ALBERT

Description: Background: Severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a rare disorder caused by ADA gene mutations, leading to lymphotoxic build-up of purine metabolites and profound immunodeficiency. Historically, enzyme replacement therapy (ERT) has been used as a bridge therapy until patients can receive an allogeneic hematopoietic stem cell transplantation (HSCT), ideally from a matched related donor (MRD) or, if none is identified, a non-matched and/or unrelated donor. We developed a self-inactivating lentiviral vector (LV), denoted EFS-ADA LV, encoding the human ADA cDNA sequence under the control of a shortened human elongation factor 1α gene promoter. A fresh or cryopreserved formulation of a drug product (OTL-101), composed of autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with EFS-ADA LV, was evaluated in 2 prospective, non-randomized Phase I/II clinical trials at 2 USA centers. We report on safety and efficacy of OTL-101 in 30 ADA-SCID pediatric gene therapy (GT) subjects treated from 2013-2017 with a median follow up (FU) of 24 months (mo; range 12-26 mo), compared to a historical cohort of 26 ADA-SCID patients treated with HSCT. Methods: UCLA Fresh Study (NCT01852071): Autologous CD34+ HSPCs were isolated from bone marrow and pre-stimulated with cytokines before transduction with EFS-ADA LV to yield OTL-101, which was infused as a fresh formulation in 20 subjects (9 male, 11 female; aged 4 mo-4.3 yrs). Single dose busulfan (4 mg/kg) was administered prior to infusion of OTL-101. Subjects were followed for 24 mo. UCLA Cryo Study (NCT02999984): 10 subjects (4 male, 6 female; aged 5-15 mo) received a cryopreserved formulation of OTL-101, which allowed for an extended shelf-life and full quality control prior to infusion. Busulfan was administered in 2 doses, the first at 3 mg/kg and the second adjusted to target a total area under the curve of 4,900 µM*min (20 ng/mL*hr). At the time of analysis, all subjects reached 12 mo FU (except 1 subject who was withdrawn from the study due to lack of engraftment); 7 subjects reached 18 mo of FU. Historical Control Group: 26 patients (aged 0.2 mo-9.8 yrs) were treated with allogeneic HSCT (MRDs n=12, non-MRDs n=14) at Great Ormond Street Hospital, UK (n=16) or Duke University Children's Hospital, USA (n=10) from 2000-2016. Results: Sustained engraftment of genetically modified HSPCs was observed in 29/30 GT subjects by 6-8 mo and persisted through FU in both studies, based on vector gene marking in granulocytes and CD3+ T cell reconstitution (Figure). Subjects who engrafted maintained long-term metabolic detoxification from deoxyadenosine nucleotides after stopping ERT approximately 1 mo post-GT. At last FU (median 24 mo; range 12-24 mo) in the GT group, overall survival (OS) was 30/30 (100%) and event-free survival (survival in the absence of ERT reinstitution or rescue allogeneic HSCT; EvFS) was 29/30 (97%). OS and EvFS were higher in the GT group at last FU compared with HSCT controls (with or without an MRD) at 2 years (Table). One of 30 OTL-101 subjects (3%) did not engraft and was restarted on ERT; the subject was withdrawn from the study at 5.9 mo and subsequently received a rescue HSCT, whereas 42% of HSCT patients required rescue HSCT, PEG-ADA ERT or died. Among the 20 OTL-101 subjects in the UCLA Fresh Study who reached 2 years FU, 18 (90%) stopped immunoglobin replacement therapy (IgRT), compared to 52% of HSCT patients. Preliminary results were observed in 5/7 (71%) OTL-101 subjects in the UCLA Cryo Study with more limited (18 mo) FU. Twelve OTL-101 subjects experienced one or more serious adverse events, most frequently infections and gastrointestinal events; only 1 of which was considered treatment-related (bacteremia due to product contamination). In the GT group, there were no events of autoimmunity with ≤24 mo FU. Due to the autologous nature of OTL-101, there was no incidence of graft vs host disease (GvHD); in contrast, 8 HSCT patients experienced GvHD events (5 acute, 3 chronic events), 1 of which resulted in death. Conclusions: Based on sustained gene correction and restoration of immune function in all subjects who engrafted, treatment of ADA-SCID with OTL-101 has a favorable benefit-risk profile. Key correlates of engraftment were consistent across the expanded cohort. Importantly, higher rates of OS and EvFS compared with HSCT (with or without an MRD) were observed. Disclosures Kohn: Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor on IP licensed from UC Regents to Orchard Therapeutics. Future royalties may occur., Research Funding; NIH: Research Funding. Shaw:Orchard Therapeutics: Consultancy, Other: Personal fees and non-financial support; NIH: Research Funding. Carbonaro-Sarracino:NIH: Other: Salary while working on project at UCLA 2013-2016, Research Funding; Orchard Therapeutics: Consultancy, Employment. De Oliveira:National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding. Terrazas:California Institute for Regenerative Medicine: Research Funding; Gene Therapy Resource Program, NHLBI/NIH: Research Funding. Hollis:Curative Therapeutics: Consultancy, Other: Personal fees. Trevisan:Orchard Therapeutics: Research Funding. Arduini:Orchard Therapeutics: Employment, Equity Ownership. Lynn:Orchard Therapeutics: Employment, Equity Ownership. Kudari:Orchard Therapeutics: Employment, Equity Ownership. Spezzi:Orchard Therapeutics: Employment, Equity Ownership. Buckley:Duke University: Research Funding. Booth:SOBI: Consultancy; GSK: Honoraria; NovImmune: Consultancy. Thrasher:Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: 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. Gaspar:Orchard Therapeutics: Employment, Equity Ownership, Patents & Royalties: Lentiviral vector for gene therapy of ADA-SCID.

