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Human CD4+25− T cells cultured in interleukin 2 (IL-2), rapamycin, and transforming growth factor β (TGFβ) along with anti-CD3 monoclonal antibody–loaded artificial antigen-presenting cells generate FoxP3+ induced regulatory T cells (iTregs) with potent suppressive function. We performed a phase 1, single-center, dose-escalation study to determine the safety profile of iTregs in adults with high-risk malignancy treated with reduced-intensity conditioning and mobilized peripheral blood stem cells (PBSCs) from HLA-identical sibling donors. Sixteen patients were enrolled and 14 were treated (2 productions failed to meet desired doses). One patient each received 3.0 × 106/kg, 3.0 × 107/kg, and 3.0 × 108/kg iTregs with corresponding T-conventional-to-iTreg ratios of 86:1, 8:1, and 1:2. After 3 patients received 3.0 × 108/kg in the presence of cyclosporine (CSA) and mycophenolate mofetil (MMF) with no dose-limiting toxicities, subsequent patients were to receive iTregs in the presence of sirolimus/MMF that favors Foxp3 stability based on preclinical modeling. However, 2 of 2 developed grade 3 acute graft-versus-host disease (GVHD), resulting in suspension of the sirolimus/MMF. An additional 7 patients received 3.0 × 108/kg iTregs with CSA/MMF. In the 14 patients treated with iTregs and CSA/MMF, there were no severe infusional toxicities with all achieving neutrophil recovery (median, day 13). Of 10 patients who received 3.0 × 108/kg iTregs and CSA/MMF, 7 had no aGVHD, 2 had grade 2, and 1 had grade 3. Circulating Foxp3+ iTregs were detectable through day 14. In summary, iTregs in the context of CSA/MMF can be delivered safely at doses as high as 3 × 108/kg. This trial was registered at www.clinicaltrials.gov as #NCT01634217.

The final harvest or wash of a cell therapy product is an important step in manufacturing, as viable cell recovery is critical to the overall success of a cell therapy. Most harvest/wash approaches in the clinical lab involve centrifugation, which can lead to loss of cells and decreased viability of the final product. Here the authors report on a multi-center assessment of the LOVO Cell Processing System (Fresenius Kabi, Bad Homburg, Germany), a cell processing device that uses a spinning filtration membrane instead of centrifugation.Four National Institutes of Health Production Assistance for Cellular Therapies cell processing facilities (CPFs) assessed the LOVO Cell Processing System for final harvest and/or wash of the following three different cell products: activated T cells (ATCs), tumor-infiltrating lymphocytes (TILs) and bone marrow-derived mesenchymal stromal cells (MSCs). Each site compared their current in-house, routinely used method of final cell harvest and/or wash with that of the LOVO device.Final harvest and/or wash of ATCs, TILs and MSCs using the LOVO system resulted in satisfactory cell viability and recovery with some substantial improvement over the in-house methods of CPFs. Processing time was variable among cell types/facilities.The LOVO Cell Processing System provides an alternative to centrifuge-based technologies. The system employs a spinning membrane filter, exposing cells to minimal g-forces compared with centrifugation, and is automated and closed. This small multi-center study demonstrated the ability of the LOVO device to yield satisfactory cell viability and recovery of T cells and MSCs.

Background aims Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a "first-in-human" clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. Methods To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. Results aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. Discussion Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.

