Abstract Recurrent glioblastoma (rGBM) is an aggressive brain tumor with median survival under one year after standard chemoradiation. Antigen heterogeneity, immune exclusion, and a suppressive tumor microenvironment (TME) limit responses to immunotherapy. A first-in-human phase 1 trial of intracerebroventricular EGFR/IL13Rα2 CAR T cells (CART-EGFR-IL13Rα2) in EGFR-amplified rGBM was feasible, produced manageable neurotoxicity, and induced radiographic tumor regressions in a subset of patients (NCT05168423).To understand how this therapy reshapes the local TME, we analyzed paired tumor resections from 6 patients enrolled in the phase 1 trial, with specimens obtained from the primary intracranial disease site at trial enrollment (pre-treatment) and at radiographic progression after CART-EGFR-IL13Rα2 infusion. Multimodal spatial profiling included regional transcriptomic and protein mapping (GeoMx), single-cell whole-transcriptome imaging (CosMx), and high-resolution spatial transcriptomics (Visium HD). We annotated tumor, myeloid, lymphoid, and stromal compartments and derived composite scores for stemness, invasion, cell death, and immune regulation. Neighborhood- and interaction-based analyses were used to compare cellular states and cell-cell communication.Across patients, post-treatment samples showed reduced expression of CAR target antigen and a shift in tumor-intrinsic programs toward less stem-like, less migratory, and more apoptotic states, despite radiographic progression. The post-treatment TME was remodeled, with fewer suppressive myeloid- and B-cell-rich niches and increases in interferon-responsive and T cell-associated activation programs. Spatial interaction analyses indicated that pre-treatment rGBM contained dense networks of myeloid-tumor and myeloid-T-cell contacts consistent with impaired antigen presentation and effector function. Post-treatment specimens, in contrast, showed partial disruption of these suppressive circuits and the emergence of microenvironments more permissive to T-cell infiltration and activity.In the parent phase 1 trial, CART-EGFR-IL13Rα2 was feasible & induced radiographic tumor regressions in a subset of patients. This correlative spatial analysis suggests that prior EGFR/IL13Rα2 CAR T exposure can leave a less suppressive, more immunologically engaged TME at the primary site, even in resections obtained at radiographic progression. Together, these data support the idea that intracerebroventricular CAR T therapy may condition rGBM for subsequent immunotherapy. Myeloid and B-cell interactions are highlighted as candidate targets for armoring next-generation CAR T cells and for designing rational combination and sequencing strategies. Citation Format: Wesley V. Wilson, MacLean P. Nasrallah, Nakial Cross, Yael A. Day, Vanessa Gonzalez, Rachel M. Leskowitz, Amy Marshall, Julie K. Jadlowsky, Gabriela Plesa, Donald L. Siegel, Elizabeth O. Hexner, Joseph A. Fraietta, Carl H. June, Stephen J. Bagley, Donald O’Rourke, Zev Binder, Andrew J. Rech. Spatial profiling of recurrent glioblastoma in a Phase I clinical trial reveals favorable immune remodeling induced by intracerebroventricular CAR T therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3444.