Description: Key Points The genetic cause of SCID impacts on survival and immune reconstitution and should be considered in tailoring HCT for individual patients. Total and naive CD4+ cell counts in SCID patients 6 and 12 months post-HCT predict long-term survival and sustained immune reconstitution.

Description: Background: Previous studies evaluating the simplified Pulmonary Embolism Severity Index (sPESI) for predicting pulmonary embolism (PE) mortality did not consistently report the timing of vital sign measurement (systolic blood pressure [SBP], heart rate [HR] and oxygen [O2] saturation) relative to the PE presentation. Objectives: To evaluate the impact of vital sign measurement timing on sPESI's ability to identify PE patients at low-risk for in-hospital all-cause mortality. Methods: This was a retrospective analysis of PE patients from a large, urban teaching hospital in the Northeastern United States. Consecutive patients, diagnosed with PE between November 2010 and May 2015, were identified using the institution's billing system. To be eligible for inclusion, patients had an International Classification of Diseases, ninth-revision, clinical modification (ICD-9-CM) code of 415.1x in the primary position. Those in whom PE could not be objectively confirmed via chart review and those receiving thrombolysis or embolectomy were excluded. Patients' first and either lowest (SBP, O2 saturation) or highest (HR) value within the first 24 hours from presentation (subsequently referred to as "least favorable" values) were recorded. We then compared sensitivity, specificity and negative predictive values (NPV) and 95% confidence intervals (CIs) and the ability of the sPESI to predict all-cause in-hospital mortality using the first and least favorable vital signs. Results: A total of 562 PE patients (18.9% 〉80 years of age, 28.5% history of cardiopulmonary disease, 29.5% history of cancer) were included and 2.1% died in-hospital. No differences in sPESI's sensitivity, specificity or NPV were observed when scored using the first or least favorable vital sign values. sPESI classified 169 (30.1%) as low-risk (sPESI=0) vs. 153 (27.2%) when the least favorable vital sign value was used. Conclusions: The sensitivity and NPV of sPESI to predict PE patients' risk for all-cause in-hospital mortality is not affected by the timing of vital sign measurement. Using the least favorable value within 24-hours of presentation does result in a smaller proportion of patients being classified as low-risk. Table 1.CharacteristicFirst% (95%CI)Least Favorable% (95%CI)P-valueSensitivity91.7 (59.8-99.6)91.7% (59.8-99.6)〉0.99Specificity30.5 (26.8-34.6)27.6% (24.0-31.6)0.31NPV99.4 (96.2-99.9)99.3% (95.9-99.9)0.94Proportion classified as low-risk, n (%)169 (30.1)153 (27.2)

