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Abstract: The optimal form of treatment for children with relapsed or refractory acute promyelocytic leukemia (APL) is unclear. We retrospectively analyzed the results of 32 (11 autologous, 21 allogeneic) hematopoietic stem cell transplants (HSCT) performed for children originally treated on either the Eastern Cooperative Group E2491 Trial or the Cancer and Leukemia Group B C9710 Trial and subsequently diagnosed with relapsed or refractory APL. For autologous HSCT, the incidence of treatment-related mortality (TRM) and relapse was 0% (95% confidence interval [CI], 0%-30%) and 27% (95% CI, 9%-57%), respectively. The 5-year event-free survival (EFS) and overall survival (OS) following autologous HSCT was 73% (95% CI, 43%-91%) and 82% (95% CI, 51%-96%), respectively. For allogeneic HSCT, the incidence of TRM and relapse was 19% (95% CI, 7%-41%) and 10% (95% CI, 2%-30%), respectively. The 5-year EFS and OS following allogeneic HSCT was 71% (95% CI, 50%-86%) and 76% (95% CI, 55%-90%), respectively. There was no significant difference in EFS or OS between autologous and allogeneic HSCT. This data demonstrates that autologous and allogeneic HSCT are both effective therapies for treatment of children with relapsed or refractory APL. Autologous HSCT is associated with a low incidence of TRM, whereas allogeneic HSCT is associated with a low incidence of relapse, suggesting a strong GVL effect against residual APL. [Copyright &y& Elsevier]

A major complication of mismatched unmanipulated hematopoietic stem cell transplant (HSCT) is Graft-versus-Host Disease (GvHD), which results in significant morbidity and increased non-relapse mortality. Novel strategies to reduce GvHD and improve long-term tolerance between mismatched donor-host pairs include regulatory T cell therapy. We are investigating a new cell product named T-allo10 which contains suppressive anergic cells and is enriched in IL-10 producing regulatory type 1 T cells (Tr1). The main advantage of adoptive immunotherapy with Tr1 compared to other regulatory T cells is the host alloantigen specificity that is established in the donor Tr1 during in vitro culture in the presence of IL-10 and host tolerogenic dendritic cells. Here we report the preliminary results of our phase I trial (IND 17292) on the use of escalating doses of T-allo10 cells for patients aged 3-45 years affected by hematological malignancies. At present, we completed the first cohort of the phase I portion of the study (Table 1). The donors were class I mismatched for patient 1 and class II HLA-mismatched for patients 2 and 3, respectively. The GvHD prophylaxis consisted of Sirolimus and Mycophenolate and was serotherapy and Calcineurin inhibitory-free. Patients received 1 × 106 T-allo10 cells/Kg on Day-1. No adverse events were observed after the T-allo10 cell infusion. All 3 patients met the safety criteria and are alive and disease-free at 2 years, 15 months and 2 months post-HSCT, respectively. Tr1, phenotypically defined as CD4+CD45RA−CD49b+LAG3+ were detectable in the peripheral blood shortly after the infusion at 21% and 26% in patient 2 and 3, respectively. At day 28 post-HSCT, Tr1 represented 12% of circulating memory CD4+ T cells in patient 1, 3% in patient 2 and 2% in patient 3. TCR sequencing of T-allo10 cells prior and after infusion showed a restricted clonotype diversity with persistence of certain clonotypes in vivo, demonstrating Tr1 cell recirculation and survival. These data show that the T-allo10 cell infusion is safe and well tolerated and demonstrate that T-allo10 cells are detectable in the recipients after the infusion and traceable by TCR clonotype analysis. [ABSTRACT FROM AUTHOR]

A major complication of mismatched unmanipulated hematopoietic stem cell transplant (HSCT) is Graft-versus-Host Disease (GvHD), which results in significant morbidity and increased non-relapse mortality. Novel strategies to reduce GvHD and improve long-term tolerance between mismatched donor-host pairs include regulatory T cell therapy. We are investigating a new cell product named T-allo10 which contains suppressive anergic cells and is enriched in regulatory type 1 T cells (Tr1). The main advantage of adoptive immunotherapy with Tr1 compared to other regulatory T cells is the host alloantigen specificity that is established in the donor Tr1 during in vitro culture in the presence of IL-10 and host tolerogenic dendritic cells. Here we report the preliminary results of our phase I trial (IND 17292) on the use of escalating doses of T-allo10 cells for patients