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8028 Background: Cilta-cel (JNJ-68284528) is a chimeric antigen receptor T (CAR-T)-cell therapy with 2 BCMA-targeting, single-domain antibodies designed to confer high avidity binding. CARTITUDE-2 (NCT04133636) is a phase 2, multicohort, open-label study assessing the efficacy and safety of cilta-cel in patients (pts) with MM in various clinical settings. Here, we describe the mitigation and management strategies implemented to identify and reduce the risk for neurologic adverse events (AEs) in Cohort A pts (progressive MM after 1−3 prior lines of therapy). Methods: Eligible pts (≥18 years of age) had MM per IMWG criteria, measurable disease, ECOG ≤1, progressive disease after 1−3 prior lines of therapy (including a PI and IMiD) and were lenalidomide refractory (no prior BCMA-targeting agent). Cilta-cel (0.75×106 [range 0.5–1.0×106] CAR+ viable T cells/kg) was given as a single infusion 5–7 days after start of lymphodepletion (cyclophosphamide 300 mg/m2 + fludarabine 30 mg/m2 daily for 3 days). Monitoring and mitigation strategies for neurologic AEs include providing more effective bridging therapy to reduce tumor burden prior to lymphodepletion, frequent assessment of CAR-T-related ICANS using the ICE tool, regular handwriting assessments to detect micrographia, and neuroimaging (brain MRI) and EEG for pts with prior neurologic disease. Management strategies include evaluating infectious and paraneoplastic etiologies upon observation of ICANS ≥Grade (gr) 1, administration of tocilizumab (if concurrent CRS, all gr of ICANS) and/or dexamethasone (gr 2/3) or methylprednisolone (gr 4). ICANS and CRS were graded by ASTCT criteria; neurotoxicities not classified as ICANS were graded per CTCAE Version 5.0. Results: As of 15 Jan 2021 (median follow-up: 5.8 months [range: 2.5–9.8 months]), 20 pts in Cohort A received cilta-cel. Median age was 60 years (range: 38–75); 65% were male. Neurotoxicities occurred in 4 pts (20%). Three pts had ICANS (gr 1/2); median time to onset of symptoms was 8 days (range: 7–11) and median duration was 2 days (range: 1–2). Two of the 3 pts received supportive measures to treat ICANS, including levetiracetam and steroids; all 3 had concurrent CRS and all recovered. One pt developed isolated facial paralysis (gr 2) on Day 29 after cilta-cel infusion, and recovered 51 days after the onset of event following treatment with dexamethasone for 28 days. No movement or neurocognitive disorders were reported. Conclusions: Neurologic AEs were generally manageable in pts with MM following treatment with cilta-cel. With a median of 5.8 months of follow-up, there were no movement or neurocognitive disorders in pts from Cohort A. These results suggest that early detection and management of neurologic AEs can lead to better treatment outcomes. Clinical trial information: NCT04133636.

The objective of this trial is to determine the safety, tolerability, and toxicity of DNX-2401 in newly diagnosed DIPG patients (NCT03178032) followed by radiotherapy. Secondary endpoints are overall survival at 12 months, percentage of responses and induced immune response against tumor. Tumor biopsy was performed through the cerebellar peduncle, followed by intratumoral injection of DNX-2401 (N=12). Three patients were treated with 1x1010vp and given the lack of toxicity we escalated to 5x1010vp. The procedure was well tolerated and reduced tumor volume was demonstrated in all patients after combined treatment (virus + radiotherapy). We performed molecular studies (RNAseq and the Oncomine Childhood Research Panel from Thermo Fisher). The immune cell composition of the biopsies pre-virus injection was assessed using multiplexed quantitative immunofluorescence. T cells were hardly detectable in these tumors while macrophages were abundant. Using a multiplexed TCR-sequencing mRNA-based assay to analyze 18 available paired pre- and post-treatment samples from the trial, we detected increased clonal T cell diversity following treatment with the virus. We also measured pre and post treatment neutralizing antibodies and their relationship with survival. Finally, we performed functional studies using 2 cell lines isolated from patients included in this trial to assess the response to the virus (infectivity, viability, T-cell recognition). In summary, the virus has shown safety and efficacy in some patients. The information obtained in this clinical study would aid understanding the response of DIPG patients to viral therapies and, therefore, to better tailor this strategy to improve the survival of these patients.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumour, being the leading cause of paediatric death caused by cancer. Despite all the advances made regarding effective therapies, the survival is dismal. Our lab has engineered the oncolytic virus Delta-24-ACT armed with the costimulatory ligand 41BBL in order to increase the antitumoral effect of the adenovirus. 41BB is a costimulatory receptor which promotes the expansion of activated T cells and the generation and maintenance of CD8 T memory cells. Therefore, we propose the use of Delta-24-ACT as a therapeutic approach for DIPG tumours. We observed that Delta-24-ACT is able to infect and replicate in NP53 and PDGFB-driven, two DIPG murine cell lines. Furthermore, 41BBL is expressed in the membranes of the infected cells and results with immunogenic cell death as shown by the different DAMPs. Injection of Delta-24-ACT in DIPG model was safe, showed no sign of toxicity and led to a significantly increase in the median overall survival, generating anti-glioma memory in long-term survivors. Mechanistic experiments, showed an increase of T cell infiltration (mainly CD8), decrease of proliferating cells and a reduction of the number of vessels in FFPE brain samples in the treated mice. We are currently performing nanostring analyses to assess the changes in the transcriptional immune phenotype of treated versus control mice. In summary, our data suggest that Delta-24-ACT is safe and induces a potent antitumor immune response in DIPG models mainly based in the activation of CD8 lymphocytes recruited by the viral activity.

