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Key Points The CD34+ compartment of human cord blood contains a range of HSPC immunophenotypes, among which the Lin−CD34+CD38+CD127+ CLP is rare. There is no correlation between the frequencies of CD34+ cells and immunophenotypic HSC in umbilical cord blood products.

Umbilical cord blood is an important graft source in the treatment of many genetic, hematologic, and immunologic disorders by hematopoietic stem cell transplantation. Millions of cord blood units have been collected and stored for clinical use since the inception of cord blood banking in 1989. However, the use of cord blood in biomedical research has been limited by access to viable samples. Here, we present a cost-effective, self-sustaining model for the procurement of fresh umbilical cord blood components for research purposes within hospital-affiliated academic institutions.

Dyskeratosis congenita is a disease of impaired tissue maintenance downstream of telomere dysfunction. Characteristically, patients present with the clinical triad of nail dystrophy, oral leukoplakia, and skin pigmentation defects, but the disease involves degenerative changes in multiple organs. Mutations in telomere-binding proteins such as TINF2 (TRF1-interacting nuclear factor 2) or in telomerase, the enzyme that counteracts age related telomere shortening, are causative in dyskeratosis congenita. We present a patient who presented with severe hypoxemia at age 13. The patient had a history of myelodysplastic syndrome treated with bone marrow transplant at the age of 5. At age 18 she was hospitalized for an acute pneumonia progressing to respiratory failure, developed renal failure and ultimately, she and her family opted to withdraw support as she was not a candidate for a lung transplant. Sequencing of the patient's TINF2 locus revealed a heterozygous mutation (c.844C > T, Arg282Cys) which has previously been reported in a subset of dyskeratosis congenita patients. Tissue sections from multiple organs showed degenerative changes including disorganized bone remodeling, diffuse alveolar damage and small vessel proliferation in the lung, and hyperkeratosis with hyperpigmentation of the skin. Autopsy samples revealed a bimodal distribution of telomere length, with telomeres from donor hematopoietic tissues being an age-appropriate length and those from patient tissues showing pathogenic shortening, with the shortest telomeres in lung, liver, and kidney. We report for the first time a survey of degenerative changes and telomere lengths in multiple organs in a patient with dyskeratosis congenita.

Background Graft-vs-host-disease (GvHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), limiting the use of this potentially curative treatment for hematological malignancies. To address this, we have developed T-allo10 cell therapy, which is enriched with type 1 regulatory T (Tr1) cells. Tr1 cells are peripherally inducible, CD49b+LAG3+IL-10+FOXP3- regulatory T cells that can confer alloantigen-specific tolerance, making them an attractive alternative to existing GvHD therapies, which non-discriminately impair both GvHD and protective immunity. T-allo10 cells are currently being evaluated in a phase I clinical trial in patients with hematological malignancies undergoing allo-HSCT (NCT03198234). Herein, we aimed to confirm that Tr1 cells are the active ingredient responsible for the T-allo10 suppressive function, and reveal the underlying molecular signatures to elucidate the mechanisms of Tr1 cell-mediated suppression. Methods T-allo10 cells were generated in a co-culture of healthy host or patient tolerogenic dendritic cells (DC-10) with allogeneic healthy donor CD4+ T cells, then tested for Tr1 phenotype, anergy, suppression and cytokine production. Sorted T-allo10-derived Tr1 cells and non-Tr1 cells, as well as control effector T cells (Teff) and parental CD4+ T cells, were analyzed by TCR- and RNA-seq. Protein expression for key differentially expressed genes were validated, and the functional roles for IL-10, CTLA-4 and PD-1 in T-allo10-mediated suppression were confirmed in a suppression assay. Results We show that the T-allo10 cell product is: i) enriched for Tr1 cells, ii) anergic in response to alloantigen re-challenge, but not to non-specific stimuli or 3rd party antigens, and iii) suppresses host-reactive T cells, but not T cell responses to other antigens. Furthermore, T-allo10-derived, isolated Tr1 cells had a restricted TCR repertoire, suggesting they clonally expand in response to alloantigens. T-allo10-derived Tr1 cells have a distinct signature compared to non-Tr1 cells, and, in addition to IL-10, express high levels of CTLA-4 and PD-1 (but not FOXP3). Notably, blockade of CTLA-4 and the PD-1 pathway completely abolishes T-allo10-mediated suppression of T cell responses. Conclusions Our data shows that Tr1 cells are the active, suppressive, and antigen-specific ingredient of T-allo10 cells. Furthermore, while the role of IL-10 in Tr1 cell-mediated suppression is well known, we uncover that Tr1 suppress in addition through CTLA-4 and PD-1. Collectively, these intriguing findings underscore the importance of CTLA-4 and PD-1 pathways in conferring cell-mediated immunological tolerance. Further, they define the key characteristics and modes of action of antigen-specific Tr1 cells, providing crucial information for the ongoing T-all10 trial and future design of novel Tr1 cell-based therapies. Ethics Approval The patient study was approved by Administrative Panels on Human Subjects in Medical Research, Stanford University, Tallo10 eProtocol # 38734. Healthy donor samples were purchased as deidentified human blood products from the Stanford Blood Center, and are thus exempt.