aged 3-45 years affected by hematological malignancies. At present, we treated two patients. The donors were class I and class II HLA-mismatched for patients one and two, respectively. The GvHD prophylaxis consisted of Sirolimus and Mycophenolate and was serotherapy and Calcineurin inhibitory-free. Patients received 1 × 106 T-allo10 cells/Kg on Day-1. No adverse events were observed after the T-allo10 cells infusion. Both patients met the safety criteria and are alive and disease-free eleven and four months post-HSCT, respectively. Tr1, phenotypically defined as CD4+CD45RA−CD49b+LAG3+ were detectable in the peripheral blood of patients after the infusion. At day 28 post-HSCT, Tr1 represented 12% of circulating memory CD4+ T cells in patient #1 and 3% in patient #2. TCR sequencing of T-allo10 cells prior and after infusion showed a restricted clonotype diversity with persistence of certain clonotypes in vivo, demonstrating Tr1 cell survival. Conclusion Our preliminary data show that the T-allo10 cell infusion is safe and well tolerated. T-allo10 cells are detectable in the recipients after the infusion and traceable by TCR clonotype analysis. [ABSTRACT FROM AUTHOR]

Successful hematopoietic cell transplantation (HCT) requires vacating recipient hematopoietic stem cell (HSC) niches to permit donor HSC engraftment to provide life-long hematopoietic and immune function. Currently HCT relies on DNA damaging radiation or chemotherapy to achieve HSC niche clearance. We have pursued a non-toxic approach to target and deplete HSC using humanized monoclonal antibody, AMG 191, that binds human CD117 (c-Kit). We opened a Phase 1 dose escalation trial using AMG 191 as the sole conditioning agent to achieve donor HSC engraftment in patients undergoing HCT for severe combined immunodeficiency (SCID). The primary endpoint is to assess the safety of administering AMG 191. Secondary endpoints include AMG 191 pharmacokinetics (PK), host HSC depletion, and determination of the dose of AMG 191 that achieves adequate donor HSC engraftment, defined as >5% donor granulocyte chimerism at 24 weeks. We have completed the first dose cohort of patients receiving 0.1 mg/kg AMG 191 and treated the first two patients in the second cohort (0.3 mg/kg). All five patients tolerated the AMG 191 infusion and the subsequent infusion of their CD34-selectd donor cells without clinical problems. Here we report efficacy in the first two patients, who have reached the 24-week timepoint. Both patients had T-B-NK+ SCID with mutations in the DCLRE1C (Artemis) gene. Both previously received unconditioned HCT as infants, failed to develop donor B cells and remained dependent on exogenous immunoglobulin. CD34-selected mobilized peripheral blood cells from the original donors were infused when the AMG 191 serum level was <100 ng/mL. Beginning at 8 weeks post-HCT both patients showed evidence of increasing peripheral blood CD19+CD20+ B cells. Extended immunophenotype analysis demonstrated that naïve B cells were present, and chimerism studies confirmed that the B cells were of donor origin. The first patient had impaired T cell function prior to his HCT. Significant increases in both naïve CD4+ and CD8+ T cells were observed with clinical improvement beginning at 22 weeks post-HCT. Importantly, starting at 8 weeks these 2 patients demonstrated donor chimerism in purified granulocytes, an indicator of HSC engraftment, which rose from 0% pre-HCT to ≥5% at 24 weeks (Table 1). Conclusion: These data are proof of concept that a humanized monoclonal antibody targeting CD117 can safely clear human HSC niches and facilitate donor HSC engraftment. This study is ongoing and open for enrollment. [ABSTRACT FROM AUTHOR]

Blood and marrow transplantation (BMT) is a standard curative therapy for patients with nonmalignant genetic diseases. Myeloablative conditioning has been associated with significant regimen-related toxicity (RRT), whereas reduced-intensity conditioning regimens have been associated with graft failure. In this prospective pilot trial conducted at 2 centers between 2006 and 2013, we report the outcome of 22 patients with nonmalignant genetic diseases who were conditioned with a novel reduced-toxicity regimen: i.v. busulfan (16 mg/kg), alemtuzumab (52 mg/m 2 ), fludarabine (140 mg/m 2 ), and cyclophosphamide (105 mg/kg). The median age of the study population was 3.5 years (range, 5 months to 26 years). No cases of sinusoidal obstruction syndrome, severe or chronic graft-versus-host disease (GVHD), or primary graft failure were reported. Median time to neutrophil engraftment (>500 cells/μL) and platelet engraftment (>20K cells/μL) were 19 (range, 12 to 50) and 23.5 (range, 14 to 134) days, respectively. The median length of follow-up was 3 years (range, .2 to 6.3). The overall survival rates were 95% at 100 days (95% confidence interval, .72 to .99) and 90% at 6 years (95% confidence interval, .68 to .98). RRT and chronic GVHD are significant barriers to BMT for patients with nonmalignant genetic diseases. This alemtuzumab-based reduced-toxicity regimen appears to be promising with durable engraftment, effective cure of clinical disease, low rates of RRT, and no observed chronic GVHD. [ABSTRACT FROM AUTHOR]

