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Background Aims: Stem and progenitor cells of hematopoietic and mesenchymal lineages reside in the bone marrow under low oxygen (O 2 ) saturation. O 2 levels used in ex vivo expansion of multipotent mesenchymal stromal cells (MSCs) affect proliferation, metabolism and differentiation., Methods: Using cell-based assays and transcriptome and proteome data, the authors compared MSC cultures simultaneously grown under a conventional 19.95% O 2 atmosphere or at 5% O 2 ., Results: In 5% O 2 , MSCs showed better proliferation and higher self-renewal ability, most probably sustained by enhanced signaling activity of mitogen-activated protein kinase and mammalian target of rapamycin pathways. Non-oxidative glycolysis-based energy metabolism supported growth and proliferation in 5% O 2 cultures, whereas MSCs grown under 19.95% O 2 also utilized oxidative phosphorylation. Cytoprotection mechanisms used by cells under 5% O 2  differed from 19.95% O 2   suggesting differences in the triggers of cell stress between these two O 2   conditions., Conclusions: Based on the potential benefits for the growth and metabolism of MSCs, the authors propose the use of 5% O 2 for MSC culture., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Subsequent malignancies are well-documented complications in long-term follow-up of cancer patients. Recently, genetically modified immune effector (IE) cells have shown benefit in hematologic malignancies and are being evaluated in clinical trials for solid tumors. Although the short-term complications of IE cells are well described, there is limited literature summarizing long-term follow-up, including subsequent malignancies. We retrospectively reviewed data from 340 patients treated across 27 investigator-initiated pediatric and adult clinical trials at our center. All patients received IE cells genetically modified with γ-retroviral vectors to treat relapsed and/or refractory hematologic or solid malignancies. In a cumulative 1027 years of long-term follow-up, 13 patients (3.8%) developed another cancer with a total of 16 events (4 hematologic malignancies and 12 solid tumors). The 5-year cumulative incidence of a first subsequent malignancy in the recipients of genetically modified IE cells was 3.6% (95% confidence interval, 1.8% to 6.4%). For 11 of the 16 subsequent tumors, biopsies were available, and no sample was transgene positive by polymerase chain reaction. Replication-competent retrovirus testing of peripheral blood mononuclear cells was negative in the 13 patients with subsequent malignancies tested. Rates of subsequent malignancy were low and comparable to standard chemotherapy. These results suggest that the administration of IE cells genetically modified with γ retroviral vectors does not increase the risk for subsequent malignancy., (© 2022 by The American Society of Hematology.)

Relapse after allogeneic hematopoietic stem cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Infusion of unselected donor lymphocytes (DLIs) enhances the graft-versus-leukemia (GVL) effect. However, because the infused lymphocytes are not selected for leukemia specificity, the GVL effect is often accompanied by life-threatening graft-versus-host disease (GVHD), related to the concurrent transfer of alloreactive lymphocytes. Thus, to minimize GVHD and maximize GVL, we selectively activated and expanded stem cell donor-derived T cells reactive to multiple antigens expressed by AML/MDS cells (PRAME, WT1, Survivin, and NY-ESO-1). Products that demonstrated leukemia antigen specificity were generated from 29 HCT donors. In contrast to DLIs, leukemia-specific T cells (mLSTs) selectively recognized and killed leukemia antigen-pulsed cells, with no activity against recipient's normal cells in vitro. We administered escalating doses of mLSTs (0.5 to 10 × 107 cells per square meter) to 25 trial enrollees, 17 with high risk of relapse and 8 with relapsed disease. Infusions were well tolerated with no grade >2 acute or extensive chronic GVHD seen. We observed antileukemia effects in vivo that translated into not-yet-reached median leukemia-free and overall survival at 1.9 years of follow-up and objective responses in the active disease cohort (1 complete response and 1 partial response). In summary, mLSTs are safe and promising for the prevention and treatment of AML/MDS after HCT. This trial is registered at www.clinicaltrials.com as #NCT02494167., (© 2021 by The American Society of Hematology.)