Successful hematopoietic cell transplantation (HCT) relies on conditioning of recipients to deplete hematopoietic stem cells (HSC) from marrow niches to allow healthy HSC to engraft. Currently, only genotoxic chemotherapy and/or radiation are used to achieve such niche clearance. We have developed a targeted non-genotoxic approach to deplete HSC using a monoclonal antibody, JSP191, that binds human CD117 (c-Kit), a receptor tyrosine kinase expressed on HSC and progenitor cells (HSPC). We have opened a phase 1 dose escalation trial using JSP191 as the sole conditioning agent in patients with SCID (ClinicalTrials.gov: NCT02963064). HCT is the only definitive cure for SCID, a uniformly lethal genetic immune disorder. Because infants with SCID lack functional T cells which can mediate transplant rejection and because of toxicity concerns, SCID infants may receive HCT without conditioning. This approach is associated with presumed engraftment of donor lymphoid progenitors but not HSC, resulting in incomplete and poorly sustained immune reconstitution, evidenced by naïve T cell production that wane over time and absent functional B cells requiring life-long immunoglobulin (Ig) replacement. Second donor HSC transplants ("boosts") without conditioning have not led to HSC engraftment and thus immune defects persist. We previously reported the initial safety, tolerability, and proof-of-concept of JSP191 as a safe and effective conditioning agent for SCID patients in whom an initial HCT failed to engraft HSC. Here, we present data supporting a recommended phase 2 dose (RP2D) of JSP191 that achieves donor HSC engraftment predictive of clinically meaningful lymphocyte reconstitution. Study endpoints include 1) donor chimerism of stringently flow-sorted CD15 + blood granulocytes ≥3% (18-24 wks post-HCT) as a measure of HSC engraftment, 2) lymphocyte reconstitution of donor-derived naïve CD4 +CD45RA + T cells >85/μL (36-104 wks post HCT), and 3) improved clinical outcomes, including reduced Ig dependence. Twelve patients with prior HCT (3 to 38 years old) of different genotypes (Table 1) were dosed in the initial dose escalation (0.1, 0.3 and 1.0 mg/kg JSP191) and at the RP2D 0.6 mg/kg. At study entry all patients lacked donor HSC engraftment evidenced by 0% donor CD15 + granulocyte chimerism. JSP191 was infused as a single IV dose and serum pharmacokinetics (PK) measured in real-time. CD34 +-selected cells obtained from the original donors were infused when the estimated JSP191 serum level fell below 500 ng/mL. JSP191 clearance displayed saturable non-linear kinetics, which was prolonged at the highest dose cohort of 1.0 mg/kg dose. JSP191 and HSPC infusions were uniformly well tolerated. Pre-and post-infusion marrow analyses in 7 evaluable patients showed a dose-dependent decline in CD117 +HSPC following JSP191 treatment (range 30-89%). Table 1a shows that 6 of 9 non-IL2RG SCID patients who are >24 wks post-HCT reached the predefined endpoint of HSC engraftment with >3% donor CD15 + blood granulocytes. This level of HSC chimerism correlated with the subsequent development of naïve donor derived T lymphocytes. Importantly, beyond HSC engraftment and naïve donor T lymphocyte production, patients who achieved HSC donor chimerism have demonstrated clinical improvement including resolution of chronic diarrhea, healthy weight gain, reduced Ig dependence, and antibody responses to vaccines. Three non-IL2RG patients did not achieve the chimerism threshold (one at 0.3 mg/kg and two at 1.0 mg/kg), potentially due to insufficient dose or prolonged JSP191 clearance and time to HSC infusion, respectively. Hence, an intermediate dose of 0.6 mg/kg which displays a shorter, more favorable half-life and higher HSC engraftment compared to the other dose cohorts was selected as the RP2D. Two of 3 IL2RG patients did not reach the donor chimerism threshold (Table 1b) reflecting the fact that re-transplanted IL2RG patients can have higher immune-mediated resistance, that may necessitate additional immune ablation. Conclusion: This study is the first to show the long-term benefit of HSC engraftment following targeted single-agent JSP191 conditioning. These results suggest JSP191 conditioning has the potential to provide benefit for HCT in a wide spectrum of disorders without the deleterious risks of genotoxic agents. This study is actively enrolling previously transplanted and newly diagnosed SCID patients. Figure 1 Figure 1. Disclosures Dvorak: Jazz Pharma: Consultancy; Alexion, Inc: Consultancy; Omeros Corp.: Consultancy. Prockop: Memorial Sloan Kettering Cancer Center: Other: S Prockop receives support for the conduct of sponsored clinical trials through MSK from Atara Biotherapeutics, Jasper and AlloVir. , Patents & Royalties: S Prockop is a co-inventor on intellectual property (IP) licensed to Atara. S Prockop has waived rights to this IP to MSK and has no personal financial interests in Atara. MSK has financial interests in Atara and IP interests relevant to this abstract. ; Atara Biotherapeutics: Other: support for the conduct of sponsored trials and Inventor; Jasper: Other: support for the conduct of sponsored trials; AlloVir: Other: support for the conduct of sponsored trials; Neovii: Consultancy; ADMA Biologics: Consultancy; MSK: Other: Inventor. Kwon: Jasper Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Long-Boyle: Jasper Therapeutics: Consultancy. Arulprakasam: Jasper Therapeutics: Current Employment. Harada: Jasper Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Prohaska: Jasper Therapeutics: Consultancy, Current holder of stock options in a privately-held company. Pang: Jasper Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Heller: NextCure: Consultancy, Current equity holder in publicly-traded company; Jasper Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Weissman: FortySeven: Patents & Royalties. Cowan: Homology Medicines, Inc: Consultancy, Current equity holder in publicly-traded company; Bluebird Bio, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Leadiant, Inc: Consultancy; Ensoma Inc.: Consultancy; Rocket Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Logan: Amgen, Pfizer, AbbVie: Consultancy; Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding. O'Reilly: Atara Biotherapeutics: Consultancy, Patents & Royalties: for EBV-specific T-Cell Bank, Research Funding. Parkman: Jasper Biotech: Consultancy. Shizuru: Jasper Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; FortySeven (Gilead): Patents & Royalties: Inventor listed on a patent, licensed by FortySeven.

