Citations

Abstract BACKGROUND Glioblastoma (GBM) is a universally lethal brain cancer with limited durable responses to immunotherapy, largely due to its immunosuppressive tumor microenvironment (TME). Ephrin receptor A3 (EphA3), a receptor tyrosine kinase minimally expressed in normal tissue, is enriched on GBM cells, glioma stem cells (GSCs), and M2-like tumor-associated macrophages (TAMs). We hypothesize that dual targeting of TAMs and tumor cells by EphA3-CAR-T is a viable treatment strategy. METHODS We developed EphA3-directed second-generation CAR-T cells (EphA3-CAR-T) incorporating the single-chain variable fragment of ifabotuzumab, an anti-EphA3 monoclonal antibody, achieving >90% transduction efficiency. We evaluated antitumor efficacy in vitro; the functional activities of EphA3-CAR-T on GSCs were assessed via selective cytotoxicity, T cell proliferation, degranulation assays, and depletion of M2-like macrophages. In vivo, 1 × 10⁶ EphA3⁺ U251 MG cells and/or 1 × 10⁶ ex vivo–polarized M2-like macrophages were implanted in the flanks of NOD-SCID-γ⁻/⁻ mice. Comparative studies with EGFRvIII-directed CAR-T cells were also performed. RESULTS Immunohistochemistry confirmed EphA3 expression across both tumor and immune compartments in patient-derived specimens. EphA3-CAR-T cells demonstrated potent, selective cytotoxicity (p<0.001), T cell proliferation (p<0.001), and CD107α degranulation (p<0.001) against EphA3⁺ GBM and GSCs, while also reversing TAM-mediated immunosuppression via depletion of ex vivo–polarized M2-like macrophages (p<0.05), compared to other validated CAR-Ts. In vivo, EphA3-CAR-T significantly reduced tumor volume (p<0.001) and improved survival (p<0.01), in the presence of TAMs. In contrast, EGFRvIII CAR-T cells were inactivated under the same TME conditions. EphA3-CAR-T cells remained active in models co-engrafted with macrophages, suggesting preserved function within the immunosuppressive TME. CONCLUSIONS EphA3-CAR-T therapy addresses a major barrier to CAR-T success in GBM: TME-mediated immunosuppression. This tumor- and TME-targeting approach demonstrated robust preclinical activity. These findings support the clinical translation of EphA3-CAR-T cells as a next-generation immunotherapy for GBM.

Abstract Glioblastoma (GBM) is the most common malignant tumor of the CNS, accounting for 80% of brain tumors. Recent advances in gene editing and immunology have led to the development of chimeric antigen receptors (CARs), including CARs incorporating chlorotoxin (CLTX) receptor domains for GBM targeting. While CAR-engineered neutrophils have shown promise, their systemic administration can induce severe off-target effects. Extracellular vesicles (EVs), secreted by these cells, retain properties of their parent cells—including CAR expression and tumor-lytic activity—yet their potential has not been explored in CAR Neutrophils. To address this, CAR-CLTX DNA was inserted into the AAV1 safe harbor locus of induced pluripotent stem cells, which were subsequently differentiated into neutrophils. Conditioned media were collected during differentiation and processed using extraPEG-based EV isolation. EVs were quantified via nanoparticle tracking analysis, electron microscopy, and western blotting. Proteomic and microRNA (miRNA) profiling of EV cargo was conducted at days 12 and 21 of differentiation. Cytotoxic effects of EVs were assessed in U87MG and LN229 glioblastoma cells in 2D culture, 3D organoids, and in vivo models. EVs were secreted in high quantities within the 100–200 nm size range, expressed canonical exosomal markers, and exhibited characteristic morphology. Proteomic analysis identified neutrophil-associated proteins involved in migration, chemotaxis, and degranulation. miRNA sequencing revealed high levels of tumor-suppressive miRNAs (e.g., miR-182) implicated in apoptotic signaling, T cell receptor pathways, cytokine-cytokine receptor interactions, Jak-STAT, Hedgehog, and NK cell-mediated cytotoxicity. EV uptake by glioma cells was demonstrated in vitro. Cytotoxic effects of CAR Neutrophil EVs were observed across glioblastoma cell lines, organoids, and in vivo. These findings demonstrate that CAR Neutrophil-derived EVs carry anti-tumor protein and miRNA cargo, are efficiently taken up by GBM cells, and mediate glioma cell cytotoxicity. This supports the feasibility of correlating EV cargo composition with function in GBM treatment.

