Search

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.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade.Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade.CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo.CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy.This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

BACKGROUND: The CFU assay is considered the only in vitro assay that assesses the biologic function of hematopoietic stem and progenitor cells (HSPC). STUDY DESIGN AND METHODS: To investigate the impact of post-thaw CFU-GM counts on the quality of umbilical cord blood (UCB), we studied transplant outcomes in 269 patients receiving single UCB transplant. We also correlated the post-thaw CFU-GM counts of 1912 units with the pre-freeze and post-thaw graft characteristics, hoping to optimize selection criteria of UCB. Data analysis included: total nucleated cells, viability, CD34+, nucleated red blood cells (NRBC), hematocrit, frozen storage time, and cord blood bank (CBB). RESULTS: We demonstrated an association between post-thaw CFU-GM dose and the speed of neutrophil and platelet engraftment (p < 0.01). Higher post-thaw CFU-GM dose showed an increased benefit for neutrophil and platelet engraftment (p < 0.01). Post-thaw CD34+ cell dose and CFU-GM dose were strongly correlated (r = 0.78). However, CFU-GM dose showed additional benefit for patients receiving the lowest quartile of CD34+ dose. HLA disparity did not adversely impact either neutrophil or platelet engraftment. Post-thaw CFU-GM/million nucleated cells plated showed moderate correlation with pre-freeze and postthaw CD34+ and weak correlation with other parameters. Post-thaw CFU-GM was not influenced by storage time, but was impacted by the CBB from which the unit is obtained (p < 0.01). CONCLUSION: Post-thaw CFU-GM is an effective measure of the quality and efficacy of the UCB graft, particularly adding valuable clinical information when the CD34+ cell dose is low. Consideration of pre-freeze CD34+ cell content and CBB as additional selection criteria is warranted.

Background Neoantigen-specific T cells isolated from tumors have shown promise clinically but fail to consistently elicit durable tumor regression. Expression of the intracellular checkpoint CISH is elevated in human tumor infiltrating lymphocytes (TIL) and has been shown to inhibit neoantigen reactivity in murine TIL. Methods To explore CISH function in human T cells we developed a CRISPR/Cas9-based strategy to knockout (KO) CISH in human T cells with high-efficiency (>90%) and without detectable off-target editing. Results CISH KO in peripheral blood T cells enhanced proliferation, cytokine polyfunctionality, and cytotoxicity in vitro. To determine if CISH KO similarly enhances TIL function, we developed a clinical-scale, GMP-compliant manufacturing process for CISH disruption in primary human TIL. In process validation runs we achieved CISH KO efficiencies >90% without detectable off-target editing while maintaining high viability and expansion. Compared to WT controls, CISH KO in patient-derived TIL demonstrated increased proliferation, T cell receptor (TCR) avidity, neoantigen recognition, and unmasked reactivity to common p53 mutations. Hyperactivation in CISH KO TIL did not increase differentiation, suggesting that CISH KO may uncouple activation and differentiation pathways. Single cell profiling identifies a pattern of CISH expression inverse to key regulators of activation, and CISH KO in human TIL increases PD1 expression. Adoptive transfer of Cish KO T cells synergistically combines with PD1 inhibition resulting in durable tumor regression in mice, highlighting orthogonal dual cell surface and intracellular checkpoint inhibition as a novel combinatorial approach for T cell immunotherapy. Conclusions These pre-clinical data offer new insight into neoantigen recognition and serve as the basis for a recently initiated human clinical trial at the University of Minnesota (NCT04426669) evaluating inhibition of the novel intracellular immune checkpoint CISH in a CRISPR-engineered, neoantigen-specific T cell therapy for solid tumors. Updates from the clinical trial will be highlighted. Trial Registration NCT04426669

