associated to toxicity (gene signature has been evaluated by bioinformatics analysis trough PubMed database, Supplemental Table\u0026nbsp;2), and the majority of them characterized by a linked signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, seven out of the 14 genes deregulated by IFNγ neutralization, were specifically upregulated by the CAR.CD19 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLastly, to confirm the data on gene expression with the phenotypic characterization of CAR.CD19 T-cells, the CAR.CD19 T-cell activation phenotype was tested upon challenge with lymphoma cells (E:T ratio of 1:1), in the presence or absence of emapalumab. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, we corroborated the gene expression data by monitoring the expression of several activation markers, including CD25, CD40L, HLA-DR, CD28, CD38, CD44 and CD69, showing no significant modulation when CAR.CD19 T-cells are stimulated by the lymphoma DAUDI cells in the presence of INFγ neutralization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIFNγ neutralization preserves CD19.CAR T-cell anti-lymphoma activity in a murine\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e \u003cb\u003emodel\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe then tested the impact of emapalumab on the anti-lymphoma activity of CAR.CD19 T cells in the \u003cem\u003ein vivo\u003c/em\u003e setting, in two different treatment settings.\u003c/p\u003e \u003cp\u003eFirst, we considered an animal model in which CAR.CD19 T cells were infused when the tumor burden was low, mimicking most of clinical applications of CAR T-cells in Bcp-ALL patients. In this setting, clinical practice has shown that the onset of toxicity is delayed and both the incidence and severity of toxicity are limited.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In light of these considerations, we administered emapalumab 7 days after CAR T-cell infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows the experimental design of the \u003cem\u003ein vivo\u003c/em\u003e model). Mice were intravenously (\u003cem\u003ei.v.\u003c/em\u003e) engrafted with Daudi FF-LUC cells on day \u0026minus;\u0026thinsp;2 and tumor burden was then monitored by \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS). After tumor engraftment, mice received \u003cem\u003ei.v.\u003c/em\u003e control NT or 10X10\u003csup\u003e6\u003c/sup\u003e CAR.CD19 T-cells, followed by the intraperitoneal (\u003cem\u003ei.p.\u003c/em\u003e) administration of either control vehicle or 100 mg/kg emapalumab on Days 7, 11 and 15, corresponding to the doses used for the non-clinical \u003cem\u003ein vivo\u003c/em\u003e pharmacology/toxicology studies of emapalumab. [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fda.gov/drugs/fda-approves-emapalumab-hemophagocytic-lymphohistiocytosis\u003c/span\u003e\u003cspan address=\"https://www.fda.gov/drugs/fda-approves-emapalumab-hemophagocytic-lymphohistiocytosis\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]. Notably, tumor was still largely detectable at the first day of emapalumab administration (Day 7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, CAR.CD19 T cells in the presence of emapalumab exerted a significant lymphoma control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, mice from #13 to #16; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D, bioluminescence average value at day 28\u0026thinsp;=\u0026thinsp;5.87E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;8.17E\u0026thinsp;+\u0026thinsp;04), which did not differ from that of mice not exposed to emapalumab (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, mice from #9 to #12; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, bioluminescence average value at day 28\u0026thinsp;=\u0026thinsp;6.18E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;9.59E\u0026thinsp;+\u0026thinsp;04; p\u0026thinsp;=\u0026thinsp;0.58).\u003c/p\u003e \u003cp\u003eIn the second animal model, we wanted to mimic the clinical situation of a high tumor burden, often associated with acute toxicity in patients treated with CAR.CD19 T-cells.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In this case, the administration of emapalumab was considered in a schedule of preventive treatment. In particular, mice were \u003cem\u003ei.v.\u003c/em\u003e engrafted with Daudi FF-LUC cells on day \u0026minus;\u0026thinsp;8 to allow a tumor burden to grow up to 10\u003csup\u003e7\u003c/sup\u003e level of bioluminescence (sec/cm2/sr) before the \u003cem\u003ei.v.