Abstract Successful transplantation of autologous gene-modified hematopoietic stem and progenitor cells (HSPCs) requires efficient ablation of resident hematopoietic stem cells. Considering that conventional myeloablative conditioning regimens are associated with nonhematologic toxicities, we evaluated CD45-directed radioimmunotherapy (RIT) using the α-emitter astatine 211 (211At) before the transplantation of ex vivo gene-edited autologous HSPCs in nonhuman primates as an alternative. We humanized the CD45 antibody, BC8, and labeled it with 211At. As a model, mobilized CD34+ HSPCs were multiplex gene-edited using an adenine base editor, modifying the HBG promoter to reactivate fetal hemoglobin and deleting CD33. Two animals each received 300 or 400 μCi/kg of 211At. In contrast to historic controls conditioned with total body irradiation, CD45-RIT animals did not show any noticeable nonhematopoietic toxicities and were almost entirely transfusion independent with rapid recovery of neutrophils and platelets. Dose-dependent engraftment of gene-edited cells was enabled by 211At. A new single-cell sequencing assay revealed up to 70% combined monoallelic and biallelic gene-editing efficiency in the blood, consistent with complete replacement of the bone marrow stem cell compartment. Assessment by bulk analysis underestimated the frequency of gene-edited cells, highlighting the importance of a single-cell readout. Single-cell sequencing further confirmed stable and unbiased contribution of multiplex-edited HSPCs to all mature lineages in the blood, providing high-resolution data assuring successful replacement upon autologous HSPC gene therapy. The levels of edited cells remained stable for the entire follow-up of >18 months. Collectively, these studies identify 211At-CD45 RIT as a targeted alternative for myeloablative conditioning for autologous gene therapy.

Hematopoietic stem cell transplantation (HSCT) is the current standard of care for a number of neurotrophic lysosomal storage disorders. The therapeutic mechanism of HSCT is believed to be mediated by engraftment of HSC progeny to the brain as microglia-like cells (MLCs). However, the engraftment of MLCs and their transcriptomic identity relative to endogenous microglia is poorly understood. Here, we utilize the autologous nonhuman primate (NHP) HSCT model to investigate the engraftment of MLCs after gene-modified autologous HSCT. We observed engraftment of gene-marked MLCs across a cohort of five NHPs. MLCs engrafted through diverse brain regions; adopted a homeostatic, ramified morphology, and upregulated core microglial transcripts. We then utilized single cell RNA sequencing to more rigorously evaluate the transcriptome of MLCs, revealing a border-associated macrophage-like phenotype. Our findings offer critical insights into the engraftment and behavior of MLCs post-HSCT, laying the groundwork for their future utilization as a directed therapeutic.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

CBFA2T3-GLIS2 (C/G) fusion is the most frequent chimeric oncogene observed in non-Down syndrome acute megakaryoblastic leukemia (AMKL) in infants. C/G-AMKL has a dismal prognosis due to resistance and relapse following standard AML chemotherapy. Our group previously reported that transduction of C/G fusion followed by coculture with an engineered endothelial cell (EC) niche was sufficient to transform human cord blood hematopoietic stem and progenitor cells (HSPC) into C/G-AMKL, providing a platform to interrogate the biology of this aggressive leukemia. To identify putative leukemic stem cells (LSC) and elucidate their resistance mechanisms, we conducted single-cell RNA sequencing on engineered C/G-AMKL during their transformation from HSPC in the EC niche. We identified a subset of cells co-expressing LSC-associated genes CD96 and CD70 that were enriched in known pediatric LSC gene signatures and signatures of dormancy. Further, a subset of C/G-AMKL cells from both engineered leukemias and patient samples co-expressed CD96 and CD70 by flow cytometry. Notably, ECs protected C/G-AMKL cells from cytarabine treatment, which enriched for the CD96+CD70+ population, suggesting niche-mediated resistance of dormant LSCs. Computational analysis of receptor-ligand interactions between ECs and C/G-AMKL LSCs suggested a prominent role for chemokine/cytokine signaling, integrin signaling, and cell adhesion, which are predicted to support LSC survival within the niche based on enriched downstream target genes. Overall, our study revealed that CD96+CD70+ LSCs may mediate resistance of C/G-AMKL through niche interactions promoting niche-retention and dormancy. Future studies using this platform could facilitate the development of therapeutic strategies to overcome treatment resistance by targeting CD96+CD70+ LSCs in this high-risk subset.