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

BACKGROUND Mesenchymal stem cells (MSCs) continue to be investigated in multiple clinical trials as potential therapy for different disorders. There is ongoing controversy surrounding the clinical use of cryopreserved versus fresh MSCs. However, little is known about how cryopreservation affects marrow as starting material. The growth kinetics of MSC cultures derived from fresh versus cryopreserved marrow were compared. STUDY DESIGN AND METHODS Data were reviewed on the growth kinetics of MSCs derived from fresh versus cryopreserved marrow of nine donors. Marrow harvested from each donor was separated into four aliquots (one fresh and three cryopreserved for culture). Data on the date of mononuclear cell cryopreservation/thaw, MSC counts at Passages 1 and 2, MSC doubling, MSC fold expansion, viability (of mononuclear cells and final MSCs), and on flow cytometry markers of mononuclear cells and final MSCs were analyzed for the fresh and cryopreserved marrow groups. RESULTS In total, 21 MSC lots (seven fresh and 14 cryopreserved) were obtained. The average age of cryopreserved mononuclear cell product was 295 days (range, 18-1241 days). There were no significant differences between MSC numbers at Passage 1 (p = 0.1), final MSC numbers (p = 0.5), MSC doubling (p = 0.7), or MSC fold expansion (p = 0.7). A significant difference was observed in viability by flow cytometry for both mononuclear cells (p = 0.002) and final MSCs (p = 0.009), with higher viability in the fresh marrow group. CONCLUSION This study demonstrates that MSCs derived from cryopreserved marrow have the same growth characteristics as fresh marrow–derived MSCs. Further studies are needed to explore potential differences in clinical efficacy.

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 Allogeneic natural killer (NK) cell adoptive immunotherapy is a growing therapeutic option for patients. Clinical-scale production of NK cells using immunomagnetic selection complies with current good manufacturing practices (cGMPs) and allows for closed-system, automated purification. We report our experience with CD3/CD19 cell-depleted (CD3/CD19dep ) NK cell production and compare to previous methods of CD3 cell depletion and CD3 cell depletion/CD56 cell enrichment. Study design and methods Nonmobilized mononuclear cells collected by apheresis were incubated with anti-CD3/anti-CD19 microbeads and depleted in an automated cell selection system (CliniMACS, Miltenyi). The NK cell-enriched products were incubated overnight in interleukin (IL)-2 or IL-15, washed, and resuspended prior to lot release testing and infusion. Results Since 2010, 94 freshly infusible CD3/CD19dep NK cell products were manufactured in support of eight clinical trials. Sixty-six products were incubated in IL-2 and 28 products in IL-15. Processing resulted in a mean NK cell recovery of 74% and viability of 95.8%; NK cells, T cells, B cells, and monocytes accounted for 47%, 0.2%, 0.08%, and 49% of the final products, respectively. Seven products required dose adjustments to meet lot release. The specification for purity changed throughout the evolution of manufacturing. IL-2 or IL-15 activation enhanced in vitro cytotoxicity compared to preactivated cells. There was no difference in final product composition or cytotoxicity between cytokine cohorts. Conclusion Clinical-scale/cGMP production of NK cells using CD3/CD19 cell-depletion effectively minimized T-cell and B-cell contamination in a single manipulation without compromise to NK-cell recovery. Cytokine activation increased in vitro cytotoxicity compared to column-depleted, preactivated NK cells.

Mesenchymal stem cells (MSCs) continue to be investigated in multiple clinical trials as potential therapy for different disorders. There is ongoing controversy surrounding the clinical use of cryopreserved versus fresh MSCs. However, little is known about how cryopreservation affects marrow as starting material. The growth kinetics of MSC cultures derived from fresh versus cryopreserved marrow were compared.Data were reviewed on the growth kinetics of MSCs derived from fresh versus cryopreserved marrow of nine donors. Marrow harvested from each donor was separated into four aliquots (one fresh and three cryopreserved for culture). Data on the date of mononuclear cell cryopreservation/thaw, MSC counts at Passages 1 and 2, MSC doubling, MSC fold expansion, viability (of mononuclear cells and final MSCs), and on flow cytometry markers of mononuclear cells and final MSCs were analyzed for the fresh and cryopreserved marrow groups.In total, 21 MSC lots (seven fresh and 14 cryopreserved) were obtained. The average age of cryopreserved mononuclear cell product was 295 days (range, 18-1241 days). There were no significant differences between MSC numbers at Passage 1 (p = 0.1), final MSC numbers (p = 0.5), MSC doubling (p = 0.7), or MSC fold expansion (p = 0.7). A significant difference was observed in viability by flow cytometry for both mononuclear cells (p = 0.002) and final MSCs (p = 0.009), with higher viability in the fresh marrow group.This study demonstrates that MSCs derived from cryopreserved marrow have the same growth characteristics as fresh marrow-derived MSCs. Further studies are needed to explore potential differences in clinical efficacy.