\u003c/em\u003e infusion of either NT or CAR.CD19 T-cells (10x10\u003csup\u003e6\u003c/sup\u003e cells per mouse) at day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE shows the experimental design of the second \u003cem\u003ein vivo\u003c/em\u003e model). Emapalumab (100 mg/kg) was administered i.p. on Days 0, 3 and 6. Also in this second animal model, we were able to provide evidence that CAR.CD19 T-cells exerted a significant lymphoma control in the presence of IFNγ neutralization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, mice #13 to #16; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, bioluminescence values \u0026ndash;dotted black lines- for each animal over time; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, bioluminescence average value at day 15\u0026thinsp;=\u0026thinsp;5.67E\u0026thinsp;+\u0026thinsp;07; p\u0026thinsp;=\u0026thinsp;0.0002 vs the cohort of mice receiving NT T-cells), which did not differ from that of untreated counterpart (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, mice #9 to #12; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, bioluminescence values \u0026ndash; black lines- for each animal over time; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, bioluminescence average value at day 15\u0026thinsp;=\u0026thinsp;2.18E\u0026thinsp;+\u0026thinsp;07; p\u0026thinsp;=\u0026thinsp;0.47).\u003c/p\u003e \u003cp\u003eAs shown in Supplementary Fig.\u0026nbsp;3A, in this latter setting, we have also monitored the IFNγ neutralization that persists until the end of the \u003cem\u003ein vivo\u003c/em\u003e experiment, without affecting the \u003cem\u003ein vivo\u003c/em\u003e CAR.CD19 T-cell expansion (Supplementary Figs.\u0026nbsp;3B, 3C, 3D and 3E).\u003c/p\u003e \u003cp\u003eThis \u003cem\u003ein vivo\u003c/em\u003e study was also performed using lower doses of CD19.CAR T-cells to investigate whether emapalumab administration affects CAR T-cell activity and proliferation in a more stringent and challenging setting characterized by a low CAR T-cell dose. For this reason, after tumor engraftment, mice received \u003cem\u003ei.v.\u003c/em\u003e infusion of 1X10\u003csup\u003e6\u003c/sup\u003e or 0.1X10\u003csup\u003e6\u003c/sup\u003e CAR.CD19 T-cells, followed by the \u003cem\u003ei.p.\u003c/em\u003e administration of 100 mg/kg of emapalumab or control vehicle on days 0, 3 and 6 (experimental setting in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, control NT conditions showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eWe did not observe any difference in the bioluminescence between emapalumab-treated or untreated mice for both CAR T-cell doses we used (Supplementary Figs.\u0026nbsp;4A and 4B), with, in particular, a significant control of the lymphoma tumor being only observed in the cohort of mice receiving 1X10\u003csup\u003e6\u003c/sup\u003e CAR T-cells and Emapalumab (Supplementary Figs.\u0026nbsp;4A and 4B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of a simplified humanized murine model effectively reproducing CRS and brain damage\u003c/h2\u003e \u003cp\u003ehGM-CSF/hIL3 NOG mice engrafted with human CD34\u0026thinsp;+\u0026thinsp;hematopoietic stem cells (HSCs) were used in order to establish an animal model recapitulating the human CRS and brain damage. hGM-CSF/hIL3 NOG mice, indeed, stably develop extensive human myeloid and lymphoid cell lineages which are present in peripheral blood, bone marrow, thymus, spleen and non-lymphoid tissue including lung and liver. Engraftment of human hematopoietic CD45\u003csup\u003e+\u003c/sup\u003e cells in the peripheral blood (Supplementary Fig.\u0026nbsp;5A), whose distribution was characterized by the presence of B cells, T cells and CD14\u0026thinsp;+\u0026thinsp;cells (Supplementary Fig.\u0026nbsp;5B), was confirmed before proceeding with the experimental plan. Notably, in order to provide a non-proliferating tumor substrate, aimed exclusively to activate CAR T-cells and to avoid developing uncontrolled tumor burden, thus confounding the CRS symptoms and impacting on mouse overall survival, humanized hGM-CSF/hIL3 NOG mice were engrafted with irradiated CD19\u0026thinsp;+\u0026thinsp;Daudi (irrDaudi) FF-LUC cell lines by \u003cem\u003ei.v.\u003c/em\u003e injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). After the infusion of Daudi cells, mice received \u003cem\u003ei.v.\u003c/em\u003e infusion of CAR.CD19 T-cells (10x10\u003csup\u003e6\u003c/sup\u003e/mouse), generated from healthy donors (HDs). CRS signs, including general suffering, as well as circulating pro-inflammatory cytokines, were monitored over the course of 15 days. We observed that humanized mice infused with irrDaudi and CAR T-cells showed a significant increase of pro-inflammatory cytokines, including IFNγ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), IL6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), TNF-α (Supplemental Fig.\u0026nbsp;6B), the INFγ-driven chemokine CXCL9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), CXCL10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) as well as IL-1β (Supplemental Fig.\u0026nbsp;6A), although this last detected to a low level of 3.96 pg/ml\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 pg/ml.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, this CRS model was characterized by a severe toxicity, all mice dying in the first 7 days after CAR.CD19 T-cell infusion, without the potentially confounding signs of sufferance due to lymphoma cell expansion, since the mice were engrafted with irradiated not-proliferating lymphoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). In addition, considering the severity of the generated model, an \u003cem\u003ead-hoc\u003c/em\u003e experiment was designed to evaluate the brain damage occurrence in this murine model of acute CAR T-cell toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), by sacrificing mice 3 days after CAR T-cell infusion. In this setting, the three doses of emapalumab were administered daily (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In mice given both irrDaudi and CAR.CD19 T-cells, we observed a significant increase in the occurrence of hemorrhagic areas in the central nervous system of mice developing CRS, as compared to control mice infused only with irrDaudi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC p\u0026thinsp;=\u0026thinsp;0.035).