Background: Fanconi anemia (FA) is a disorder of defective deoxyribonucleic acid (DNA) repair, progressive bone marrow failure (BMF), and a predisposition to hematologic malignancies and solid tumors. Approximately 60 to 70% of all cases result from a mutation in the Fanconi Anemia Complementation Group A (FANCA) gene (FA-A). 80% of FA patients experience BMF within the first decade of life. Allogeneic hematopoietic stem cell transplant (alloHSCT) is potentially curative for BMF; however, its efficacy is limited by human leukocyte antigen (HLA)-matched sibling donor availability and transplant-related toxicities. Lentiviral mediated gene therapy utilizing autologous FA-A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs) confers a proliferative advantage to gene-corrected HSPCs as demonstrated in preclinical studies and the FANCOLEN-I clinical trial conducted in Madrid, Spain. We report results from ongoing RP-L102 studies using "Process B" manufacturing optimizations including transduction enhancers, commercial grade vector, and modified cell processing. Methods: Patients (pts) with a FANCA gene mutation, age ≥1 year with no HLA-matched sibling donor and at least 30 CD34+ cells/µL in bone marrow (BM) are eligible. Peripheral blood (PB) mononuclear cells are collected via leukapheresis on 2 consecutive days after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. CD34+ HSPCs are enriched, transduced with a lentiviral vector encoding for the FANCA gene (PGK-FANCA-WPRE) and infused without cryopreservation or conditioning. Patients are followed for 3 years post-infusion for safety assessments (replication competent lentivirus [RCL], insertion site analysis [ISA]) and to ascertain evidence of efficacy (increasing PB and BM vector copy number [VCN] and mitomycin-C [MMC] resistance in BM colony forming units [CFUs]), along with stabilization/correction of cytopenias. Results: As of March 2021, 9 pts (aged 2 to 6 years) have received RP-L102. Evidence of engraftment has been identified in 6 pts with ≥6 months of follow up as indicated by PB VCN. 2 of 3 pts with follow up of ≥12 months have shown increased MMC resistance in BM CFUs. 1 pt developed BMF requiring alloHSCT after influenza B infection. 1 pt had a serious Grade 2 transient RP-L102 infusion-related reaction. Updated safety and efficacy data for pts with ≥12 months of follow-up will be presented. Conclusions: RP-L102's safety profile remains favorable. Increasing evidence of engraftment has been confirmed in 6 subjects as demonstrated by PB VCN; without conditioning, 12+ months of follow up is likely required to observe the proliferative advantage of transduced HSPCs. Disclosures Czechowicz: Editas Medicine: Current equity holder in publicly-traded company; Rocket Pharmaceuticals, Inc.: Research Funding; Global Blood Therapeutics: Current equity holder in publicly-traded company; Stemodontics: Consultancy, Current equity holder in publicly-traded company; GV: Consultancy, Current equity holder in publicly-traded company; Spotlight Therapeutics: Consultancy, Current equity holder in publicly-traded company; Beam Therapeutics: Consultancy, Current equity holder in publicly-traded company; Magenta Therapeutics: Current equity holder in publicly-traded company, Other: Intellectual property rights; Decibel Therapeutics: Current equity holder in publicly-traded company; Forty Seven Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences: Other: intellectual property rights; Jasper Therapeutics: Other: Intellectual property rights. Sevilla: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Sevilla is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, and may be entitled to receive financial benefits from the licensing of such patents., Patents & Royalties: J. Sevilla is a consultant and has licensed medical products from Rocket Pharmaceuticals, Inc. ; Amgen: Consultancy; Novartis: Consultancy; Miltenyi: Consultancy; SOBI: Consultancy. Booth: GSK: Honoraria; Orchard Therapeutics: Consultancy, Honoraria; SOBI: Consultancy, Honoraria; Takeda: Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Río: Rocket Pharmaceuticals: Current equity holder in publicly-traded company, Other: Paula Rio has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Research Funding. Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: Dr. Navarro has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Beard: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Wagner: Magenta Therapeutics: Consultancy; Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company; Vertex Pharmaceuticals: Consultancy; ASC Therapeutics: Consultancy; Bluebird Bio: Consultancy. Rao: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Schwartz: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Bueren is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, may be entitled to receive financial benefits from the licensing of such patents and receives funding for research., Patents & Royalties, Research Funding.

Solid organ transplantation (SOT) treats over 1500 children/year in the US. While short-term outcomes have improved, long-term outcomes still remain poor. With the rare exception of monozygotic twins as donors, recipients require lifelong immunosuppression with its accompanying toxicities, the risk of nonadherence, and chronic rejection. Further, up to half of recipients require a second transplant. We have pioneered translational approaches that abrogate rejection and the need for post-transplant immunosuppression. Previous attempts in adult patients utilizing allogeneic hematopoietic stem cell transplantation (HSCT) to eliminate the need for post-SOT immunosuppression in non-histocompatible kidney transplant (KT) recipients have failed(Lowsky R, BMT 2019). The establishment of mixed lymphoid chimerism has permitted a reduction in the number of immunosuppressive drugs needed, but not their discontinuation, whereas complete donor engraftment after HSCT has resulted in fatal graft-versus-host disease in some patients (Leventhal JR, Eur J Pediatr 2000). The use of HSCT to eliminate the need for immunosuppression following pediatric KT has not been evaluated. We have pioneered sequential haploidentical HSCT followed by KT in three patients with Schimke immuno-osseous dysplasia (SIOD) to abrogate kidney rejection and the need for post-transplant immunosuppression. SIOD is a monogenic disorder with progressive nephropathy correctable by KT and a T-cell immunodeficiency curable by HSCT. All three patients received αβT-cell/CD19 B-cell depleted HSCT (αβhaplo-HSCT) from a parent prior to a KT from the same parental donor. The pre-HSCT reduced intensity preparative regimen consisted of fludarabine (a starting dose of 1 mg/kg x 4 days, which was adjusted based on AUC), total body irradiation (TBI) 200 cGy, cyclophosphamide 1200 mg/m 2, anti-thymocyte globulin (ATG) thymoglobulin® 7.5 mg/kg, and rituximab 200 mg/m 2. Immunosuppressive drugs were not prophylactically administered after HSCT. After confirmation of full donor lymphoid chimerism post-HSCT, the patients received a living donor KT from their parental HSCT donor. Post-KT immunosuppression included intraoperative methylprednisolone and post-operative low-dose oral prednisone (0.5 mg/kg/day with taper) and tacrolimus (target serum level of 3-5 ng/ml) to reduce potential reperfusion inflammation. All immunosuppressive drugs were tapered off by Day +30 post-KT. To demonstrate functional tolerance after KT, Mixed Lymphocyte Cultures (MLC) were performed between peripheral blood mononuclear cells (PBMC) and patient/parent-derived irradiated EBV transformed lymphoblastoid cells lines (EBV-LCL) as stimulators (Figure 1). PBMC were isolated from SIOD patients (n=3) by Ficoll Hypaque separation and were labelled with Invitrogen™ CellTrace Violet Proliferation Kit (CTV). EBV-LCL were established from SIOD patients, parents and healthy donors. The assay was performed in round bottom microtiter plates with 100,000 labelled PBMC as responder cells (R) and 50,000 irradiated (30gy) EBV-LCL in RPMI medium with 10% fetal calf serum. Cells were harvested on Day 6, and T-cell proliferation determined by CTV expression of CD3+ T cells using a Novocyte Penteon flow cytometer (Agilent). Donor-derived T cells isolated from the peripheral blood of all three recipients > 1-year post-HSCT/KT, did not respond to the donor cells, while they did proliferate to the non-donor parental and control cells (Fig. 1). These results demonstrate that post-HSCT/KT the circulating donor-derived T cells are functionally tolerant to the transplanted kidney and, therefore, are unable to mediate graft rejection even in the absence of immune suppression. We have achieved two goals: first, ablating the recipient immune system before HSCT we safely established a new donor-derived immune system - that could also, if required, cure a patient's underlying disease- and, second, functional tolerance to the transplanted kidney. At 12 to 24 months after KT, all patients have full donor lymphoid and myeloid chimerism, normal renal function without immunosuppression, MLC demonstrated tolerance towards donor cells, and normal proliferative responses to non-donor parental cells. These findings suggest that the combination of αβhaplo-HSCT and SOT could be extended to other solid transplantation like liver and small intestine. Figure 1 Figure 1. Disclosures Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees. Shah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Parkman: Jasper Biotech: Consultancy.

