Cell Therapy Laboratory Research Articles

Expanding the reach of commercial cell therapies requires changes at medical centers

The clinical application of cell therapies is becoming increasingly important for the treatment of cancer, congenital immune deficiencies, and hemoglobinopathies. These therapies have been primarily manufactured and used at academic medical centers. However, cell therapies are now increasingly being produced in centralized manufacturing facilities and shipped to medical centers for administration. Typically, these cell therapies are produced from a patient’s own cells, which are the critical starting material. For these therapies to achieve their full potential, more medical centers must develop the infrastructure to collect, label, cryopreserve, test, and ship these cells to the centralized laboratories where these cell therapies are manufactured. Medical centers must also develop systems to receive, store, and infuse the finished cell therapy products. Since most cell therapies are cryopreserved for shipment and storage, medical centers using these therapies will require access to liquid nitrogen product storage tanks and develop procedures to thaw cell therapies. These services could be provided by the hospital pharmacy or transfusion service, but the latter is likely most appropriate. Another barrier to implementing these services is the variability among providers of these cell therapies in the processes related to handling cell therapies. The provision of these services by medical centers would be facilitated by establishing a national coordinating center and a network of apheresis centers to collect and cryopreserve the cells needed to begin the manufacturing process and cell therapy laboratories to store and issue the cells. In addition to organizing cell collections, the coordinating center could establish uniform practices for collecting, labeling, shipping, receiving, thawing, and infusing the cell therapy.

Going with the Flow: Moving Cells and Changing Values in Biomedical Practice

Science and technology studies have devoted considerable attention to the economic implications of biomedical technoscience. This article enriches these studies by offering a new reading of several strands of literature to explore how the valuation processes of gifts, commodities, and assets are intertwined in practice. We derive this approach from an empirical analysis of autologous blood donation in the case of a cell therapy called “extracorporeal photopheresis.” Combining ethnographic fieldwork with semi-structured interviews, focus groups, and document analysis in a cell therapy laboratory at a Belgian university hospital, we followed cells in motion from their original donation to their reinjection into the body, observing the practices that constitute and shape their value. We find that the various qualifications as “gift,” “commodity,” or “asset” that cells acquire, accumulate, or relinquish, as well as the consequences of these qualifications, are accessible only by observing the valuation practices that configure living processes. Our analysis highlights the interrelations between the economic forms that living entities can take and rejects the idea of a watertight boundary between them. By emphasizing the entanglement of logics specific to donation, commodification, or assetization, we contribute to linking the value shifts observed at the level of the laboratory to broader capitalist transformations.

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.

Collection and processing of hematopoietic progenitor cell products at risk of presenting with cold agglutination

Background aimsCold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3–4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo, complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. Case reportsFour cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. ConclusionsWhile uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection.

Human nasal olfactory stem cells, purified as advanced therapy medicinal products, improve neuronal differentiation.

Olfactory ecto-mesenchymal stem cells (OE-MSC) are mesenchymal stem cells derived from the lamina propria of the nasal mucosa. They display neurogenic and immunomodulatory properties and were shown to induce recovery in animal models of spinal cord trauma, hearing loss, Parkinsons's disease, amnesia, and peripheral nerve injury. As a step toward clinical practice, we sought to (i) devise a culture protocol that meets the requirements set by human health agencies and (ii) assess the efficacy of stem cells on neuron differentiation. Nasal olfactory mucosa biopsies from three donors were used to design and validate the good manufacturing process for purifying stem cells. All processes and procedures were performed by expert staff from the cell therapy laboratory of the public hospital of Marseille (AP-HM), according to aseptic handling manipulations. Premises, materials and air were kept clean at all times to avoid cross-contamination, accidents, or even fatalities. Purified stem cells were cultivated for 24 or 48 h and conditioned media were collected before being added to the culture medium of the neuroblastoma cell line Neuro2a. Compared to the explant culture-based protocol, enzymatic digestion provides higher cell numbers more rapidly and is less prone to contamination. The use of platelet lysate in place of fetal calf serum is effective in promoting higher cell proliferation (the percentage of CFU-F progenitors is 15.5%), with the optimal percentage of platelet lysate being 10%. Cultured OE-MSCs do not show chromosomal rearrangement and, as expected, express the usual phenotypic markers of mesenchymal stem cells. When incorporated in standard culture medium, the conditioned medium of purified OE-MSCs promotes cell differentiation of Neuro2a neuroblastoma cells. We developed a safer and more efficient manufacturing process for clinical grade olfactory stem cells. With this protocol, human OE-MSCs will soon be used in a Phase I clinical based on their autologous transplantation in digital nerves with a neglected injury. However, further studies are required to unveil the underlying mechanisms of action.