Abstract Secondary central nervous system lymphoma (SCNSL) is defined as the diagnosis of CNS lymphoma diagnosed concurrently with systemic lymphoma or after prior treatment. Due to the poor prognosis associated with SCNSL, the lack of randomized trial data, and the need for international consensus guidelines, there is a high unmet need for effective therapy options for SCNSL. Here we present safety and efficacy data on 2 SCNSL patients treated with the combination of emavusertib and ibrutinib. Both patients had directly progressed on their prior BTKi treatment. One patient achieved an unconfirmed complete response (CRu) and remained on treatment for over 4 mos, while the other patient maintained stable disease (SD) with a 38% reduction in disease burden from baseline and was on treatment for more than 8 mos. The patient with a durable SD, who received 5 prior lines of therapy along with an ASCT, presented with 3 related AEs Grade ≥ 3 that were reversible and manageable. These included elevated ALT, AST, and bilirubin levels that resolved. The patient received emavusertib 300 mg BID and ibrutinib 560 mg QD. The CRu patient received 6 prior lines of therapy and experienced CK elevation, fatigue, hypoglycemia, sepsis, urinary retention, neutropenia, and constipation that resolved with either a dose reduction and/or supportive care. The initial dose for this patient was 150 mg emavusertib BID and ibrutinib 560 mg QD. Subsequently, the dose of emavusertib was decreased to 150 mg BID due to Grade 4 neutropenia, then increased to 200 mg AM and 150 mg PM once resolved. This patient achieved CRu within 60 days on treatment. The 2 patients presented here are supportive data for using the combination of emavusertib and ibrutinib in SCNSL.

Abstract There are no approved treatments for relapsed/refractory (R/R) PCNSL. We show promising efficacy in the salvage setting using emavusertib in combination with ibrutinib, which targets the BCR and TLR pathways. These 2 major pathways are associated with activation of NF-κB. Updated safety and efficacy data of this combination in R/R PCNSL from the ongoing Phase 1/2 TakeAim Lymphoma trial (NCT03328078) will be presented. As of 01 May 2025, 33 patients with R/R PCNSL were treated with emavusertib in combination with ibrutinib. Eligible patients receive emavusertib in combination with ibrutinib until tumor progression or unacceptable toxicity. Median age was 63 years (range 43-81); 16 patients were male. Median prior anti-cancer therapy was 2 (range 1-5); 26 (78.8%) patients had prior BTKi exposure. Median duration of emavusertib/ibrutinib treatment was 51 days (range 5-798). Grade ≥ 3 treatment-related adverse events (TRAEs) were reported in 11 (33.3%) patients; neutropenia in 5 (15.2%) patients, amylase increased, lipase increased, and leukopenia in 2 (6.1%) patients each. The majority of Grade ≥ 3 TRAEs were reversible and manageable. Among the 19 BTKi-experienced patients treated for ≥ 28 days, there were 5 CRs and 2 PRs, with an ORR of 36.8%. Three patients with CR were on treatment for 10+ mos, with 1 CR patient ongoing with DOT of 26.2+ mos. Among the 5 BTKi-naïve patients treated for ≥ 28 days, 1 achieved CR and 3 had PR, with ORR of 80%; the CR patient is ongoing with DOT of 18.1+ mos. The ORR in the combined population was 45.8% (6 CRs; 5 PRs). The combination of emavusertib and ibrutinib demonstrates promising clinical activity and an acceptable safety profile in patients with R/R PCNSL. This regimen appears capable of overcoming resistance to prior BTKi therapy. Patient enrollment in this trial continues.