Background HSC dose and HLA match are risk factors that impact mortality using cord blood units (CBU) in transplant. Low CD34+ doses result in prolonged cytopenia and higher graft failure risk. A minimum cell dose of 3.0 × 107 total nucleated cells (TNC)/kg has generally been required in CBU selection; however, cell dose limits often require the use of a 2nd unit and markedly limits the availability of 7-8/8 HLA-matched units in larger patients. MGTA-456 is a cell therapy product utilizing an aryl hydrocarbon receptor antagonist for expansion of CD34+ HSCs in vitro. In prior studies with fresh MGTA-456, 36 patients with hematologic malignancies demonstrated rapid neutrophil recovery and 100% engraftment. This study (NCT03674411) will evaluate the safety and efficacy of cryopreserved MGTA-456 and the effectiveness of lowering the TNC threshold of the selected CBU to 1.0 × 107 TNC/kg before expansion to improve HLA match. Methods 13 patients aged 2-47 years (12-159 kg) with high-risk hematologic malignancy were enrolled with 11 transplanted to date. Patients received cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2 and total body irradiation 1320 cGy or a busulfan-based regimen for children ≤3 years of age prior to MGTA-456 with cyclosporine/mycophenolate mofetil prophylaxis. G-CSF began the day after infusion until the neutrophil count exceeded 2500/uL for 3 days. Results MGTA-456 contained a median 2.3 × 109 CD34+ cells (range, 0.65-8.0) after expansion (422-fold expansion of CD34+ cells [range, 219-1313]). Neutrophil recovery occurred in all patients with a median of 15 days (range, 0-31), similar to recipients of fresh MGTA-456 in a prior study (median 14 days) and significantly faster than in recipients of unmodified CBUs (median 25 days). Median platelet recovery was 41 days (range 24-49) vs 64 days with unmodified CBUs. MGTA-456 CD34+CD90+ content strongly correlated with speed of neutrophil recovery (Fig. 1), consistent with preclinical murine data showing CD34+CD90+ cells represent the true engraftable HSC population. Lowering the TNC requirement to 1.0 × 107 TNC/kg for CBU selection pre-expansion improved HLA match and/or eliminated the need for double CBU transplant in all 5 patients weighing >80 kg. Three patients received 8/8 grafts who would have otherwise received 1 or 2 6/8 units (n=2) or two 7/8 units (n=1) which may account for the low incidence of aGVHD overall (3 cases of Grade 1-2 and no Grade 3-4). With a follow-up of 5.2 months (0.4-8.5 months), all patients are alive. Conclusion Cryopreserved MGTA-456 cell therapy resulted in rapid and 100% engraftment with speed of neutrophil recovery correlating with CD34+CD90+ cell dose in patients with high risk hematologic malignancy. Expansion of smaller CBUs increases the chance of finding a better HLA match, particularly for adults, thus reducing the barriers associated with low cell dose and poor HLA match in CBU transplantation.

Clinical application of umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by low CD34+ cell dose, increased risk of graft failure and slow hematopoietic recovery. While the cell dose limitation is partially mitigated by using two UCB units, larger dosed single units would be preferable. We have evaluated the feasibility and safety of StemRegenin-1 (SR-1), an aryl hydrocarbon receptor antagonist that expands CD34+ cells, by placing one of the two units in expansion culture. SR-1 produced a 330-fold increase in CD34+ cells and led to engraftment in 17/17 patients at a median of 15 days for neutrophils and 49 days for platelets, significantly faster than in patients treated with unmanipulated UCB. Taken together, the marked expansion, absence of graft failure and enhanced hematopoietic recovery support testing of SR-1 expansion as a stand-alone graft and suggest it may ameliorate a limitation of UCB transplantation.

Background Patient access to well-matched cord blood (CB) containing high doses of stem cells remains a challenge for successful transplants. Low numbers of CD34+ cells in CB has resulted in delayed neutrophil recovery and increased risk of graft failure. MGTA-456 is a cell therapy that consists of CD34+ cells expanded in a 15-day culture in the presence of an aryl hydrocarbon receptor antagonist (AHRa) and the CD34 depleted fraction obtained from the same CB unit. To date, 41 patients with hematological malignancies (n=36) and non-malignant diseases (n=5) have received MGTA-456 with a median follow-up of 2.5 years (range 0.1 to 5 years) and 75 days (80 to 203 days), respectively. All patients engrafted at a significantly faster rate as compared to similarly treated historical controls (p Results After the selected CD34+ cells are cultured, the expanded product is ∼35% CD34+ and 65% CD34- (Figure 1). The CD34+ cells can be further fractionated into CD34+CD133-CD90- cells (late progenitors), CD34+CD133+CD90- cells (early progenitors) and CD34+CD133+CD90+ (progenitors and stem cells) as shown in Figure 1. The CD34- cells within the expanded product contained erythroid (CD71+) and megakaryocyte progenitors (CD41+), CD33+, CD14+, CD15+, and CD11b+ myeloid cells and CD56+ cells. CD3+, CD8+, CD4+, CD16+, CD19+ as well as CD10+ cells were not present ( Conclusion In these studies, we demonstrate that the expanded CD34+ cell fraction of MGTA-456 contains large numbers of CD34+CD90+ HSC, which are critical for long term engraftment in NSG mice and correlated with rapid neutrophil recovery clinically.