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: Interlaboratory scoring performances were determined using a traditional 14-day colony-forming unit (CFU) assay and a new 7-day CFU assay. STUDY DESIGN AND METHODS: Digital images of colonies were utilized to train personnel at each site. A central laboratory inoculated methylcellulose with progenitors and sent the samples by overnight courier to participating labs for plating. RESULTS: Colony counts from two digital images showed greater variability by novice counters (coefficients of variation [CV], 18.5 and 23.0%; n = 8) than for experienced staff (CV, 7.3 and 4.8%; n = 5). CFU assays plated immediately, 24 and 48 hours after methylcellulose inoculation displayed 39.5 CFU, 37.1 ± 10.6 (CV, 28%) and 34.8 ± 8.5 (CV, 24%) colonies for the 7-day assay and 39.5 CFU, 39.1 ± 9.9 (CV, 25%) and 37.1 ± 10.6 (CV, 28%) colonies for the 14-day assay, respectively. Overall, no significant differences in colony counts were noted between assays (p = 0.68). Also, no differences in CFU counts were seen when assays were set up immediately, 24 and 48 hours after methylcellulose inoculation (14-day p = 0.695; 7-day p = 0.632). CONCLUSION: Total CFUs obtained in 7- and 14-day CFU assays are comparable and show similar levels of interlaboratory variability. The major source of this variability is due to differences in how CFU plates are scored by individuals at different sites. UCB progenitor cells can be maintained in methylcellulose-based media at room temperature for up to 48 hours prior to transport without a significant loss in CFUs.

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.

Background: Patient access to well-matched 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 a risk of graft failure relative to other hematopoietic stem cell (HSC) sources. 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. Thus far, 40 patients with hematological malignancy (n=36) and non-malignant diseases (n=4) have received MGTA-456 with a median follow-up of 2.5 years (range 0.1 to 5 years) and 75 days (20 to 143 days) respectively. All patients engrafted at a significantly faster rate as compared to similarly treated historical controls (p Methods: The expansion culture consisted of StemSpan SFEM supplemented with SCF, FLT- 3L, TPO and IL-6 (all at 50 ng/mL) and the AHRa without the addition of antibiotics. After a 10 or 15 day culture, the CD34 expanded product was characterized by cell surface markers (CD34, CD90, CD133, CD41, CD71, CD235a, CD3, CD4, CD8, CD14, CD15, CD16, CD11b, CD33, CD19, CD56, and CD10), as well as colony-forming unit (CFU) capacity and engraftment potential in NOD-scid IL2Rgammanull (NSG) mice in a subset of products. Results: The expanded CD34+ cell fraction of MGTA-456 consisted of 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 fraction 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 doses of CD34+CD90+ HSC and progenitors, which are critical for long term engraftment in NSG mice and correlated with rapid neutrophil recovery clinically. Disclosures Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding.

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.