Autologous T cells targeting Epstein-Barr virus (EBV) latent membrane proteins (LMPs) have shown safety and efficacy in the treatment of patients with type 2 latency EBV-associated lymphomas for whom standard therapies have failed, including high-dose chemotherapy followed by autologous stem-cell rescue. However, the safety and efficacy of allogeneic donor-derived LMP-specific T cells (LMP-Ts) have not been established for patients who have undergone allogeneic hematopoietic stem-cell transplantation (HSCT). Therefore, we evaluated the safety and efficacy of donor-derived LMP-Ts in 26 patients who had undergone allogeneic HSCT for EBV-associated natural killer/T-cell or B-cell lymphomas. Seven patients received LMP-Ts as therapy for active disease, and 19 were treated with adjuvant therapy for high-risk disease. There were no immediate infusion-related toxicities, and only 1 dose-limiting toxicity potentially related to T-cell infusion was seen. The 2-year overall survival (OS) was 68%. Additionally, patients who received T-cell therapy while in complete remission after allogeneic HSCT had a 78% OS at 2 years. Patients treated for B-cell disease (n = 10) had a 2-year OS of 80%. Patients with T-cell disease had a 2-year OS of 60%, which suggests an improvement compared with published posttransplantation 2-year OS rates of 30% to 50%. Hence, this study shows that donor-derived LMP-Ts are a safe and effective therapy to prevent relapse after transplantation in patients with B cell- or T cell-derived EBV-associated lymphoma or lymphoproliferative disorder and supports the infusion of LMP-Ts as adjuvant therapy to improve outcomes in the posttransplantation setting. These trials were registered at www.clinicaltrials.gov as #NCT00062868 and #NCT01956084., (© 2018 by The American Society of Hematology.)

The clinical success achieved using CD19-directed CAR-T cells has stimulated many academic institutions to explore the feasibility of manufacturing these, and other CAR-T cells, in-house. This article reviews the issues that must be addressed in order to achieve this goal. It includes the manufacturing infrastructure, the regulatory environment, practical aspects of production, and the costs involved., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an "open-access" manner, such as through publication or database collection., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantify the immune-modulatory capacity of bone marrow-derived mesenchymal stromal cells (BM-MSCs)., Methods: Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third-party BM-MSCs. After stimulation with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression., Results: The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16-h) assay that was based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation between different responder T cells. Suppression by BM-MSCs on different responders correlated with suppression by K299. We therefore used K299 suppression as the reference to define suppression potency of BM-MSCs in K299 Suppression Units. We found that inter-donor variability, passage number, method of manufacture and exposure of BM-MSCs to steroids or interferon-γ all affected BM-MSC potency of suppression., Conclusions: This method provides a platform for standardizing suppressor function to facilitate comparisons between laboratories and for use as a cell product release assay., (Published by Elsevier Inc.)

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103., (© 2015 by The American Society of Hematology.)

Background Aims: The T-cell suppressive property of bone marrow-derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T-cell suppression., Methods: At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center, we developed an in vitro quality control T-cell suppression immunopotency assay (IPA) that uses anti-CD3 and anti-CD28 antibodies to stimulate T-cell proliferation. We measured MSC-induced suppression of CD4+ T-cell proliferation at various effector-to-target cell ratios with the use of defined peripheral blood mononuclear cells and in parallel compared with a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product., Results: Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T-cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T-cell proliferation, with immunopotency assay values ranging from 27% to 88%. However, MSC suppression did not correlate with human leukocyte antigen-DR expression., Conclusions: We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohn's disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times., Methods: We evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials., Results: After only two passages, we were able to expand a mean of 6.6 × 10(8) MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10(8) cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active., Conclusions: The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892., (© 2014 by The American Society of Hematology.)