Purpose/Objective(s) Late toxicity of treatment remains a major consideration among survivors of childhood leukemia. Pediatric patients with leukemia who receive fractionated total body irradiation (fTBI) as conditioning prior to stem cell transplantation (SCT) are frequently treated with a 4 Gy testicular boost in addition to fTBI to 12-13.2 Gy to reduce the risk of testicular relapse in this “sanctuary site.” However, total body irradiation can be associated with risk of impaired hormonal function; it is unclear if the 4 Gy testicular boost is needed to reduce risk of testicular relapse, and whether it contributes to the toxicity beyond that imparted by fTBI. This study investigated patients both with and without a boost for risk of recurrence, hormonal function, and fertility. Materials/Methods We retrospectively reviewed records of boys treated for leukemia with fTBI as part of conditioning for SCT at our institution from 1989-present. We included patients with both AML and ALL in our analysis. We excluded patients who died in the peri-transplant period. Cumulative incidence was tested using Gray's Test for Equality of Cumulative Incidence Functions. Endocrine outcomes were tested using chi square test. Results We identified 91 boys (age range 9 months – 22 years), 62 with ALL and 29 with AML. Median follow up was 43.4 months. In addition to fTBI to 12-13.2 Gy, 52 patients received a midplane testicular dose of 4 Gy (49 with ALL, 3 with AML), while 39 patients did not receive the testicular boost (13 with ALL, 26 with AML). There was no difference in time to first relapse or cumulative incidence of relapse between the boost and no-boost groups. There were 2 testicular relapses, one ALL patient who received boost and one AML patient who did not receive boost. The risk of abnormal luteinizing hormone (LH) was 65% in the boost group vs 26% in the non-boost group, P = 0.0063. Serum follicle-stimulating hormone, total testosterone, free testosterone, abnormal sperm studies did not differ between groups, nor did the percentage of patients who required endocrine or fertility treatments. We further investigated the subset of patients with ALL and similarly found no difference in risk of relapse between patients who had received a testicular boost and those who had not boost. Conclusion Omission of boost was associated with comparable risk of recurrence and improved endocrine outcomes, specifically LH levels. Our data suggest that omission of testicular boost in the fTBI program is oncologically safe and results in improved hormonal function.

Background 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. Objective 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. Design/Methods 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. Results 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). Conclusions 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.

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.

We describe 6 pediatric patients (12 to 18 y) with relapsed or refractory Hodgkin lymphoma treated with consolidative Brentuximab vedotin (Bv) following reinduction chemotherapy and autologous stem cell transplantation. The progression-free survival after autologous stem cell transplantation was 12, 18, 22, 24, 30, and 30 months. Most patients tolerated Bv well although 2 patients developed grade 3 neuropathy that prevent them from completing the scheduled 16 doses of Bv. Consolidative Bv in children and adolescents, as currently recommended for adult patients with early relapsed or refractory Hodgkin lymphoma, is feasible but with some significant toxicities.

We studied 1110 children, adolescents and young adults with β thalassemia major transplanted with grafts from HLA-matched relative (n=677; 61%), HLA-mismatched relative (n=78; 7%), HLA-matched unrelated (n=252; 23%) and HLA-mismatched unrelated (n=103; 9%) donors in 2000 - 2016. The median age at transplantation was 6 years (range Disclosures Dvorak: Jazz Pharmaceuticals: Consultancy; Chimerix, Inc.: Membership on an entity's Board of Directors or advisory committees. Shenoy:Novartis, Vertex, Bluebird Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees. Walters:Sangamo Therapeutics: Consultancy; bluebird bio: Research Funding; ViaCord Processing Lab: Other: Medical Director; AllCells Inc.: Other: Medical Director.