Natural killer (NK) cells represent a lineage of immune cells capable of direct cytotoxicity against tumor cells and are a critical source of key inflammatory cytokines such as interferon (IFN)-γ and tumor necrosis factor (TNF). NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system. The unique biological attributes of NK cells, including multifaceted effector function, tumor cell recognition independent of antigen presentation and target cell selectivity independent of HLA-matching, has enabled NK cells from a donor to be adoptively transferred to a patient for the treatment of cancer. This safe and effective administration of donor NK cells to patients validates their potential for broad use as part of an off-the-shelf cancer immunotherapy strategy, including in combination with monoclonal antibody and checkpoint inhibitor therapies. We have previously shown that human induced pluripotent stem cells (hiPSC) can be clonally-selected, cryopreserved and banked, and that these master pluripotent cell lines (MPCLs) can be used to renewably generate large clonal populations of NK cells. The use of MPCLs represents a highly-promising, off-the-shelf approach to cell-based cancer immunotherapy, with the potential to overcome many of the challenges and limitations of patient-sourced and donor-derived cell therapies. However, to clinically and commercially enable this off-the-shelf strategy, it is essential to efficiently and reproducibly differentiate MPCLs to fully-functional NK cells using a robust and scalable process that meets regulatory requirements. Here we describe a novel paradigm for the manufacture of hiPSC-derived NK (iNK) cells consisting of a well-defined, small molecule-driven, staged protocol that enables clinical translation and is compatible with current good manufacturing practice (cGMP) requirements. The manufacturing protocol is currently being transferred from the laboratories of Fate Therapeutics to Molecular and Cellular Therapeutics at the University of Minnesota, which is a state-of-the-art GMP/GTP compliant, full-service developer and manufacturer of cell- and tissue-based products. iNK cell therapy manufacture consists of four unique steps including: 1) the derivation and master cell banking of a clonal pluripotent cell line (> 95% SSEA4+/TRA181+ hiPSCs); 2) differentiation of the clonal pluripotent cell line towards hematopoietic progenitor cells (enriched for > 80% CD34+ cells); 3) differentiation and expansion of iNKs (Figure 1A, approximately 1,000-fold expansion in 14 days); and 4) freeze and thaw of drug product, comprised of a sufficiently pure homogenous population of iNKs (Figure 1B, > 95% CD45+, > 90% CD56+, minimal CD3+ T cells). Importantly, testing at both the molecular and culture stages demonstrate that no hiPSCs exist in the final drug product (limit of detection 1 hiPSC in 1.25 million iNK cells). This novel manufacturing paradigm supports the generation of significant numbers of iNK cells: approximately 1 million-fold cell expansion is achieved in less than 50 days, such that a very small population of hiPSCs can readily produce 1x1012 iNK cells. We estimate that this represents hundreds of doses of drug product per each manufacturing run (Figure 1A). The iNK cells display markedly augmented effector function relative to ex vivo expanded primary peripheral blood or cord blood NK cells with respect to cytokine release (IFN-γ and TNF) and cellular cytotoxicity against various leukemic and solid tumor-derived target cells including K562, Raji, A549 and SKOV3 (Figure 1C). To enable centralized manufacturing, we established a freeze and thaw strategy that supports greater than 85% viability with a recovery of greater than 80% iNK cells at twenty-four hours post-thaw. Because the freeze process uses an infusible medium formulation, we demonstrated in vitro and in vivo that the iNK cells maintain their efficacy post-thaw and can be immediately infused into patients. The manufacturing data presented herein support the filing of an Investigational New Drug application for an off-the-shelf iNK cell therapy product to treat advanced hematologic and solid tumor malignancies alone or in combination with monoclonal antibody and checkpoint inhibitor therapies. Disclosures Bjordahl: Fate Therapeutics: Employment, Equity Ownership. Gaidarova: Fate Therapeutics Inc.: Employment, Equity Ownership. Rogers: Fate Therapeutics Inc.: Employment, Equity Ownership. Clarke: Fate Therapeutics Inc.: Employment, Equity Ownership. Groff: Fate Therapeutics Inc.: Employment. Moreno: Fate Therapeutics Inc.: Employment. Abujarour: Fate Therapeutics Inc.: Employment. Robinson: Fate Therapeutics Inc.: Employment. Bonello: Fate Therapeutics Inc.: Employment. Lee: Fate Therapeutics Inc.: Employment, Equity Ownership. Lan: Fate Therapeutics Inc.: Employment, Equity Ownership. Rezner: Fate Therapeutics, Inc.: Employment. Abbot: Fate Therapeutics Inc.: Employment. Wolchko: Fate Therapeutics Inc.: Employment. Kaufman: Fate Therapeutics: Consultancy, Research Funding. Valamehr: Fate Therapeutics: Employment, Equity Ownership. Miller: Oxis Biotech: Consultancy; Celegene: Consultancy; Fate Therapeutics: Consultancy, Research Funding.

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. The use of UCB has been substantially limited by the low finite number of CD34+ cells, resulting in prolonged periods of cytopenia and high risk of graft failure, restricting its widespread application particularly for adults. SR1 is an aryl hydrocarbon receptor antagonist that enables CD34 cell proliferation in the presence of SCF, Flt-3L, IL-6 and TPO without differentiation. Therefore, we tested the safety and efficacy SR1 expanded UCB (referred to as ‘HSC835’). Patients and Methods: Patients with high-risk hematologic malignancy were enrolled in a Phase 1/2 dose escalation study. All were treated with cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2 and total body irradiation 1320 cGy with cyclosporine and mycophenolate mofetil post-transplant immune suppression. Seventeen patients received HSC835 along with its CD34 depleted fraction and an unmanipulated UCB unit and two patients thus far received HSC835 as a stand-alone graft. Results: For the first 17, SR-1 expansion culture yielded a median of 1,440 x 106 CD34+ cells (range, 140.2-6361.9) as compared to the input number of 4.4 x 106 (range, 2.1-15.6) after CD34 selection – a 328-fold (range, 65.9-844.0) expansion of CD34+ cells. Relative to recipients of a standard double UCB graft without manipulation (n=111) containing a median of 0.5 x 106 CD34+ cells/kg (0.1-2.1) with 87% engraftment, HSC835-treated patients received a median of 12.3 x 106 CD34+ cells/kg (2.3-48.5) with 100% engraftment. Of the 11/17 patients in whom the HSC835 predominated, neutrophils recovered at a median of 11 days (6-23) as compared to 23 days (14-30) for those in whom the unmanipulated unit predominated (Figure). Similarly, as compared to the 111 double UCBT controls, both neutrophil and platelet recoveries were remarkably shortened in HSC835 recipients (p Conclusion: HSC835 dramatically accelerates hematopoietic recovery, abrogating the principal barrier to the successful use of UCB. Such impressive CD34+ expansion potentially heralds a paradigm shift for both UCB transplantation and cord blood banking. With such high CD34+ cell doses, all patients will have an adequate single UCB unit with an improved chance of identifying a 5-6/6 HLA matched unit since the entire UCB inventory will be available irrespective of weight. Furthermore, units with cell counts Figure 1 Figure 1. Disclosures Wagner: Novartis: Research Funding. Maahs:Novartis: Employment. Laughlin:Novartis: Employment. Perry:Novartis: Employment. Boitano:Novartis: Employment. Cooke:Novartis: Employment. Bleul:Novartis: Employment.