Adoptive transfer of T lymphocytes expressing a CD19-specific chimeric antigen receptor (CAR.CD19) induces complete tumor regression in patients with lymphoid malignancies. Although in vivo persistence of CAR-T cells correlates with clinical responses, it remains unknown whether specific cell subsets within the CAR-T-cell product correlate with their subsequent in vivo expansion and persistence. We analyzed 14 patients with B-cell malignancies infused with autologous CAR.CD19-redirected T cells expanded ex vivo using IL-2, and found that their in vivo expansion only correlated with the frequency within the infused product of a CD8(+)CD45RA(+)CCR7(+) subset, whose phenotype is closest to "T-memory stem cells." Preclinical models showed that increasing the frequency of CD8(+)CD45RA(+)CCR7(+) CAR-T cells in the infused line by culturing the cells with IL-7 and IL-15 produced greater antitumor activity of CAR-T cells mediated by increased resistance to cell death, following repetitive encounters with the antigen, while preserving their migration to secondary lymphoid organs. This trial was registered at www.clinicaltrials.gov as #NCT00586391 and #NCT00709033., (© 2014 by The American Society of Hematology.)

Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control.

Virus-specific T cell (VST) lines could provide useful antiviral prophylaxis and treatment of immune-deficient patients if it were possible to avoid the necessity of generating a separate line for each patient, often on an emergency basis. We prepared a bank of 32 virus-specific lines from individuals with common HLA polymorphisms who were immune to Epstein-Barr virus (EBV), cytomegalovirus, or adenovirus. A total of 18 lines were administered to 50 patients with severe, refractory illness because of infection with one of these viruses after hematopoietic stem cell transplant. The cumulative rates of complete or partial responses at 6 weeks postinfusion were 74.0% (95% CI, 58.5%-89.5%) for the entire group (n = 50), 73.9% (95% CI, 51.2% -96.6%) for cytomegalovirus (n = 23), 77.8% for adenovirus (n = 18), and 66.7% (95% CI, 36.9%-96.5%) for EBV (n = 9). Only 4 responders had a recurrence or progression. There were no immediate infusion-related adverse events, and de novo graft-versus-host disease developed in only 2 patients. Despite the disparity between the lines and their recipients, the mean frequency of VSTs increased significantly postinfusion, coincident with striking decreases in viral DNA and resolution of clinical symptoms. The use of banked third-party VSTs is a feasible and safe approach to rapidly treat severe or intractable viral infections after stem cell transplantation. This study is registered at www.clinicaltrials.gov as NCT00711035.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytes, osteoblasts and chondroblasts as well as secreting a vast array of soluble mediators. This potentially makes MSCs important mediators of a variety of therapeutic applications. They are actively under evaluation for immunomodulatory purposes such as graft-versus-host disease and Crohn's disease as well as regenerative applications such as stroke and congestive heart failure. We report our method of generating clinical-grade MSCs together with suggestions gathered from manufacturing experience in our Good Manufacturing Practices facility., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available., Methods: We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex)., Results: Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation., Conclusions: We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.

Infections with a range of common community viruses remain a major cause of mortality and morbidity after allogeneic hematopoietic stem cell transplantation. T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenoviruses can safely prevent and infections with these three most common culprits, but the manufacture of individual T cell lines for each virus would be prohibitive in terms of time and cost. We have demonstrated that T cells specific for all three viruses can be manufactured in a single culture using monocytes and EBV-transformed B lymphoblastoid cell lines (LCLs), both transduced with an adenovirus vector expressing pp65 of CMV, as antigen-presenting cells. Trivirus-specific T cell lines produced from healthy stem cell donors could prevent and treat infections with all three viruses, not only in the designated recipient, but in unrelated, partially-HLA-matched third party recipients. We now provide the details and logistics of T cell manufacture.

