Introduction Chemotherapy and TBI-based conditioning for stem cell transplant and gene therapy (GT) cause significant toxicities. Antibody-drug conjugates (ADCs) offer a targeted alternative, depleting hematopoietic stem and progenitor cells (HSPCs) by antigen specificity. CD117, expressed primarily on HSPCs, is an appealing target, but prior attempts at safe and effective CD117-ADCs were limited by mast cell degranulation and payload-related toxicities. We developed a novel CD117-ADC (YTD005) with four design features: (1) An antagonistic anti-CD117 clone identified by phage display; (2) Monomeric fragment lacking Fc-effector function to reduce mast cell activation; (3) Fab’ design enabling rapid systemic clearance to limit infused stem cell loss; and (4) Incorporation of the tubulin inhibitor payload MC-MMAF at a drug-to-antibody ratio of 4. MC-MMAF is a non-cell-permeable toxin with low bystander effect and uses a non-cleavable linker, further reducing off-target payload effects. Objective To evaluate the safety and engraftment outcomes with YTD005 in a non-human primate (NHP) model of GT using CD34+ cells transduced with BCH-BB694, a lentiviral vector targeting BCL11A. Methods Three NHP underwent autologous transplantation with BCH-BB694–transduced CD34+ cells after YTD005 (1.5 mg/kg/day continuous infusion, day –9 to –2). Following 2 washout days, the GT product was infused. Animals were monitored up to 1-year for toxicity and engraftment; primary engraftment data obtained at Day +30. Results YTD005 achieved robust depletion of Lin– CD34+ HSPCs (median 98.2%; range, 97.8-99.6%) and Lin–CD34+CD90+CD45RA– cells (median 96.8%; range 94-99.6%). Conditioning was well tolerated, with no organ toxicities or mast cell activation. Animals received median 5.7×10⁶ CD34+ cells/kg (mean product VCN 4.2 per diploid genome; range 3.6-4.6c/dg). All developed transient pancytopenia: neutrophil nadirs ∼100/µL (range 100-300) at day +9 (range, day +7 - +11) with recovery by day +13 (range, +13 - +14); platelet nadirs ∼56K (range 43-102K) at day +3 (range, -4 - +7) with engraftment by day +13 (range, +7 - +14); anemia was mild.Engraftment was assessed by peripheral blood (PB) VCN and the % of individually isolated hematopoietic bone marrow progenitor CD34-derived colonies with a VCN >1. At day +30, mean PB VCN was 0.082 ± 0.028 (∼8% of PB cells), and mean colony engraftment 15.6% (range, 13.4-20%). At 3 months (n=3), mean PB VCN was 0.094± 0.024 (∼9% of PB cells) and colony engraftment was 15.2% (range, 14.1-15.9%). One animal has been followed to 1 year, demonstrating stable engraftment (blood VCN 0.1; 15.4% colonies), with 2 animals ongoing. Conclusions YTD005 incorporates rational design features to optimize safety and efficacy. A single infusional treatment with this ADC leads to engraftment of gene-modified cells, with mixed chimerism stable up to >1-year post-infusion.

Background Relapse remains the leading cause of treatment failure after allogeneic hematopoietic stem cell transplant (HCT) in pediatric myeloid malignancies. The Maintenance for Relapse Elimination (MoRE; NCT05796570) trial evaluates the feasibility of post-HCT prophylaxis with decitabine and granulocyte colony-stimulating factor (G-CSF). Children with myeloid neoplasms, including those with germline leukemia predisposition syndromes, are eligible. A negative binomial design was employed to capture both eligibility for maintenance and the ability to deliver therapy. Objective To assess the feasibility of delivering planned post-HCT decitabine/G-CSF maintenance and to characterize peri-HCT complications. Methods This single-arm, single-center pilot trial enrolls pediatric and young adult patients with myeloid malignancies. The primary endpoint is feasibility, defined as delivery of planned maintenance therapy. Secondary endpoints include leukemia-free survival, overall survival, and relapse at 24 months. Patients are enrolled pre-HCT; post-HCT complications precluding treatment initiation are recorded. The study plans to enroll 37 patients to account for 40% attrition, ensuring >80% probability of meeting feasibility thresholds. Immune reconstitution analyses are ongoing. Results As of 10/9/2025, 24 patients consented; three did not proceed to HCT due to grade >2 toxicity from prior cancer-directed therapy, competing trial enrollment, or uncontrolled leukemia. Diagnoses included high-risk AML (n=9), relapsed AML (n=3), therapy-related myeloid neoplasm (n=4), and germline-associated myeloid neoplasm (n=4). Germline predisposition variants were identified in five patients (GATA2 n=3, RUNX1 n=1, TP53 n=1; the latter developed therapy-related AML). All patients received myeloablative conditioning; donors included umbilical cord blood (n=12), haploidentical (n=4), unrelated (n=4), and fully matched sibling (n=1). Five children are pre-HCT or awaiting engraftment. Among 16 evaluable post-HCT patients, 13 (81%) initiated decitabine/G-CSF; nine completed all six planned cycles, three remain on treatment, and one discontinued after early relapse. Median therapy initiation was day +49 (range 40–108). No non-relapse mortality occurred. Flow cytometry and single-cell sequencing reveal expansion of CD8⁺ T cells characterized by proliferative and activation signatures. Conclusion The negative binomial design captures feasibility of post-HCT maintenance more comprehensively than traditional approaches. Inclusion of patients with germline predisposition syndromes provides novel insight into peri-transplant complications and regimen tolerability in a population often excluded from studies. Findings will help guide maintenance strategies to prevent relapse and contribute the understanding of the graft versus leukemia effect.

