Introduction Obe-cel, an autologous, fast off-rate 4-1BB-ζ CD19-directed CAR T, demonstrated long-term efficacy and low severe immunotoxicity in adult R/R B-ALL (Roddie C, et al. NEJM 2024). In the Phase I CARPALL study (NCT02443831), obe-cel showed promising efficacy and safety in pediatric R/R B-ALL (Ghorashian S, et al. Nat Med 2019). Objective To evaluate the preliminary safety/efficacy of obe-cel in the R/R B-ALL cohort of the Phase Ib/II CATULUS study (NCT06173518). Methods CATULUS is a single-arm, open-label, international multi-center study. Eligible pts (<18 years, with primary refractory R/R B-ALL/high-risk first relapse/≥2 relapses) underwent lymphodepletion (fludarabine 4 × 30 mg/m2; cyclophosphamide 2 × 500 mg/m2), followed by a single obe-cel infusion (target dose: 1.0 × 106/kg CAR T-cells). The Phase Ib primary endpoints include frequency/severity of treatment-emergent adverse events. Secondary endpoints include overall remission rate (ORR; complete remission [CR]/CR with incomplete hematologic recovery [CRi]), CAR T-cell expansion, CAR T-cell persistence, and B-cell aplasia. Results As of April 30, 2025, 20/21 (95.2%) enrolled pts with R/R B-ALL received obe-cel at the target dose; all products were in specification. Baseline characteristics are shown in Table 1. Post infusion, Grade ≥3 cytokine release syndrome (CRS)/immune effector cell-associated neurotoxicity syndrome (ICANS) each occurred in two pts; five pts had Grade ≥3 infection (Table 2). One pt died from progressive disease. At data cut-off (median follow-up: 6.7 months [range: 0.9–15.2 months]), the ORR (CR/CRi) was 95.0% (n=19; 95% confidence interval: 75.1–99.9). Following obe-cel infusion, the geometric mean for Cmax was 92,773 copies/µg DNA (coefficient of variation [CV%]: 156.7) and 860,815 copies/µg DNA × days (CV%: 169.6) for AUC0–28d. The median Tmax was 14 days (range: 9–21 days). All responders (n=19) achieved measurable residual disease (MRD)-negative remission (<10–4 leukemic cells). Of the responders, 17/19 (89.5%) were still in ongoing remission at data cut-off. While in remission, 3/19 (15.8%) pts received stem cell transplant (SCT); 14/16 (87.5%) pts who did not receive SCT post obe-cel were in ongoing B-cell aplasia at data cut-off. Pts with morphological relapse (n=1)/emergence of MRD (n=1) received further anti-leukemic therapy. Conclusions Manufacture of obe-cel was successful for all pts. Obe-cel effectively expanded after a single infusion of 1.0 × 106/kg CAR T-cells. The pediatric safety profile of obe-cel was consistent with that previously reported for adults, with low rates of high-grade CRS/ICANS. ORR was high at 95%; nearly 90% of responders had ongoing remission at data cut-off. While longer follow-up is needed, obe-cel holds potential as treatment in pediatric R/R B-ALL. Additional Phase Ib data will be presented and the Phase II expansion trial is being planned.