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.

Abstract 513 UCB transplantation (UCBT) is associated with greater risk of graft rejection and, in particular in double UCBT, with graft vs. host disease (GVHD). Our group has shown in mouse models that ex vivo activated and expanded Tregs improve engraftment and reduce and treat GVHD. We here report on the first clinical trial to study the safety of the infusion of UCB Tregs in patients receiving a nonmyeloablative double UCB transplant. We enrolled 19 patients (pts) (age 25-65 yrs; weight 52-133; male 9; CMV seropositive 6) with high risk or advanced leukemia (n=12) and lymphoma/CLL (n=7). All patients received the same nonmyeloablative conditioning consisting of cyclophosphamide 50mg/kg/×1day, fludarabine 40 mg/m2/×5d, and total body irradiation 200 cGy/×1d; immunosuppression was cyclosporine A/mycophenolate mofetil (MMF) in the first 17 and sirolimus (Siro)/MMF in the last 2 pts. Tregs were obtained from a 3rd UCB unit and after CD25+ selection (Miltenyi CliniMACS) were activated and expanded in the presence of anti-CD3/CD28 mAb coated beads and IL-2 for 18±1 days. Treg dose escalation levels were 1, 3, 10, 30 × 105/kg on day +1, and 30 × 105/kg on days +1 and +15 after UCBT. After CD25+ selection the median % CD4+/CD25+ was 62% (range , 11-87%) and following culture was 86% (r, 62-97%). Median fold expansion was 198 (r, 13-1169); median mixed lymphocyte reaction suppression at a 1:4 ratio post-culture was 86% (39-95%). All products achieved lot release. Pts were monitored for 48h post-infusion and no dose limiting or serious adverse event was observed. After infusion an increase in the proportion of peripheral blood comprised of CD4+FoxP3+CD127- cells was observed. Donor Tregs were clearly detected in all pts receiving Tregs that were HLA disparate with the pt and graft donor units (Figure 1). Mixed day 21 donor graft UCB chimerism was observed in 59% (10/19) of pts vs. 35% (38/107) in historical controls. The cumulative incidence of grades II-IV acute GVHD was 47% (95%CI, 24-71) and III-IV 16% (95%CI, 0-33). In pts receiving ≥ 30 × 105/kg (n=14), grades II-IV acute GVHD was 43% (95%CI, 16-70) and III-IV 7% (95%CI, 0-21). Compared to historical controls receiving the same conditioning regimen, the median time to the development of acute GVHD was longer (Figures 2), although not statistically significant. Neutrophil recovery was 95% (95%CI, 79-100) at median of 8.5 days (r, 3-36), platelet recovery >50,000/mL was achieved in 74% (95%CI, 53-95) at a median 43 days (r, 27-57), cumulative incidence of opportunistic infections (fungal + viral) at 100 days was 32% (95%CI, 11-53), treatment related mortality at 100 days was 11% (95%CI, 0-25), and disease free-survival was 40% (95%CI, 15-65%). None differed from historical controls. Based on our results, the co-infusion of ex vivo expanded and activated UCB-derived Treg to recipients of nonmyeloablative UCBT 1) is safe at the tested dose levels, 2) leads to detectable increased in Treg-donor unit derived circulating CD4+FoxP3+CD127- cells, and 3) results in an increased the proportion of mixed chimerism at day +21. Identification of the Treg MTD in the context of Siro/MMF immunosuppression will set the stage for the planned definitive studies that will establish the true impact of Tregs on engraftment and GVHD and for future studies in the treatment of autoimmune disease. Disclosures: No relevant conflicts of interest to declare.