OBJECTIVES: Newborn screening for severe combined immunodeficiency (SCID) was instituted in California in 2010. In the ensuing 6.5 years, 3 252 156 infants in the state had DNA from dried blood spots assayed for T-cell receptor excision circles (TRECs). Abnormal TREC results were followed-up with liquid blood testing for T-cell abnormalities. We report the performance of the SCID screening program and the outcomes of infants who were identified. METHODS: Data that were reviewed and analyzed included demographics, nursery summaries, TREC and lymphocyte flow-cytometry values, and available follow-up, including clinical and genetic diagnoses, treatments, and outcomes. RESULTS: Infants with clinically significant T-cell lymphopenia (TCL) were successfully identified at a rate of 1 in 15 300 births. Of these, 50 cases of SCID, or 1 in 65 000 births (95% confidence interval 1 in 51 000–1 in 90 000) were found. Prompt treatment led to 94% survival. Infants with non-SCID TCL were also identified, diagnosed and managed, including 4 with complete DiGeorge syndrome who received thymus transplants. Although no cases of typical SCID are known to have been missed, 2 infants with delayed-onset leaky SCID had normal neonatal TREC screens but came to clinical attention at 7 and 23 months of age. CONCLUSIONS: Population-based TREC testing, although unable to detect immune defects in which T cells are present at birth, is effective for identifying SCID and clinically important TCL with high sensitivity and specificity. The experience in California supports the rapid, widespread adoption of SCID newborn screening.

Invasive fungal disease (IFD) remains a major cause of morbidity and mortality in pediatric patients after allogeneic hematopoietic stem cell transplant (HSCT). We analyzed the outcome of 152 consecutive pediatric patients who underwent allogeneic HSCT from 2005 to 2012: 126 of these without a history of IFD and 26 with IFD before HSCT. Antifungal prophylaxis agent was determined by the primary transplant attending. The rate of IFD after HSCT among patients with or without prior IFD was similar (7.7% with and 7.1% without a history of fungal disease before transplant). Mortality in these 2 populations did not differ (35% vs. 28%, P=0.48, χ). Patients deemed at higher risk for IFD were generally placed on voriconazole prophylaxis; however, this did not affect rates of posttransplant IFD. All-cause mortality in patients with posttransplant IFD was significantly higher than those without posttransplant IFD (67% vs. 21%, P

Successful hematopoietic stem cell transplantation (HSCT) requires vacating recipient hematopoietic stem cell (HSC) niches in the bone marrow to permit donor HSC engraftment that can provide life-long hematopoietic and immune function. Currently, HSCT in SCID relies on DNA damaging chemotherapy to eliminate recipient HSC and achieve niche clearance. We have pursued a non-toxic approach to target and deplete HSC using a humanized monoclonal antibody, JSP191, that binds human CD117 (c-Kit). We previously showed the safety and successful HSC engraftment in a Phase 1 trial of the first 6 patients with severe combined immunodeficiency (SCID), who underwent a second transplant because of HSC engraftment failure and poor immunity after their first transplantation. In these re-transplant patients even a low level of stringently measured myeloid chimerism resulted in significant and sustained generation of naive T cells and clinical improvement. Based on these results, the study of JSP191 (NCT#02963064)has opened a cohort of newly diagnosed infants with SCID. Here we report data from the first patient in this cohort, a SCIDX1 patient who received a primary HSCT with haploidentical CD34+ cells after conditioning with JSP 191. The patient had a c.270-15A>G variant in the IL2RG gene, which is predicted to cause a null phenotype. Besides a T- B+ NK- phenotype typical of SCIDX1 including dysfunctional B cells, the patient had anemia and intermittent neutropenia and thrombocytopenia. Despite evidence of maternal T cell engraftment, the patient had no clinical graft-versus-host disease (GVHD). The patient was initially enrolled in a trial of lentiviral gene therapy, but harvested bone marrow cells died in vitro during transduction and culture. The patient also mobilized poorly with G-CSF/Plerixafor. Further investigation revealed heterozygosity for loss-of-function mutations in two genes involved in DNA repair, BRCA1 and RAD51; Diepoxybutane (DEB) breakage study showed greater than normal pathologic chromosomal breaks, but less than that seen in Fanconi anemia. Because of concern for possible hypersensitivity to alkylating agent-based conditioning, the patient was referred for transplant with JSP191 conditioning. The patient received a CD34+ peripheral blood HSCT from his father after conditioning with 0.3 mg/kg of JSP 191 antibody intravenously over an hour on Day -8 and rATG (Thymoglobulin) on Day -5, -4, -3 and -2 (3.5 mg/kg total) to prevent rejection by the maternal T cells. The cryopreserved donor CD34+ cells were administered after sufficient clearance of the JSP191 serum level. The antibody infusion was well tolerated without toxicity, and the post-transplant course was uneventful without acute toxicities or GVHD. As a surrogate marker for HSC engraftment, CD15+ myeloid cells from peripheral blood were stringently sorted by flow cytometry and donor levels were quantified by short-tandem repeat (STR) analysis. Progressive levels of myeloid engraftment were observed beginning at Week 4. The level of donor chimerism at 12 weeks was 8% in the sorted CD15+ blood cells, and a marrow aspirate showed 25% donor CD34+ cells. By 3 months pre-existing abnormal CD19-CD20+ host B lymphocytes were significantly reduced, and CD19+ donor-derived B lymphocytes were emerging. At 2 months, CD4+ recent thymic emigrant and naïve T lymphocytes were observed, and by 3 months, overall T and NK lymphocyte numbers were 390/uL and 117/uL, respectively. Normal blastogenic responses to the T cell mitogen PHA were observed at 3 months. These first-in-class results provide proof of concept of the safety and efficacy of the use of JSP191 antibody to clear host marrow niche space to enable sufficient donor HSC engraftment and immune reconstitution as primary therapy of SCID. Non-genotoxic conditioning with JSP191 may replace conventional conditioning for newly diagnosed infants with SCID, thereby avoiding toxicities of chemotherapy. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. De Oliveira:Orchard Therapeutics: Research Funding; bluebird bio, Inc.: Research Funding. Czechowicz:Rocket Pharmaceuticals, Inc.: Research Funding. Brown:Merck: Membership on an entity's Board of Directors or advisory committees; Ansun: Membership on an entity's Board of Directors or advisory committees; Cidara: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Cellerant Therapeutics: Membership on an entity's Board of Directors or advisory committees. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