Background. HSC dose and HLA match are independent risk factors that impact non-relapse mortality in children and adults undergoing umbilical cord blood (UCB) transplant for acute leukemia (Eapen et al. Blood 2014 123:133-140). Low number of CD34+ HSCs results in prolonged periods of cytopenia and higher risk of graft failure. To reduce these risks, a minimum cell dose threshold, e.g. 3.0 x 107 total nucleated cells (TNC)/kilogram (kg), has generally been required in CBU selection. While beneficial in terms of hematopoietic recovery, this cell dose threshold markedly limits the number of available cord blood units (CBUs) particularly for larger adolescent and adult recipients, thus reducing the probability of identifying a 7-8/8 HLA-matched graft. In addition, a second UCB unit is often required for adults as a single unit may not meet the cell dose threshold. MGTA-456 is an expanded CD34+ HSC product utilizing an AHR antagonist in the presence of SCF, Flt-3L, IL-6 and TPO. In previous studies with fresh MGTA-456, 36 patients with hematologic malignancies demonstrated rapid neutrophil recovery and sustained engraftment in all patients. The aims of this study (NCT03674411) were to evaluate the safety and efficacy of cryopreserved MGTA-456 as well as the effectiveness of lowering the minimum cell dose threshold of the selected CBU from 3.0 x 107 to 1.0 x 107 TNC/kg to improve donor-recipient HLA match. Patients and Methods: Ten patients with high-risk hematologic malignancy were enrolled with 9 transplanted to date. Conditioning consisted of cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2 and total body irradiation 1320 cGy (total doses) with cyclosporine and mycophenolate mofetil as immunoprophylaxis. G-CSF was initiated on the day after infusion and continued until the neutrophil count exceeded 2500/uL for 3 consecutive days. Results: Cryopreserved MGTA-456 contained a median of 1.9 x 109 CD34+ cells (range, 1.1-6.2) after expansion culture (a 491-fold expansion of CD34+ cells [range, 219-672]). As shown in Table 1, neutrophil recovery occurred in 100% of patients (with one pending after recent transplant) at a median of 15 days (range, 0-31), similar to recipients of fresh MGTA-456 in a prior study (median 14 days, range 7-32) and significantly faster than in recipients of unmodified UCB (median 25 days). Platelet recovery (>20,000/uL for 7 days without transfusion) was also comparable in recipients of cryopreserved and fresh MGTA-456 (median 42 [range 27-53] vs 45 days [range 28-54], respectively), and again faster relative to recipients of unmodified CBUs (median 64 days). In line with preclinical experiments in NSG murine recipients that demonstrates all engrafting cells are retained in the CD34+CD90+ subpopulation, CD34+CD90+ content strongly correlated with speed of neutrophil recovery in recipients of MGTA-456 (cryopreserved and fresh) as shown in Figure 1. As expected, lowering the cell dose requirement from 3.0 x 107 to 1.0 x 107 TNC/kg for UCB unit selection prior to expansion culture improved HLA match and/or eliminated the need for double UCB transplant in 5 of 6 adults (Table 1). As a result, all but one patient received an 8/8 (n=5) and 7/8 (n=4) HLA matched UCB graft, potentially contributing to the low incidence of acute GVHD with only one patient of the 7 out >42 days having grade 2 acute GVHD. This low rate of GVHD compares favorably to that observed in the prior study of fresh MGTA-456. With a follow-up of 19-187 days (median 89), all patients are alive. Conclusion: Transplantation of cryopreserved MGTA-456 resulted in complete engraftment and rapid recovery with speed of neutrophil recovery correlating with the CD34+CD90+ cell dose. Based on the marked expansion that is now possible, units with fewer cells can now be considered, increasing the probability of finding a better HLA matched unit, particularly for adults. Availability of MGTA-456 could reduce the barriers associated with cell dose and poor HLA match previously limiting the successful use of UCB in transplantation. Disclosures Stefanski: Novartis: Consultancy, Speakers Bureau. Brunstein:Magenta: Research Funding; Gamida: Research Funding; Astex: Research Funding. McKenna:Icahn School of Medicine, New York, New York: Consultancy; CIBMTR BMT CTN (NIH): Other: Medical Monitor; National Eye Institute (NIH): Other: DSMB (2); Magenta Therapeutics: Research Funding; Gamida: Research Funding; NMDP: Other: Donor and Patient Safety Monitoring Advisory Group; Fate Therapeutics: Research Funding; Intima: Patents & Royalties: Royalities, Research Funding. Miller:Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees. Blazar:KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Raffel:Magenta Therapeutics: Employment, Equity Ownership. Davis:Magenta Therapeutics: Employment, Equity Ownership. Wagner:Rocket Pharmaceuticals: Consultancy; Magenta: Consultancy, Research Funding; BlueRock: Research Funding; Gadeta: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding.