Abstract PCNSL is a rare and aggressive NHL with no approved treatments for R/R patients, representing a critical unmet need. MyD88 mutations in ~70% of PCNSL patients drive IRAK4 activation, promoting NF-κB signaling, inflammation, and tumor progression. Emavusertib, a potent oral IRAK4 inhibitor, crosses the blood-brain barrier and shows preclinical synergy with BTKis, and has the ability to re-sensitize BTKi-resistant cell lines. This study evaluates the molecular and PK data associated with responses to emavusertib + ibrutinib combination therapy in R/R PCNSL patients. The safety, clinical activity, and potential biomarkers of emavusertib in R/R PCNSL are being investigated in the ongoing TakeAim Lymphoma trial (NCT03328078). Pre-dose and 1.5-hour post-dose plasma samples were collected on Day 1 of Cycles 3, 5, and 7. CSF samples were obtained via a lumbar puncture within 1.5 hrs of collection of the post-dose plasma PK sample on Cycle 3 Day 1. As of 06 December 2024, CSF concentration was available for 7 patients. The mean emavusertib concentration in CSF was 81.3 ng/ml (54.7-104.0) in patients receiving 100 mg emavusertib BID (n=4). In patients receiving 200 mg emavusertib BID (n=3), the mean emavusertib concentration in CSF was higher at 175.7 ng/ml (114.8-209.4), which is 2.2 × the mean value in patients who received 100 mg emavusertib BID (p-value = 0.02). MyD88 mutation status was available for 7 patients of which all had prior BTKi exposure. Among these patients, 6 had MyD88 mutations of which 4 had responded (3 CRs and 1 PR) to the combination with DOR up to 18.9 mos with data collection ongoing. Preliminary CNS PK data demonstrate that emavusertib concentration in CSF increases with increasing emavusertib dose. Patients with MyD88 mutations showed expected promising preliminary efficacy to emavusertib + ibrutinib combination and may overcome BTKi resistance. Enrollment is ongoing.

Abstract BACKGROUND The phase III ECOG-ACRIN E3F05 trial tested whether combined therapy with radiation and concomitant followed by adjuvant temozolomide is more effective than radiation alone in patients with grade II gliomas. The study incorporated 1p/19q analysis but predated assessment of IDH mutational status. We previously reported that the addition of temozolomide to radiotherapy significantly improved overall survival in grade II gliomas, with similar hazard ratios in co-deleted (0.56) and non-codeleted (0.53) tumors. Herein we incorporate the results of DNA methylation profiling to examine outcomes in IDHmt, non-codeleted subjects as well as co-deleted ones. METHODS All 172 subjects underwent 1p/19q co-deletion analysis as a stratification factor, 1p and 19q deletion analysis was performed on formalin-fixed paraffin-embedded (FFPE) gliomas by Fluorescent In Situ Hybridization (FISH) following standard methods, 97/172 (56.3%) had tissue with 60% or more tumor content available for DNA methylation profiling using Heidelberg classifier v12 and Bethesda Classifier 2.0 to ascertain classification as IDH mutant glioma and alternative evaluation of 1p/19q co-deletion status. RESULTS Of the 172 subjects, 74 (43.0%) had 1p/19q codeletion by FISH, 38 (22.1%) were IDHmt without codeletion, 14 (8.1%) were IDHwt, and 46 (26.7%) were non-codeleted with unknown IDH status (these include n=4 pts originally classified as 1p/19q undetermined). With a median follow-up of 130 months, the addition of TMZ to RT improved OS in patients with IDHmt, non-codeleted tumors (HR 0.15 (0.03-0.74)) and 10-year OS from 39% to 80%. In codeleted tumors, the addition of TMZ improved OS (HR 0.51 (0.19-1.42)) and 10-year OS from 68% to 90%. CONCLUSION The addition of temozolomide to radiation improves overall survival in both co-deleted and IDHmt, non-co-deleted grade II gliomas.

