Harvest Quality, Nucleated Cell Dose and Clinical Outcomes in Bone Marrow Transplantation: A Retrospective Study.

Higher doses of infused nucleated cells (NCs) are associated with improved clinical outcomes in bone marrow transplantation (BMT) recipients. Most clinicians recommend infusing at least 2.0 × 108 NCs/kg. BMT clinicians request a target NC dose, but the harvested NC dose may be below the requested NC dose even before cell processing. We conducted this retrospective study to investigate the quality of bone marrow (BM) harvest and factors that influence infused NC doses at our institution. We also correlated infused NC doses with clinical outcomes. The study population included 347 BMT recipients (median age, 11 years; range, <1 to 75 years) at the University of Minnesota between 2009 and 2019. Underlying diagnoses mainly included 39% malignant and 61% nonmalignant diagnoses. Requested, harvested, and infused NC doses, as well as cell processing data, were obtained from the Cell Therapy Laboratory; clinical outcomes data were obtained from the University of Minnesota BMT Database. BM harvests were facilitated either by our institution (61%) or by the National Marrow Donor Program (39%). Associations of infused doses with baseline characteristics were assessed using the general Wilcoxon test/Pearson's correlation coefficient. The association of infused dose with neutrophil engraftment (absolute neutrophil count >500) by day 42, platelet engraftment (>20,000) by 6 months, acute graft-versus-host disease grade II-IV, and overall survival (OS) at 5 years were evaluated using regression and Kaplan-Meier curves. The median requested NC dose was 3.0 × 108/kg (range, 2 to 8 × 108/kg), and the median harvested and infused NC doses were 4.0 × 108/kg and 3.6 × 108/kg, respectively. Only 7% of donors had a harvested dose below the minimum requested dose. Moreover, the correlation between requested doses and harvested doses was adequate, with a harvested/requested dose ratio <.5 observed in only 5% of harvests. Additionally, the harvest volume and cell processing method were significantly correlated with the infused dose. Harvest volume exceeding the median of 948 mL was related to a significantly lower infused dose (P < .01). Moreover, hydroxyethyl starch (HES)/buffy coat processing (used to reduce RBCs with major ABO incompatibility) led to a significantly lower infused dose (P < .01). Donor age (median, 19 years; range, <1 to-70 years) and sex did not significantly influence the infused dose. Finally, the infused dose was significantly correlated with neutrophil and platelet engraftment (P < .05) but not with 5-year OS (P = .87) or aGVHD (P = .33). In our program's experience, BM harvesting is efficient and meets the requested minimum dose for 93% of recipients. Harvest volume and cell process play significant roles in determining the final infused dose. Minimizing harvest volume and cell processing could lead to increased infused dose and thus improved outcomes. Moreover, a higher infused dose leads to a better rate of neutrophil and platelet engraftment but not to improved OS, which may be linked to the sample size of our study.

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. CONCLUSIONS: 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. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Assessment of the LOVO device for final harvest of novel cell therapies: a Production Assistance for Cellular Therapies multi-center study.

BACKGROUND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

First-in-human phase 1 trial of induced regulatory T cells for graft-versus-host disease prophylaxis in HLA-matched siblings.

Human CD4+25- T cells cultured in interleukin 2 (IL-2), rapamycin, and transforming growth factor ß (TGFß) 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.

Evaluation of post-thaw CFU-GM: clinical utility and role in quality assessment of umbilical cord blood in patients receiving single unit transplant.

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 post-thaw 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.

Clinical-scale production of cGMP compliant CD3/CD19 cell-depleted NK cells in the evolution of NK cell immunotherapy at a single institution.

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.

Impact of starting material (fresh versus cryopreserved marrow) on mesenchymal stem cell culture.

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.

Optimization of cGMP purification and expansion of umbilical cord blood-derived T-regulatory cells in support of first-in-human clinical trials.

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.

Umbilical cord blood-derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect.

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.

Phase I/II Trial of StemRegenin-1 Expanded Umbilical Cord Blood Hematopoietic Stem Cells Supports Testing as a Stand-Alone Graft.

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 transplant.

Clinical mesenchymal stromal cell products undergo functional changes in response to freezing.

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.

Complement fragment 3a priming of umbilical cord blood progenitors: safety profile.

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.

Massive ex vivo expansion of human natural regulatory T cells (T(regs)) with minimal loss of in vivo functional activity.

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.

Interlaboratory assessment of a novel colony-forming unit assay: a multicenter study by the cellular team of Biomedical Excellence for Safer Transfusion (BEST) collaborative.

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.

Shipping of therapeutic somatic cell products.

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.

CD34(+) cell selection using small-volume marrow aspirates: a platform for novel cell therapies and regenerative medicine.

BACKGROUND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Loss of integrity of umbilical cord blood unit freezing bags: description and consequences.

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 6 1/2-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.

Good manufacturing practices production of natural killer cells for immunotherapy: a six-year single-institution experience.

BACKGROUND: Natural killer (NK) cells, a subset of lymphocytes and part of the innate immune system, play a crucial role in defense against cancer and viral infection. Herein is a report on the experience of clinical-scale, good manufacturing practices (GMPs) production of NK cells to treat advanced cancer. STUDY DESIGN AND METHODS: Two types of NK cell enrichments were performed on nonmobilized peripheral blood mononuclear cell apheresis collections with a cell selection system (CliniMACS, Miltenyi): CD3 cell depletion to enrich for NK cells and CD3 cell depletion followed by CD56 cell selection to obtain a more pure NK cell product. After overnight incubation with interleukin-2 (IL-2), cells were washed, resuspended in 5 percent human serum albumin, and then released for infusion. RESULTS: A total of 70 NK cell therapy products have been manufactured for patient infusion since 2000. For the CD3 cell-depleted NK cell products, the mean purity, recovery, and viability were 38, 79, and 86 percent, respectively. For the CD3 cell-depleted/CD56 cell-enriched NK cell products, the mean purity, recovery, and viability were 90, 19, and 85 percent, respectively. Gram stain, sterility, and endotoxin testing were all within acceptable limits for established lot release. Compared to the resting processed cells, IL-2 activation significantly increased the function of cells in cytotoxicity assays. CONCLUSION: Clinical-scale production of NK cells is efficient and can be performed under GMPs. The purified NK cell product results in high NK cell purity with minimal contamination by T cells, monocytes, and B cells, but it requires more time for processing and results in a lower NK cell recovery when compared to NK cell enrichment with CD3 cell depletion alone. Additional laboratory studies and results from clinical trials will identify the best source and type of NK cell product.