We studied the safety and clinical outcomes of patients treated with umbilical cord blood (UCB)-derived regulatory T cells (Tregs) that expanded in cultures stimulated with K562 cells modified to express the high-affinity Fc receptor (CD64) and CD86, the natural ligand of CD28 (KT64/86). Eleven patients were treated with Treg doses from 3-100 × 10(6) Treg/kg. The median proportion of CD4(+)FoxP3(+)CD127(-) in the infused product was 87% (range, 78%-95%), and we observed no dose-limiting infusional adverse events. Clinical outcomes were compared with contemporary controls (n = 22) who received the same conditioning regimen with sirolimus and mycophenolate mofetil immune suppression. The incidence of grade II-IV acute graft-versus-host disease (GVHD) at 100 days was 9% (95% confidence interval [CI], 0-25) vs 45% (95% CI, 24-67) in controls (P = .05). Chronic GVHD at 1 year was zero in Tregs and 14% in controls. Hematopoietic recovery and chimerism, cumulative density of infections, nonrelapse mortality, relapse, and disease-free survival were similar in the Treg recipients and controls. KT64/86-expanded UCB Tregs were safe and resulted in low risk of acute GVHD.

Background aims Shipment of therapeutic somatic cells between a current good manufacturing practice (cGMP) facility and a clinic or between different cGMP facilities requires validated standard operating procedures (SOP). Under National Heart Lung & Blood Institute (NHLBI) sponsorship, the Production Assistance for Cellular Therapies (PACT) group conducted a validation study for the shipping SOP it has created, including shipments of cryopreserved somatic cells, fresh peripheral blood specimens and apheresis products. Methods Comparisons of pre- and post-shipped cells and cell products at the three participating facilities included measurements of viability, phenotypic profiles and cellular functions. The data were analyzed at the University of Pittsburgh Biostatistics Facility. Results No consistent shipping effects on cell viability, phenotype or functions were detected for cryopreserved and shipped peripheral blood mononuclear cells (PBMC), monocytes, immature dendritic cells (iDC), NK-92 or cytotoxic T cells (CTL). Cryopreserved mesenchymal stromal cells (MSC) had a significantly decreased viability after shipment, but this effect was in part because of inter-laboratory variability in the viable cell counts. Shipments of fresh peripheral blood and apheresis products for the generation of CTL and dendritic cells (DC), respectively, had no significant effects on cell product quality. MSC were successfully generated from fresh bone marrow samples shipped overnight. Conclusions This validation study provides a useful set of data for guiding shipments of therapeutic somatic cells in multi-institutional clinical trials.