Abstract: Allogeneic hematopoietic stem cell transplantation for patients with a hemoglobinopathy can be curative but is limited by donor availability. Although positive results are frequently observed in those with an HLA-matched sibling donor, use of unrelated donors has been complicated by poor engraftment, excessive regimen-related toxicity, and graft-versus-host disease (GVHD). As a potential strategy to address these obstacles, a pilot study was designed that incorporated both a reduced-intensity conditioning and mesenchymal stromal cells (MSCs). Six patients were enrolled, including 4 with high-risk sickle cell disease (SCD) and 2 with transfusion-dependent thalassemia major. Conditioning consisted of fludarabine (150 mg/m2), melphalan (140 mg/m2), and alemtuzumab (60 mg for patients weighing > 30 kg and .9 mg/kg for patients weighing <30 kg). Two patients received HLA 7/8 allele matched bone marrow and 4 received 4-5/6 HLA matched umbilical cord blood as the source of HSCs. MSCs were of bone marrow origin and derived from a parent in 1 patient and from an unrelated third-party donor in the remaining 5 patients. GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. One patient had neutropenic graft failure, 2 had autologous hematopoietic recovery, and 3 had hematopoietic recovery with complete chimerism. The 2 SCD patients with autologous hematopoietic recovery are alive. The remaining 4 died either from opportunistic infection, GVHD, or intracranial hemorrhage. Although no infusion-related toxicity was seen, the cotransplantation of MSCs was not sufficient for reliable engraftment in patients with advanced hemoglobinopathy. Although poor engraftment has been observed in nearly all such trials to date in this patient population, there was no evidence to suggest that MSCs had any positive impact on engraftment. Because of the lack of improved engraftment and unacceptably high transplant-related mortality, the study was prematurely terminated. Further investigations into understanding the mechanisms of graft resistance and development of strategies to overcome this barrier are needed to move this field forward. [Copyright &y& Elsevier]

Adoptive immunotherapy using banked virus-specific T-lymphocytes (VSTs) derived from partially HLA-matched donors has been successful in restoring antiviral immunity after hematopoietic stem cell transplantation (HSCT), although broad availability of this therapy is currently limited, and best methods for donor-recipient matching remain unclear. To determine the feasibility and efficacy of third-party VSTs for the treatment of immunocompromised pediatric patients with refractory viral infections in a multicenter phase I/II consortium study. There are two arms to this study. Arm A are pediatric subjects who had infections with CMV, EBV, and/or adenovirus after hematopoietic stem cell transplant (HSCT) and had failed antiviral therapy, or who had toxicities related to anti-viral drug-related. Arm B enrolls patients with primary immunodeficiency disorders with refractory viral infection prior to HSCT. A VST bank containing products generated from 40 donors at Children's National was utilized, and antiviral HLA mapping was applied to identify the most suitable product for each patient referred. Patients received a dose of 2 × 10^7 VSTs/m2, with optional additional doses given 28 days after the initial VST infusion. Viral PCRs were monitored for efficacy, and VST persistence and expansion in vivo was measured via IFN-g ELISpot and flow cytometry. Twenty-three patients were enrolled from 11 study sites to date. Twenty-two were treated in Arm A (after HSCT), at a median time of 95 days post-HSCT (range 16-476). Fourteen patients were treated for adenovirus and 9 for CMV. Patients received a median of 1 infusion (range 1-3). Infused VSTs were matched at a median of 3/10 HLA alleles (range 2-5), and 22 had confirmation of antiviral activity through at least one