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo study the relevance of our experimental model for the occurrence of HLH/MAS, we analyzed bone marrow samples obtained from mouse tibiae. We observed a marked \u0026ldquo;starry sky\u0026rdquo; appearance, representing phagocytes that have engulfed apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, middle panels). HLH/MAS histological findings was never observed in control mice infused only with irrDaudi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, top panels).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEmapalumab is highly effective in controlling acute and fatal toxicity related to CAR.CD19 T-cells\u003c/h2\u003e \u003cp\u003eThe humanized model was instrumental to study the ability of emapalumab to control CAR.CD19 T-cell toxicity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, emapalumab infusion was associated with a significant reduction in the serum levels of the INFγ-driven chemokine CXCL9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD; p\u0026thinsp;=\u0026thinsp;0.007 at Day\u0026thinsp;+\u0026thinsp;3 and p\u0026thinsp;=\u0026thinsp;0.01 at Day\u0026thinsp;+\u0026thinsp;4), CXCL10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE; p\u0026thinsp;=\u0026thinsp;p\u0026thinsp;=\u0026thinsp;0.002 at Day\u0026thinsp;+\u0026thinsp;3 and p\u0026thinsp;=\u0026thinsp;0.005 at Day\u0026thinsp;+\u0026thinsp;4) and IL6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC; p\u0026thinsp;=\u0026thinsp;0.009 at Day\u0026thinsp;+\u0026thinsp;3 and p\u0026thinsp;=\u0026thinsp;0.01 at Day\u0026thinsp;+\u0026thinsp;4). Most importantly, mice engrafted with irrDaudi and treated with the concomitant infusion of CAR.CD19 T-cells and emapalumab showed an overall survival of 100%, even extending the follow-up to 15 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, p\u0026thinsp;=\u0026thinsp;0.02 vs control untreated mice). Moreover, in the animals receiving CAR T-cells and emapalumab, we observed a significant reduction of the hemorrhagic areas in brains compared to mice not given emapalumab, their histology being similar to that observed in control animals that did not receive CAR T-cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, p\u0026thinsp;=\u0026thinsp;0.045). To deeply evaluate the impact of IFNγ neutralization in reducing brain damage in mice developing CRS, brain tissue sections were analyzed for the expression of 770 human genes involved in the cellular stress and injury response, glial regulatory pathways, inflammation and peripheral immune invasion, glial cell homeostasis and activation, and neurotransmission. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and Supplementary Table\u0026nbsp;3, 32 genes were significantly downregulated in emapalumab-treated mice compared to their untreated counterparts. Notably, these genes are characterized by a linked signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), significantly associated to biological pathways relevant for our setting, namely oxidative stress, IFNγ signaling, chemokine- and cytokine-mediated inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLastly, histopathologic evaluation of tibia bone marrow from mice infused with CAR.CD19 T-cells and treated with emapalumab showed a significant reduction in phagocytic cells or mitoses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, bottom panels) compared to mice not given emapalumab which develop severe CRS.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs CAR T-cell field is exponentially growing, scientists are seeking for novel strategies to mitigate, cure or even prevent the toxicities associated with treatment, therefore increasing the therapeutic window of the approach. However, the lack of comprehensive animal models able to recapitulate human toxicities represents a relevant obstacle. A humanized model of CRS was previously published,\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e but the complexity of the approach employed has limited its large-scale application to further investigate CAR T-cell toxicity/pathophysiology and therapeutic interventions.