We generated MHC-independent chimeric antigen receptors (CARs) directed to the GD2 antigen expressed by neuroblastoma tumor cells and treated patients with this disease. Two distinguishable forms of this CAR were expressed in EBV-specific cytotoxic T lymphocytes (EBV-CTLs) and activated T cells (ATCs). We have previously shown that EBV-CTLs expressing GD2-CARs (CAR-CTLs) circulated at higher levels than GD2-CAR ATCs (CAR-ATCs) early after infusion, but by 6 weeks, both subsets became low or undetectable. We now report the long-term clinical and immunologic consequences of infusions in 19 patients with high-risk neuroblastoma: 8 in remission at infusion and 11 with active disease. Three of 11 patients with active disease achieved complete remission, and persistence of either CAR-ATCs or CAR-CTLs beyond 6 weeks was associated with superior clinical outcome. We observed persistence for up to 192 weeks for CAR-ATCs and 96 weeks for CAR-CTLs, and duration of persistence was highly concordant with the percentage of CD4(+) cells and central memory cells (CD45RO(+)CD62L(+)) in the infused product. In conclusion, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. This study is registered at www.clinialtrials.gov as #NCT00085930.

Background Aims: The Food and Drug Administration (FDA) currently recommends at least 4 h of recipient monitoring after T cell infusions to detect early infusion reactions. Recent catastrophic reactions to 'first-in-man' biologic agents have emphasized the importance of this rule for initial studies of new products. The value of such monitoring for better established agents is less obvious., Methods: We reviewed infusion-related adverse events (AE) following administration of ex vivo-expanded T cell products (antigen-specific cytotoxic T lymphocytes, allodepleted T cells, and genetically modified T cells) on investigational new drug (IND) studies in our center., Results: From 1998 to 2008, we infused 381 T cell products to 180 recipients, enrolled on 18 studies, receiving T cells targeting malignancies or post-transplant viral infections. There were no grade 3-4 infusion reactions during initial monitoring or 24-h follow-up. Twenty-four mild (grade 1-2) AE occurred in 21 infusions either during or immediately following infusion (up to 6 h), most commonly nausea and vomiting (10/24, 41.6%), probably because of the dimethyl sulfoxide cryoprotectant, and hypotension (20.8%), attributable to diphenhydramine pre-medication. Twenty-two additional non-severe events were reported within 24 h of infusion, most commonly culture-negative fever, chills and nausea. An increased risk of adverse events was associated with age [incidence rate ratio (IRR) 0.98; 95% confidence interval (CI) 0.96-1.00, P = 0.05], while an increased risk of immediate infusion-related events was higher in patients reporting allergies (IRR 2.72, 95% CI 1.00-7.40, P = 0.05); sex, disease type and T cell source (allogeneic or autologous) had no effect on frequency of adverse events., Conclusions: Infusion of these T cell products was safe in the outpatient setting and associated with no severe reactions, so monitoring for 1 h after infusion is probably sufficient. As many of the AE were attributable to diphenhydramine premedication, a lower dose (0.25 mg/kg) should be selected.

Abstract Background aims. Multicenter cellular therapy clinical trials require the establishment and implementation of standardized cell-processing protocols and associated quality control (QC) mechanisms. The aims here were to develop such an infrastructure in support of the Cardiovascular Cell Therapy Research Network (CCTRN) and to report on the results of processing for the first 60 patients. Methods. Standardized cell preparations, consisting of autologous bone marrow (BM) mononuclear cells, prepared using a Sepax device, were manufactured at each of the five processing facilities that supported the clinical treatment centers. Processing staff underwent centralized training that included proficiency evaluation. Quality was subsequently monitored by a central QC program that included product evaluation by the CCTRN biorepositories. Results. Data from the first 60 procedures demonstrated that uniform products, that met all release criteria, could be manufactured at all five sites within 7 h of receipt of BM. Uniformity was facilitated by use of automated systems (the Sepax for processing and the Endosafe device for endotoxin testing), standardized procedures and centralized QC. Conclusions. Complex multicenter cell therapy and regenerative medicine protocols can, where necessary, successfully utilize local processing facilities once an effective infrastructure is in place to provide training and QC.