Abstract Patients with relapsed or refractory B-cell lymphoma who experience disease progression after CD19-directed CAR T-cell therapy have poor outcomes and few effective treatment options. Subsequent therapies provide only modest benefit, with complete remission (CR) rates near 20% and limited durability. huCART19-IL18, an IL-18-secreting CD19 CAR T-cell product, produced an overall response rate of 81% and a CR rate of 52% after prior anti-CD19 CAR T failure in a phase I trial, but the spatial determinants of durable response within the tumor microenvironment (TME) remain incompletely defined. We performed multimodal spatial profiling of paired pre- and post-huCART19-IL18 lymph node biopsies from 11 patients (6 with CR, 5 without CR) enrolled in the phase I study. GeoMx whole-transcriptome atlas and protein profiling were integrated with CosMx 6000-plex single-cell spatial imaging to map transcriptional and cellular remodeling across tumor, myeloid, and lymphoid compartments. CoPro, a computational framework for detecting coordinated progression of cell states in space, was applied to identify spatially coordinated gene expression programs within and between myeloid and T-cell compartments. Post-infusion samples from responders showed increased T-cell and NK-cell infiltration, frequent tertiary lymphoid structures, and induction of interferon (IFN)- and tumor necrosis factor-responsive chemokine and immune effector programs, including enhanced antigen presentation, together with coordinated loss of B-cell identity and signaling. These changes localized to regions enriched for CAR T cells, effector memory CD4+ and CD8+ T cells, and IFN-polarized macrophages, consistent with IL-18-driven recruitment and reprogramming of myeloid and T-cell compartments. Pre-infusion TMEs in responders showed baseline myeloid chemokine signatures associated with clinical response. CoPro revealed that myeloid functional heterogeneity is organized along spatial gradients that are independent of lineage identity and coordinated with T-cell effector programs in CAR T-infiltrated regions. These findings support a model in which IL-18 armoring promotes spatially organized remodeling of the TME through coordinated myeloid and T-cell activation associated with durable remission after CD19 CAR T failure, nominating myeloid recruitment, antigen presentation, and T-cell exhaustion programs as critical biomarkers and rational engineering targets for next-generation armored CAR T strategies in lymphoma. Citation Format: Nakial C. Cross, Yael A. Day, Sonia Ndeupen, Vanessa E. Gonzalez, Zhen Miao, Sam I. Kim, Rachel M. Leskowitz, Amy Marshall, Julie K. Jadlowsky, Gabriela Plesa, Donald L. Siegel, Elizabeth O. Hexner, Jakub Svoboda, Stephen J. Schuster, Nancy R. Zhang, Joseph A. Fraietta, Andrew J. Rech, Carl H. June. Spatial remodeling of the tumor microenvironment by IL-18-armored CD19 CAR T cells is associated with durable remission in relapsed or refractory lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6470.

Chimeric antigen receptor (CAR) T-cell therapy holds great promise for patients with cancer, and the identification of predictive biomarkers is crucial in finding new ways to guide therapy. Major challenges to the application of informatics and machine learning in CAR T-cell therapy include limited sample sizes and non-uniformity in data generation across cancer indications and trials. Here we took a global, pan-haematologic cancer approach, analysing 256 patients across 5 cancer types and 13 clinical trials. We generated data using a framework that included pre-infusion clinical features, over 2 million apheresis T cells analysed by flow cytometry using 17 unique markers, ex vivo T-cell expansion during CAR T-cell manufacture, more than 90,000 measurements of 30 serum markers and serial tracking of circulating CAR T cells using qPCR. From this data resource, we demonstrate the potential of pan-cancer predictive biomarkers that capture generalizable characteristics of treatment response and non-response in CAR T-cell therapy.

6501 Background: Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with myelofibrosis (MF) and anemia. Chronic hepcidin elevation limits iron availability for red blood cell (RBC) production and contributes to anemia onset and severity. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin, a co-receptor in the bone morphogenetic protein-signaling pathway driving hepcidin expression. Methods: RALLY-MF (NCT05320198) is an ongoing Phase 2, open-label study conducted in the US. The primary aim is to evaluate DISC-0974 efficacy in participants with MF and anemia. Secondary aims include evaluating safety, pharmacokinetics (PK), and pharmacodynamics (PD). Eligible participants are ≥18 years of age with primary or secondary MF and hemoglobin (Hgb) <10 g/dL or RBC transfusion requirement. Participants enroll into 3 cohorts based on RBC units transfused in the 84 days prior to screening: non-transfusion dependent (nTD, 0 units and Hgb <10 g/dL), low transfusion burden (TD low, 1-2 units), high transfusion burden (TD high, 3-12 units). Stable dose of concomitant Janus kinase inhibitor (JAKi) is allowed. DISC-0974 is administered subcutaneously at 50 mg monthly for up to 6 doses, with escalation to 75 mg for inadequate or lost response. Primary endpoints include major hematologic response defined as transfusion independence (TI) during any consecutive 16 weeks (TD low) or 12 weeks (TD high), or a mean Hgb increase of ≥1.5 g/dL from baseline for ≥12 weeks (nTD). Secondary endpoints include PK/PD markers of iron regulation and safety assessments. Data were summarized using descriptive statistics. Results: At data cut (16 Oct 2025), 47 participants enrolled in the nTD (n=30), TD low (n=10), and TD high (n=7) cohorts, with 53% on concomitant JAKi therapy. DISC-0974 led to sustained hepcidin reduction and iron mobilization across cohorts. DISC-0974 resulted in meaningful hematologic responses among evaluable participants: 50% of nTD participants achieved a mean Hgb increase of ≥1.5 g/dL for ≥12 weeks, 71% of TD low participants achieved TI for ≥16 weeks, and 67% of TD high participants experienced a ≥50% reduction in transfusion burden, with follow-up ongoing. Major hematologic response rates were 50% for participants on and off concomitant JAKi therapy (n=18 and n=16, respectively). DISC-0974 was associated with meaningful improvement in FACIT-Fatigue scores for nTD and TD low participants. Serious adverse events (AEs, n=9) and ≥Grade 3 AEs (n=16) were considered unrelated to DISC-0974. There were no early withdrawals due to AEs. Conclusions: DISC-0974 was well tolerated and shows a favorable safety profile. Anemia response and fatigue scores improved across patients with MF and anemia, including those receiving JAKi therapy. These data validate hepcidin reduction as a promising approach to treating anemia in MF. Clinical trial information: NCT05320198 .