This study was initiated to determine whether CD34(+) cell selection of small-volume bone marrow (BM) samples could be performed effectively on the Isolex(R) 300i Magnetic Cell Selection System device and whether the results obtained from these samples were comparable with results from large standard-volume samples. The impact on CD34(+) recovery using a full versus half vial of Isolex(R) CD34 reagent and the effects of shipping a post-selection product were evaluated.A protocol to evaluate CD34(+) cell selection with two ranges of smaller volume BM samples (c. 50 mL and c. 100 mL) was developed and instituted at three Production Assistance for Cellular Therapies (PACT) facilities. The study was performed in two phases.In phase I, the mean post-selection CD34(+) recoveries from the two sizes of samples were 104.1% and 103.3% (smallest and largest volumes, respectively), and mean CD34(+) recoveries were 115.6% and 88.7%, with full and half vials of reagent, respectively. Mean CD34(+) recoveries for post-shipment smaller volume samples were 106.8% and for larger volume samples 116.4%; mean CD34(+) recoveries were 99.9% and 127.4% for post-shipment samples processed with full and half vials of reagent, respectively. In phase II, mean CD34(+) recovery was 76.8% for post-selection samples and 74.0% for post-shipment samples.The results suggest that smaller volume BM sample processing on the Isolex(R) system is as efficient or more efficient compared with standard-volume sample processing. Post-processing mean CD34(+) recovery results obtained using a full or half vial of CD34 reagent were not significantly different.

Background. The use of umbilical cord blood (UCB) in transplant has been limited by the low number of CD34+ cells, resulting in prolonged periods of cytopenia for patients and high risk of graft failure, thereby restricting its widespread application. MGTA-456 is the hematopoietic cell product after cord blood CD34+ cells are placed in expansion culture for 15 days with an aryl hydrocarbon receptor (AHR) antagonist in the presence of SCF, Flt-3L, IL-6 and TPO. In a prior Phase 1/2 safety study, 18 patients received MGTA-456, its accompanying CD34negfraction and a larger, unexpanded UCB unit. All patients engrafted at a median of 14.5 days (range, 7-23), significantly faster than similarly treated historical controls (p Patients and Methods : Twenty patients with high-risk hematologic malignancy and a partially HLA-matched CBU were enrolled; 10 were treated with cyclophosphamide (CY) 120 mg/kg, fludarabine (FLU) 75 mg/m2 and total body irradiation (TBI) 1320 cGy (MAC) and 10 with CY 50 mg/kg, FLU 200 mg/m2 and TBI 200 cGy (NMAC). All patients received cyclosporine and mycophenolate mofetil post-transplant immunoprophylaxis. Expansion was low in 2 UCB units, therefore 18 patients received MGTA-456 and its CD34neg fraction. Results : Expansion culture yielded a median of 1,227 x 106 CD34+ cells (range, 201-8969) as compared to the input number of 4.2 x 106 (range, 1.4-16.3) after CD34 selection - a 324-fold (range, 42-1643) expansion of CD34+ cells. As transplant results vary by intensity of the conditioning, patient outcomes were compared to similarly treated historical cohorts between 2006 and 2015 (n=151 MAC; n=132 NMAC). For both groups, demographics were similar except for more recent year of transplant for recipients of MGTA-456. For recipients of MAC, MGTA-456 engrafted in all patients at a median of 14 days (range, 7-32) as compared to 89% engraftment at a median of 23 days (range, 19-31) in the control population (p200/uL was achieved at day 60 (median) in recipients of MGTA-456 regardless of conditioning regimen. Results were also encouraging for other transplant outcomes. For recipients of MGTA-456 compared to the historical cohort after MAC, incidence of acute GVHD (aGVHD) grade 3-4 was 22% vs 24%; chronic GVHD (cGVHD), 11% vs 21%; transplant-related mortality (TRM), 11% vs 34%; and overall survival (OS), 67% vs 55%. After NMAC, results were similar between cohorts except for a higher risk of aGVHD in recipients of MGTA-456 (aGVHD 3-4, 43% vs 15%; cGVHD, 0% vs 19%; TRM, 22% vs 20%; and OS, 44% vs 49%). The increased rate of aGVHD in the NMAC cohort likely reflects non-compliance with prescribed GVHD immunoprophylaxis in 2 of 9 recipients. Conclusion : In these studies, MGTA-456 significantly accelerated hematopoietic recovery and abrogated the engraftment barrier typically associated with UCB transplantation. The marked expansion of CD34+ cells in MGTA-456 suggests that a significant number of patients will have an adequate single CBU and better HLA matched graft since a greater proportion of the cord blood inventory will be available irrespective of weight. Given these promising results, additional studies are being planned. Disclosures Wagner: Novartis: Research Funding; Magenta Therapeutics: Research Funding. Brunstein: Novartis: Research Funding; Magenta Therapeutics: Research Funding. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McKenna: Magenta Therapeutics: Research Funding; Novartis: Research Funding. Sanna: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Bleul: Hoffmann-La Roche AG: Employment. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