IntroductionNewborn screening (NBS) for severe combined immunodeficiency (SCID) leads to early diagnosis and hematopoietic cell transplant (HCT). Limited studies are available on the quality of life (QoL) of SCID patients treated with HCT. We prospectively evaluated the QoL of SCID patients and families to assess the overall impact of HCT at the time of transplant and in follow-up.MethodsPrimary Immune Deficiency Consortium (PIDTC) Protocol 6901 is a prospective natural history study of infants with SCID. The health-related QoL of infants/young children was measured using the Pediatric Quality of Life Inventory (PedsQL) completed by parent-proxy, while parental and family burden was assessed using the PedsQL Family Impact Module. Age-appropriate questionnaires were offered at baseline (pre-HCT) and 12, 24, and 48 months post-HCT. Patients transplanted between 2010 and 2021 who completed ≥1 baseline or post-HCT survey were included in this analysis. Scores were transformed to a 0–100 scale, with higher scores indicating better QoL or family functioning.ResultsOf 249 families, 144 (57.8%) completed baseline QoL surveys (supplementary table). Population characteristics did not differ significantly between patients with completed QoL surveys and those without. At baseline, parent-proxy overall QoL mean was 78.75 (n = 141, SD = 15.36). The association of trigger for diagnosis with baseline overall QoL was significant (p = 0.0002). Lower overall QoL was reported by patients diagnosed by clinical symptoms such as infection compared with patients diagnosed by family history (95% confidence interval [CI]: −17.49, −1.76, p = 0.02) or by NBS (95% CI: −18.44, −6.77, p < 0.0001). Families reported significantly higher mean overall Family Impact scores, including subscales at 12, 24, and 48 months post-transplant, compared with baseline (Figure 1 A), reflective of decreased burden of chronic illness on parents and families. Improved mean parent-proxy overall QoL and physical health scores were reported at 12 months post-transplant (Figure 1 B).Figure 1.(A) Mean and confidence interval plots for PedsQL Family Impact Module scores from pretransplant (“baseline”) to 48 months post-transplant reported by families or caregivers of children with SCID. The PedsQL Family Impact Module is a tool used to measure the impact of chronic health conditions on parents/families. Scores are reported on a scale of 0–100, with higher scores indicating better family functioning. (B) Mean and confidence interval plots for PedsQL Scores (parent-proxy) from pretransplant (“baseline”) to 48 months post-transplant reported by families/caregivers of children with SCID. The PedsQL (and PedsQL Infant scales in patients 0–24 months old) are tools designed to measure health-related QoL in infants and children. Scores are reported on a scale of 0–100, where higher values reflect better health-related QoL as reported by parents and/or patients.ConclusionPatients diagnosed with SCID through NBS reported higher pre-transplant QoL than those diagnosed by family history or clinical presentation. HCT was associated with sustained improved parent and family functioning, indicated by higher Family Impact scores post-transplant. Further analysis is forthcoming to identify pre- and peri-transplant variables as well as post-transplant complications, such as graft-versus-host disease, that may impact patient QoL following treatment.Tabular data are included as downloadable supplement files.

Achieving an adequate dose of genetically modified hematopoietic stem cells (HSCs) for gene therapy in patients with sickle cell disease (SCD) remains a challenge due to limitations related to stem cell mobilization using plerixafor alone, reduced apheresis collection efficiency, and losses during ex vivo cell manipulation. To date, 39 SCD patients were enrolled in NHLBI-funded Phase I (NCT03282656; PMC7962145) and NHLBI/CIRM-funded multi-site Phase II (NCT05353647) gene therapy trials to receive autologous CD34+ HSCs transduced with a lentiviral vector encoding a short hairpin RNA embedded in a microRNA (shmiR) targeting BCL11A. To collect HSCs as the starting material for drug product manufacture (minimum 4×10⁶ cells/kg), apheresis (minimum 4 blood volumes or up to 8 hours) was performed within 3 hours of daily plerixafor for 2 consecutive days. A third day of collection was used to generate a back-up product as needed. Mean mobilized, pre-apheresis peripheral blood (PB) CD34+ count was 40 cells/μL (range 7–126). Among 35 evaluable patients (4 excluded: 1 pending release, 3 withdrew), 74% (26/35) reached target CD34+ collection in a single mobilization cycle (mean 2.6 procedures, range 2–6). Manufacturing was achieved with a mean of 11.92×10⁶ CD34+ cells/kg collected resulting in a drug product of 7.04×10⁶ CD34+ cells/kg and a net cell recovery from apheresis to drug product of 62% (range 38–91%). Drug products were successfully generated for all patients with a mean product vector copy number of 4.04 copies/cell (range 1-7). The time interval from first collection cycle to completion of product testing was a median of 39 days (mean 56) for all patients, and a median of 37 days (mean 38) for those collected in 1 cycle. Preparative transfusions were given before collections to bridge patients after stopping hydroxyurea or to mitigate stress erythropoiesis. We examined the impact of preparative transfusion on mobilization and apheresis efficiency in 16 evaluable patients at a single site. All underwent ≥ 3 months of preparative transfusion with a HbS target of ≤30% before mobilization using simple transfusion or automated red cell exchange. The mean HbS% at mobilization was 10.8% (range 2.9–21.5%). Overall, the preparative transfusion regimen reduced PB reticulocyte percentage by 50% to a mean of 7.2% (range 3.7–12.3), indicating decreased stress erythropoiesis. Resting PB CD34+ counts before and after 3-months of transfusion were 10.8 cells/μL (range 2–21) and 6.2 cells/μL (range 1–13), respectively. A total of 39 mobilization/collection procedures were performed in 16 patients with a mean post-plerixafor CD34+ count of 43 cells/μL (range 13–85) with collections performed between the two darkest color preferences of the manufacturer. Cell collection preference was monitored using real-time intraprocedural CD34+ cell sampling. A mean of 4.3 total blood volumes was processed (range 2.5–6.4), with mean collection efficiency (CE) (CD34+ cells collected ÷ [pre-apheresis CD34+ count × total blood processed]) of 48% (range 8.2–107.1). The mean reticulocyte percent in patients with CE <30% (11.4%, range 2.5 –18.9) was significantly higher than in those with CE >30% (6.2%, range 1.8–11.9, p=0.0026). Two patients had a history of delayed hemolytic transfusion reactions and/or multiple RBC alloantibodies before study entry that limited preparative transfusions to reach a HbS of 30% for 3 months. They were successfully collected, and products were generated after simple transfusions with a single limited-volume red cell exchange immediately prior to collection. The success of this study was driven by optimizing apheresis strategies, including suppression of stress erythropoiesis through transfusion, real-time instrument adjustments, and efficient manufacturing that allowed a collection target of nearly half the CD34+ cells recommended in the FDA-approved gene product. Strikingly, the highly efficient ex vivo manipulation platform described here enables successful mobilization and manufacturing of gene therapy products for SCD in several months, easing the path to autologous gene therapy for SCD.

Introduction: HCT is an established curative treatment for children, adolescents, and young adults (CAYA) with high-risk/relapsed B-ALL. Inclusion of TBI in HCT conditioning has been shown to be superior to non-TBI approaches for B-ALL, but is associated with significant late effects. Based upon retrospective data showing low rates of relapse, we hypothesized that patients with negative pre-HCT MRD by next-generation-sequencing of IgH B-cell receptor rearrangements (NGS-MRD) could achieve 2-year EFS exceeding 75% with a non-TBI regimen, an outcome comparable to those receiving TBI-based regimens. Methods: The Pediatric Transplantation and Cellular Therapy Consortium (PTCTC) conducted a phase II prospective trial at 45 Centers in North America (ONC1701 EndRAD: NCT03509961) between 2018 and 2025 to evaluate outcomes of myeloablative non-TBI conditioning regimens for allogeneic HCT in B-ALL patients at lower risk for relapse defined by absence of NGS-MRD (Clonoseq) of B-cell receptor rearrangements (BCR) just prior to HCT. Eligibility criteria included age >/= 1 year to <31 years, first or second complete remission status (CR1/CR2), and no isolated or combined CNS disease at relapse. Prior blinatumomab, inotuzumab ozogamicin, or CAR-T treatments were allowed. All graft sources were permitted. Mismatched related/haploidentical grafts received post-transplant cyclophosphamide or TCRαβ/CD19 depletion according to institutional preference. All patients received myeloablative non-TBI conditioning. Graft-versus-host disease (GVHD) prophylaxis was according to graft source and institutional