shared HLA allele. Of 18 evaluable patients, 14 had antiviral responses at 1-month post-infusion (4/5 with CMV, and 10/13 with adenovirus, Figure 1), including 7 who had complete resolution of viremia. No GVHD was observed after infusion, and 17 of 23 (74%) are virus free up to 15 months post-infusion. Virus-specific T-cell activity was higher by day 45 (mean 177 IFN-g SPW) post-infusion than at baseline (mean 13 IFN-g SPW, p=0.045). VSTs remained detectable for up to 3 months via flow cytometry staining of discordant HLA alleles (Figure 2). Third-party VSTs utilized from a single bank can be distributed and administered in a multicenter consortium setting, and are safe and feasible for the treatment of refractory viral infections in pediatric patients. Preliminary efficacy data shows high rates of response and long-term survival. Studies of targeting strategies and characterization of T-cell epitopes are ongoing. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-matched donor, either related or unrelated, has been extensively used to treat patients with genetic disorders. However, only 25% of patients eligible to receive allogeneic HSCT have an HLA-identical sibling, and an HLA-matched unrelated donor can be identified for less than 60% of other patients. Mismatched HSCT has been associated with a high risk of graft loss, graft-versus-host disease (GvHD), and transplant-related mortality. Graft manipulation strategies have been used over the last decade to reduce these risks. HLA-haploidentical HSCT after ab T-cell/CD19 B-cell depletion (abhaplo-HSCT) has been shown to be effective in curing up to 90% of children with a variety of non-malignant disorders [Bertaina A, Blood 2014]. However, a scarcity of data are available for patients with rare and complex genetic diseases, including Tregopathies, an emerging new class of primary immunodeficiencies. Here we report preliminary data on the single center experience developed at Lucile Packard Children's Hospital, Stanford, CA, USA on the use of abhaplo-HSCT for rare and complex monogenic diseases (Table 1). All the cell products were manufactured at the Laboratory for Cell and Gene Medicine (LCGM) at Stanford (Table 2). Between 05/2018 and 08/2019, 5 patients with complex monogenic disorders were referred to our Center for HSCT including: a 23 year-old with Fanconi Anemia, two with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) and two with Schimke Immuno-Osseous Dysplasia (SIOD). Remarkably, all these patients were previously considered not eligible for a HSCT, and one of the IPEX patients was in palliative care. At a median follow-up of 240 days (range 39-520), all patients are alive and disease-free. One patient (SIOD) developed Grade I/stage II skin only graft-versus-host disease (GvHD), while none of the evaluable patients had developed chronic GvHD. Five months after abhaplo-HSCT and with 100% of the circulating T cells being donor derived, the first SIOD patient received a living kidney transplant from the stem cell donor (mother) using minimal immunosuppression with tacrolimus and steroid. Four weeks after the kidney transplant all immunosuppression was stopped, and the patient remains off immunosuppression 12 weeks post-kidney transplantation. Our preliminary data on the use of abhaplo-HSCT in patients with rare and complex monogenic diseases provide a strong rationale for expanding this therapeutic approach to diseases not previously considered as an indication for allogeneic HSCT, to patients in very poor clinical condition and to patients requiring both HSCT and solid organ transplant. [ABSTRACT FROM AUTHOR]

Patients who fail to adequately reconstitute the donor-derived immune system after allogeneic hematopoietic stem cell transplantation (HSCT) are at increased risk for infections and leukemia relapse. In the past, pan T-cell depleted haploidentical grafts were associated with delayed immune reconstitution (IR). Recently, the majority of patients receiving αβ T-cell/B-cell depleted haploidentical HSCT (αβhaplo-HSCT) reach a threshold of 200 CD3+ T cells/mcl by 100 days after HSCT (Bertaina A et al. Blood 2014 Jul 31;124(5):822-6). However, a proportion of patients experience a slower IR with consequent higher morbidity and mortality. Early prediction of delayed IR may permit prompt clinical intervention such as infusion of donor lymphocytes or of virus-specific cytotoxic T cells. Flow cytometry, the most widely applied approach for IR analysis, suffers from intrinsic limitations, such as high lymphocyte number requirement, degradation of samples, and