Chronic graft-versus-host disease (cGVHD) affects up to 54% of allogeneic hematopoietic cell transplantation recipients and is the leading cause of late non-relapse morbidity and mortality. Despite the availability of National Institutes of Health consensus criteria, Foundation for the Accreditation of Cellular Therapy accreditation standards, and CIBMTR reporting requirements, standardized screening practices remain inconsistent. The Engraft Learning Health Network (LHN) conducted a multicenter assessment to characterize cGVHD screening practices across participating transplant centers. To characterize current cGVHD screening practices across participating transplant centers within the Engraft LHN, identify areas of practice variation, and inform development of a standardized screening approach. This multicenter qualitative study included 10 transplant centers (8 pediatric and 2 adult) within the Engraft LHN. Thirty-three structured interviews were conducted with clinician representatives using a standardized guide addressing eight screening domains: assessment schedules, documentation practices, and organ-specific screening of the skin, oral cavity, ocular, pulmonary, gastrointestinal, genitourinary, and musculoskeletal systems. Interview summaries were validated by participants and analyzed to quantify screening practices across centers using descriptive statistics and heat map visualization. Marked heterogeneity was observed across all screening domains, with no uniform approach identified across centers. Assessment schedules ranged from weekly to as-needed evaluations, with only 52% of respondents reporting monthly evaluations in the first 6 mo following day 100 post-transplant and 37% continuing quarterly assessments beyond 1 yr. Only 10% incorporated the Lee Symptom Scale into routine documentation. Skin was universally included in the review of systems, but comprehensive examination (12%) and structured scoring (21%) were infrequent. Pulmonary function test monitoring showed significant gaps at intermediate time points, with most centers performing PFTs at annual milestones but fewer than 25% reporting assessment at quarterly intervals recommended by current guidelines. Genitourinary screening demonstrated the greatest inconsistency, with 42% and 63% of respondents not routinely discussing symptoms with female and male patients, respectively. Rare manifestations including neuropathy and serositis were screened for by fewer than 10% of centers. cGVHD screening practices vary widely across transplant centers in timing, documentation, organ-specific assessment, and utilization of standardized tools. Existing assessment frameworks, while clinically detailed, pose practical challenges for routine use outside specialized GVHD clinics. These findings highlight the need for a streamlined, evidence-informed screening bundle with defined assessment intervals, guideline-recommended PFT surveillance, and integration of patient-reported outcome measures. The Engraft LHN is leveraging these findings to develop and implement such a bundle at the provider, patient, and center levels using rapid-cycle quality improvement methodology.

Background Chronic graft-versus-host disease (cGVHD) remains the most common cause of late morbidity following pediatric hematopoietic cell transplantation (HSCT). The National Institutes of Health (NIH) consensus criteria for cGVHD diagnosis and staging added rigor and consistency across trials. We hypothesized these criteria may not fully encompass the physiological and clinical nuances of pediatric HSCT recipients. To capture current practice patterns and identify areas for pediatric-specific refinement, we conducted a multinational survey of pediatric transplantation providers through the Pediatric Transplantation and Cellular Therapy Consortium (PTCTC). Methods Between May and July 2025, PTCTC members completed a structured electronic survey addressing diagnostic approaches, organ-specific assessment tools, and perspectives on common challenges in cGVHD diagnosis. Quantitative items were summarized descriptively, while free-text responses underwent thematic analysis to identify shared challenges and consensus directions. Results Eighty-two respondents from 54 pediatric transplant centers participated. Nearly all reported routine use of the 2014 NIH criteria. For skin involvement, 94% emphasized the need for standardized pediatric body surface area tools and noted considerable inter-clinician variability in scoring. Opinions on the diagnostic role of skin biopsy were mixed, with 85% not endorsing for diagnosis. Age-appropriate Lansky performance measures were recommended. In the gastrointestinal domain, 59% supported distinguishing late-acute from chronic GVHD, favoring growth-curve or failure-to-thrive metrics over absolute weight loss, with suggestions to incorporate biomarkers such as fecal calprotectin and malabsorption tests. For hepatic involvement, respondents recommended adding GGT, AST, and alkaline phosphatase to current metrics; 70% supported including biopsies in liver cGVHD staging. Pulmonary assessment was considered particularly difficult in young children, with participants supporting relative FEV1 decline, serial testing, six-minute walk evaluation, and/or restrictive physiology parameters. Although opinions diverged on adding renal, neurologic, or serositis domains, there was broad agreement on the need for pragmatic, harmonized tools across centers (78%). Conclusions This multinational survey highlights challenges with current NIH cGVHD diagnostic criteria related to childhood physiology and practice realities for pediatric HSCT recipients. Priority domains include standardized pediatric BSA scoring, growth-based gastrointestinal metrics, expanded hepatic and pulmonary assessments, and age-appropriate functional measures. Findings support collaborative validation studies to inform future pediatric-specific refinements to international cGVHD consensus guidelines.