standards. Results: Fifty-one patients (51% males) in CR1 (49%) or CR2 (51%) status received HCT. Median age (range) at initial diagnosis and HCT were 11.9 (1.2-28.1) and 13.5 (2.3-32.5) years, respectively. Of patients enrolled, 33% were White/Non-Hispanic, 37% Hispanic, 12% Black or African American, and 18% other. Prior to HCT, 28 patients (55%) received blinatumomab, 1 (2%) received inotuzumab, while 11 (21%) received CAR-T, 7 (14%) received 2 prior immunotherapies, and 4 (8%) had no prior immunotherapy.Forty-four patients (86%) received the preferred study non-TBI conditioning regimen (busulfan, fludarabine, thiotepa); 2 comparable allowed regimens were received: fludarabine, melphalan, and thiotepa by 3 patients (6%) and melphalan, fludarabine, clofarabine, and thiotepa by 4 patients (8%). Donors included HLA matched siblings (41%), mismatched related/haploidentical (33%), matched unrelated (18%), or unrelated cord blood (8%). Related and unrelated donor graft sources were 71% bone marrow and 21% peripheral blood stem cells. Transplant-related mortality in the first 100 days post-HCT was low at 2%. At a median follow up of 2.3 (range: 0.2-6.0) years, the 2-year OS and EFS (alive/relapse-free) were 82% (95% CI: 67.1%, 90.6%) and 76.3% (95% CI: 61.1%, 86.1%), respectively. Five patients (10%) who were pre-HCT NGS-MRD negative by BCR had detectable T-cell receptor sequences (BCR-/TCR+); all 5 are alive and relapse-free. Non-relapse mortality (NRM) was 12% (6 patients, 0.1-2.9 years from HCT to NRM) and occurred predominantly in older children (4 (67%) >/=14 yrs old). Relapses occurred in 6 children, with 4 (67%) undergoing HCT in CR2. Four of the six relapses occurred after matched sibling donor HCT. Acute GVHD occurred in 20 patients (39%, with 15 (75%) grade 1-2 and 5 (25%) grade 3-4). Chronic GVHD occurred in 13 patients (25%) (11 (85%) requiring systemic immunosuppressive treatment). Conclusions: The primary endpoint of our study was met with the 2-year EFS exceeding 75% following non-TBI conditioning and allogeneic HCT in pre-HCT NGS-MRD negative B-ALL. OS in our cohort is comparable to published Center for International Blood & Marrow Transplant Research (CIBMTR) results in B-ALL where patients receive TBI-based conditioning. Our results show that pre-HCT NGS-MRD can be used to allow the choice of myeloablative non-TBI preparative regimens for CAYA undergoing allogeneic HCT that may result in decreased late effects. Additional analyses to be reported at the meeting will more fully investigate factors that impact post-HCT outcomes, including baseline cytogenetics/genomics and post-HCT bone marrow and peripheral blood NGS-MRD, along with planned comparisons to an observational cohort (n=146) enrolled on the trial including infants and older patients treated non-TBI approaches and older children treated with TBI-based regimens.

BACKGROUND:Severe combined immunodeficiency (SCID) comprises rare inherited disorders of immunity that require definitive treatment through hematopoietic cell transplantation (HCT) or gene therapy for survival. Despite successes of allogeneic HCT, many SCID patients experience incomplete immune reconstitution, persistent T-cell lymphopenia, and poor long-term outcomes.OBJECTIVE:We hypothesized that CD4+ T-cell lymphopenia could be associated with a state of T-cell exhaustion in previously transplanted SCID patients.METHODS:We analyzed markers of exhaustion in blood samples from 61 SCID patients at a median of 10.4 years after HCT.RESULTS:Compared to post-HCT SCID patients with normal CD4+ T-cell counts, those with poor T-cell reconstitution showed lower frequency of naive CD45RA+/CCR7+ T cells, recent thymic emigrants, and TCR excision circles. They also had a restricted TCR repertoire, increased expression of inhibitory receptors (PD-1, 2B4, CD160, BTLA, CTLA-4), and increased activation markers (HLA-DR, perforin) on their total and naive CD8+ T cells, suggesting T-cell exhaustion and aberrant activation, respectively. The exhaustion score of CD8+ T cells was inversely correlated with CD4+ T-cell count, recent thymic emigrants, TCR excision circles, and TCR diversity. Exhaustion scores were higher among recipients of unconditioned HCT, especially when further in time from HCT. Patients with fewer CD4+ T cells showed a transcriptional signature of exhaustion.CONCLUSIONS:Recipients of unconditioned HCT for SCID may develop late post-HCT T-cell exhaustion as a result of diminished production of T-lineage cells. Elevated expression of inhibitory receptors on their T cells may be a biomarker of poor long-term T-cell reconstitution.