Abstract BACKGROUND STELLAR was a phase III randomized, open-label trial of eflornithine (ornithine decarboxylase inhibitor) with lomustine versus lomustine alone in patients with recurrent anaplastic astrocytoma (AA). We previously reported an overall survival (OS) and progression free survival (PFS) of the combination in the patients with grade 3 IDH-mutant astrocytoma. Here we report updated OS and PFS outcomes based on additional molecular classification and blinded independent central review (BICR). METHOD Eligibility included: age ≥ 18 years, AA (2016 WHO criteria) with first recurrence ≥ 6 months after radiation, KPS ≥ 70, and no imaging findings consistent with grade 4 glioblastoma. Patients received eflornithine (2.8 g/m2 TID Q 2/3 weeks) with lomustine (90 mg/m2 Q 6 weeks) (Arm A) or lomustine alone (110 mg/m2 Q 6 weeks) (Arm B). Primary endpoint was OS. RESULTS Updated molecular data resulted in re-classification of the 343 AA patients enrolled into 3 WHO 2021 defined tumor subtypes, 196 tumors were IDHmut, CDKN2A/B intact, consistent with diagnosis of astrocytoma, IDHmut, WHO grade 3, 33 tumors had CDKN2A/B loss consistent with astrocytoma, IDHmut, WHO grade 4, 106 tumors were IDHwt consistent with diagnosis of glioblastoma, IDHwt, WHO grade 4. Updated results strengthen initial conclusion that eflornithine plus lomustine resulted in significant survival benefit in the astrocytoma, IDHmut, WHO grade 3 group (mOS 34.9 vs, 23.5 months, HR = 0.64, stratified log rank p =0.0136). PFS with eflornithine was also improved (15.8 vs, 7.2 months, HR = 0.57, stratified log rank p =0.0113). BICR demonstrated a high correlation between investigator and centrally determined PFS (p=0.0305), and high correlation between OS and PFS (p=.0001). CONCLUSION The clinically meaningful OS and PFS benefits observed with eflornithine in the molecularly defined 2021 WHO CNS grade 3 astrocytomas is further confirmed and expanded based on additional molecular classification and BICR of PFS.

Abstract Pontine H3 K27-altered diffuse midline gliomas (DMGs) are rare, fatal pediatric brain tumors resistant to systemic therapies due to the blood-brain barrier (BBB) and efflux transporters (ETs), which actively clear drugs from the brain. Convection-enhanced delivery (CED) bypasses the BBB by directly infusing therapeutics into the tumor, but maintaining adequate drug concentrations remains a challenge. This study explores strategies to enhance drug-tumor residence time and improve treatment efficacy. Using chemical modeling, FDA-approved drugs identified in prior high-throughput screening efforts were classified based on physiochemical properties impacting CED pharmacokinetics, including BBB penetration and ET substrate status. Drug behavior was validated in patient-derived in vitro and in vivo DMG models. The impact of systemic drug co-administration on drug retention and therapeutic efficacy was evaluated using multiple patient-derived orthotopic xenograft (PDOX) models. ET substrate drugs exhibited rapid elimination from the brain post-CED, limiting their therapeutic potential. However, systemic everolimus administration prolonged drug retention and potentiated the effects of efflux substrate drugs delivered via CED (e.g., topotecan, ponatinib). This combinatorial approach significantly enhanced anticancer effects in multiple PDOX models, demonstrating improved tumor control compared to monotherapy. Overall, these findings suggest that pharmacological inhibition of ETs via systemic everolimus administration can enhance drug retention in DMG when combined with CED. The observed synergistic effects highlight a promising strategy to overcome drug clearance limitations and improve treatment outcomes. Further clinical investigation is warranted to validate this approach in patients.