Graft-versus-host disease (GVHD) and poor immune reconstitution (IR) cause morbidity and mortality after hematopoietic cell transplant (HCT). CTLA-4 blockade with abatacept (Aba) prevents severe acute GVHD (aGVHD) in patients (pts) post-unrelated donor (URD) HCT for malignancy. Following our pilot trial (Aba NMD), we sought to confirm safety and efficacy of Aba in pediatric (peds) non-malignant disease (NMD) pts post URD HCT.The multicenter, single arm ASCENT clinical trial (NCT03924401) enrolled NMD pts <22 years (yr) lacking a matched related donor. GVHD prophylaxis was Aba 10 mg/kg IV on days (d) −1, +5, +14, +28, +56, +84, +112, and +150 with standard calcineurin inhibitor and mycophenolate mofetil. Reduced intensity conditioning was standardized for sickle cell disease (SCD), severe aplastic anemia (SAA), and Fanconi anemia (FA); for others, investigators chose from two options. Pts are followed to 2 yr for clinical endpoints; this interim analysis reports on IR to 1 yr. Events for severe GVHD, rejection-free survival (GRFS) were death, severe aGVHD (d100) or moderate-severe chronic (c) GVHD (1 yr).Thirty pts underwent URD HCT (63% 7/8 donor) and engrafted (Table). EBV viremia was more common than CMV, but viral disease was rare. Five pts (17%) developed severe aGVHD, and 4 (13%) recipients developed severe cGVHD. Two pts died: 1 due to resistant CMV disease (complicated by aGVHD) and 1 due to TA-TMA. At 1 yr, overall and rejection-free survival were both 93%, and GRFS was 60%. IR is shown in Figures 1-2, with no significant difference post-HCT seen by cohort. For CD4 T cell recovery, 79% had recovery by d100 (CD4>50 cells/μL), 71% by d180 (CD4>200 cells/μL), and 79% by 1 yr (CD4>400 cells/μL). Poor CD4 IR was not associated with viremia, viral disease, severe aGVHD, moderate-severe cGVHD, or death. Compared to our published Aba NMD cohort (non-SCD; n=10), ASCENT pts had significantly earlier CD8 T and NK cell recovery (data not shown). Similar to our published Aba2 peds cohorts (Figure 3), ASCENT/Aba NMD pts recovered a higher % of naïve (and lower effector memory) T cells, amongst CD4 and CD8 T cells (CD4 count and CD8 data not shown).In this interim 1-yr analysis, peds NMD pts post URD HCT had excellent rejection-free survival. IR kinetics were not impaired, and there was earlier recovery of naïve T cells (relative to TEM), consistent with peds pts receiving Aba for malignancy. Impact of donor matching and HCT indication on our primary endpoint of GRFS to 2 yr post-HCT is ongoing.

Tessera Therapeutics is pioneering a novel approach to in vivo genome editing that uses RNA Gene Writers and delivery of all RNA-based components to introduce precise edits into the genome of cells in vivo by target-primed reverse transcription. Our RNA Gene Writers have been engineered to enable a wide range of genomic modifications, from transgene insertion to single nucleotide changes, offering a highly versatile platform for the treatment of inherited and acquired diseases. In this study, we applied RNA Gene Writers to correct the HBB E6V mutation, the genetic cause of sickle cell disease (SCD), in hematopoietic stem cells (HSCs), with the aim of achieving therapeutically relevant levels of gene correction in vivo. We report that intravenous administration of the RNA-based Gene Writer, delivered via targeted lipid nanoparticles (LNPs), achieved therapeutic levels of HBB editing in HSCs in both SCD patient-derived mouse models and non-human primates (NHPs). Our approach would eliminate the need for HSC mobilization, intensive myeloablative conditioning, and ex vivo HSC manipulation, offering a potentially curative treatment that is safer, simpler, and more scalable for patients with SCD. We first assessed the efficiency and impact of our RNA Gene Writers to edit the HBB locus in HSCs by introducing the HBB Makassar variant (E6A) into ex vivo cultured wild-type human CD34+ cells and we achieved an average editing efficiency of 74%. Edited cells transplanted into immunodeficient mice demonstrated robust engraftment and stable maintenance of approximately 70% HBB editing in long-term HSCs (LT-HSCs) and multi-lineage progeny at 16 weeks post-transplantation. Similar editing levels were sustained following secondary transplantation, confirming the successful and durable editing of HSCs. We then evaluated RNA Gene Writers designed to make the wild-type correction in cultured HSCs derived from SCD donors and observed ~70% correction. Upon in vitro erythroid differentiation, these cells exhibited a 98% restoration of adult hemoglobin (HbA) levels, as measured by Liquid Chromatography-Mass Spectrometry (LC-MS) and reduced sickling under hypoxic conditions. To edit HSCs in vivo, we developed HSC-targeted LNPs that we have previously shown could deliver GFP mRNA to ~95% of LT-HSCs (Lin-CD34+CD38-CD90+CD45RA-) in both humanized mice and non-human primates (NHPs) following intravenous administration. Using humanized NBSGW mice engrafted with human CD34+ cells, we first demonstrated in vivo installation of the HBB Makassar variant in 62% of LT-HSCs. We repeated the experiment with HSCs from multiple donors and consistently observed editing efficiencies of > 50% across all samples. Comparable editing levels were maintained in LT-HSCs and their multi-lineage progeny after transplantation of in vivo edited CD34+ cells into secondary recipients, providing additional evidence of successful in vivo targeting of LT-HSCs and preservation of stem cell function. Importantly, using this same approach in cynomolgus NHPs, we achieved an average of 24% in vivo HBB Makassar editing in LT-HSCs by day 14 post-treatment. Editing levels remained stable in LT-HSCs for up to nine months, with edited cells detected across multiple hematopoietic lineages above 20%. Longitudinal tracking of edited cells revealed early and sustained multi-lineage output, with stable contributions observed as early as two months post-treatment, providing key insights into the kinetics of hematopoietic reconstitution following in vivo editing. Collectively, these results demonstrate the robustness of our Gene Writer and HSC delivery platforms to target and durably edit multiple loci in LT-HSCs in vivo in both murine and NHP models, supporting their therapeutic potential in the treatment of SCD and other hematologic disorders.