Viral infections and leukemic relapse account for the majority of treatment failures in patients with B-cell acute lymphoblastic leukemia (B-ALL) receiving allogeneic hematopoietic stem cell (HSC) or cord blood (CB) transplants. Adoptive transfer of virus-specific cytotoxic T lymphocytes (CTLs) provides protection against common viruses causing serious infections after HSC transplantation without concomitant graft-versus-host disease. We have now generated CTL lines from peripheral blood (PB) or CB units that recognize multiple common viruses and provide antileukemic activity by transgenic expression of a chimeric antigen receptor (CAR) targeting CD19 expressed on B-ALL. PB-derived CAR(+) CTLs produced interferon-gamma (IFNgamma) in response to cytomegalovirus-pp65, adenovirus-hexon, and Epstein-Barr virus pepmixes (from 205 +/- 104 to 1034 +/- 304 spot-forming cells [SFCs]/10(5) T cells) and lysed primary B-ALL blasts in (51)Cr-release assays (mean, 66% +/- 5% specific lysis; effector-target [E/T] ratio, 40:1) and the CD19(+) Raji cell line (mean, 78% +/- 17%) in contrast to nontransduced controls (8% +/- 8% and 3% +/- 2%). CB-derived CAR(+) CTLs showed similar antiviral and antitumor function and both PB and CB CAR(+) CTLs completely eliminated B-ALL blasts over 5 days of coculture. This approach may prove beneficial for patients with high-risk B-ALL who have recently received an HSC or CB transplant and are at risk of infection and relapse.

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.

Viral infection or reactivation remains a major cause of morbidity and mortality after allogeneic stem cell transplantation. We now show that infusions of single cytotoxic T lymphocyte (CTL) lines (5 x 10(6)-1.35 x 10(8) cells/m(2)) with specificity for 2 commonly detected viruses, Epstein-Barr virus (EBV) and adenovirus, can be safely administered to pediatric transplantation recipients receiving partially human leukocyte antigen-matched and haploidentical stem cell grafts (n = 13), without inducing graft-versus-host disease. The EBV-specific component of the CTLs expanded in vivo and persisted for more than 12 weeks, but the adenovirus-specific component only expanded in vivo in the presence of concomitant adenoviral infection. Nevertheless, adenovirus-specific T cells could be detected for at least 8 weeks in peripheral blood, even in CTL recipients without viral infection, provided the adenovirus-specific component of their circulating lymphocytes was first expanded by exposure to adenoviral antigens ex vivo. After infusion, none of these 13 high-risk recipients developed EBV-associated lymphoproliferative disease, while 2 of the subjects had resolution of their adenoviral disease. Hence, bispecific CTLs containing both EBV- and adenovirus-specific T cells can safely reconstitute an antigen responsive "memory" population of CTLs after human leukocyte antigen-mismatched stem cell transplantation and may provide antiviral activity. This trial was registered at www.clinicaltrials.gov as #NCT00590083.

The naive phenotype of cord blood (CB) T cells may reduce graft-versus-host disease after umbilical cord blood transplantation, but this naivety and their low absolute numbers also delays immune reconstitution, producing higher infection-related mortality that is predominantly related to CMV, adenovirus (Adv), and EBV. Adoptive immunotherapy with peripheral blood-derived virus-specific cytotoxic T lymphocytes (CTLs) can effectively prevent viral disease after conventional stem cell transplantation, and we now describe the generation of single cultures of CTLs from CB that are specific for multiple viruses. Using EBV-infected B cells transduced with a clinical-grade Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv, and CMV antigens, we expanded virus-specific T cells even from CB T cells with a naive phenotype. After expansion, each CTL culture contained both CD8(+) and CD4(+) T-cell subsets, predominantly of effector memory phenotype. Each CTL culture also had HLA-restricted virus-specific cytotoxic effector function against EBV, CMV, and Adv targets. The CB CTLs recognized multiple viral epitopes, including CD4-restricted Adv-hexon epitopes and immunosubdominant CD4- and CD8-restricted CMVpp65 epitopes. Notwithstanding their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a single culture of CB, which may be of value to prevent or treat viral disease in CB transplant recipients. This study is registered at www.clinicaltrials.gov as NCT00078533.