Background: Fanconi anemia (FA) is a rare inherited disorder of defective cellular deoxyribonucleic acid (DNA) repair, associated with developmental abnormalities and characterized by progressive bone marrow failure (BMF) and a predisposition to hematologic malignancies and solid tumors. Approximately 60-70% of all cases result from mutations in the Fanconi Anemia Complementation Group A (FANCA) gene (FA-A). 80% of FA patients develop BMF within the first decade of life. Although allogeneic hematopoietic stem cell transplant (allo-HSCT) is a potentially curative treatment for BMF, its utilization and efficacy are limited by availability of suitable human leukocyte antigen (HLA)-matched donors, risk of graft-versus-host disease (GVHD) and transplant-related toxicities. Ex-vivo lentiviral mediated gene therapy of autologous FA-A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs) has been shown to confer a survival advantage to gene-modified HSPCs in preclinical studies and, most recently, in the investigator initiated Phase 1/2 FANCOLEN-I clinical trial conducted in Madrid, Spain. Based on the highly favorable safety profile and promising preliminary efficacy data, global studies using "Process B" optimization including transduction enhancers, commercial-grade vector, and modified cell processing are underway. Herein, we report updated results from the US Phase 1 clinical trial and preliminary data from the global Phase 2 study in US and EU. Design and Methods: Subjects with a confirmed FANCA gene mutation aged 1 year or older, with no HLA-matched sibling donor and at least 30 CD34+ cells/µL in bone marrow (BM) were eligible for enrollment. Peripheral blood (PB) mononuclear cells were collected via leucocytapheresis on two consecutive days after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. CD34+ HSPCs were enriched, transduced with a lentiviral vector (PGK-FANCA-WPRE) and infused fresh (not cryopreserved) without any antecedent conditioning. Patients are being followed for 3 years post-infusion for safety assessments (replication competent lentivirus (RCL), insertion site analysis (ISA)) and to ascertain evidence of efficacy (increasing PB vector copy number (VCN) and BM mitomycin-C (MMC) resistance), along with stabilization/correction of cytopenias. Results: As of August 2020, 2 subjects (aged 5 and 6 years) have received RP-L102 infusion on the Phase 1 study with over 12 months of follow up. Preliminary evidence of gene marking in PB post-RP-L102 infusion at various timepoints has been observed in both subjects. Increased bone marrow (BM) mitomycin-C (MMC) resistance post treatment has also been identified in at least 1 subject. Subject L102-001-1001 has had blood count stabilization over the 12 months following gene therapy administration. Subject L102-001-1002's course has been complicated by influenza B infection with concomitant decreases in blood counts requiring red blood cell transfusions. Transfusion requirements have decreased following resolution of infection. Since November 2019, 5 additional subjects have been enrolled onto the global Phase 2 study and received investigational infusion. Updated preliminary safety and efficacy data including PB VCN, blood counts and BM MMC resistance will be available at the time of presentation for subjects with over 12 months of follow up; drug product (DP) information (VCN and CD34+ cell dose) will be available for all treated subjects. Conclusions: DP has been successfully manufactured in the Phase I (N=2) and Phase 2 (N=5) to meet the required specificationsSafety profile of RP-L102 continues to be highly favorable.Evidence of engraftment has been seen in at least 1 subject with follow up of at least 12 months as indicated by PB genetic markings and increasing BM CFC MMC resistance; 12+ months of follow-up may be required to observe the proliferative advantage of transduced HSPCs. Disclosures Czechowicz: Rocket Pharmaceuticals, Inc.: Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Río:Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: PR has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Wagner:BlueRock: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; Gadeta: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding.

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.

Introduction 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. Objectives 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. Methods 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. Results 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. Conclusions 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.

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.

Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become a curative treatment option for non-malignant diseases in children. Despite improvements in preparative regimens and support therapies, graft failure, graft-versus-host disease (GvHD) and infection are still a challenge. Historically, the risk of graft failure and GvHD have been described to be correlated with the presence of mixed chimerism, mainly in hemoglobinopathies and severe aplastic anemia. Objective Our aim is to describe the chimerism kinetic in whole blood and leukocyte subsets after allo-HSCT. Methods We evaluated 80 patients with non-malignant disorders transplanted between January 1999 to December 2016 at Lucile Packard Children's Hospital, Stanford, CA in whom the chimerism has been evaluated at least once between the engraftment and one year after alloHSCT. Results Median age at transplant was 6.9 years (0.3-21 years) and 53.7% were male. Underlying diseases were classified into five groups: hemoglobinopathies (HP, n = 13, 16.2%), severe aplastic anemia (SAA, n = 28, 35%), Fanconi anemia (FA, n = 10, 12.5%), metabolic disorders (MD, n = 11, 13.8%) and primary immunodeficiency (PID, n = 18, 22.5%). Bone marrow was the graft source in 73.7%. Unrelated donors were employed in 41 (51%) cases, 61% of whom with. Non-m, while 41% and 9% received a fully myeloablative and a reduced-intense regimen, respectively. Acute and chronic GvHD occurred in 38.7% and 28.7% of patients, respectively. Chimerism analysis were performed by short tandem repeat (STR) on DNA extracted from whole peripheral blood (WB) and CD3+, CD19+ and CD34+ leukocyte subsets, at 30 days (D+30), 100 days (D+100), 6 months and 1-year post-HSCT. was available in 71 patients: it was a mixed chimerism ( Conclusion Chimerism kinetic performed on sorted populations at different time points post allo-HSCT can predict outcomes in non-malignant disorders. Based on our preliminary results, D+30, D+100 and the chimerism on CD3+ are informative and should be monitored carefully in patients with non-malignant disorders.