As curative therapies for pediatric acute myleoid leukemia (AML) remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the major histocompatibility complex-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial (clinicaltrials gov. Identifier: NCT01371981). Thirty-eight percent of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower white blood cells and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event-free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro preclinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AML at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

Immunosuppressed bone marrow transplant patients with pulmonary infiltrates routinely undergo bronchoscopy with bronchoalveolar lavage (BAL) to investigate potential etiologies. Cytokine release syndrome after BAL is unreported in the literature in general and in this patient population. We report on an allogeneic bone marrow transplant patient with non-infectious organizing pneumonia of the lungs who developed delayed and rapidly progressive shock and hypoxia post-procedure over the course of 12 h resulting in intensive care unit admission for supportive care. BAL was characterized by a marked lymphocytic, cytotoxic T cell infiltrate on pathology and flow cytometry without clear evidence of infection. The patient’s clinical status improved quickly only after the initiation of high dose intravenous steroids and returned to baseline as an outpatient. The patient’s clinical data and course suggest a cytotoxic T cell response from the lung and BAL as the etiology. With an increasing number of cellular therapies for cancer entering the clinic, the potential for unusual but morbid complications from routine bronchoscopy should be considered.

Chimeric antigen receptor (CAR) T cell therapy was a resounding success in CD19+ ALL despite the fact that it is essential for B-cell maturation because it is not expressed during normal myeloid hematopoiesis. Normal and leukemic B-cells are targeted by CD19+ CAR T-cells without causing myeloid hematopoietic toxicity. Advancing such therapy to acute myeloid leukemia (AML) has been challenging in part due to a paucity of “dispensable” targets or targets whose expression is limited to the leukemic cells. Using a large AML and normal hematopoiesis transcriptome database, we pursued discovery of “AML-Restricted Targets”; genes that are silent in normal hematopoiesis but expressed in AML. This broad discovery effort yielded a library of AML-restricted targets that included PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein expressed via MHC on the cell surface, to be a highly expressed in AML including the aggressive KMT2A-rearranged (KMT2A-r) AML. We used a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface, to develop a PRAME directed TCR mimic CAR T (PRAME mTCRCAR T) for pre-clinical studies. Studies in cell lines and CDX models have shown significant efficacy of this CAR T (Kirkey, Bld Adv. 2022). To conduct final IND-enabling studies, we generated a KMT2A-r HLA-A2+/PRAME+ patient-derived xenograft (PDX) model to treat with PRAME mTCRCAR T-cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against this unique PDX AML model. VL and VH sequences from the PRAME specific TCR mimic antibody (Pr20) were used to construct the single-chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME +/HLA-A2 + pediatric patient with KMTA2-r AML. PDX cells were transduced with lentiviral ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. Mice were transplanted with 1x10 6 PDX leukemia cells then 1 week later, PDX leukemia-bearing mice were treated with unmodified T cells or PRAME mTCRCAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse. Leukemia burden was measured by IVIS imaging and regular peripheral blood analysis. Mice who received unmodified T cells had rapid disease progression by day 50 and all died by day 105. In contrast, PRAME mTCRCAR T-treated mice rapidly cleared disease and all remained alive and leukemia-free >180 days post-treatment (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T-cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75- and 429.6-fold increase in radiance by IVIS at 6 and 11 weeks post-treatment, respectively. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. We further evaluated hematopoietic toxicity of PRAME mTCRCAR T cells in humanized mice. Sub-lethally irradiated NSG-SGM3 mice were reconstituted with HLA-A2+ CD34 selected human cord blood stem cells with simultaneous and allowed to engraft. Human hematopoietic cell engraftment was evaluated following treatment with unmodified T cells vs. PRAME mTCRCAR T cells. Evaluation of human CD45+ cells in the peripheral blood via flow cytometry showed no difference in the levels of hematopoietic engraftment in mice treated with PRAME mTCRCAR T cell (8%) vs. unmodified T cells (6%) vs. an untreated control cohort (5%), demonstrating lack of toxicity of PRAME mTCRCAR T cells against HLA-A2 hematopoiesis. We demonstrate that PRAME mTCRCAR T cells can irradicate AML in a target specific manner in aggressive KMT2A-r AML without toxicity. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival, without hematopoietic toxicity. These results provide strong rationale for advancing this therapeutic approach to clinical development.