insufficient standardization due to technical and operator variability. To overcome these limitations, we used a DNA methylation-based quantitative PCR that detects the epigenetic signature of different peripheral blood immune cell subsets (epigenetic quantification). This technique provides relative and absolute immune cell counts applicable to fresh, frozen, or paper-spotted dried blood. Epigenetic measurements are based on a per cell DNA copy number and provide a clear positive or negative signal rather than arbitrarily defined thresholds for "positivity" as in flow cytometry. We hypothesize that epigenetic quantification at day 15 after αβhaplo-HSCT could predict flow-based IR at day 100. Patients were consented at Lucile Packard Children's Hospital (Stanford, CA). Blood was collected between days 10-17 for epigenetic quantification and days 82-124 for flow cytometry. Bisulfite treated DNA underwent qPCR quantification of cell type-specific DNA regions of de-methylation (Baron U et al. Sci Transl Med 2018 Aug 1;10(452):eaan3508). Flow cytometry was performed using directly conjugated antibodies. Absolute cell counts were determined, plotted, and then analyzed using a linear regression model. In the first 5 αβhaplo-HSCT patients evaluated, we found a direct correlation between the epigenetic quantification at day 15 and flow cytometry at day 100 for CD4+ T cells (P=0.01), while the early epigenetic quantification of CD3+ and CD8+ T cells was not informative (Fig. 1). Preliminary data suggest that the use of epigenetic quantification early after αβhaplo-HSCT can predict the IR of CD4+ T cells at day 100 in αβhaplo-HSCT recipients. Ongoing analysis on a larger cohort of both αβhaplo-HSCT and unmanipulated HSCT recipients, will confirm if epigenetic quantification results obtained early post-HSCT can be used as a clinical biomarker of delayed IR and guide physicians in the use of post-HSCT adoptive immunotherapy. [ABSTRACT FROM AUTHOR]

Umbilical cord blood (UCB) is a vital source of hematopoietic stem and progenitor cells (HSPCs). Since 1991, UCB has been collected and stored in public cord blood banks to be primarily used as a source for allogeneic hematopoietic stem cell transplantation (HSCT). Since the seminal UCB transplant reported in 1988 by Gluckman et al., UCB has contributed to advances in HSCT and clinical trials. However, UCB-derived cells remain costly and difficult to procure for pre-clinical research. Here we present a protocol for a multidisciplinary, self-sustaining cord blood program dedicated to biomedical research: The Binns Program for Cord Blood Research, piloted at the Stanford University School of Medicine. The primary objective of this study was to educate others on the benefits of implementing a self-sustaining cord blood program that increases biomedical research throughout the medical community. Eligible donors are identified using IRB-approved inclusion criteria. UCB samples are then collected in utero, with informed consent, by obstetric providers at the time of delivery. Within 24-48 hours of isolation, deidentified samples are processed into three fractions: (1) plasma, (2) hematopoietic stem and progenitor cells (HSPCs) by immunomagnetic separation, and (3) non-HSPC cellular flow-through. UCB-derived fractions are then distributed to researchers upon request. Between November 2015 and April 2019, 1,385 women gave informed consent to donate their cord blood following delivery. Of those participants, 1,147 (83%) samples were collected and 927 (81%) distributed for research purposes. The median post-enrichment cell count per cord following positive selection for CD34+ HSPCs was 1.25 × 106 (Figure 1). Corresponding median post-enrichment sample purity was 90% following single-step purification. By October 2016, a protocol had been developed and optimized for the Binns Program to begin providing UCB-derived products to six laboratories. Now, the program supports 47 laboratories across Stanford. The research being supported spans a broad array of topics including normal and abnormal hematopoiesis, genetic engineering, and novel approaches to HCST. After initial start-up funding, the Binns Program became self-sustaining, representing a feasible model for cord blood banking to support the advancement of research by providing fresh or cryopreserved UCB-derived cells to the research community. We hope this model may be useful for other institutions and centers where the need to support research is high and the traditional model for public cord blood banking may not be a suitable option. [ABSTRACT FROM AUTHOR]