In chronic-phase chronic myeloid leukemia (CP-CML) patients treated with frontline imatinib, failure to achieve early molecular response (EMR; EMR failure: BCR-ABL1 >10% on the international scale at 3 months) is predictive of inferior outcomes. Identifying patients at high-risk of EMR failure at diagnosis provides an opportunity to intensify frontline therapy and potentially avoid EMR failure. We studied blood samples from 96 CP-CML patients at diagnosis and identified 365 genes that were aberrantly expressed in 13 patients who subsequently failed to achieve EMR, with a gene signature significantly enriched for stem cell phenotype (eg, Myc, β-catenin, Hoxa9/Meis1), cell cycle, and reduced immune response pathways. We selected a 17-gene panel to predict EMR failure and validated this signature on an independent patient cohort. Patients classified as high risk with our gene expression signature (HR-GES) exhibited significantly higher rates of EMR failure compared with low-risk (LR-GES) patients (78% vs 5%; P < .0001), with an overall accuracy of 93%. Furthermore, HR-GES patients who received frontline nilotinib had a relatively low rate of EMR failure (10%). However, HR-GES patients still had inferior deep molecular response achievement rate by 24 months compared with LR-GES patients. This novel multigene signature may be useful for selecting patients at high risk of EMR failure on standard therapy who may benefit from trials of more potent kinase inhibitors or other experimental approaches.

Prospective/retrospective protocols of the PIDTC have collected detailed data from children undergoing allogeneic hematopoietic cell transplantation (HCT) for SCID at 33 North American centers since 1982. We previously reported that from 1982 to 2009, overall survival (OS) after HCT was unchanged. Newborn screening (NBS) for SCID, initiated in 2010, was hypothesized to improve SCID OS following HCT. Children born and receiving HCT during 4 time intervals (1982–89; 1990–99; 2000–09; and 2010–18) were analyzed for categorial variables (chi-square test), continuous outcomes (Kruskal-Wallis test), and OS (Kaplan-Meier method). 902 children with typical (n = 747) and atypical (n = 155) SCID were included. Unlike years 1982–2009 when 5-year OS after HCT was stagnant (72–73%), improvement was seen for the first time beginning in 2010, when 5-year OS increased to 87% (p < 0.001). To determine what contributed to this finding, multiple factors were evaluated to identify differences between children transplanted between 2010–18 compared to prior time intervals. Significant differences (all p < 0.001) were found in the trigger for diagnosis being NBS or family history as opposed to clinical illness, age at HCT, active infection at HCT, and transplant characteristics including conditioning intensity, stem cell source, and donor source. On multivariable analysis, which excluded matched sibling donors due to their consistently high rates of OS (≥92%) in all time intervals, active infection (HR 2.41, 95% CI 1.53–3.72; p < 0.001), age ≥3.5 months at HCT (HR 2.21, 95% CI 1.38–3.24; p = 0.001), certain genotypes (ADA, DCLRE1C/LIG4/NHEJ, and other rare genotypes) (HR 2.22–3.67, 95% CI 1.23–7.45; p < 0.001), and Black/African American race (HR 2.33, 95% CI 1.56–3.54; p < 0.001) were associated with lower survival rates. A subgroup multivariable analysis examining the effect of trigger for diagnosis in the era of NBS, adjusting for genotype and race, confirmed that improved OS with NBS was related to earlier age and fewer infections at time of HCT. This study confirmed for the first time the direct benefit of population-based SCID NBS in improving OS after HCT. Our findings support adoption of SCID NBS worldwide, but also point out that persistent racial disparities in survival despite universal availability of screening must be addressed.