\u003c/p\u003e \u003cp\u003eIn order to study CAR T-cell-related complications, we developed a humanized mouse model that reproduces the most relevant human acute toxicities observed in patients after CAR T-cell infusion, namely CRS, ICANS and HLH/MAS. After humanization, mice were engrafted with a high tumor burden and received a standard dose of CAR T-cells. Thanks to the sub-lethal tumor cell irradiation, we were able to discern signs of CAR T-cell-derived toxicity, minimizing those related to lymphoma engraftment and progression. The developed model is able to recapitulate severe CAR T-cell-related acute toxicities, and all mice died in the first 7 days following CAR T-cell infusion. Moreover, to the best of our knowledge, this is the first animal model in which a clear CAR T cell-induced brain damage has been reported. In particular, after infusing a high number of tumor cells and high dose of CAR T-cells, mice developed multifocal brain hemorrhages, which are well-known manifestation of loss of cerebral vascular integrity with evidence of endothelial activation. Although brain hemorrhages are not formally included in the definition of ICANS, endothelial activation and multifocal vascular disruption are commonly found in the brain of patients developing fatal neurotoxicity after CAR T-cell infusion.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Indeed, it has been documented that patients with severe ICANS (grade\u0026thinsp;\u0026ge;\u0026thinsp;3) show evidence of endothelial activation, disseminated intravascular coagulation, capillary leak, and increased BBB permeability, leading to increased risk of intracranial bleeding.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e The permeable BBB fails to protect the cerebrospinal fluid (CSF) from accumulation of systemic cytokines, including IFNγ, which induces brain vascular pericyte stress and secretion of endothelium-activating cytokines, ultimately promoting multifocal brain bleeding.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn our animal model recapitulating the CAR-T cell-related toxicities, we decided to investigate the role of INFγ neutralization. We focused our study on the use of emapalumab, because this humanized monoclonal antibody, targeting both free and receptor-bound INFγ, is already clinically available and has shown a very promising toxicity profile in children with primary HLH relapsing, refractory or intolerant to conventional therapies, therefore representing an easily clinically translatable approach.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e We documented the emapalumab ability to neutralize high concentration of IFNγ produced by CAR T-cells upon the engagement with tumor cells; we also showed that this neutralization does not affect the anti-tumor activity of CAR.CD19 T-cells. This finding was first obtained \u003cem\u003ein vitro\u003c/em\u003e, through the functional study of CAR.CD19 T-cells cultured with lymphoma cells in the presence of high concentration of emapalumab, in standard co-culture conditions (E:T ratio of 1:1) and then confirmed with very low E:T ratio. Even in this unfavourable condition, the presence of emapalumab did not affect CAR T-cell activity. Proliferation assay and analysis of cytotoxicity at shorter timepoints confirm that CAR T-cell efficacy is preserved in presence of emapalumab. It is noteworthy that these findings were obtained independently from the costimulatory domain included in the second-generation CAR construct targeting CD19, namely either 4.1BB or CD28, the latter being reported to be associated with the occurrence of more severe acute toxicities than those triggered by 4-1BB CAR.CD19 T-cells.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMoreover, we have performed gene expression analysis to compare eight essential pathways related to CAR T-cell biology in either the presence or absence of IFNγ neutralization, showing that the only 14 genes (out of the 780 investigated) modulated in the presence of emapalumab were mainly genes associated to IFNγ signalling and toxicity pathways, as expected. This result is also corroborated by the flow-cytometry analysis of CAR.CD19 T-cell with respect to the expression of activation markers, which reveals an identical activation profile between CAR.CD19 T-cells exposed to emapalumab and the untreated counterpart. The preserved functionality of CAR T-cells in presence of emapalumab was confirmed also \u003cem\u003ein vivo\u003c/em\u003e, in a lymphoma animal model, in the presence of high doses of emapalumab, administered with a schedule of 3 infusions, 4-day apart, concomitant with the onset of toxicity or concurrent with the infusion of CD19.CAR T-cells. No significant reduction of the activity of CAR.CD19 T-cell against Daudi cells was observed in mice infused with emapalumab. In addition, in the NSG mouse model, our data indicate that CAR T-cell persistence and expansion were unaffected by IFNγ neutralization. Encouraged by these data, we then tested the possibility to control toxicity of CAR T-cells by using emapalumab in a comprehensive humanized model. Notably, the administration of emapalumab induced a complete neutralization of IFNγ with significant reduction of other inflammatory cytokines and chemokines, including CXCL-9, a molecule shown to be highly correlated with HLH/MAS activity. In particular, we measured in mice blood, human IL-6, one of the most elevated cytokines during CRS, likely released by activated endothelial cells, as well as by activated macrophages stimulated by IFNγ, which represents the most relevant therapeutic target to manage CRS in humans. In patients, IL-6 elevation can cause capillary leakage, hypotension, activation of complement pathway and coagulation cascades, and myocardial dysfunction.