Supplemental methods that provides additional detail on statistical methods and laboratory protocols and reagents.

Preferentially Expressed Antigen in Melanoma (PRAME), a cancer-testis antigen, provides an ideal target for immunotherapy in acute myeloid leukemia (AML). We have shown expression of PRAME in a significant subset of childhood and adult AML and lack of expression in normal hematopoiesis. Although an intracellular antigen, we developed a novel approach to target PRAME using a chimeric antigen receptor (CAR) construct encoding a targeting domain based on T-cell receptor (TCR) mimic antibodies that target the peptide-HLA complex. We used the antibody sequence from a previously designed TCR mimic (mTCR) antibody, Pr20, that recognizes the PRAME ALY peptide in complex with HLA-A*02 and verified expression of PRAME in AML cell lines and primary AML blasts. Using the Pr20 antibody sequence, we developed CAR T cells (PRAME mTCRCAR T) to be tested against primary samples from patients with AML and AML cell lines that express the PRAME antigen in the context of HLA-A2 expression. In contrast to appropriate controls, PRAME mTCRCAR T cells demonstrate target-specific and HLA-mediated in vitro activity in OCI-AML2 and THP-1 cell lines, HLA-A2 cell lines expressing the PRAME antigen, and against primary AML patient samples. In vivo cell-derived xenograft models treated with PRAME mTCRCAR T cells demonstrated potent leukemia clearance and improved survival compared with unmodified T-cell controls. Furthermore, the cytolytic activity of PRAME mTCRCAR T cells was enhanced by treating the target cells with interferon gamma, which increases PRAME antigen expression. These results demonstrate the feasibility and efficacy of targeting PRAME with novel PRAME mTCRCAR T cells.

2553 Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment, but has had limited success against AML in part due to overlap of cell surface antigens expressed in AML and normal hematopoietic cells. To identify AML-restricted targets, we interrogated the transcriptome from over 3000 AML patients and found PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein, to be a highly expressed AML-restricted target. Using a novel approach to target intracellular antigens, we developed a PRAME CAR T cell using a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface. To conduct final IND-enabling studies, we generated an in vivo patient derived xenograft (PDX) model to treat with PRAME mTCR CAR T cells. Here we demonstrate the in vivo activity of PRAME mTCR CAR T cells against a PDX AML model. Methods: We used the VL and VH sequences from the PRAME TCR mimic antibody (Pr20) to construct the single chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME + /HLA-A2 + pediatric patient with AML. PDX cells were transduced with ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. PDX leukemia-bearing mice were treated with unmodified T-cells or PRAME mTCR CAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse 1 week after leukemia injection. Leukemia burden was measured by IVIS imaging and peripheral blood analysis. Results: Treatment with PRAME mTCR CAR T cells led to eradication of leukemia with all mice alive >100 days post-treatment, while control mice (unmodified T cells) had disease progression at Day 50 (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75 and 429.6 fold increase in radiance by IVIS at 6 and 11 weeks post-treatment. No significant increase in radiance was seen in the PRAME mTCR CAR T cell treated group. Human T cells (CD45+/CD3+) were detectable in the peripheral blood at Day 7 and 14 post-treatment in both the unmodified and CAR T cell treated groups. Conclusions: We demonstrate the therapeutic potential of targeting PRAME with mTCR CAR T cells in AML. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCR CAR T cells resulting in prolonged survival. These results provide a novel approach to target PRAME with CAR T cells and provide compelling data to evaluate PRAME mTCR CAR T cells for use in clinical trials against AML. [Table: see text]