Objectives: Cellular kinetic (CK) measurement of CAR-T cell expansion in-vivo by quantitative polymerase chain reaction (qPCR) has been measured in units of transgene copy number/μg of DNA. We propose a formula to convert the qPCR, and flow cytometry (FC) measurements to interpretable numbers of CAR T-cells/μL blood. Further, after the conversion CK parameters were correlated with efficacy/safety endpoints. Methods: CK data measured using both qPCR and FC assays were utilized from the ELIANA trial in pediatric and young adult patients with relapsed and refractory acute lymphoblastic leukemia (pALL). qPCR measures the presence of CAR transgene in cells with units of copies of CAR-DNA/μg of genomic DNA, while FC quantifies surface expression of CAR T-cells as the % of either T-cells or white blood cells (WBCs) that express CAR. Neither measurement accounts for the typical significant increase in WBCs following CAR-T infusion. We propose equations for converting FC (Eq1) or qPCR (Eq2) into concentration of CAR-cells/μL of blood, using WBC counts from the complete blood count. For qPCR, the equation relies on 3 additional parameters M·F/N: amount of DNA/WBC (M), average number of copies of CAR-DNA/CTL019 cell (N), and fraction of cells with CAR-DNA that express CAR receptor (F). To estimate M·F/N, we performed regression of the cells/μL estimate from FC vs qPCR. We fit the CK model [1] to obtain the model parameters. Finally, CK parameters with safety/efficacy were correlated to compare the converted estimates to the native CK units. • (CD3+CAR+cells)/μl blood= WBC/μl blood × (CD3+CAR+cells)/WBC (1) • (CAR+cells)/μl blood=WBC/μl blood × CAR DNA copies/μg DNA × M μg DNA/WBC × 1 CAR cell/N CAR DNA copies×F (2) Results: There was high correlation between FC and qPCR estimates of CAR-T in the blood of pALL patients (r2=0.775). The M·F/N value derived based on FC and qPCR results was estimated as 2.68e-6μg DNA/CAR copies. Assuming M=6.6e-6μg DNA/WBC [2] and F=1, this predicts that N=2.46 CAR copies/CAR-T cell. There was also high correlation between the copies/μg and cells/μL estimates using qPCR (r2=0.752). The relationship between CK parameters and safety/efficacy endpoints was not improved when cells/μL was used. This can be attributed to high correlation between these metrics. Using the CK model, 11x greater fold expansion was predicted using the cells/μL estimate compared to copies/μg. This is because cells/μL estimate accounts the expansion of both CAR-T and WBC numbers following lymphodepletion. Conclusions: The conversion of CK into cells/μL allows for a physiological interpretation of CK data with a high correlation between the cells/μL and copies/μg indicating either metric can be used to predict safety/efficacy. References: 1 Stein M et al. CPT: Pharmacometrics & Systems Pharmacology(2019) 2 Gillooly JF et al. Cold Spring Harbor Perspectives in Biology vol.7,7 a019091(2015) Citation Format: Anwesha Chaudhury, Andrew Stein, Stephan Grupp, John Levine, Michael Pulsipher, G Doug Myers, Edward Waldron, Xu Zhu, Fraser McBlane, Rakesh Awasthi, Edmund K. Waller. Conversion of cellular kinetic data for chimeric antigen receptor T-cell therapy (CAR-T) into interpretable units [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 509.

Optimal post-remission therapy for adolescents and young adults (AYAs) with Ph-negative acute lymphoblastic leukemia (ALL) in first complete remission (CR1) is not established. We compared overall survival (OS), disease-free survival (DFS), relapse, and non-relapse mortality (NRM) for patients receiving post-remission therapy on CALGB 10403 to a cohort undergoing myeloablative (MA) allogeneic hematopoietic cell transplantation (HCT) in CR1. In univariate analysis, OS was superior with chemotherapy compared to MA allogeneic HCT (3-year OS 77% vs. 53%, P < 0.001). In multivariate analysis, allogeneic HCT showed inferior OS (HR 2.00, 95% CI 1.5–2.66, P < 0.001), inferior DFS (HR 1.62, 95% CI 1.25–2.12, P < 0.001), and increased NRM (HR 5.41, 95% CI 3.23–9.06, P < 0.001) compared to chemotherapy. A higher 5-year relapse incidence was seen with chemotherapy compared to allogeneic HCT (34% vs. 23%, P = 0.011). Obesity was independently associated with inferior OS (HR 2.17, 95% CI 1.63–2.89, P < 0.001), inferior DFS (HR 1.97, 95% CI 1.51–2.57, P < 0.001), increased relapse (1.84, 95% CI 1.31–2.59, P < 0.001), and increased NRM (HR 2.10, 95% CI 1.37–3.23, P < 0.001). For AYA ALL patients in CR1, post-remission therapy with pediatric-style chemotherapy is superior to MA allogeneic HCT for OS, DFS, and NRM.