The use of systemic corticosteroids is recommended only for grades II to IV acute graft-versus-host disease (GVHD), but many patients are often treated at the onset of grade I in real-world practice. We retrospectively analyzed outcomes of 1143 patients with grade I GVHD from 24 Mount Sinai Acute GVHD International Consortium (MAGIC) transplant centers. Approximately half received "up-front" systemic steroids, whereas the remainder received topical steroids alone; GVHD progressed to grade II to IV in only one-third of these patients. Up-front steroids decreased the risk for grade II to IV GVHD but not grade III to IV acute GVHD or the need for a second line of treatment. Systemic steroids were equally effective at treating grade I or II GVHD; however, up-front therapy exposed 3 times more patients to systemic steroids, with a corresponding threefold increase in GVHD that required additional therapy and a threefold rise in infection-related deaths. In multivariable analyses, up-front steroids were significantly associated with higher nonrelapse mortality (NRM; P = .026). MAGIC biomarkers identified most patients with grade I GVHD as low risk and unlikely to progress to severe GVHD regardless of treatment. Nevertheless, low-risk patients who received up-front steroids experienced a threefold increase in infectious deaths compared with those treated topically. This study supports the consensus recommendation of topical therapy for patients with low-risk grade I GVHD, a strategy that can be supported by biomarkers to avoid unnecessary steroid exposure and increased NRM.

ABSTRACT:Advanced systemic mastocytosis (AdvSM), a clonal hematologic neoplasm driven predominantly by D816V-mutant KIT, is often characterized by organ damage. Associated hematologic neoplasms (AHNs; usually myeloid) are often present, leading to poor survival. We report on the oral, highly selective, potent KIT D816V inhibitor avapritinib (200 mg once daily, starting dose) with >4 years follow-up from the fully enrolled PATHFINDER study. End points included overall response rate (ORR; primary), duration of response (DOR), progression-free survival (PFS), overall survival (OS), changes in objective biomarkers of disease, and safety (all secondary). Of 107 patients with AdvSM (including 71 [66%] with SM-AHN; overall population median follow-up, 49 months), 83 were response-evaluable. ORR was 73% (95% confidence interval, 63-83). Median DOR was 58 months; PFS, 51 months, and OS, 62 months. Disease progression occurred in 21 of 107 patients (20%), predominantly in SM-AHN and largely driven by the AHN. Reductions in objective biomarkers of disease were observed. Most frequent (≥30% patients) treatment-emergent adverse events (TEAEs; any grade; grade ≥3) were thrombocytopenia (58%; 31%); periorbital edema (57%; 6%), anemia (54%; 33%), peripheral edema (48%; 2%), and diarrhea (36%; 5%). Adverse events of special interest were cognitive effects (34%; 8%) and intracranial bleeds (4%; 2%). Eleven (10%) patients experienced TEAEs leading to death, of which 1 was deemed related to avapritinib by the principal investigator. With 4-year follow-up, patients with AdvSM treated with avapritinib experienced deep and durable responses and a favorable benefit-risk profile. This trial was registered at www.ClinicalTrials.gov as #NCT03580655.