Background: Severe acute GVHD (aGVHD) remains a major cause of mortality after transplant, especially with CNI/MTX prophylaxis. However, the molecular mechanisms driving breakthrough aGVHD with CNI/MTX remain largely unknown. The ABA2 trial led to FDA approval of abatacept, a CD28:CD80/86 costimulation blocker, as an adjunct to CNI/MTX that significantly prevented aGVHD. Using ABA2 samples, we combined multiparameter flow cytometry with RNA-seq to identify the cells and pathways that drive aGVHD under CNI/MTX, and how abatacept controls them. We have discovered the transcription factor, ZNF683 (HOBIT), classically defined as regulating cytotoxicity and tissue resident memory programming, but recently identified as mediating T cell responses to Immune Checkpoint Inhibitors (ICI), as a central regulator of breakthrough alloproliferation during aGVHD, which can be controlled with CD28:CD80/86 blockade. Methods: PBMCs from ABA2's 8/8 HLA-matched arm [n=69 in CNI/MTX/Placebo (PBO) n=73 in CNI/MTX/abatacept (ABA)] were studied. Multiparameter flow tracked 308 immune populations, including 227 naïve/memory T cell subtypes/cell states. Flow-sorted Day+21 to +28 CD4 and CD8 T cells from 88 patients underwent bulk RNA-Seq, and Day+28 T cells from 36 patients (18/arm) underwent scRNA-seq; those developing aGVHD before sample collection (median aGVHD onset Day+36) were excluded. The role of HOBIT in T cell proliferation was interrogated with CellTrace-labeled mixed lymphocyte reactions (MLR) and CD3/CD28 stimulation, comparing non-modified T cells, lentivirus-mediated HOBIT overexpression (HOBIT-OE), and transduction controls. The Division Index (‘DI’, the average # divisions a cell has undergone) was calculated with FlowJo. Results: In PBO, the major immunologic signature of aGVHD was T cell proliferation, with multiplexed flow analysis identifying Ki67+ CD4 Tcm and Tem as significantly associated with Grade (Gr) 2-4 aGVHD. Gene Set Enrichment Analysis (GSEA) of CD4+ and CD8+ bulk RNA-Seq from Gr 2-4 vs Gr 0-1 aGVHD revealed that, of the top 20 C2cp GSEA signatures (ranked by Normalized Enrichment Score, 'NES'), 18/20 and 19/20, respectively, were associated with proliferation/cell cycle. scRNA-Seq of PBO proliferating T cells further identified type-I interferon signaling enriched in Gr 2-4 aGVHD (Reactome 'Interferon alpha/beta Signaling': NES=2.6, p <0.002). Pseudo-bulk differential expression (DE) analysis (Gr 2-4 vs Gr 0-1 aGVHD, fold-change > 2; adjusted p < 0.05) identified candidate drivers of inflammatory proliferation. Five genes were DE in proliferating CD4+ cells (Up:IL2RA, PELI1, BCL2L11; Down:HOBIT, HLA-DR5,) and two in CD8+ cells (Down:HOBIT, MXRA7). The transcription factor HOBIT was the only DE gene in both, downregulated 5.9x in CD4+ and 4.5x in CD8+ Gr 2-4 aGVHD proliferating T cells (p=0.038, p=0.008). Importantly, abatacept controlled both the inflammatory proliferation of T cells, and normalized HOBIT expression in Gr 2-4 aGVHD. A dynamic role for HOBIT in T cell proliferation was validated using bead-based stimulation assays and MLRs. With CD3/CD28 stimulation, HOBIT-OE significantly increased the proportion of T cells entering division vs controls (Day 3 DI for CD4+: 1.73 vs 1.21; CD8+: 1.79 vs 1.23, n=6, p=0.03 for each). However, when non-gene-modified T cells (capable of modulating HOBIT expression) were assessed in MLRs, significant transcriptional downregulation of HOBIT occurred as alloproliferation progressed, with >20-fold HOBIT downregulation in highly proliferating vs non-proliferating T cells (n=8 biologic replicates, p=0.008). These experiments reveal complex transcriptional control of HOBIT during T cell activation, with its expression enhancing the probability that T cells enter division, followed by HOBIT downregulation as alloproliferation proceeds. Conclusions: We have discovered a prominent role for the transcription factor HOBIT in regulating inflammatory T cell alloproliferation that drives CNI/MTX breakthrough aGVHD, and its control with abatacept. These results are the first to identify HOBIT as a key regulator of alloreactivity, uncovering a new link between the mechanisms that harness the nascent T cell activation potential unleashed by ICI, and those that drive aGVHD. They nominate HOBIT as a critical regulator of multiple facets of T cell activation, and underscore the central role that CD28:CD80/86 signaling plays in modulating HOBIT expression, and its downstream effects.