\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e We clearly showed that the neutralization of IFNγ represents a valid approach to mitigate the elevation of this key component of the inflammatory cascade downstream of IFNγ itself. Indeed, we detected a significant reduction of circulating human IL6 levels in mice treated with emapalumab, as compared to untreated mice. Moreover, in our animal model of CRS, we measured circulating levels of two human chemokines, CXCL9 (also known as MIG) and CXCL10 (also known as IP-10), specifically induced by IFNγ and synthesized and secreted by histiocytes and dendritic cells, thus allowing quantification of IFNγ-inhibition.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Both chemokines have been found to be significantly increased in patients experiencing grade 4\u0026ndash;5 versus grade 0\u0026ndash;3 CRS.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e In our model, we found a significant increase of CXCL9 and CXCL10 in mice developing CRS after CAR.CD19 infusion. Notably, IFNγ neutralization was able to inhibit their elevation. Most importantly, emapalumab administration was able to completely protect mice from acute CRS, as all mice treated with emapalumab survived behind the time-line of the experimental plan (15 days), while untreated mice died within 7 days after CAR.CD19 T-cell infusion. We also studied the brain damage in mice developing acute toxicity, and proved that emapalumab administration is able to significantly control the multifocal bleeding in CNS, which has been associated to high-grade neurotoxicity in patients treated with CAR T-cells, \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e as well as to downregulate the expression of key signalling molecules associated to IFNγ pathway and inflammation mediated by chemokines/cytokines. Altogether, these results provide the biological rationale for considering the use of emapalumab as pathogenetic treatment of ICANS.\u003c/p\u003e \u003cp\u003eOur data are in line with a recent experimental study demonstrating that an anti-INFγ approach, performed using a no azide/low endotoxin (NA/LE)-produced mouse anti-human IFNγ monoclonal antibody, abrogates macrophage activation in an \u003cem\u003ein vitro\u003c/em\u003e model of CRS.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e However, in the study of Bailey and colleagues\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, the impact of IFNγ neutralization on severe systemic CRS, BBB damage or secondary HLH was not investigated \u003cem\u003ein vivo\u003c/em\u003e. The potential importance of INFγ neutralization in management of CAR T-cell toxicities is supported by a recently published case report of a patient treated with emapalumab for life-threatening grade 4 CRS and neurotoxicity refractory to tocilizumab.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Emapalumab (administered at the dosage of 1 mg/kg) was infused on day 9 after numerous interventions, including repeated doses of tocilizumab, corticosteroids and siltuximab. Although it is not possible to attribute definitive causality to any particular intervention, patient fully recovered from CRS at day\u0026thinsp;+\u0026thinsp;18, and one week later from neurotoxicity. Notably, this patient maintained clinical remission and sustained B-cell aplasia for 12 months after CAR T-cell infusion, this observation corroborating our experimental observation that INFγ neutralization does not interfere with CAR T-cell efficacy.\u003c/p\u003e \u003cp\u003eIn summary, in a simplified but comprehensive humanized model of CRS that overcomes the experimental limitations of previously reported CRS models, our study provides, for the first time, consistent evidence that emapalumab is a promising drug able to contain the CAR T-cell inflammatory cascade associated to high-grade CRS, MAS/HLH and brain injury, while sparing anti-tumor activity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures\u003c/h2\u003e \u003cp\u003eCD19 positive human Burkitt's lymphoma cell lines Daudi and Raji (ATCC, USA), genetically modified with firefly luciferase (FF-Luc), were cultured in RPMI 1640 medium (EuroClone, Italy) supplemented with 10% heat-inactivated foetal bovine serum (EuroClone), 2mM l-glutamine (GIBCO, USA), 25 IU/mL of penicillin, and 25 mg/mL of streptomycin (EuroClone), in a humidified atmosphere containing 5% CO2 at 37\u0026deg;C. All cell lines were authenticated by PCR-single-locus-technology (Promega, USA. PowerPlex 21 PCR) performed in \"BMR Genomics s.r.l.\" (Italy), and routinely checked for mycoplasma (Venor\u0026reg;GeM Advance, MB Minerva biolabs, UK) and surface marker expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCAR.CD19 T-cells generation\u003c/h2\u003e \u003cp\u003ePBMC derived from buffy coats of HDs were isolated, activated, transduced and expanded as previously described.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e HDs signed a written informed consent, in accordance with rules set-up by the Institutional Review Board (IRB) of Bambino Ges\u0026ugrave; Children Hospital, IRCCS, Rome, Italy (OPBG; Approval of Ethical Committee N\u0026deg;969/2015 prot.N\u0026deg;669LB, and N\u0026deg;1422/2017 prot.N\u0026deg;810).