Hematopoietic stem cell transplantation is a well-known treatment for hematologic malignancies, wherein nascent stem cells provide regenerating marrow and immunotherapy against the tumor. The progeny of hematopoietic stem cells also populate a wide spectrum of tissues, including the brain, as bone marrow-derived macrophages similar to microglial cells. We developed a sensitive and novel combined immunohistochemistry (IHC) and XY fluorescence in situ hybridization assay to detect, quantify, and characterize donor cells in the cerebral cortices of 19 female patients who underwent allogeneic stem cell transplantation. We showed that the number of male donor cells ranged from 0.14% to 3.0% of the total cells or from 1.2% to 25% of microglial cells. Using tyramide-based fluorescent IHC, we found that at least 80% of the donor cells expressed the microglial marker ionized calcium-binding adapter molecule-1, consistent with bone marrow-derived macrophages. The percentage of donor cells was related to pretransplantation conditioning; donor cells from radiation-based myeloablative cases averaged 8.1% of microglial cells, whereas those from nonmyeloablative cases averaged only 1.3%. The number of donor cells in patients conditioned with busulfan- or treosulfan-based myeloablation was similar to that in total body irradiation-based conditioning; donor cells averaged 6.8% of the microglial cells. Notably, patients who received multiple transplantations and those with the longest posttransplantation survival had the highest level of donor engraftment, with donor cells averaging 16.3% of the microglial cells. Our work represents the largest study characterizing bone marrow-derived macrophages in patients after transplantation. The efficiency of engraftment observed in our study warrants future research on microglial replacement as a therapeutic option for disorders of the central nervous system.

PURPOSE Graft-versus-host disease (GVHD) causes morbidity and mortality following allogeneic hematopoietic cell transplantation. Naive T cells (T N ) cause severe GVHD in murine models. We evaluated chronic GVHD (cGVHD) and other outcomes in three phase II clinical trials of T N -depletion of peripheral blood stem-cell (PBSC) grafts. METHODS One hundred thirty-eight patients with acute leukemia received T N -depleted PBSC from HLA-matched related or unrelated donors following conditioning with high- or intermediate-dose total-body irradiation and chemotherapy. GVHD prophylaxis was with tacrolimus, with or without methotrexate or mycophenolate mofetil. Subjects received CD34-selected PBSC and a defined dose of memory T cells depleted of T N . Median follow-up was 4 years. The primary outcome of the analysis of cumulative data from the three trials was cGVHD. RESULTS cGVHD was very infrequent and mild (3-year cumulative incidence total, 7% [95% CI, 2 to 11]; moderate, 1% [95% CI, 0 to 2]; severe, 0%). Grade III and IV acute GVHD (aGVHD) occurred in 4% (95% CI, 1 to 8) and 0%, respectively. The cumulative incidence of grade II aGVHD, which was mostly stage 1 upper gastrointestinal GVHD, was 71% (95% CI, 64 to 79). Recipients of matched related donor and matched unrelated donor grafts had similar rates of grade III aGVHD (5% [95% CI, 0 to 9] and 4% [95% CI, 0 to 9]) and cGVHD (7% [95% CI, 2 to 13] and 6% [95% CI, 0 to 12]). Overall survival, cGVHD-free, relapse-free survival, relapse, and nonrelapse mortality were, respectively, 77% (95% CI, 71 to 85), 68% (95% CI, 61 to 76), 23% (95% CI, 16 to 30), and 8% (95% CI, 3 to 13) at 3 years. CONCLUSION Depletion of T N from PBSC allografts results in very low incidences of severe acute and any cGVHD, without apparent excess risks of relapse or nonrelapse mortality, distinguishing this novel graft engineering strategy from other hematopoietic cell transplantation approaches.