Current literature lacks details on the impact of pediatric chronic graft-versus-host disease (cGVHD) on long-term survivorship after allogeneic hematopoietic cell transplantation (HCT). Nonetheless, cGVHD remains a leading cause of post-transplant morbidity and mortality in children and adolescents, which is particularly relevant given the longer life-expectancy after HCT (measured in decades) compared to older adults. To address this knowledge gap, leaders of the Pediatric Transplant and Cellular Therapy Consortium convened a multidisciplinary taskforce of experts in pediatric cGVHD and HCT late effects known as RESILIENT after Chronic GVHD (Research and Education towards Solutions for Late effects to Innovate, Excel, and Nurture after cGVHD). Our goals were to define: (1) the current state of understanding about how cGVHD impacts long-term survivorship in children transplanted <18 yr of age; (2) practical aspects of care to help clinicians managing long-term pediatric cGVHD survivors; and (3) develop a research framework for the next decade to further our knowledge. Four working groups were formed, each tasked with addressing a unique theme: (1) cGVHD natural history (phases of cGVHD) and its impact on clinicians' ability to taper and durably discontinue systemic therapy; (2) organ dysfunction and immune reconstitution in relation to survivorship; (3) how cGVHD and its treatment impact growth, metabolism, and development in children; and (4) psychosocial health and patient reported outcomes. The 4 groups met before the 2024 BMT Tandem Meeting in San Antonio, Texas, and then convened a larger in-person RESILIENT conference held on February 20, 2024, at the Tandem meeting to put forth recommendations from their respective working groups and garner feedback. These recommendations are now presented in a series of 4 manuscripts. This current manuscript focuses on the first theme and discusses the phases of cGVHD, challenges in differentiating clinically active from quiescent cGVHD in clinical practice, and the resultant difficulties in determining when and if to taper systemic therapy. To overcome these challenges, we propose revised categorization of long-term cGVHD outcomes and practical recommendations for clinicians and researchers around the long-term follow-up for these patients, including determining when and if to taper systemic therapy, along with the integration of non-immunosuppressive supportive care interventions. (c) 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Hematopoietic cell transplantation (HCT) for pediatric sickle cell disease (SCD) has steadily increased, however data on long-term effects is limited. Previous registry studies focused on survival, rejection and GVHD. Data on long-term health-related quality of life (HRQoL) post-HCT in SCD is limited by small sample sizes and short follow-up. The objective of Project Sickle Cure (PSC) is to measure HRQoL and decisional regret (DR) in children with SCD, hypothesizing that HCT will improve HRQoL longitudinally with low rates of DR. PSC is a prospective observational multicenter study of SCD participants undergoing allogeneic HCT. Since 2021, 15 STAR sites in North America have enrolled 89 participants, with 65 having >6 mo post-HCT follow-up and included in this analysis. Baseline demographics, disease, and HCT characteristics were collected and summarized. Participants and their primary caregivers complete HRQoL measures (PedsQL Generic Core [GC], PedsQL Family Impact Module [FIM], and Patient Reported Outcomes Measurement Information System [PROMIS]) measures alongside a new DR Scale (DRS; higher scores indicate greater regret). Data are collected pre-HCT and post-HCT at 30, 90, and 180 days (d) and 1, 2, and 3 years (yrs). General linear mixed-effects models were used to evaluate preliminary changes over time. Results are presented as least-square means (LSM) and differences in least-square means (ΔLSM) from baseline. In this interim exploratory analysis, no statistical adjustments to p-values (e.g., alpha spending or corrections for multiple testing) were applied; results may differ after full enrollment and completion of planned analyses. Mean duration of follow up was 2.3 yrs (SD=1.0), with 85% >1 yr post-HCT. Participants were 46% male with mean age at HCT of 10.1 yrs (SD=5.3). Most had HbSS genotype (94%), and disease severity was severe, less severe, and mild in 35%, 35%, and 30%, respectively. Donor was matched related in 75%, bone marrow graft source in 68%, and conditioning was myeloablative in 53%, reduced intensity in 38%, and nonmyeloablative in 9%. Acute GVHD of any severity was reported in 34%, and any chronic GVHD in 5%. Many participantshad significant improvement from baseline in multiple domains of HRQoL, some with sustained improvement and from the earliest timepoint. For PedsQL GC, patient (pt) and parent-proxy HRQoL significantly increased from LSM=71.7 and 73.7 at baseline starting d 180 and 1 yr to 78.3 and 81.2 by yr 2, respectively (pt ΔLSM (95% CI)=6.6 (0.6,12.6), p=.032; parent-proxy ΔLSM (95% CI)=7.5 (0.1,14.9), p=.046). For PedsQL FIM, parent-reported total family functioning significantly increased from baseline of LSM=76.3 to 88.3 starting d 180 to yr 3 (ΔLSM (95% CI)=12.1 (2.9, 21.2), p=.011). Patient PROMIS scores significantly improved from baseline for anxiety (starting d 30) and fatigue up to yr 1 (anxiety ΔLSM (95% CI) =-6.0 (-10.5,-1.5), p=.009; fatigue ΔLSM (95% CI)=-6.8 (-12.4,-1.2), p=.019), as well as mobility from yr 2 to yr 3 (ΔLSM (95% CI)=7.2 (0.3,14.1), p=.040) and pain interference