Oncolytic viruses have become promising therapeutic candidates to treat gliomas. Our group has developed Delta-24-ACT, an oncolytic adenovirus armed with the positive costimulatory ligand 4-1BBL which is capable to trigger the activation of T cells and thereby increase their antitumor immune response. Here we evaluate the anti-glioma effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor. We observed that Delta-24-ACT was able to infect and kill murine glioma (GL261-5 and CT-2A) and also human glioma cell lines (U87-MG and U251-MG), while maintaining its replication in the latter. Of importance, Delta-24-ACT infection resulted in 4-1BBL expression on the membrane of glioma cells. Moreover, this ligand was functional and was able to stimulate CD8 lymphocytes in vitro, suggesting the potential of Delta-24-ACT to trigger an effective immune response. Furthermore, in vivo Delta-24-ACT showed anti-tumour effect in two murine glioma models by significantly increasing the median survival time and leading to long-term survivors. Mechanistic studies demonstrated an increase of the T cell infiltration and the activation of different immune cell populations by flow cytometry and a decrease of proliferative cells and tumour vessels by immunohistochemistry on FFPE brain samples. Importantly, the infiltrating lymphocytes also showed signs of exhaustion increasing the amount of IL-10 and the expression of PD-1. To overcome this exhaustion we combined Delta-24-ACT with an anti-PD-1 antibody. Evaluation of this combination in vivo further increased the median survival time of treated tumor-bearing mice and resulted in 50% long-term survivors. Rechallenge studies with the same cell line showed that combination treatment effectively protected these animals of developing tumors and therefore, the acquisition of immune memory. In summary, our data demonstrated that Delta-24-ACT induces a potent anti-tumour effect in vitro and in vivo as a result of the recruitment of immune cell populations modulating the immunosuppressive tumour microenvironment of glioma.

Despite our increased understanding of Diffuse Intrinsic Pontine Glioma (DIPG) the outcome remains dismal. Recently we showed that the virus Delta-24-RGD (DNX-2401 in the clinic) was effective in preclinical models of DIPG and had the ability to trigger an antitumor immune response. These data allowed us to propel a phase I clinical trial for newly diagnosed DIPGs (NCT03178032) where the patients received an intratumoral viral injection followed by standard radiotherapy. The main objective is to determine the safety, tolerability, and toxicity of DNX-2401. Secondary endpoints are overall survival at 12 months, percentage of responses and induced immune response against tumor. Tumor biopsy was performed through the cerebellar peduncle, followed by intratumoral injection of DNX-2401 (N=12). Three patients were treated with the D1=1x1010vp and because the lack of toxicity we escalated to the D2= 5x1010vp. The procedure was well tolerated and safe. All patients displayed a reduced tumor volume after combined treatment. We performed molecular studies in 9 out of the 12 patients. The immune cell composition of the biopsies was assessed using multiplexed quantitative immunofluorescence. T cells were hardly noticeable in these tumors while macrophages were abundant. We detected increased clonal T cell diversity following treatment with virus in peripheral blood lymphocytes when compared paired pre- and post-treatment samples from the trial. In addition, we measure pre and post treatment neutralizing antibodies and its relationship with survival. Finally, we performed functional studies using 2 cell lines isolated from patients included in this trial to assess the response to the virus (infectivity, viability, T-cell recognition). Overall, the administration of DNX2401 was safe, feasible and therapeutically beneficial in a subgroup of patients. This trial constitutes a proof of principle that aids to understand the response of DIPGs to viral therapies allowing to set the bases to improve this strategy for DIPG.