BACKGROUND: Umbilical cord blood (UCB) is now a commonly used resource for hematopoietic stem cell (HSC) transplantation; great effort has been put forth in standardizing protocols for processing, storage, and testing of UCB units. Because UCB units are selected on an individual basis to maximize the chance of engraftment, loss of container integrity may have adverse effects on patient outcome. STUDY DESIGN AND METHODS: All bag breaks involving UCB units thawed for transplantation at our institution between January 1, 2000, and May 31, 2006, were identified. Information on various laboratory variables and the clinical consequences of UCB bag breaks was obtained from the deviation database of the Clinical Cell Therapy Laboratory (CCTL). Patient medical charts were reviewed for infusion-related data. RESULTS: The incidence of bag breaks over a 6½-year period was 3.5 percent. A majority of cases of loss of container integrity occurred in units that had been cryopreserved for more than 2 years (75%) and resulted in minimal loss of product. There were no significant decreases in quantity or quality of UCB, as determined by various quality control tests; no adverse clinical outcomes related to receiving a broken UCB unit were noted except increased antibiotic usage. CONCLUSION: There was a relatively low incidence of UCB bag breaks in this study that did not result in significant loss of UCB or adverse clinical outcomes. With the FDA considering licensure of UCB for hematopoietic reconstitution, improvement in container design and possibly guidelines limiting length of storage will likely be addressed in detail.

Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA.Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing.Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases.The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

Background Errors and accidents, or deviations from standard operating procedures, other policy, or regulations must be documented and reviewed, with corrective actions taken to assure quality peiformance in a cellular therapy laboratory. Though expectations and guidance for deviation management exist, a description of the framework for the development of such a program is lacking in the literature. Here we describe our deviation management program, which uses a Microsoft Access database and Microsoft Excel to analyze deviations and notable events, facilitating quality assurance (QA) functions and ongoing process improvement. Methods Data is stored in a Microsoft Access database with an assignment to one of six deviation type categories. Deviation events are evaluated for potential impact on patient and product, and impact scores for each are determined using a 0–4 grading scale. An immediate investigation occurs, and corrective actions are taken to prevent future similar events from taking place. Additionally, deviation data is collectively analyzed on a quarterly basis using Microsoft Excel, to identify recurring events or developing trends. Results Between January 1, 2001 and December 31,2001 over 2500 products were processed at our laboratory. During this time period, 335 deviations and notable events occurred, affecting 385 products and/or patients. Deviations within the ‘technical error’· category were most common (37%). Thirteen percent of deviations had a patient and/or a product impact score ≥ 2, a score indicating, at a minimum, potentially affected patient outcome or moderate effect upon product quality. Discussion Real-time analysis and quarterly review of deviations using our deviation management program allows for identification and correction of deviations. Monitoring of deviation trends allows for process improvement and overall successful functioning of the QA program in the cell therapy laboratory. Our deviation management program could serve as a model for other laboratories in need of such a program.