from yr 1 up to yr 2 (ΔLSM (95% CI)=-7.9 (-14.8, -1.0), p=.024). Parent-proxy scores significantly improved from baseline up to yr 3 for anxiety (from d 180; ΔLSM (95% CI)=-8.6 (-15.4,-1.8), p=.013), fatigue (from 1 yr; ΔLSM (95% CI)=-9.2 (-16.1,-2.3), p=.009), mobility (from yr 2; ΔLSM (95% CI)=6.6 (1.3,12.0), p=.016), and pain interference (from d 90; ΔLSM (95% CI)=-11.0 (-17.8,-4.2), p=.002). Pt and parent-proxy depression did not significantly differ from baseline. Though pt peer relationship scores significantly decreased from baseline to d 180 (ΔLSM (95% CI)=-4.8 (-9.1,-0.4),p=.031), all scores remained above cut-off for impairment. For DRS, pt and parent-proxy scores indicated low regret up to yr 3 (LSM=17.5 and 11.2, respectively), which did not significantly differ from baseline at d 90. Most PROMIS HRQoL scores were normal pre-HCT in our cohort. Previous studies with smaller sample sizes have demonstrated both low and high baseline scores. However, it is the favourable change from baseline which is of greater relevance to patients and providers. In conclusion, our interim analysis demonstrated significant improvements in multiple domains of HRQoL at longitudinal timepoints, as early as 30 d and lasting up to 3 yrs post-HCT with low DR. Our findings are strengthened by our large cohort, duration of follow-up and the use of diverse measures, including a novel DRS.

ABSTRACT:We report on 8 patients with ankyrin repeat domain 26 (ANKRD26)-related thrombocytopenia 2 (ANKRD26-RT) with elevated bone marrow myeloblasts and dysmegakaryopoiesis, without somatic genetic abnormalities or progression to malignancy during long-term observation, findings which may constitute inherent ANKRD26-RT biology rather than progression to myeloid malignancy.

Chronic graft-versus-host disease (cGVHD) has a profound impact on the endocrinologic and cardiovascular health of survivors of transplantation performed in childhood. The impact of cGVHD is long-lasting and contributes to morbidity and early mortality through multiple mechanisms. Organs and tissues may be direct targets of alloreactive donor-derived immune cells. Corticosteroids and other cGVHD-directed therapies influence hormonal actions, alter bone metabolism, and negatively impact cardiometabolic health. Pediatric survivors are particularly vulnerable to the endocrinologic and cardiovascular effects of cGVHD as it develops during periods of intense growth and development, although little is known about the direct contribution to late effects. The Research and Education Toward Solutions for Late Effects to Innovate, Excel, and Nurture after cGVHD (RESILIENT after cGVHD) effort brought together content experts to determine the state of the science, develop clinical recommendations, and propose a research agenda in endocrine, cardiovascular, and metabolic cGVHD survivorship, which are detailed in this report.

Background: Chimeric antigen receptor T-cell therapy (CAR-T) has revolutionized treatment for B-cell malignancies, yet >50% of patients ultimately relapse, highlighting the need to overcome barriers to durable responses. While CAR-T dysfunction is often driven by T cell-intrinsic inhibitory pathways, how these pathways regulate in vivo proliferation, tissue infiltration, and persistence remains poorly understood. To identify key regulators, we performed the first in vivo CRISPR/Cas9 loss-of-function screen in a non-human primate (NHP) model of B cell-directed CAR-T therapy. This screen enabled high-resolution, cross-tissue dissection of the dominant regulators of CAR-T efficacy in an immunocompetent model that recapitulates human CAR-T biology. This competitive screen uncovered key gene targets that differentially constrain CAR-T expansion, persistence, and trafficking across tissues, offering new opportunities for fine-tuning of CAR-T function. Methods: NHP T-cells were virally transduced to express CD20.BBz CAR and a CRISPR/Cas9 library targeting 1,502 T-cell relevant genes and positive/negative controls, followed by Cas9 electroporation. 6x10⁶ autologous edited CAR-Ts/kg (>1,000x guide coverage) were infused into two NHPs after lymphodepletion chemotherapy. Guide enrichment was analyzed in sorted CD4⁺ and CD8⁺ CAR-Ts from peripheral blood (PB), bone marrow (BM), lymph node (LN), and CSF across CAR-T expansion (days 8-9), contraction (d16-26), and late persistence (d70-90). Top candidates were functionally validated using in vitro cytotoxicity and proliferation assays, and in vivo testing in the NHP CD20 CAR-T model. Results: The two in vivo screen CAR-T recipients demonstrated robust concordance in guide distribution. The infusion products showed expected distribution of positive and negative controls with an ROC AUC >0.8, and importantly, similar distribution of targets to the plasmid library, emphasizing the ability to identify targets that were selected in vivo and not during the in vitro manufacturing process. CAR-T expansion was comparable to non-genetically modified CAR-T controls (58.2+/-13.9% vs 42.3+/-4.7% of total T-cells) and recipients experienced comparable low-grade CRS and ICANS to control recipients. This in vivo screen uncovered a dynamic regulatory network comprising 221 high-confidence gene targets (FDR<0.05) consistently enriched across both animals. These targets stratified into distinct categories: a core set of 15 genes shared across multiple timepoints and compartments, likely representing potent universal regulators of CAR-T cell function; a late-emerging subset enriched during contraction and late time points (95 genes) likely