Phase I Trial of CD8-Depleted Human Leukocyte Antigen (HLA) Mismatched Unrelated Donor Lymphocyte Infusion (DLI) to Achieve Remissions in Myelodysplastic Syndrome (MDS) and Secondary Acute Myeloid Leukemia (sAML)

Background Hypomethylating agents (HMAs) are the standard of care in higher risk MDS, yielding a median survival of 12-18 months. After HMA failure or progression to sAML. median survival is only 4-6 months. We developed a novel cellular therapy using HLA mismatched, unrelated, CD8-depleted, CD4+ DLI to induce remission in patients with MDS and sAML who have not previously undergone allogeneic hematopoietic cell transplant (HCT). The DLI is intentionally class II HLA mismatched to activate donor CD4+ T cells which then provide immunologic "help" to reverse recipient immune exhaustion and, thus, promote a potent antitumor response (Symons. BBMT. 2008). Further, HLA mismatched donors are easily identified and allow the potential for an "off the shelf" product. By depleting CD8+ T cells, it is intended that the DLI does not engraft, thus negating the risk of graft-versus-host disease (GVHD). Instead, this therapy is intended to induce remission as a bridge to curative allogeneic HCT. Methods This completed phase I trial enrolled patients with MDS refractory to at least 4 cycles of HMA or sAML at the Moffitt Cancer Center from 2020-2022. Patients who received prior allogeneic HCT were excluded. DLI donors were selected from a pre-existing pool of unrelated donors at the New York Blood Center (NYBC) on the basis of class II HLA mismatch, CMV compatibility, and ABO compatibility. Patients were treated with induction chemotherapy as chosen by their primary physician. In parallel, donors underwent leukapheresis at the NYBC and the cell product was shipped to the Moffitt Cancer Center. The DLI was depleted of CD8+ T cells using the CliniMACS® system with CliniMACS® CD8 reagent in the Moffitt Cell Therapy Laboratory. HLA-mismatched CD4+ DLI was infused fresh within 24-36 hours after completing induction chemotherapy. The trial followed a 3+3 design to determine the recommended phase II dose (RP2D) of DLI where dose level (DL) 1 was 1x106 CD4 cells/kg, DL2 was 1x107 CD4 cells/kg, and DL3 was 5x107 CD4 cells/kg. Dose limiting toxicities (DLTs) were defined as grade 2-4 acute GVHD, grade 3-4 cytokine release syndrome (CRS), grade 2-4 infusion reactions, aplasia beyond 56 days in the presence of detectable donor chimerism, or grade 4 organ toxicity by CTCAE 5.0. Mechanistic studies were pursued to evaluate markers of immune tolerance, cytokine profiles, and confirm the DLI did not engraft. Results Nine patients (8 sAML, 1 MDS) were treated. All patients had progressed through at least one prior line of therapy (Table 1). The median age was 66. All AML patients had adverse risk disease by ELN risk stratification including 4 patients with TP53 mutation and complex cytogenetics. The MDS patient had very high-risk disease by R-IPSS. A suitable donor was identified for all patients despite a donor pool of only 30 donors. Cell collections, shipments, and processing for all patients were successful with all products achieving <3% CD8+ T cells and >70% cell viability. No patients experienced DLTs at any dose level. One patient at DL3 experienced grade 2 CRS that resolved with tocilizumab. There were no cases of grade 3-4 CRS nor GVHD (Table 2). Complete remission (CR) was achieved in 2/3 (66%) patients at DL3, 1/3 (33%) patients at DL2, and 0/3 (0%) patients at DL1. All 3 patients who reached CR proceeded to allogeneic HCT. At the time of marrow recovery post-treatment, donor DNA in the blood and marrow was no longer detectable in any of the patients. Patients who achieved CR (n=3) had significantly higher levels of IL6 on day +1 (p=0.01) and a trend towards higher levels of IL2, IL15, IFN-gamma, and TNF-alpha in the first 7 days after DLI compared to patients who did not achieve CR. Conclusions Induction chemotherapy followed by CD8-depleted HLA mismatched, unrelated donor DLI was feasible and well tolerated with no DLTs, confirming the RP2D of 5x107 CD4 cells/kg. At DL3, 2 of 3 patients achieved CR, including one with TP53 mutation and complex cytogenetics, which is generally resistant to induction chemotherapy and compares favorably to 20-30% historical CR rates with chemotherapy alone in those pts. Mechanistic studies correlated increased post-treatment cytokine levels with depth of remission. Assessment of markers of immune tolerance are pending and will be presented at the conference. Based on these promising results, this novel cellular therapy is being investigated now in a phase II clinical trial at the Moffitt Cancer Center. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Thawing and infusion of CAR-T cell products

Some patients with hematologic malignancies may benefit from chimeric antigen receptor-T (CAR-T) cell treatment. Usually, the manufacture of this cells is carried out by centralized and specialized cell therapy laboratories that cryopreserve the CAR-T cells in bags and send it for infusion in the patient treatment center. In this context, the thawing and infusion of the CAR-T cell products are critical steps in this custody chain. This article aims to discuss the most relevant points related with the CAR-T cell thawing and infusion and proposes a kind of standard operational protocol (SOP) for these processes.