Introduction We recently showed that ID-PTCy (25 mg/kg/day on days +3/+4) effectively prevents graft-versus-host disease (GVHD) after myeloablative (MAC) HLA-haploidentical (Haplo) bone marrow HCT. Moreover, compared with high-dose (HD) PTCy, ID-PTCy had clinical benefits, including 5-day earlier engraftment, markedly faster T-cell recovery, less severe BK virus cystitis, and fewer cases of CMV reactivation. Objectives Here, we investigated the use of ID-PTCy in a two-institution clinical trial of RIC Haplo or HLA-mismatched unrelated (≥5/10; mMUD) bone marrow HCT. Methods RIC consisted of fludarabine, cyclophosphamide, and total body irradiation (400 cGy). Beyond ID-PTCy, GVHD prophylaxis included sirolimus (d+5 to 60) and mycophenolate mofetil (d+5 to 35). Two cohorts were enrolled: 1) patients ≥60 years and 2) patients <60 years, but with comorbidities precluding MAC. Each cohort enrolled an initial 6 patients in phase I, and, if ≤1 grade III-IV acute GVHD events by day +60 (the primary endpoint), then 14 evaluable patients in phase II. Additional patients were enrolled if graft failure, relapse, or death occurred prior to day +60. All patients were included in the analyses, even if unevaluable for the primary endpoint. Accrual was completed in November 2024. Results Forty-four patients received Haplo (n=31) or mMUD (n=13) HCT. Median patient age was 60 years (range, 20-78): 34% had KPS ≤80, 66% had HCT-CI ≥3 (range, 0-8), and 27% had Disease-Risk Index that was high or very high. Acute leukemia was the HCT indication in 77%, of whom 53% were CR≥2 and 32% were MRD+ at HCT. Median follow-up of survivors was 2.3 (range 0.9-3.9) years. Engraftment occurred at a median of 15 days for neutrophils and 25 days for platelets. There was one primary graft failure and one secondary graft failure. By day +60, 98% of evaluable patients (n=40) had donor myeloid and CD3 chimerisms >95%. Among all 44 patients, cumulative incidence (CuI) of grade III-IV acute GVHD at 200 days was 9% (95% CI: 3-20). 1-year CuI of moderate-severe chronic GVHD and chronic GVHD requiring systemic therapy were both 9% (95% CI: 3-20). 1-year estimates for OS, DFS, and GRFS were 75% (95% CI: 59-85), 70% (95% CI: 55-82), and 64% (95% CI: 48-76), respectively. 1-year CuI of relapse and non-relapse mortality were 9% (95% CI: 3-20) and 21% (95% CI: 10-34), respectively. Primary causes of NRM (n=9) in the first post-HCT year were infection (1), infection in the setting of immunosuppression (2), GVHD (2), bleeding (1), and organ failure (3). Conclusions ID-PTCy provides excellent GVHD protection after bone marrow HCT. Despite a high-risk cohort, survival outcomes were favorable, and relapse rates were remarkably low. These findings suggest that ID-PTCy is not only safe, but with advantages, such as the enhancement of GVL, perhaps through more robust immune recovery. Ultimately, these results should motivate a randomized comparison with HD-PTCy.