\u003c/p\u003e \u003cp\u003eThe retroviral CAR.CD19 construct includes the anti-human CD19-scFv derived from FMC63 clone, in frame with 16aa sequence of the human CD34 antigen, the CD8 stalk domain, the CD8 transmembrane domain and the 4.1BB and CD3ζ cytoplasmic domains.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e The retroviral CD28 based-CAR.CD19 construct includes the anti-human CD19-scFv derived from FMC63 clone, in frame with 16aa sequence of the human CD34 antigen, the CD8 stalk domain, the CD8 transmembrane domain and the CD28 and CD3ζ cytoplasmic domains.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhenotypic analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExpression of cell surface molecules was determined by flow-cytometry using standard methodology. The following monoclonal antibodies (mAbs) were used: CD3, CD4, CD8, CD14, CD19, CD25, CD28, CD34 (for CAR detection), CD38, CD40L, CD44, CD45, CD69 and HLA-DR (BD Bioscience). Samples were acquired with a BD LSRFortessa X-20 and analysed using the FACSDiva software (BD Biosciences). For each sample, we analysed a minimum of 20,000 events.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCytokine analysis\u003c/h2\u003e \u003cp\u003eIFNγ, Granzyme B, IL-2, TNF-α, IL-6, IL-1β, CXCL9/MIG and CXCL10/IP-10 levels found either after 24h co-culture assays or in peripheral blood of mice were analyzed by the enzyme-linked lectin assay Ella Automated Immunoassay System (Bio-Techne, California, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity Assays\u003c/h2\u003e \u003cp\u003eThe IncuCyte S5 Live Cell Assay System (Sartorius, Michigan, MI, USA) was used for kinetic monitoring of CAR T-cell cytotoxicity against tumor in presence or absence of emapalumab. Effector T-cells (NT or CAR.CD19) and FF-LUC Daudi tumor cells were plated at 0.25 x10\u003csup\u003e6\u003c/sup\u003e cells/well on 48-well plates at 1:1 E:T ratio in presence or absence of 100\u0026micro;g/ml emapalumab and incubated for 64 hours at 37\u0026deg;C in a humidified atmosphere (5% CO2). Growth curves were generated by the algorithm in the \u0026ldquo;2021C\u0026rdquo; software (Schr\u0026ouml;dinger; New York, NY, USA) evaluating GFP values ​​expressed by Daudi tumor cells from data points acquired during imaging at 2-hour intervals. All samples were plated in triplicate.\u003c/p\u003e \u003cp\u003eShort-term (6 hours) CAR.CD19 T-cell cytotoxic assay was performed with a luciferase-based test. Briefly, effector T-cells (NT or CAR.CD19) and FF-LUC-expressing Raji tumor cells were plated, at 0.5 x10\u003csup\u003e6\u003c/sup\u003e cells/well, on 24-well plates, at 1:1 E:T ratio, either in the presence or in the absence of 100\u0026micro;g/ml emapalumab, and incubated at 37\u0026deg;C in a humidified atmosphere (5% CO2). After 6 hours, 15\u0026micro;g/ml of luciferin (Xenolight D-luciferin; Perkin Elmer) was added to the media and bioluminescence (BLI) was quantified after 10 min of incubation at 37\u0026deg;C in a humidified atmosphere (5% CO2) on an ENSPIRE Multimode plate reader (Perkin Elmer).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis Assay\u003c/h2\u003e \u003cp\u003eApoptosis of Raji target cells was evaluated by flow-cytometry-based assay. In particular, effector T-cells (NT or CAR.CD19) and Raji tumor cells were plated at 0.5 x10\u003csup\u003e6\u003c/sup\u003e cells/well, on 24-well plates, at 1:1 E:T ratio, either in the presence or in the absence of 100\u0026micro;g/ml emapalumab, and incubated at 37\u0026deg;C in a humidified atmosphere (5% CO2). After 6 hours, cells were labeled with Annexin V BUV-395 (BD Bioscences)/ 7-Amino-Actinomycin D (7-AAD; BD Bioscences) according to the manufacturer's instructions and evaluated by flow-cytometry, as specified in the phenotypic analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation assay was performed by labeling cells with the CellTraceTM Cell Proliferation Kit (Far Red, Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, CAR.CD19 T-cells were washed with PBS and resuspended at 10\u003csup\u003e6\u003c/sup\u003e cells/mL in working dye solution (1\u0026micro;M in 1xPBS) for 20 minutes at 37\u0026deg;C. Thereafter, labeled cells were resuspended in five volumes of cell culture medium, centrifuged, and resuspended in culture media to be co-cultured with Daudi tumor cells (at 1:1 E:T ratio) in presence or absence of 100\u0026micro;g/ml emapalumab for up to three days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNanostring analysis\u003c/h2\u003e \u003cp\u003eRNA was isolated from samples using the RNeasy mini kit (Qiagen) according to supplier\u0026rsquo;s instructions. Total RNA was quantified with NanoDrop ND-100. We analyzed the expression of 780 genes (including 10 reference genes) related to components of CAR-T biology using the nCounter CAR-T Characterization Panel\u0026trade; (XT CAR-T Code-Set Panel, Nanostring, Seattle, WA) and of 770 genes (including 10 reference genes) related to components of glial biology using the nCounter Human Glial Profiling panel (XT Human Glial Profiling Code-Set Panel, Nanostring, Seattle, WA). Total