Description: Engineered adoptive immunotherapies have shown unprecedented activity in the treatment of cancer and chronic viral infections. Current approaches rely on individualized ex vivo genetic modification of autologous T cells due to the risk of graft-versus-host disease from allogeneic T cells. These processes furthermore require activation and prolonged expansion of T cells, which may reduce in vivo efficacy and persistence. Direct in vitro differentiation of engineered T cells from hematopoietic stem and progenitor cells (HSPCs) may overcome these problems by permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. Existing methods of in vitro human T cell differentiation are subject to wide experimental variability and do not adequately support the positive selection of immature T cell precursors to mature T cells, and thus have not been suitable for clinical-scale production of engineered T cells. We report here the preclinical development of an artificial thymic organoid (ATO) system using off-the-shelf, serum-free components and a standardized stromal cell line that supports highly efficient in vitro differentiation and positive selection of native and TCR-engineered human T cells from cord blood (CB), bone marrow, and mobilized peripheral blood CD34+ HSPCs, and purified CD34+CD38- hematopoietic stem cells. ATOs closely recapitulated thymic T cell commitment and differentiation, resulting in greater than 80% CD7+CD5+ T-lineage cells and 50% CD4+CD8+ double positive (DP) T cell precursors by 4 weeks. By 6 weeks, 30-40% of ATO cells were CD3+TCRαβ+ T cells, of which 20-30% were mature CD8 single positive (SP) T cells. CD4SP cells were generated at a lower frequency and later in culture (2-14% of CD3+TCRαβ+ cells). ATO-derived T cells exhibited a naïve CD45RA+CD27+CCR7+CD62L+ phenotype, a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. Transduction of CB CD34+ HSPCs with an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell output per ATO (〉400-fold, relative to input HSPCs) and enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. Positive selection of TCR-engineered naïve T cells could be further enhanced by expression of cognate HLA-A*02:01 in ATO stromal cells. ATO-derived TCR-engineered T cells exhibited a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. ATO-derived engineered T cells underwent antigen-specific cytotoxic priming, polyfunctional cytokine release, and proliferation in response to artificial APCs; and exhibited antigen-specific killing of NY-ESO-1+ tumor cells in vitro and in vivo. ATOs thus present a highly efficient off-the-shelf platform for the generation of clinically relevant numbers of naïve and potentially non-alloreactive engineered T cells for adoptive immunotherapy. Clinical translation of the ATO system will be aided by its simplicity, scalability, use of serum-free components, and compatibility with irradiated stromal cells. In addition, genetic manipulation of stem or stromal cell components can be easily incorporated into the system to further enhance downstream T cell engraftment or function. Disclosures Seet: Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property. Montel-Hagen:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property. Crooks:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property, Research Funding.

Description: Key Points Active infection pretransplant adversely impacts survival (81% in patients with active infection vs 95% in infection-free patients; P = .009). Preparative chemotherapy improved 1-year post-HCT median CD4 counts (P = .02) and freedom from IV immunoglobulin (P 〈 .001).

Description: Key Points IL2RG/JAK3-deficient B cells remain intrinsically defective posttransplant despite follicular helper T-cell reconstitution. In vitro response of B cells to IL-21 is a potential biomarker for humoral immunity in patients with IL2RG/JAK3 SCID after transplantation.

Description: Key Points Delivery of ZFNs and donor templates results in high levels of gene correction in human CD34+ cells from multiple sources, including SCD BM. Modified CD34+ cells are capable of engrafting immunocompromised NSG mice and produce cells from multiple lineages.