Abstract BACKGROUND Access to in-person evaluations is challenging for patients with glioma due to disease-related disability and distance from specialized neuro-oncology centers. Telehealth is an alternative to in-person evaluations, but acceptability of and therapy compliance with this model has not been prospectively evaluated. METHODS In this single-arm non-randomized pragmatic clinical trial, we compared patient satisfaction with telehealth and in-person assessments among patients with glioma undergoing chemotherapy with temozolomide. Participants acted as their own controls, alternating between telehealth and in-person visits. Patient satisfaction with care delivered was evaluated by Press-Ganey surveys with Likert response scales (1-5, 1=very poor, 5=very good). A digital pill diary (DPD) was implemented to track chemotherapy compliance. RESULTS We accrued 30 patients (median age 59, 38% female) between October 2024 and May 2025. Half of the patients (n=15) reported a primary residence greater than 150 miles from our institution. Surveys from 25 participants were reviewed, with 17/25 completing one or more surveys following an in-person visit, and 21/25 completing one or more surveys following a telehealth visit. Overall average Press-Ganey scores were similar between the two visit modalities with high scores (telehealth=4.8; in-person=4.7). Median scores for specific modalities were similarly highly rated: likelihood of recommending practice to others (telehealth=5.0; in-person=5.0), concern the care provider showed (telehealth=5.0, in-person=4.7), provider’s discussion of treatment options (telehealth=5.0, in-person=5.0), provider effort to include patients in decision-making (telehealth=5.0, in-person=5.0). DPDs used to monitor treatment compliance indicated adherence to all prescribed doses of temozolomide in 75/79 cycles; the other 4 (of 79) treatment cycles were halted per provider instructions. CONCLUSION This interim assessment demonstrates feasibility of treating patients with similar patient satisfaction of care delivered between telehealth and in-person visits. The DPD was effective in tracking chemotherapy administration. Additional results including visit type preference and acute care utilization data are maturing.

Abstract BACKGROUND Mirdametinib, a MEK1/2 inhibitor, is the first FDA-approved medicine for both adults and children (≥2y) with NF1-PN not amenable to complete resection. In the phase 2b ReNeu trial (NCT03962543), confirmed objective response rate (ORR) was 41% in adults and 52% in children. This analysis explored relationships between baseline characteristics and mirdametinib responses in ReNeu. METHODS Confirmed ORR was the proportion of patients with ≥20% reduction from baseline in target PN volume by magnetic resonance imaging on consecutive scans during the 24-cycle treatment phase (“responders”) as assessed by blinded independent central review. Characteristics included age, body surface area (BSA), sex, race, ethnicity, target PN location, PN type (typical, nodular, or solitary nodular), target PN volume, and progression status. Rates of dose reductions and treatment-related adverse events (TRAEs) were evaluated. These factors were descriptively analyzed for potential relationships with confirmed objective response (data cutoff: 20September2023). RESULTS With mirdametinib, there was no clear difference between responders (n=53) and non-responders (n=61) in age (mean, 22.89 and 22.67y), BSA (mean, 1.42 and 1.51 m2), sex (60.4% and 57.4% female), race (83.0% and 68.9% White), or ethnicity (83.0% and 85.2% non-Hispanic/Latino). The majority of responders and non-responders had head/neck for target PN location (50.9% and 47.5%), typical PN type (81.1% and 63.9%), and baseline progression status (60.4% and 55.7%). Mean (95% CI) target PN volume was 283.43 (155.19, 411.66) and 415.74 (211.71, 619.77) mL, respectively. Dose reduction (11.3%; 18.0%) and TRAE rates (96.2%; 96.7%) were comparable. CONCLUSIONS This analysis suggests mirdametinib provides clinical benefit across a broad range of patient and NF1-PN tumor characteristics; a relationship between analyzed baseline characteristics and confirmed objective response was not established. Mean PN volume was higher in non-responders, though CIs overlapped. Dose reductions and emergence of TRAEs did not appear to impact response to mirdametinib. SUPPORT SpringWorks Therapeutics, Inc.