involved in persistence; and compartment-specific hits (23 in PB, 33 in CSF, 9 in BM, 11 in LN) potentially driving tissue-resident activity and infiltration. Among these hits, genes involved in immune synapse formation and T activation were preferentially enriched in PB (MEF2D, CD2, PTPN2, SLAMF6); regulators of cytokine signaling dominated in BM (IFNAR1, IL2RA, IL2RB); cell trafficking and cytoskeletal modulator genes were enriched in LN and CSF (PLXNA4, CTNNAL1, TNS2). Among the top hits consistently enriched across timepoints and tissues were TET2, PI3Kδ, PTPN2, and MEF2D, representing key universal regulators of CAR-T cell efficacy. Functional validation confirmed that deletion of these targets significantly enhanced CAR-T proliferation, increased secretion of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-2), and improved leukemia-targeted cytotoxicity in vitro. In vivo, PTPN2-KO CD20 CAR-T cells demonstrated superior dose-dependent expansion (82.4% vs. 42.3% CAR+ T cells) and more rapid B-cell aplasia compared to WT counterparts, supporting their enhanced therapeutic potency. Notably, the screen also identified enrichment of guides targeting canonical pathways involved in cytokine signaling and T cell activation/effector function (IL2RA, IL2RB, IFNGR, GZMB, and CD2), underscoring the need to fine-tune, rather than simply amplify, CAR-T activation to maximize efficacy while avoiding overstimulation-induced dysfunction. Conclusions: This study pioneers the first in vivo CRISPR-Cas9 screen in an immunocompetent NHP CAR-T model, generating a comprehensive atlas of genetic regulators that shape CAR-T expansion, trafficking to sites of disease, and persistence within the complex in vivo microenvironment, building a roadmap for rational next-generation CAR-T engineering.

B-cell targeting CAR-T cell therapies achieve high remission rates, yet durable responses occur in fewer than 40% of patients. Deletion of negative T-cell regulators, such as PTPN2, a key inhibitor of TCR and cytokine signaling, represents a promising strategy to enhance the efficacy of CAR-T cells. While transfer of PTPN2 knockout (KO) T cells has demonstrated antitumor benefits in murine models, its impact on human-derived CAR-T cells and, importantly, the associated in vivo efficacy and toxicity remain unclear. Here, we demonstrate that PTPN2-KO human CD19 CAR-T cells exhibit enhanced cytokine production, cytotoxicity, TCR and CAR affinity and signaling, leading to superior in vitro elimination of leukemic cells with low CD19 expression. To assess in vivo efficacy and toxicity, we performed a dose-escalation study using a non-human primate (NHP) model of B-cell-targeting CD20 CAR-T cell therapy. We demonstrated that PTPN2-KO CD20 CAR-T cells exhibited superior in vivo expansion and B-cell depletion compared to WT CAR-T cells, in a dose-dependent manner. At the highest dose level, CAR-T expansion was associated with increased toxicities, particularly ICANS, compared to PTPN2 WT CD20 CAR-T cells driven by enhanced CNS-infiltration. Transcriptional profiling revealed a dominant effector and proliferative signature, with cytotoxic CNS-infiltrating CD8+ PTPN2-KO CAR-T cells implicated in ICANS pathogenesis. This study details the comprehensive evaluation of PTPN2-KO CAR-T cells in an immunocompetent model, demonstrating their enhanced on-target functionality, while highlighting increased toxicity risks, underscoring the need for rigorous preclinical assessment of potent genetic modifications in CAR-T therapy. Key points:PTPN2-KO CAR-T cells exhibit enhanced effector functionIn a dose escalation study in rhesus macaques, PTPN2-KO mediated enhanced proliferation and CNS infiltration was associated with increased ICANS.

ABSTRACT:Rapid CD137 upregulation on alloreactive T cells upon allogeneic stimulation suggests that their selective elimination could prevent acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HCT). Here, we developed a novel aGVHD prophylactic regimen consisting of a single dose of an anti-CD137 antibody-drug conjugate (CD137-ADC) administered on the day of transplant without additional immunosuppression. The CD137-ADC depleted both human and nonhuman primate (NHP) activated T cells and proved highly effective in preventing xenogeneic aGVHD in mice receiving human peripheral blood mononuclear cells, as well as in NHP undergoing major histocompatibility complex (MHC)-haploidentical HCT. Flow cytometry analysis of NHP T cells indicated specific depletion of activated PD-1+ CD4 and CD8 T cells, while sparing naïve and PD-1-OX40+ memory T-cell subsets during the first week after HCT. CD137-ADC-treated NHP recipients demonstrated robust hematopoietic and immune reconstitution. Hallmarks of T-cell recovery after CD137-ADC, which were associated with long-term aGVHD-free survival, included reconstitution of CD4 memory T cells expressing TRAIL, terminally differentiated CD8 T cells expressing CX3CR1, and CD4 FoxP3+ regulatory T cells, cell types not expected to be involved in aGVHD pathogenesis. CD137-ADC-treated recipients demonstrated a higher risk of reactivation of rhesus lymphocryptovirus (the rhesus macaque Epstein-Barr virus analog), which was associated with reconstitution of follicular helper T cells, interferon signaling-associated memory, and γδT-cell subsets. This reactivation was controllable with rituximab administration. These results document effective depletion of alloreactive T cells and prevention of aGVHD after a single dose of CD137-ADC, suggesting that clinical translation should be carefully explored.