Description: Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising clinical strategy for the treatment of genetic diseases. Clinical trials are currently underway to treat hemoglobinopathies using a lentivirus expressing anti-sickling β‐ or γ-globin genes. However, clinical scale production of globin vectors has proven difficult due to the large size and complexity of the human β-globin gene expression cassette. Vector products often have low titers and require large manufacturing volumes to treat a single patient. We hypothesized that less vector per patient could be used by further purifying hematopoietic stem cells (HSC) beyond standard CD34+ selection prior to gene modification and transplantation. Here, we have optimized and characterized an immunomagnetic bead (IB) based cell sorting method to enrich CD34+CD38- cells from human bone marrow (BM). Our results suggest we can use clinically available technology (IB-based cell sorting) to achieve a ~10-fold reduction in vector requirements while still retaining the HSC required for clinical benefit after transplant. We first used competitive transplant studies to determine the SCID repopulating activity of different subpopulations of BM CD34+ cells after 2-day ex-vivo culture and LV transduction. The CD34+ BM cells were sorted into 3 intervals of increasing CD38 expression, each marked with a distinct fluorescent LV vector, and competitively transplanted into NSG mice. At 〉16 weeks post-transplant, 〉90% of all hCD45+hCD34+ cells in NSG marrow were derived from the lowest ~6% of CD38 negativity. We next optimized small-scale (50-100 mL of BM) enrichment of CD34+CD38- cells using an IB-based cell isolation method. CD38+ cells were first depleted, and CD34+38- cells were subsequently selected. This double-step CD34+CD38- purification non-specifically enriched granulocytes, which represented up to 50% of total isolated cells. Thus, we modified the first step of our protocol to include co-depletion of both CD15+ myeloid cells and CD38+ cells, which increased the purity of CD34+38- cells up to 2-fold. The dual IB-based CD34+CD38- purification and standard CD34+ purification were performed in parallel on 4 independent BM samples. Recovery of CD34+CD38- cells from starting material (MNCs) was assessed by each method. The single-step IB CD34+ purification recovered 91.8±6.5% (mean±SD) of CD34+CD38- cells while double-step IB CD34+CD38- purification recovered 72±1.4% of CD34+CD38- cells. An additional 20±6% of CD34+CD38- cells remained in the CD38+ fraction. When compared to single-step IB CD34+ purification, CD34+CD38- two-step purification reduced the total number of isolated cells by a range of 7.6-17.8-fold. We next compared the long-term engraftment capabilities of IB-purified CD34+ and CD34+CD38- cells transduced with a LV expressing mCitrine. CD34+ or CD34+CD38- cells purified from an equivalent volume of marrow were transplanted into NSG mice at limiting cell doses. Purified cells were transduced at the same cell density and vector concentration, with the CD34+CD38- cell grafts requiring 11.8-fold less LV than CD34+ cells. At 20 weeks, recipients of CD34+ or CD34+CD38- purified cells had similar levels of human chimerism indicating retention of engraftment capacity by the enriched CD34+CD38- cells. Additionally, hCD45+ cells in engrafted NSG mice exhibited similar levels of mCitrine expression (MFI), suggesting that IB enrichment of CD34+CD38- cells does not affect gene transfer into HSC. One potential disadvantage of using purified CD34+CD38- cells in a clinical setting is delayed myeloid recovery in the post-transplant period. NSG mice transplanted with purified CD34+CD38- cells demonstrated reduced circulating human myeloid cells at 3 weeks post-transplant as compared to mice transplanted with CD34+ cells. In order to overcome this potential clinical hurdle, we explored a strategy of adding back non-transduced CD38+ cells, which restored early levels of circulating human myeloid cells. We are currently investigating the effects of adding non-transduced CD38+ cells on long-term engraftment of gene-marked HSC. In summary, we have developed a clinically applicable method to enrich HSCs and reduce vector requirements. This strategy could directly contribute to clinical trials for hemoglobinopathies by addressing the factors currently limiting gene therapy methods. Disclosures No relevant conflicts of interest to declare.