RNA was used as input and sample hybridization was performed according to the manufacturer's instructions. Sample detection and analysis were completed on an nCounter\u0026reg; Digital Analyzer. Raw data processing, quality control, and normalization were performed using the nSolver\u0026trade; 4.0 analysis software (NanoString nCounter Technologies, Seattle, WA). Background subtraction from raw transcript counts was performed through negative input controls. Normalization to 10 housekeeping genes and differential expression analysis were completed using the Advanced Analysis software plugin (version 2.0.115). For differential expression analysis, a p-value of \u0026le;\u0026thinsp;0.05, were applied as cut-offs. Gene set enrichment was evaluated with the Enrichr (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://maayanlab.cloud/Enrichr/\u003c/span\u003e\u003cspan address=\"https://maayanlab.cloud/Enrichr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) web tool. The basic interaction unit in STRING has been used to evaluate possible network between genes differentially expressed (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vivo\u003c/span\u003e \u003cb\u003eCAR\u0026thinsp;+\u0026thinsp;lymphoma mouse model\u003c/b\u003e\u003c/p\u003e \u003cp\u003e All procedures were performed in accordance with the Guidelines for Animal Care and Use of the National Institutes of Health (Ethical committee for animal experimentation Prot. N 088/2016-PR). To perform anti-tumor \u003cem\u003ein vivo\u003c/em\u003e study, Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) female mice were purchased from Charles Rives Laboratories and maintained in the Plaisant animal facility in Castel Romano, Rome, Italy. Mice were \u003cem\u003ei.v.\u003c/em\u003e engrafted with 0.25x10\u003csup\u003e6\u003c/sup\u003e FF-Luciferase positive Daudi cells at Day-2 or at Day-8, and treated with 10x10\u003csup\u003e6\u003c/sup\u003e un-transduced (NT) or CAR.CD19 T-cells/mouse at Day 0. At Day 7, 11, 15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) or at Day 0, 3, 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) respectively, mice received 100mg/Kg Emapalumab. Tumor growth was monitored weekly by IVIS Imaging System,\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e after D-Luciferin (PerkinElmer, D-Luciferin potassium salt) intraperitoneal (i.p.) administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHumanized murine model of CRS and brain injury after CAR.CD19 T-cell administration\u003c/h2\u003e \u003cp\u003ehGM-CSF/hIL3 NOG mice were purchased from Taconic Laboratories and maintained at the Plaisant animal facility in Castel Romano, Rome, Italy. Mice were engrafted with human umbilical cord blood-derived CD34\u0026thinsp;+\u0026thinsp;hematopoietic stem cells (HSCs). Mice were aged 10 weeks post engraftment and quality checked for human leukocyte reconstitution by flow-cytometry. Only mice reaching\u0026thinsp;\u0026ge;\u0026thinsp;25% hCD45\u0026thinsp;+\u0026thinsp;cells were used in the experiments. Humanized mice were infused with irradiated (30cGy) FF-Luciferase positive Daudi cells at Day-2 and treated either with 10x10\u003csup\u003e6\u003c/sup\u003e un-transduced (NT) or with CAR.CD19 T-cells/mouse either in the presence or absence of 100mg/Kg/day emapalumab, according to the treatment schedules. Mice were subject to blood bleeding at Day 3 and Day 4 for the monitoring of cytokines. For brain injury evaluation, 3 days after CAR.CD19 T-cell infusion, animals were sacrificed, and brains were fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) and paraffin embedded. The histopathologic haematoxylin and eosin staining was performed on the middle axial sections.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e For HLH/MAS bone marrow analysis, tibiae of experimental mice were collected at sacrifice, and tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. Deparaffined sections were stained with hematoxylin and eosin (Thermo Fischer Scientific). Images were acquired on a ScanScope XT scanner and digitized to scalable images up to a 20\u0026times; objective (Leica Biosystems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eUnless otherwise noted, data are summarized as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The Student\u0026rsquo;s t-test or Mann-Whitney test (two-sided) were used to determine statistically significant differences between samples, with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating a significant difference.