Background aims Current methods of mesenchymal stromal cell (MSC) cryopreservation result in variable post-thaw recovery and phenotypic changes caused by freezing. The objective of this investigation was to determine the influence of ex vivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing. Methods Human bone marrow aspirate was used. MSCs were isolated and cells were assessed for total count, viability, apoptosis and senescence over 6 passages (8–10 doublings/passage) in ex vivo culture. One half of cells harvested at each passage were re-plated for continued culture and the other half were frozen at 1°C/min in a controlled-rate freezer. Frozen samples were stored in liquid nitrogen, thawed and reassessed for total cell count, viability and senescence immediately and 48 h after thaw. Results Viability did not differ significantly between samples before freeze or after thaw. Senescence increased over time in pre-freeze culture and was significantly higher in one sample that had growth arrest both before freeze and after thaw. Freezing resulted in similar initial post-thaw recovery in all samples, but 48-h post-thaw growth arrest was observed in the sample with high senescence only. Conclusions High pre-freeze senescence appears to correlate with poor post-thaw function in MSC samples, but additional studies are necessary to obtain a sample sizes large enough to quantify results.

Preclinical data showed that priming CD34+ hematopoietic progenitor cells with complement fragment 3a (C3a) improved homing and engraftment. Thus, we hypothesized that priming of umbilical cord blood (UCB) hematopoietic progenitors with C3a would facilitate homing and could potentially be used to address the need for improved engraftment after UCB transplantation. We primed 1 of 2 UCB units for double UCB transplantation after nonmyeloablative conditioning. This design provided adequate safety and the potential to observe skewed long-term chimerism in favor of the C3a-primed unit as a surrogate measure of efficacy. C3a priming of 1 UCB unit did not result in infusional toxicity. Increased grades 1 to 3 hypertension were the only infusional adverse events observed in 9 (30%) patients. We observed no activation of inflammatory or coagulation pathways downstream of C3a. As tested, C3a priming did not impair engraftment, but did not skew chimerism toward the treated unit. As compared with historical controls, mortality and survival were not adversely affected. Thus, before any additional clinical studies, C3a priming to promote engraftment will require further preclinical optimization.

Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4 + CD25 + regulatory T cells (nT regs ) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nT regs has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nT regs could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nT regs in UCB units is limited, and average yield after expansion was only 1 × 10 9 nT regs . Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nT regs . PB nT regs were purified under GMP conditions and expanded 80-fold to yield 19 × 10 9 cells using anti-CD3 antibody–loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. A single restimulation increased expansion to ~3000-fold and yield to >600 × 10 9 cells while maintaining Foxp3 expression and suppressor function. nT reg expansion was ~50 million–fold when flow sort–purified nT regs were restimulated four times with aAPCs. Indeed, cryopreserved donor nT regs restimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune-deficient mice. The capability to efficiently produce donor cell banks of functional nT regs could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.

Background Despite enhanced tolerability of HLA mismatch, the reduced number of HSPC in an UCB graft limits the use of this stem cell source because of delayed hematopoietic recovery and increased risk of graft failure, particularly in adults. For this reason, we explored the effectiveness of SR1, an aryl hydrocarbon receptor antagonist, in the presence of cytokines to expand HSPC ex vivo prior to transplantation. Patients and Methods Nine patients with high-risk lympho-hematopoietic malignancy and two partially HLA (4-6/6)-matched UCB units were treated with cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2 and total body irradiation 1320 cGy followed by double UCB transplantation. Unit1 (the unmanipulated larger of the two units) was infused followed by SR1-expanded UCB HSPC (derived from CD34 selected Unit2 cells cultured for 15 days) as well as rethawed CD34 negative Unit2 cells. GVHD prophylaxis was cyclosporine A and mycophenolate mofetil. Results Culture in the presence of SR1 resulted in a median of 248-fold (range, 66-446) expansion of CD34+ cells. The median number of CD34+ cells and CD3+ cells infused for Unit1 and Unit2 were 0.3 x 106/kg (range, 0.2-0.9) and 11.0 x 106/kg (range, 1.4-48.9), and 7.3 x 106/kg (range, 4.6-10.6) 2.8 x 106/kg (range, 0.4-4.9), respectively. There were no infusional toxicities noted. Based on a presumed graft-graft interaction, the HSC835 product predominated in 5 of 9 patients and resulted in sustained hematopoiesis for a median follow up of 303 days (range 140-401). The median time to neutrophil recovery (days to absolute neutrophil count of ≥500/uL) was shorter in recipients of HSC835 (i.e., 16 days [range, 6-23] versus 24 days [range, 22-30]) with the speed of neutrophil recovery correlating with the number of CD34+ cells infused (r2 = -0.87, p Conclusion The AHR antagonist SR1 is a potent inhibitor of HSC differentiation, resulting in marked expansion of HSPC. Hematopoietic reconstitution as early as day 6 is dependent on CD34+ cell dose in the expanded product, HSC835. Based on the long-term engraftment potential of HSC835 in half of the patients using the double UCB platform and the accelerated neutrophil recovery seen in patients recovering with HSC835, future patients will receive HSC835 alone eliminating the confounding graft-graft effects and potentially further reducing the time to neutrophil recovery. Disclosures: Wagner: Novartis: Research Funding. Brunstein:Novartis: Research Funding. McKenna:Novartis: Research Funding. Sumstad:Novartis: Research Funding. Maahs:Novartis: Employment. Boitano:Novartis: Employment. Cooke:Novartis: Employment. Bleul:Novartis: Employment.