Background Allogeneic HSCT is a well-established treatment for children with AL. For pts lacking a compatible matched related or unrelated donor, HLA-haplo-HSCT represents an alternative. Promising results were reported with selective depletion of αβ T and B cells (Locatelli, Blood 2017). PX-501 is an allogeneic product consisting of T cells modified to express the inducible caspase-9 (iC9) safety switch and truncated CD19 to allow monitoring and expansion of BPX-501 following transplant. BPX-501 provides broad virus and tumor-specific immunity; the safety switch provides the unique ability to promptly and durably resolve graft-versus-host disease (GvHD) symptoms following the administration of rimiducid. Aims Evaluate the safety and efficacy of BPX-501 in pediatric pts with AL by determining whether BPX-501 infusion can increase efficacy outcomes through an enhanced graft-versus-leukemic (GvL) effect, while maintaining a low risk of GvHD. Methods A subset of pts had high-risk ALs. BPX-501 was planned to be infused on day14±4 after the allograft with no post-transplant GvHD prophylaxis allowed. Pts who developed steroid-resistant GvHD could receive ≥1 dose of rimiducid. Results As of June 30, 2018, 100 pts with AL (described in Table 1) were efficacy evaluable. Median time for neutrophil and platelet engraftment was 16 and 12 days, respectively. Four pts (4.1%) experienced primary graft failure. Of 96 evaluable pts, 5 (3.1%) developed Grade III-IV aGvHD. Of 82 evaluable pts, 12 developed cGvHD (18.1%), with 3 moderate-severe. Rimiducid was administered to 10 pts. Best overall clinical response (CR/PR) post-rimiducid was 80% (8 pts). Among responding patients, 7 (87.5%) had a CR. Six (6.6%) pts died after transplantation. Efficacy outcomes in AL subsets are in Table 2. CD3+ and CD3+CD4+ T cells above 500 cells/ml were achieved by 180 and 270 days, respectively. IgA and IgM levels achieved normal values by 180 days. Conclusion BPX-501 following αβ-T and B-cell depleted haplo-HSCT represents a highly effective transplantation strategy for pediatric pts with AL. Rimiducid was an effective treatment for pts with steroid-resistant GvHD.

Hematopoietic stem-cell transplantation (HSCT) requires highly specialized, resource-intensive care. Myeloablative conditioning regimens used before HSCT generally require inpatient stays and are more intensive than other preparative regimens, and may therefore be more costly.To estimate the costs associated with inpatient HSCT according to the type of the conditioning regimen used and other potential contributors to the overall cost of the procedure.We used data from the Truven Health MarketScan insurance claims database to analyze healthcare costs for pediatric (age18 years) and adult (age ≥18 years) patients who had autologous or allogeneic inpatient HSCT between January 1, 2010, and September 23, 2013. We developed an algorithm to determine whether conditioning regimens were myeloablative or nonmyeloablative/reduced intensity.We identified a sample of 1562 patients who had inpatient HSCT during the study period for whom the transplant type and the conditioning regimen were determinable: 398 patients had myeloablative allogeneic HSCT; 195 patients had nonmyeloablative/reduced-intensity allogeneic HSCT; and 969 patients had myeloablative autologous HSCT. The median total healthcare cost at 100 days was $289,283 for the myeloablative allogeneic regimen cohort compared with $253,467 for the nonmyeloablative/reduced-intensity allogeneic regimen cohort, and $140,792 for the myeloablative autologous regimen cohort. The mean hospital length of stay for the index (first claim of) HSCT was 35.6 days in the myeloablative allogeneic regimen cohort, 26.6 days in the nonmyeloablative/reduced-intensity allogeneic cohort, and 21.8 days in the myeloablative autologous regimen cohort.Allogeneic HSCT was more expensive than autologous HSCT, regardless of the regimen used. Myeloablative conditioning regimens led to higher overall costs than nonmyeloablative/reduced-intensity regimens in the allogeneic HSCT cohort, indicating a greater cost burden associated with inpatient services for higher-intensity preparative conditioning regimens. Pediatric patients had higher costs than adult patients. Future research should involve validating the algorithm for identifying conditioning regimens using clinical data.

Purpose To evaluate survival and neurocognitive outcomes in pediatric acute lymphoblastic leukemia (ALL) patients with central nervous system (CNS) involvement treated according to an institutional protocol with stem cell transplantation (SCT) and a component of craniospinal irradiation (CSI) in addition to total-body irradiation (TBI) as preparative regimen. Methods and Materials Forty-one pediatric ALL patients underwent SCT with TBI and received additional cranial irradiation or CSI because of CNS leukemic involvement. Prospective neurocognitive testing was performed before and after SCT in a subset of patients. Cox regression models were used to determine associations of patient and disease characteristics and treatment methods with outcomes. Results All patients received a cranial radiation boost; median total cranial dose was 24 Gy. Eighteen patients (44%) received a spinal boost; median total spinal dose for these patients was 18 Gy. Five-year disease-free survival (DFS) for all patients was 67%. Those receiving CSI had a trend toward superior DFS compared with those receiving a cranial boost alone (hazard ratio 3.23, P=.14). Patients with isolated CNS disease before SCT had a trend toward superior DFS (hazard ratio 3.64, P=.11, 5-year DFS 74%) compared with those with combined CNS and bone marrow disease (5-year DFS 59%). Neurocognitive testing revealed a mean post-SCT overall intelligence quotient of 103.7 at 4.4 years. Relative deficiencies in processing speed and/or working memory were noted in 6 of 16 tested patients (38%). Pre- and post-SCT neurocognitive testing revealed no significant change in intelligence quotient (mean increase +4.7 points). At a mean of 12.5 years after transplant, 11 of 13 long-term survivors (85%) had completed at least some coursework at a 2- or 4-year college. Conclusion The addition of CSI to TBI before SCT in pediatric ALL with CNS involvement is effective and well-tolerated. Craniospinal irradiation plus TBI is worthy of further protocol investigation in children with CNS leukemia.