Introduction Chimeric antigen T-cell receptor (CART) therapy is highly effective for patients (pts) with relapsed/refractory (R/R) lymphoma/multiple myeloma (MM). Due to toxicity concerns, older pts are underrepresented in CART trials and CART is underutilized in older adults. Fried’s frailty phenotype (FP) combines subjective (exhaustion, weight loss, activity level) and objective (gait speed, grip strength) measures to categorize older adults as fit, pre-frail, or frail and as we have previously shown, predicts survival in stem cell transplant (SCT) recipients. Objectives We hypothesize that FP is associated with PFS and OS in older adults receiving CART. Methods We performed a prospective study measuring FP prior to CART infusion and at 7 days (d), 14d, 21d, 1 month (mo), 3mo, 6mo and 12mo post-infusion in pts ≥ 60 years. Results 42 pts were enrolled with a median age at CART infusion of 69 (60-81). 55% had MM (61% with intermediate/high-risk by RISS). The remainder had lymphoma (DLBCL or follicular lymphoma) with IPI > 2 in 58%. Idecabtagene vicleucel (n=12), tisagenlecleucel (n=12) and ciltacabtagene autoleucel (n=11) were the most frequently administered products. Median follow up was 23mo. Median number of prior therapies was 3 (range 1-7) and 48% had prior autoSCT. The majority of pts had 0-1 ECOG performance status (83%), of whom 63% were categorized as frail or prefrail by FP. Prior to CART, 35% were fit (score 0), 43% were prefrail (score 1-2), and 23% were frail (score 3-5). Frail pts were more likely to be admitted for >7d (OR 5.3, p=0.04). Pre-infusion FP was significantly associated with inferior OS and PFS (p values <0.001). Median PFS in fit, prefrail, and frail pts was 23.4mo (95% CI 17.1-NR), 18.4mo (95% CI 6.8-31.8) and 4.0mo (95% CI 2.5-8.4), respectively. 2-year OS was 100%, 87% and 13% in fit, prefrail, and frail pts respectively. Subgroup analysis in pts with high-risk disease by IPSS or IPI showed frail status predicted for worse OS (p<0.001), indicating FP may be an independent predictor of outcome. At 21d, 25% of pts were fit, 50% were prefrail, and 25% were frail. At 1mo, 21% were fit, 58% were prefrail, and 21% were frail. In landmark analyses, being frail at 21d (p=0.04) or 1mo (p=0.05) was associated with inferior OS and PFS from that time point. 17 pts (40.5%) maintained or improved their FP from pre-infusion to 1mo; 8 received physical therapy while in hospital. Notably, pts who maintained or improved FP had better OS than pts who had declines in their scores. Conclusion In pts ≥ 60 with R/R lymphoma/MM undergoing CART, frailty status by FP at pre-infusion, 21d, and 1mo were associated with OS, with more disease-related deaths in frail patients independent of traditional disease risk features. Future studies to reverse frailty with exercise interventions and to uncover the biologic mechanisms of frailty’s association with adverse disease outcomes may lead to novel targets for intervention.

2013 Background: We previously reported the safety, bioactivity, and preliminary efficacy of bivalent CAR T cells targeting EGFR and IL13Rα2 in 18 patients with recurrent GBM (Bagley, et. al, Nat Med 2025). Given that median follow-up time was only 8.1 months at the time of the data cut-off for that publication, we now report updated overall survival (OS) and safety data with longer follow-up time, as well as neurologic function outcomes. Methods: Eighteen patients with recurrent EGFR-amplified GBM were enrolled using a 3+3 design (dose levels: 5.0 x 10 6 , 1.0 x 10 7 , and 2.5 x 10 7 cells). Patients received a single intracerebroventricular (ICV) dose of CART-EGFR-IL13Rα2 cells. OS was defined as the time from the date of initial study treatment to the date of death from any cause, or censored at the last date of contact if the patient was not known to have died at the date of analysis cut-off (January 16, 2026). To capture changes in neurologic function over the course of the trial, patients were assessed prospectively using the Neurologic Assessment in Neuro-Oncology (NANO) scale. NANO is an objective clinician-reported outcome of neurologic function scored on nine domains, with a higher score indicating worse neurologic function. NANO scores were assessed at baseline prior to CART (day 0), on days 1, 4, 7, 10, 14, 21, and at the 1-month and 2-month visits post-CART. Results: With median follow-up time of 18.5 months, median OS was 12.0 months (95% confidence interval, 7.4 – 23.2 months) with 3 patients surviving greater than 18 months. Other than one patient with prolonged grade 1 neurotoxicity as previously described, no prolonged, late-onset, or unexpected toxicities were observed within this longer follow-up time, including no evidence of on-target off-tumor toxicity or secondary cancers. On day 1 post infusion, an increase in NANO scores was observed (mean increase in NANO score from baseline of 1.9; p=0.09), which resolved to baseline by day 4. Patient NANO scores were unchanged from baseline at one month (mean change 0.41; p=0.998) and two months (mean change 0.54; p=0.992) post infusion. Those who underwent retreatment with a second dose of CAR-T cells at the time of tumor progression (n=7) did not experience an increase in NANO score on day 1 (mean change 0.33, p=0.99), despite having a significant increase on day 1 following the first infusion (mean change 2.9, p=0.008). Conclusions: With over 18 months of median follow-up time, ICV delivery of CART-EGFR- IL13Ra2 cells in patients with recurrent GBM has not demonstrated long-term or delayed toxicities and is associated with promising OS outcomes that warrant continued clinical development. Neurologic function worsens immediately following infusion but recovers to pre-treatment baseline by one month following CAR T cell infusion. Clinical trial information: NCT05168423 .