Background: Both graft versus host disease (GVHD) and infections remain major causes of morbidity and non-relapse mortality after hematopoietic cell transplant (HCT). As acute GVHD (aGVHD) is a T cell mediated alloreactive process, several T cell depleting, suppressing, and modulating strategies have been employed to prevent GVHD. Abatacept modulates T cell activity through CD28:CD80/86 costimulation blockade. In the ABA2 trial, a 4-dose regimen of abatacept (10mg/kg on days -1, +5, 14, 28) when added to CNI/MTX (the ‘ABA’ arm) significantly reduced aGVHD without increasing infections, when compared to CNI/MTX/Placebo (‘PBO‘), after unrelated-donor HCT. Understanding the biology underpinning ABA's success in preventing aGVHD, as well as its potential immunologic trade-offs, is crucial to advance prevention and treatment of GVHD. Here, we studied the size and diversity of the T cell repertoire in the ABA2 trial. Methods: We performed T cell receptor (TCR) sequencing using TCRB Immunosequencing (Adaptive Biotechnologies) of samples from ABA2's 185 participants. We analyzed peripheral blood TCRs at baseline (prior to conditioning) and post-HCT on days 28, 63, 100, 180, and 365. In addition, TCR sequencing data from select transplant product bags was available. A total of 889 samples were analyzed with a median of 3,725,249 TCRs profiled per time point. To assess TCR diversity, we calculated Shannon clonality, for which 0 represents a completely even sample and 1 a monoclonal sample. We also assessed richness (the number of unique TCRs in a sample) and singleton TCRs after performing clone abundance-based down-sampling. Finally, to assess repertoire stability and turnover, we calculated Morisita indices, which also range 0-1, with 1 representing complete repertoire overlap between timepoints. Results: Both PBO and ABA patients demonstrated high TCR diversity, which was evident early and persisted throughout the first year post-HCT. Notably, rather than decreasing TCR diversity vs CNI/MTX, as has been demonstrated for PT-Cy/Tac/MMF, CNI/MTX/ABA actually increased TCR diversity compared to PBO: For example, on day 28, median Shannon Clonality for PBO = 0.095 vs ABA = 0.036 (p <0.001); on day 60 median Shannon Clonality for PBO = 0.155 vs ABA = 0.074 (p = 0.04). Down-sampled singleton TCR analysis at Day 28 similarly revealed a significantly higher number of singletons in those receiving ABA vs PBO (median singleton TCR count of 730 for PBO vs 863 for ABA when data statistically down-sampled to 1000, p = 0.003; median singleton count of 2882 for PBO vs 3846 for ABA when data statistically down-sampled to 5000, p<0.001). Similarly, richness was higher in ABA compared to PBO at Day 28 (median richness 799 for PBO vs 904 for ABA when data statistically down-sampled to 1000, p = 0.003; 3381 for PBO vs 4205 for ABA when data statistically down-sampled to 5000, p<0.001). Together, these measurements provide a consistent illustration of intact TCR repertoire diversity in ABA patients despite significant control of aGVHD. Analysis of TCR sharing between two successive time points demonstrated no difference in the achievement of repertoire stability between PBO and ABA, with no difference in Morisita indices when comparing graft vs day 28, day 28 vs 60, and day 60 vs 100. Notably, ABA patients demonstrated more repertoire turnover between Days 100-180 compared to PBO (median Morisita index 0.79 for PBO vs 0.54 for ABA, p=0.01), suggesting increased T cell dynamics after abatacept levels waned. Conclusions: Here we demonstrate that ABA did not reduce either the size or diversity of the T cell repertoire in HCT recipients, despite its ability to control aGVHD. These results are consistent both with the body of work establishing the mechanism of ABA as being immune modulating rather than T cell depleting, and with the intact protective immunity observed in ABA2. Notably, the higher number of singletons and higher richness at day 28 in those receiving ABA is consistent with our previous results demonstrating that ABA preserves naïve T cells. This naïve T cell preservation, along with repertoire turnover after Day 100, is consistent with the control of aGVHD with ABA without a beneficial impact on chronic GVHD.Together, these findings underscore the ability of ABA to preserve the TCR repertoire, providing a key biologic explanation for its ability to control aGVHD without increasing the risk of infectious complications after HCT.

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage(-)Sca1(+)cKit(+) cells within 48 hours. Fanca(-/-) recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc(-/-) and Fancg(-/-) recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca(-/-) recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.