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

Successful hematopoietic cell transplantation (HCT) requires vacating recipient hematopoietic stem cell (HSC) niches to permit transplanted HSC to engraft. Currently, DNA damaging radiation or chemotherapy are used to eliminate recipient HSC and achieve niche clearance. We have pursued a non-genotoxic approach to target and deplete HSC using a humanized monoclonal antibody, AMG 191, that binds human CD117 (c-Kit), a receptor tyrosine kinase expressed on the surface of HSC and progenitor cells (HSPC). We have shown that AMG 191 suppresses human hematopoiesis in vitro, depletes human HSC in mice xenografted with human cells, and safely depletes HSC of non-human primates. We have initiated a Phase I dose escalation trial to test AMG 191 as the sole conditioning agent to achieve donor CD34-enriched HSPC engraftment in patients undergoing HCT for severe combined immunodeficiency (SCID) (ClinicalTrials.gov: NCT02963064). SCID is a severe genetic immune disorder curable only by HCT. Because of toxicity concerns, infants with SCID often receive donor hematopoietic grafts without conditioning, resulting in a lack of donor HSC engraftment. Instead, mature T lymphocytes and possibly lymphoid progenitors engraft but support only donor T cell development. This approach is associated with incomplete and poorly sustained immune reconstitution, and many patients have either no donor B cells and/or poor B cell function requiring life-long immunoglobulin replacement therapy. Second unconditioned donor HSC "boosts" can be performed, but they do not result in HSC engraftment and immune defects may persist. The primary endpoint of our study is to assess the safety and tolerability of AMG 191 as a conditioning agent in SCID patients. Secondary endpoints include AMG 191 pharmacokinetics (PK), host HSC depletion, and the determination of the dose of AMG 191 that achieves adequate donor HSC engraftment, defined as >5% donor blood granulocyte chimerism in peripheral blood at 24 weeks. Seven patients have been treated to date who are >12 weeks post-HCT (Table 1): three in each of the first two dose cohorts (0.1 and 0.3 mg/kg AMG 191), and one patient in the third cohort (1.0 mg/kg). An eighth patient has been treated at the 1.0 mg/kg dose and is three weeks post-HCT. Patients have a mixture of SCID genotypes. All patients treated to date had prior HCT with lack of donor HSC engraftment as evidenced by 0% donor sorted granulocyte chimerism at study entry. AMG 191 administration and infusion of original donor CD34+-selected cells were uniformly well tolerated. Pre- and post-infusion marrow analyses in five evaluable patients demonstrated dose-dependent decline in CD117+HSPC following AMG 191 treatment. Table 1 shows that four of six patients, who are >24 weeks post-HCT, reached the predefined endpoint of >5% granulocyte chimerism at 24 weeks, demonstrating donor HSC engraftment. The two patients who did not show donor engraftment at 24 weeks had detectable, low level (36 weeks show the production of recent thymic emigrants and/or de novo production of naïve T and/or B cells. In addition to improved lymphocyte values, patients have demonstrated clinical improvement including resolution of chronic diarrhea, significant weight gain, and reduced IVIG requirements. Conclusion: This study is the first demonstration of HSC engraftment following monoclonal antibody-based conditioning of patients without chemo(radio)therapy. Specifically, this first-in-human HCT trial shows that an anti-CD117 antibody safely clears HSC niches and facilitates donor HSPC engraftment in patients with SCID. Clinical benefit has been observed with minimal to no toxicity. Four of six evaluable patients have sustained evidence of donor myeloid engraftment along with T and B lymphopoiesis, indicative of engraftment of multipotent HSC. These results suggest that antibody conditioning for HCT may be preferable to traditional chemo(radio)therapy conditioning, especially in patients with non-malignant diseases and/or increased risk of toxicities due to such agents, such as certain forms of SCID, Fanconi anemia and sickle cell disease. Anti-CD117 antibody conditioning may also be applicable to gene therapy with genetically corrected autologous HSC. The AMG 191 study is actively enrolling previously transplanted SCID patients and newly diagnosed SCID patients. Disclosures Dvorak: Alexion Inc: Consultancy; Jazz Pharmaceuticals: Consultancy. Prohaska:Forty Seven Inc: Equity Ownership, Patents & Royalties. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Cowan:Rocket Pharma: Consultancy; Homology Medicine: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; California Institute Of Regenerative Medicine: Research Funding; UpToDate: Honoraria; Leadiant: Consultancy; NIH NIAD: Research Funding. Logan:Kadmon: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Research Funding; TeneoBio: Consultancy; Novartis: Consultancy; Astellas: Research Funding; Abbvie: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Kiadis: Consultancy; Jazz: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees. Weinberg:U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Shizuru:Forty Seven Inc: Equity Ownership, Patents & Royalties.

e19012 Background: High-dose therapy followed by autologous stem-cell transplantation (ASCT) is standard of care for patients with relapsed Hodgkin lymphoma (HL). Approximately 50% of relapsed HL patients are cured after ASCT. However, most patients with unfavorable risk factors (refractory disease, early relapse < 12 months and extranodal involvement prior to ASCT) progress after ASCT. Brentuximab Vedotin (BV) improves progression-free survival (PFS) when given as early consolidation after ASCT in adults. In pediatrics, the use of consolidative BV for relapsed HL has not been studied. Methods: Retrospective review of relapsed HL patients who received post ASCT consolidative BV from January 2016 to January 2017. We evaluated time of relapse, sites of involvement, presence of extranodal disease, clinical response after salvage therapy, response, PFS and BV toxicity. Results: During the study period 6 patients (age 12 -18 years) had relapsed HL. Of those, five had high risk (IIIB n = 1 , IVA n = 2, IVB n = 2) and one intermediate risk (IIA) disease. All had disease progression 0 – 3 months after completion of frontline therapy. High risk patients received ABVE-PC regimen and intermediate risk received Stanford V. Four patients received radiation therapy (RT) based on anatomical response as per protocol. Following relapse, 2 cycles of salvage Gemcitabine and Vinorelbine was given to 5 patients. Due to partial response, two of these patients received 2 additional cycles of BV-Bendamustine. One patient was re-induced with BV-Gemcitabine. FDG-PET/CT status prior to ASCT was negative for all 6 patients (Deauville score equal to or less than 3). High dose therapy prior to ASCT consisted of BEAM or BEC. Four patients received additional RT. All patients received consolidative BV every 3 weeks up to 16 cycles. Two patients experienced grade 3 motor and sensorial neuropathy and discontinued BV after 12 cycles. Two patients experienced grade 3 neutropenia. At present the PFS for the six patients is 12, 16, 23, 24, 28 and 30 months. Conclusions: Early consolidation with BV after ASCT is feasible in patients less than 18 years of age. Toxicity included grade 3 peripheral neuropathy and neutropenia. Larger studies are needed to determined safety and efficacy of consolidative BV post ASCT.

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 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.