Approximately 30% of patients with acute graft-versus-host disease (GVHD) develop steroid-refractory disease and have very poor outcomes. Ruxolitinib has become the standard of care for steroid-refractory acute GVHD, but it is unclear which patients derive benefit. The MAGIC Composite Score (MCS), an algorithm that combines clinical symptoms and biomarkers, has been validated to predict outcomes at the start of primary GVHD treatment. Here, we evaluated its performance at the initiation of second-line treatment in 278 patients. MCS stratified patients into three risk groups (MCS1-3), with the majority (88%) classified as intermediate or high risk. Increasing MCS score was associated with progressively higher 1-year non-relapse mortality (NRM) rates (16%, 41%, and 73%; p<0.001), lower 1-year survival (77%, 56%, and 24%; p<0.001), and lower complete response (CR) rates at day 28 (47%, 38%, and 20%, respectively; p<0.01). The area under the receiver operating characteristic curve (AUROC) for 1-year NRM was significantly higher with MCS compared to clinical symptoms alone (0.70 vs. 0.63; p=0.023). Among patients treated with ruxolitinib, higher MCS similarly predicted higher NRM and lower survival and CR rates. Patients classified as MCS2/3 had poor outcomes despite ruxolitinib, underscoring the need for novel therapies in this patient population. In conclusion the MCS is an accurate predictor of outcomes for patients who require second-line treatment and may be of use as an eligibility criterion for future clinical trials in this high-risk population.

Acute graft-versus-host disease (GVHD) remains a lethal barrier to successful allogeneic hematopoietic cell transplantation, yet pre-transplant donor selection entirely ignores hypervariable T-cell receptor (TCR) architecture. Here, by characterizing over 64,000 alloreactive clonotypes, we demonstrate that human alloreactivity is dictated by a constrained, predictable baseline structural signature. Pathogenic, tissue-infiltrating alloreactive T cells exhibit significantly shortened CDR3β regions, altered antigen-facing biophysical features, biased VJ gene usage and extensive inter-donor sharing originating from public anti-pathogen memory reservoirs. These potent clones natively cluster within the high-frequency fraction of the unstimulated baseline donor repertoire. We introduce R50, an assay-independent metric quantifying this clonal dominance, which independently predicted a six-fold increased risk of acute GVHD in a cross-institutional cohort. This scalable in silico platform shifts pre-transplant risk stratification from HLA typing and demographic surrogates to precision immune-receptor modeling.

Patients with relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) have a dismal prognosis largely mediated by non-sustained responses to chemotherapy and few targeted therapy options. Surface antigen targets in T-ALL include CD2, CD5, CD7, and CD38; however, ongoing clinical development of these targets is challenged by (1) T-cell fratricide during manufacturing, (2) T-cell depletion during treatment, and (3) high frequency of target negative relapse. Here, we use a combination of flow-cytometry, single-cell genomics, and bulk RNA-sequencing to identify CCR4 as a novel surface target in T-ALL. We analyzed the T-ALL microenvironment from 40 T-ALL cases treated on the AALL0434 clinical trial and identified a subpopulation of bone-marrow-enriched CCR4+ FOXP3+ T-regulatory cells which express immune checkpoints (PD-1 and TIGIT) and could be targeted with anti-CCR4 therapy. Lastly, we describe the preclinical efficacy of an anti-CCR4 CAR-T in in vitro and in vivo models, paving the way for future translational efforts in chemotherapy refractory T-ALL.