Treatment of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) with EBV-specific cytotoxic T cells (EBV-specific CTL) has been promising, producing clinical responses. However, infused EBV-specific CTL did not expand in vivo, likely limiting their antitumor activity. Lymphodepleting patients with chemotherapy before T-cell transfer enhances in vivo T-cell expansion, but results in nonspecific destruction of the resident immune system and can have significant toxicity. To evaluate if monoclonal antibodies (mAbs) can produce a more selective lymphodepletion, we conducted a clinical study in which NPC patients received a pair of lymphodepleting mAbs targeted to the CD45 antigen (CD45 mAbs) before EBV-specific CTL infusion. Eight patients with recurrent NPC received CD45 mAbs followed by escalating doses of autologous EBV-specific CTL. Infusion of CD45 mAbs resulted in transient lymphopenia in all patients and an increase in interleukin-15 (IL-15) levels in 6 out 8 patients. All patients had an increase in their peripheral blood frequency of EBV-specific T cells after CTL infusion. Three patients with a persistent increase had clinical benefits including 1 complete response (> 24 months) and 2 with stable disease (for 12 and 15 months). Lymphodepleting mAbs prior CTL transfer may represent an alternative to chemotherapy to enhance expansion of infused CTL. This study is registered at (http://www.clinialtrials.gov) as NCT00608257.

Epstein-Barr virus (EBV)-associated tumors developing in immunocompetent individuals present a challenge to immunotherapy, since they lack expression of immunodominant viral antigens. However, the tumors consistently express viral proteins including LMP2, which are immunologically "weak" but may nonetheless be targets for immune T cells. We previously showed that a majority of cytotoxic T lymphocytes (CTLs) reactivated using EBV-transformed B-lymphoblastoid cells lines (LCLs) contained minor populations of LMP2-specific T cells and homed to tumor sites. However, they did not produce remissions in patients with bulky disease. We have now used gene transfer into antigen-presenting cells (APCs) to augment the expression and immunogenicity of LMP2. These modified APCs increased the frequency of LMP2-specific CTLs by up to 100-fold compared with unmodified LCL-APCs. The LMP2-specific population expanded and persisted in vivo without adverse effects. Nine of 10 patients treated in remission of high-risk disease remain in remission, and 5 of 6 patients with active relapsed disease had a tumor response, which was complete in 4 and sustained for more than 9 months. It is therefore possible to generate immune responses to weak tumor antigens by ex vivo genetic modification of APCs and the CTLs so produced can have substantial antitumor activity. This study is registered at http://www.cancer.gov/clinicaltrials (protocol IDs: BCM-H-9936, NCT00062868, NCT00070226).

We have investigated the in vivo safety, efficacy, and persistence of autologous Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) for the treatment of solid organ transplant (SOT) recipients at high risk for EBV-associated posttransplantation lymphoproliferative disease (PTLD). EBV-CTLs generated from 35 patients expanded with normal kinetics contained both CD8 and CD4 lymphocytes and produced significant specific killing of autologous EBV-transformed B lymphoblastoid cell lines (LCLs). Twelve SOT recipients at high risk for PTLD, or with active disease, received autologous CTL infusions without toxicity. Real-time polymerase chain reaction (PCR) monitoring of EBV-DNA showed a transient increase in plasma EBV-DNA suggestive of lysis of EBV-infected cells, although there was no consistent decrease in virus load in peripheral-blood mononuclear cells. Interferon-gamma enzyme-linked immunospot (ELISPOT) assay and tetramer analysis showed an increase in the frequency of EBV-responsive T cells, which returned to preinfusion levels after 2 to 6 months. None of the treated patients developed PTLD. One patient with liver PTLD showed a complete response, and one with ocular disease has had a partial response stable for over one year. These data are consistent with an expansion and persistence of adoptively transferred EBV-CTLs that is limited in the presence of continued immunosuppression but that nonetheless produces clinically useful antiviral activity.