Description: Background: Both the simplified Pulmonary Embolism Severity Index (sPESI) and the multivariable In-hospital Mortality for Pulmonary embolism using Claims daTa (IMPACT) rule classify patients' risk of early post-pulmonary embolism (PE) complications. Objective: To externally validate sPESI and IMPACT for predicting 90-day all-cause mortality and readmission rates among PE patients treated within the Veterans Health Administration (VHA). Methods: We used VHA data from 10/1/2010-9/30/2015 to identify adult patients with: (1) ≥1 inpatient diagnosis for acute PE (International Classification of Diseases-9th Revision-Clinical Modification codes=415.1x), (2) continuous medical and pharmacy enrollment for ≥12-months prior to the index PE (baseline period), (3) a minimum of 90-days of post-event follow-up or until death (whichever came first), and (4) ≥1 claim for an anticoagulant during the index PE stay. Patients were excluded if they had a claim for PE or an anticoagulant during the baseline period. We classified patients as low-risk for early post-PE complications if their sPESI score=0 or their absolute in-hospital mortality risk estimated by IMPACT was 90% and NPVs 〉96% for all-cause 90-day mortality, but low specificity and PPVs (Table). IMPACT's sensitivity for all-cause readmission was numerically higher than sPESI, but both had comparable NPVs. Similar trends were observed for accuracy in predicting readmissions due to recurrent VTE or major bleeding. Conclusion: In this external validation study utilizing VHA data, IMPACT classified patients for 90-day post-PE outcomes with similar accuracy as sPESI. While not recommended for prospective clinical decision-making, IMPACT appears useful for identification of PE patients at low-risk for early mortality or readmission in retrospective claims-based studies. Table. Test characteristics for sPESI and IMPACT for 90-day post-pulmonary embolism outcomes CI= confidence interval; IMPACT=In-hospital Mortality for Pulmonary embolism using Claims data; NPV=negative predictive value; PPV=positive predictive value; sPESI=simplified Pulmonary Embolism Severity Index; VTE=venous thromboembolism Table. Test characteristics for sPESI and IMPACT for 90-day post-pulmonary embolism outcomes CI= confidence interval; IMPACT=In-hospital Mortality for Pulmonary embolism using Claims data; NPV=negative predictive value; PPV=positive predictive value; sPESI=simplified Pulmonary Embolism Severity Index; VTE=venous thromboembolism Disclosures Kumar: Johnson & Johnson: Employment. Wells:Itreas: Other: Served on a Writing Committee; Janssen Pharmaceuticals: Consultancy; Bayer Healthcare: Other: Speaker Fees and Advisory Board; BMS/Pfizer: Research Funding. Peacock:Comprehensive Research Associates LLC: Equity Ownership; Cardiorentis: Consultancy, Research Funding; The Medicine's Company: Consultancy, Research Funding; Banyan: Research Funding; Emergencies in Medicine LLC: Equity Ownership; Abbott: Research Funding; Alere: Consultancy, Research Funding; Prevencio: Consultancy; Janssen: Consultancy, Research Funding; Portola: Consultancy, Research Funding; Pfizer: Research Funding; Roche: Research Funding; ZS Pharma: Consultancy, Research Funding; Ischemia Care: Consultancy; Phillips: Consultancy. Fermann:Janssen Pharmaceuticals: Other: Advisory Board, Speakers Bureau; Pfizer: Research Funding. Wang:Janssen Pharmaceuticals: Research Funding. Baser:Janssen Pharmaceuticals: Research Funding. Schein:Johnson & Johnson: Employment, Equity Ownership, Other: Own in excess of $10,000 of J&J stock. Crivera:Johnson & Johnson: Employment, Equity Ownership, Other: Owns excess of $10,000 in stock. Coleman:Boehringer-Ingelheim Pharmaceuticals, inc.: Consultancy, Research Funding; Bayer Pharmaceuticals AG: Consultancy, Research Funding; Janssen Pharmaceuticals: Consultancy, Research Funding.