\u003c/p\u003e \u003cp\u003eMouse survival data were analyzed using Kaplan-Meier survival curves and the log-rank test was used to measure differences between groups. No valuable samples were excluded from the analyses. Neither randomization nor blinding were performed during \u003cem\u003ein vivo\u003c/em\u003e studies. However, mice were matched based on the tumor signal for control and treatment groups before infusion of NT or CAR T-cells. To compare the growth of tumours over time, bioluminescence signal intensity was collected blindly. Bioluminescence signal intensity was log-transformed and, then, compared using a two-sample t-test. We estimated the sample size considering no significant variation within each group of data. The principle of using the smallest sample size possible was adopted in planning the animal experiments. We estimated the sample size in order to detect a difference in averages of 2 standard deviations at the 0.05 level of significance with an 80% power. Graphic representations and statistical analysis were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental work was supported by grants awarded by\u0026nbsp;Accelerator Award – Cancer Research UK/AIRC – INCAR project (F.L.), Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC)-Special Project 5×1000 no. 9962 (F.L.), AIRC IG 2018 id. 21724 (F.L.), MFAG 21979 (C. Quintarelli), id.26915-2021 AIRC Fellowships Call (S.M.), Ricerca Corrente (C. Q., B.D.A.),\u0026nbsp;Ministero dell’Università e della Ricerca (Grant PRIN 2017 to F.L.); Italian Healthy Ministry project on CAR T RCR-2019-23669115 (Coordinator\u0026nbsp;F.L.),\u0026nbsp;GR-2016-02364546 (B.D.A), RF-2016-02364388 (F.L.), Independent Research grant AIFA (F.L.: 2016 call), “PNRR M4C2- Investment 1.4-CN00000041”– NextGenerationEU, PNC HLS-TA 2022 (F.L.). We are very grateful to\u0026nbsp;SOBI for the collaboration in providing emapalumab used for the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.L and P.M. on September 2022 participated into an advisory board on primary HLH organized by Sobi, receiving honoraria.\u0026nbsp;All the other authors do not have any conflicting financial interests to disclose. SOBI has not supported the study, beside the provision of the emapalumab drug.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.Q., B.D.A., and F.L. designed experimental studies, supervised the project conduction, analysed the data and wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS.M., S.R., Z.A., M.G., S.C., S.D.C., M.S., R.C., L.I. developed the \u003cem\u003ein vitro\u003c/em\u003e models and performed the \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e\n\u003cp\u003eS.M., F.D.B, M.C., and B.D.A. performed the \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e\n\u003cp\u003eS.R. and D.A.S. performed bioinformatics analysis on NanoString data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC.Q. and B.D.A. cloned the retroviral vector.\u003c/p\u003e\n\u003cp\u003eS.M. and M.S. performed FACS analysis.\u003c/p\u003e\n\u003cp\u003eR.D.V. performed HLA/MAS evaluation in the \u003cem\u003ein vivo\u003c/em\u003e model.\u003c/p\u003e\n\u003cp\u003eF.D.B., P.M., A.M., M.C.L., M.G.C. and F.L. provided healthy donor material, medical advices and expertise in CAR T-cell toxicity, as well as in the use of emapalumab.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eLee, D. 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Cancer Res \u003cstrong\u003e78\u003c/strong\u003e, 3337\u0026ndash;3349, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/0008-5472.CAN-17-3140\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1482837/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1482837/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChimeric antigen receptor (CAR) T-cell therapy represents a revolutionary approach to induce long-lasting remission in patients with B-cell malignancies not responding to conventional therapies. Nevertheless, possible severe side effects, including cytokine release syndrome (CRS), neurotoxicity and macrophage activation syndrome, whose management is still challenging, as well as lack of pathophysiological experimental models to investigate novel interventions, limit the widespread use of this therapy. In light of these considerations, we developed a comprehensive humanized mouse model to investigate the role of IFNγ neutralization, provided by the clinically approved monoclonal antibody, emapalumab, in controlling severe toxicity related to CAR T cells. We demonstrated that emapalumab reduces the pro-inflammatory environment in the animal model, allowing severe CRS control and preventing brain damage, characterized by multifocal hemorrhages. Furthermore, we proved that IFNγ inhibition does not affect the ability of CAR.CD19 T cells to eradicate CD19+ lymphoma cells, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Neutralization of IFNγ improves the safety profile of CAR T-cells while maintaining unaffected efficacy against B-cell malignancies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-01 14:54:16","doi":"10.21203/rs.3.rs-1482837/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c061987d-ae31-49a9-a4b9-78449134f089","owner":[],"postedDate":"March 1st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-06-10T07:07:26+00:00","versionOfRecord":{"articleIdentity":"rs-1482837","link":"https://doi.org/10.1038/s41467-023-38723-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2023-06-09 04:00:00","publishedOnDateReadable":"June 9th, 2023"},"versionCreatedAt":"2023-03-01 14:54:16","video":"","vorDoi":"10.1038/s41467-023-38723-y","vorDoiUrl":"https://doi.org/10.1038/s41467-023-38723-y","workflowStages":[]},"version":"v1","identity":"rs-1482837","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1482837","identity":"rs-1482837","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}