Analysis of EGR2 and type I IFN pathway regulation in CAR T-cells. (A-C) ATAC-seq tracks of genes associated with dysfunction, type I IFN signaling, and memory differentiation are shown, with differentially accessible regions indicated. D, Heatmap showing expression of exhaustion genes in tonically signaling GD2 and functional CD19 naïve CD8+ CAR-T cells (GSE136891). E, Comparison of EGR2 and type I IFN gene signature scores in central memory CD8+ T-cells expressing a tonically signaling GD2 CAR and control CD19 CAR. CAR T-cells were generated using T-cells from a healthy donor (GSE136891). F, Top transcription factor co-expression signatures overexpressed in CD19 CAR T-cell products of CLL complete responders. G, Comparison of EGR2 and type I IFN signature scores in unstimulated CD19 CAR T-cell products from CLL patients (CR: complete response, PRTD: very good partial response, PR: conventional partial response, NR: no response). H, Comparison of type I IFN and EGR2 module scores in PSMA CAR T-cells from lymphodepleted prostate cancer patients, and their association with in vivo CAR T-cell proliferation and PSA decline.

Supplemental tables: (1) Subject characteristics. (2) Cytogenetic profiles and high-risk features. (3) Prior treatment exposures and refractoriness. (4) CAR T cell product characteristics. (5) Products that did not meet target dose. (6) Adverse events of grade 3-4. (7) Cytokine release syndrome and ICANS. (8). Maintenance therapy. Supplemental Figures: (1) Study schematic and subject disposition, (2) Correlates of manufacturing success, (3) Hematopoietic recovery, (4) Post-infusion T cell phenotypes, (5) Correlates of in vivo expansion and manufacturing success, (6) Late post-infusion CAR T cell re-expansion, (7) Soluble BCMA, (8) Late-onset clinical responses, (9) MM cell BCMA expression, (10) Pre- and post-treatment Sox2-specific T cell responses in CART-BCMA monotherapy patients, (11) Pre- and post-treatment Sox2-specific T cell responses in CART-BCMA + huCART19 combination therapy patients, (12) Sustained post-treatment SOX2-specific T-cell responses.

Patients with advanced pancreatic ductal adenocarcinoma (PDAC) have a median survival of less than a year, highlighting the urgent need for treatment advancements. We report on a phase 1 clinical trial assessing the safety and feasibility of intravenous and local administration of anti-mesothelin CAR T cells in patients with advanced PDAC. While therapy is well tolerated, it demonstrates limited clinical efficacy. Analyses of patient samples provide insights into mechanisms of treatment resistance. Single-cell genomic approaches reveal that post-infusion CAR T cells express exhaustion signatures, including previously identified transcription factors ID3 and SOX4, and display enrichment for a GZMK+ phenotype. Single knockout of ID3 or SOX4 enhances efficacy in xenograft models, though with donor-dependent variability. However, single-knockout cells eventually fail. Conversely, ID3 and SOX4 double-knockout CAR T cells exhibit prolonged relapse-free survival, demonstrating a sustained therapeutic effect and a potential avenue for engineering more potent CAR T cells in PDAC. This study was registered at ClinicalTrials.gov (NCT03323944).