Abstract 1260 The optimal threshold for prophylactic platelet transfusion in hypoproliferative thrombocytopenic patients has been evaluated in multiple studies involving hematology and oncology patients. For patients without additional risk factors for bleeding, a trigger of 10 × 109/L is now accepted as safe and recommended in different guidelines. In the original studies, platelet transfusion above this trigger was allowed according to variable predetermined risk factors for bleeding. But, no studies have evaluated the clinicians' decision-making process leading to a change in the trigger for platelet transfusion. We report on the evaluation of trigger change for platelet transfusion following an initial threshold of 10 × 109/L and the relation with the presence of bleeding according to WHO grades and systems. Eighty-patients that were enrolled at University of Minnesota Medical Center for the previously published SPRINT Trial represent the patient population for the current analysis. Seventy-four patients (93%) had a starting trigger for platelet transfusion of 10 × 109/L and the majority (46/74; 62%) had an increase in their trigger during their hospitalization. Adherence to this starting trigger varied widely between treatment groups. Only a minority of patients treated with chemotherapy alone (3/12; 25%) and autologous transplant (6/15; 40%) had a change in their initial trigger of 10 × 109/L in contrast to the majority of allogeneic transplant patients (37/47; 79%) (p=0.001 compared to chemotherapy group and p=0.009 compared to autologous transplant group, respectively). The main reason reported by clinicians for a change after a starting trigger of 10 × 109/L was bleeding (32/46; 70%). However, trigger changes were not associated with more clinically significant bleeding. The occurrence of WHO grade 2–4 bleeding was very similar in patients with a trigger change and patients without a trigger change (51% and 54%; p=1.00). Even when limiting the analysis to trigger changes for a bleeding reason the overall distribution of the highest bleeding grades (WHO grade 0–4) leading to that change was similar to the highest bleeding grades of patients without a trigger change (p=0.205). Clinicians were influenced by the WHO organ system in which bleeding occurred when making their decision whether or not to raise the trigger for platelet transfusion. There was an overrepresentation of grade 1 mucocutaneous bleeding leading to an increase in the trigger (62% of the cases). And, there was an overrepresentation of grade 2 genitourinary bleeding not leading to an increase in the trigger (57% of cases). In conclusion, having a universal trigger of 10 × 109/L for platelet transfusion in hypoproliferative thrombocytopenic patients is probably not adequate because of the diversity of the patient population and their respective bleeding risk factors. Also, these multiple complicating factors for bleeding that lead to trigger changes need better validation to support clinicians in their decision-making process when addressing the optimal trigger for platelet transfusion. Otherwise, a seemingly valid trigger of 10 × 109/L becomes useless if misused or changed for any kind of reasons. Disclosures: No relevant conflicts of interest to declare.