Temporally-regulated alternative splicing choices are vital for proper development yet the wrong splice choice may be detrimental. Here we highlight a novel role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in both humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis and forced over-expression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring TRK fusions. Affinity purification-mass spectrometry (AP-MS) proteomic analysis reveals TrkB.T1 has distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, TGF-ß, Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.

Currently, there is no convincing evidence that the grade of follicular lymphoma (FL) impacts patient outcome. We correlated grades in 33 925 patients with nodal FL during 1992-2018 in the SEER database with disease-specific survival (DSS) and overall survival (OS). Patients with FL grade 3 had lower DSS and OS as compared to FL grades 1-2. During 1992-2005, the 10-year DSS for patients with FL grades 3 and grades 1-2 were 68.6%, and 71.4%, respectively, and in 2006-2018, they were 77.7% and 82.6%, respectively. The 10-year OS estimates in 1992-2005 were 49.9% and 54.2% for grade 3 and grades 1-2 respectively, and in 2006-2018, they were 59.1% and 63.5% for grade 3 and grades 1-2, respectively. After adjustment for stage and age, the hazard ratios for death due to FL and death from any cause for patients with FL grade 3 during 1992-2005 were 1.09 (1.02-1.16) and 1.07 (1.02-1.12), respectively, compared to FL grades 1-2; and during 2006-2018, the hazard ratios for death due to FL and death from any cause for patients with FL grade 3 were 1.34 (1.22-1.45) and 1.16 (1.10-1.23), respectively compared to FL grades 1-2. The grade of FL is an important determinant of disease biology.

A safe, effective, and inclusive gene therapy will significantly benefit a large population of patients with hemophilia. We used a minimally invasive transcutaneous ultrasound-mediated gene delivery (UMGD) strategy combined with microbubbles (MBs) to enhance gene transfer into 4 canine livers. A mixture of high-expressing, liver-specific human factor VIII (hFVIII) plasmid and MBs was injected into the hepatic vein via balloon catheter under fluoroscopy guidance with simultaneous transcutaneous UMGD treatment targeting a specific liver lobe. Therapeutic levels of hFVIII expression were achieved in all 4 dogs, and hFVIII levels were maintained at a detectable level in 3 dogs throughout the 60-day experimental period. Plasmid copy numbers correlated with hFVIII antigen levels, and plasmid-derived messenger RNA (mRNA) was detected in treated livers. Liver transaminase levels and histology analysis indicated minimal liver damage and a rapid recovery after treatment. These results indicate that liver-targeted transcutaneous UMGD is promising as a clinically feasible therapy for hemophilia A and other diseases.

Mutations characterize diverse human cancers; there is a positive correlation between elevated mutation frequency and tumor progression. One exception is acute myeloid leukemia (AML), which has few clonal single nucleotide mutations. We used highly sensitive and accurate Duplex Sequencing (DS) to show now that AML, in addition, has an extensive repertoire of variants with low allele frequencies, < 1%, which is below the accurate detection limit of most other sequencing methodologies. The subclonal variants are unique to each individual and change in composition, frequency, and sequence context from diagnosis to relapse. Their functional significance is apparent by the observation that many are known variants and cluster within functionally important protein domains. Subclones provide a reservoir of variants that could expand and contribute to the development of drug resistance and relapse. In accord, we accurately identified subclonal variants in AML driver genes NRAS and RUNX1 at allele frequencies between 0.1% and 0.3% at diagnosis, which expanded to comprise a major fraction (14-53%) of the blast population at relapse. Early and accurate detection of subclonal variants with low allele frequency thus offers the opportunity for early intervention, prior to detection of clinical relapse, to improve disease outcome and enhance patient survival.