CD40L generates immune responses in leukemia-bearing mice, an effect that is potentiated by IL-2. We studied the feasibility, safety, and immunologic efficacy of an IL-2- and CD40L-expressing recipient-derived tumor vaccine consisting of leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 (hIL-2) and hCD40L. Ten patients (including 7 children) with high-risk acute myeloid (n = 4) or lymphoblastic (n = 6) leukemia in cytologic remission (after allogeneic stem cell transplantation [n = 9] or chemotherapy alone [n = 1]) received up to 6 subcutaneous injections of the IL-2/CD40L vaccine. None of the patients were receiving immunosuppressive drugs. No severe adverse reactions were noted. Immunization produced a 10- to 890-fold increase in the frequencies of major histocompatibility complex (MHC)-restricted T cells reactive against recipient-derived blasts. These leukemia-reactive T cells included both T-cytotoxic/T-helper 1 (Th1) and Th2 subclasses, as determined from their production of granzyme B, interferon-gamma, and interleukin-5. Two patients produced systemic IgG antibodies that bound to their blasts. Eight patients remained disease free for 27 to 62 months after treatment (5-year overall survival, 90%). Thus, even in heavily treated patients, including recipients of allogeneic stem cell transplants, recipient-derived antileukemia vaccines can induce immune responses reactive against leukemic blasts. This approach may be worthy of further study, particularly in patients with a high risk of relapse.

Epstein-Barr virus (EBV) is a common viral infection in pediatric liver transplant patients and can lead to development of post-transplant lymphoproliferative disorder (PTLD). Differing studies have used immunosuppression reduction, antiviral medications or i.v. CMV-immunogloublin for EBV prevention and treatment. The purpose of this study was to determine whether implementation of a protocol for frequent EBV monitoring and EBV viral load-driven immunosuppression reduction could decrease the incidence of PTLD in our patient population. All data were prospectively collected between 2001 and 2004 at a single institution. Seventy-three patients were entered into the study. Patients were divided into a historical control group (pre-2001, 30 patients) and a treatment group (post-2001, 43 patients). Approximately 1271 blood samples of 73 patients were collected between 2001 and 2004. Eleven out of 43 patients received immunosuppression tapering due to high EBV viral loads (>4000 copies/microg DNA). One patient developed allograft rejection after immunosuppression modulation. Prior to 2001, the incidence of PTLD at our institution was 16%. After instituting a protocol for EBV monitoring, the incidence of PTLD decreased to 2% (p-value<0.05). These findings illustrate that frequent EBV viral load monitoring and preemptive immunosuppression modulation have an integral role in preventing PTLD in the pediatric liver transplant population.

Conventional treatment for nasopharyngeal carcinoma (NPC) frequently fails and is accompanied by severe long-term side effects. Since virtually all undifferentiated NPCs are associated with Epstein-Barr virus (EBV), this tumor is an attractive candidate for cellular immunotherapy targeted against tumor-associated viral antigens. We now demonstrate that EBV-specific cytotoxic T-cell (CTL) lines can readily be generated from individuals with NPC, notwithstanding the patients' prior exposure to chemotherapy/radiation. A total of 10 patients diagnosed with advanced NPC were treated with autologous CTLs. All patients tolerated the CTLs, although one developed increased swelling at the site of pre-existing disease. At 19 to 27 months after infusion, 4 patients treated in remission from locally advanced disease remain disease free. Of 6 patients with refractory disease prior to treatment, 2 had complete responses, and remain in remission over 11 to 23 months after treatment; 1 had a partial remission that persisted for 12 months; 1 has had stable disease for more than 14 months; and 2 had no response. These results demonstrate that administration of EBV-specific CTLs to patients with advanced NPC is feasible, appears to be safe, and can be associated with significant antitumor activity.