BACKGROUND:Relapse of B-cell acute lymphoblastic leukemia (B-ALL) with CD19-antigen loss after CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has a dismal prognosis. Novel immunotherapeutic strategies for this patient population are urgently needed. METHODS:We tested a novel, fully human anti-CD22/4-1BB CAR T-cell construct, CART22-65s, in parallel phase I studies for pediatric and adult B-ALL. After lymphodepletion, CART22-65s was infused using a 3-day fractionated dosing scheme, allowing for omission of the second and third doses in cases of early cytokine release syndrome (CRS). RESULTS:Twenty-two patients, all with relapse after prior CD19-directed immunotherapy, were enrolled. Of 19 infused patients (pediatric, n=17; adult, n=2), 14 (74%) achieved a complete remission (CR), including 4 of 6 (67%) patients refractory to prior inotuzumab. Five of 14 patients in a CR proceeded to consolidative hematopoietic cell transplantation (HCT). With a median follow-up of 38 months, the 12-month relapse-free survival rate was 38.4% (95% CI 19.3% to 76.5%) and overall survival rate was 52.6% (95% CI 34.3% to 80.6%). Two patients received additional CART22-65s treatments for subsequent CD22-positive relapses; one achieved another CR. All CRS (n=17, 89%) and neurotoxicity (n=4, 21%) events after initial infusion were grades 1-2. The only grade 3 CRS/neurotoxicity and the only high-grade immune effector cell-associated hemophagocytic lymphohistocytosis-like syndrome occurred in the retreatment setting. In vivo cellular kinetic data revealed robust CART22-65s proliferation by quantitative PCR peaking at a median of 20 days postinfusion, with the cells persisting out to month 42 in one patient who achieved a long-term remission with CART22-65s alone. CONCLUSIONS:The favorable safety profile and high remission rates in exceedingly refractory B-ALL support the continued development of CART22-65s but also highlight the need to use the product in combination with HCT or other novel strategies. TRIAL REGISTRATION NUMBERS:NCT02650414 and NCT03620058.

Analysis of survival outcomes and EGR2 gene expression in CD19 CAR T-cell products. The figure presents the P values and hazard ratio of different EGR2 molecular marker stratification points in relation to A, overall survival and B, event-free survival The black arrows indicate the stratification points used in the study. C, EGR2-targeted gene expression scores in CD19 CAR T-cell products from responders and non-responders in pediatric ALL. D, Summary of how EGR2 regulates resistance to CAR T-cell therapy through the type I IFN pathway.

Marker gene expression in CAR T-cell clusters. A, Uniform manifold approximation and projection (UMAP) plot of AAVS1 and EGR2 knockout (KO) CAR T-cell samples is shown. B, UMAP plots showing expression levels of CD4 and CD8. C, Violin plot depicting expression of cluster-defining markers in CD4+ T-cells. D, Differentially expressed genes in IL7R+ versus CTLA4+ CD4+ T-cells. E, Violin plots showing expression levels of cluster-defining markers in CD8+ T-cells. F, Heatmap displaying differentially expressed genes between CD8+ cell clusters. G, Cell cycle scores mapped on UMAP plots.

Epigenetic remodeling of CAR T-cells by EGR2 knockout and effect of type I IFN signaling on the development of memory and exhaustion. A, Volcano plots showing differentially accessible chromatin regions within genes between KLF2+ and MKI67+ CD8+ T-cells. B, Volcano plots depicting differentially accessible chromatin regions within genes between EGR2 and AAVS1 knockout (KO) CD8+ CAR T-cells. C, Representative contour plots showing frequencies of TIM3- and LAG3-expressing CD8+ CAR-T cells after exposure to IFNβ (1ng/mL) following chronic CAR stimulation. D, Proportions of CD27+ (left) or CD62L+ (right) CD8+ CAR-T cells after exposure to IFNβ. E, Representative contour plots showing frequencies of CD45RO+CD27+ CD8+ CAR-T cells after IFNAR blockade (Anifrolumab, 1µg/mL) during chronic antigen stimulation. F, Frequencies of TIM3+LAG3+ CD8+ CAR-T cells after IFNAR blockade. G, Cytolytic capacity of CAR T-cells as measured by normalized cell index kinetics using the xCELLigence real-time cytotoxicity assay following chronic stimulation with target cancer cells in the setting of either IFNβ or IFNAR blockade. H, Normalized cell index at 75 hours after challenge with target cancer cells. All experiments were conducted using healthy donor T-cells from independent donors (Mann-Whitney test, n = 4). *P < 0.05, *P < 0.01, ***P < 0.001, ns.: not significant.