Posttransplantation lymphoproliferative disorders (PTLDs) caused by uncontrolled expansion of Epstein-Barr virus (EBV)-infected B cells after hematopoietic stem cell transplantation (HSCT) can be predicted by an increase in EBV DNA in peripheral blood mononuclear cells. We used real-time quantitative polymerase chain reaction (RQ-PCR) analysis to determine whether frequent monitoring of EBV DNA to allow preemptive treatment is truly of value in patients after HSCT. More than 1300 samples from 85 recipients were analyzed. No patient with consistently low EBV DNA levels developed PTLD. Nine patients had a single episode with a high EBV load (more than 4000 EBV copies/microg peripheral blood mononuclear cell [PBMC] DNA), and 16 patients had high EBV loads detected on 2 or more occasions. Only 8 of these developed symptoms consistent with PTLD, and all were promptly and successfully treated with EBV-specific cytotoxic T cells or CD20 monoclonal antibody. Hence, quantitative measurement of EBV DNA may best be used to enable the prompt rather than the preemptive treatment of PTLD.

The increasing utilization of cell and gene therapy in the treatment of human diseases, particularly cancers, has led lawmakers to institute stringent rules to regulate this new area of medicine and optimally protect patients. Cell and gene therapy centers, often located within academic institutions, are becoming subject to rules previously applied only to pharmaceutical companies. Based on the US experience, we review here the regulations that currently regulate or are being proposed to cover cell and gene therapy centers. We describe the principles of good manufacturing practices, quality assurance and quality control and accreditation. Finally, we discuss the future of cell and gene therapy in the context of the increasingly rigorous regulatory environment implemented in the US.

Chronic active Epstein-Barr virus (CAEBV) infection syndrome is a heterogeneous EBV-related disorder characterized by chronic fatigue, fever, lymphadenopathy, and/or hepatosplenomegaly, associated with abnormal patterns of antibody to EBV. CAEBV can range from disabling mild/moderate forms to rapidly lethal disorders. Even patients with mild/moderate disease frequently suffer adverse effects from long-term anti-inflammatory agents and have a quality of life that progressively deteriorates. It is still unknown why these individuals are unable to produce an effective immune response to control EBV, and no effective treatment is currently available. Since ex vivo-expanded EBV-specific cytotoxic T lymphocytes (EBV-CTLs) can safely restore EBV-specific cellular immune responses in immunodeficient patients, we assessed the possibility that adoptive immunotherapy might also effectively treat CAEBV infection. Following stimulation with irradiated EBV-transformed lymphoblastoid cell lines (LCLs), EBV-CTLs were successfully generated from 8 of 8 patients with the mild/moderate form of CAEBV infection. These CTLs were predominantly CD3(+) CD8(+) cells and produced specific killing of the autologous LCLs. There were 5 patients with 1- to 12-year histories of disease who were treated with 1 to 4 injections of EBV-CTLs. Following infusion, there was resolution of fatigue and malaise, disappearance of fever, and regression of lymphadenopathy and splenomegaly. The pattern and titers of anti-EBV antibodies also normalized. No toxicity was observed. There were 4 patients who did not show any relapse of disease within 6 to 36 months follow-up; one patient had recurrence of fatigue and myalgia one year after CTL infusion. We suggest that adoptive immunotherapy with autologous EBV-CTLs may represent a safe and feasible alternative treatment for patients affected with mild/moderate CAEBV infection and that this approach should be evaluated in the more severe forms of the disease.

Cellular therapies offer new promise for the treatment of a variety of diseases. In order to protect patients and donors, the Food and Drug Administration in the USA has been developing specific regulations. These have undergone several iterations, however, a broad-based strategy has recently emerged. These proposals are reviewed and their impact on academic institutions is assessed.