Meeting abstracts from the 15th international conference on neonatal and childhood pulmonary vascular disease

Meeting abstracts from the 15th international conference on neonatal and childhood pulmonary vascular disease

Study of a new device for washing and concentrating cryopreserved hematopoietic stem cells and mononuclear cells: a single center experience

Background aimsCryopreserved cellular products, as parts of hematopoietic progenitor cell (HPC) transplants, mononuclear cell reinjections for donor lymphocyte infusion or extracorporeal photopheresis, can be washed before being reinjected into the patient or infused directly, depending on local practices. The aim of washing is to reduce the incidence and severity of adverse reactions (ARs) due to the dimethyl sulfoxide (DMSO) used as a cryoprotective agent and other factors, such as dead cell debris. At the authors’ cell therapy laboratory (CTL) in Poitiers, France, as in 76% of Etablissement Français du Sang (EFS) CTLs, all cryopreserved products undergo thawing in a water bath followed by washing with the COBE 2991. As this device will soon cease to be available, an alternative process needs to be assessed. MethodsThe authors compared two closed systems: the authors’ semi-automatic system using the traditional centrifugation method (COBE 2991) and an automated device using spinning membrane filtration (Lovo). A total of 72 HPC bags available for research were used. The authors first performed a paired comparison, processing one or two HPC bags washed by each device. A second study was carried out to compare two different washing solutions generally used by EFS CTLs along with variable storage conditions. Finally, the authors studied the efficiency of the Lovo with three or four thawed bags. The main parameters studied were viable CD34+ cell recovery and viability, CD3+ cell recovery, stability up to 6 h after washing, DMSO elimination and center feasibility. ResultsThe Lovo device showed better CD34+ cell recovery compared with the COBE 2991 while maintaining CD34+ viability and stability over 6 h. Moreover, Lovo efficiency seemed to be independent of the number of thawed bags processed and washing solution used in the authors’ study. CD3+ cell recovery met the authors’ internal specifications (cell recovery >50%), with similar results seen when processing with either the COBE 2991 or Lovo. Additionally, on average, 97% of DMSO was removed after washing with Lovo, minimizing the risk of ARs. The storage conditions post-processing indicated preferred storage conditions of 7 ± 3°C. Finally, if processing time seemed shorter using COBE 2991 for one bag washed, the Lovo device required only one staff member regardless of the number of HPC bags processed. ConclusionsThe Lovo device seems to provide an opportunity to standardize HPC processing, ensuring patient safety, with, on average, 97% of DMSO removed, while improving recovery of cells of interest and maintaining viability over time in case of delayed transplant. The Lovo device consequently seems to be a serious alternative to the COBE 2991.

Allogeneic transplant procurement in the times of COVID-19: Quality report from the central European cryopreservation site

BackgroundBecause of limitations of transportation imposed by the COVID-19 pandemic, current recommendation calls for cryopreservation of allogeneic stem cell transplants before patient conditioning. A single cell therapy laboratory was selected to function as the central cryopreservation hub for all European registry donor transplants intended for the Australian-Pacific region. We examined properties of these transplants to ascertain how quality is maintained.MethodsWe analyzed 100 pandemic-related allogeneic mobilized blood-derived stem cell apheresis products generated at 30 collection sites throughout Europe, shipped to and cryopreserved at our center between April and November of 2020. Products were shipped in the cool, subsequently frozen with DMSO as cryoprotectant. Irrespective of origin, all products were frozen within the prescribed shelf-life of 72 h.ResultsPrior to cryopreservation, viable stem cell and leukocyte count according to the collection site and our reference laboratory were highly concordant (r2 = 0.96 and 0.93, respectively) and viability was > 90% in all instances. Median nominal post-thaw recovery of viable CD34+ cells was 42%. Weakly associated with poorer CD34+ cell recovery was higher leukocyte concentration, but not time lag between apheresis or addition of cryopreservant, respectively, and start of freezing. The correlation between pre- and post-thaw CD34+ cell dose was high (r2 = 0.85), hence predictable. Neutrophil and platelet engraftment were prompt with no evidence of dose dependency within the range of administered cell doses (1.31–15.56 × 106 CD34+ cells/kg).ConclusionsGeneral cryopreservation of allogeneic stem cell transplants is feasible. While more than half of the CD34+ cell content is lost, the remaining stem cells ensure timely engraftment.

Fresh Versus Cryopreserved Peripheral Stem Cell for Autologous Transplantation in Multiple Myeloma: An Analysis of Short-Term Outcomes

BACKGROUND: The current standard practice in North America is to cryopreserve mobilized autologous stem cells for use after high dose chemotherapy for multiple myeloma. Fresh autologous stem cell infusions are used infrequently around the world for this same purpose with comparable efficacy. This study aims to compare short term outcomes after autologous transplant with fresh versus (vs) cryopreserved (cryo) stem cells. METHODS: Sixty-four (64) consecutive patients who underwent autologous stem cells for multiple myeloma at our center between June 2018 and December 2019 were grouped based on whether fresh or cryopreserved product was used for transplantation. The primary outcomes included the number of days until neutrophil and platelet recovery. The secondary outcome was length of hospital stay (LOS). After mobilization with G-CSF with or without plerixafor, peripheral blood stem cells were harvested over 1 day. For the fresh group, the collected cells were stored at 4 °C in the cell therapy laboratory for up to 48 hours. Meanwhile, the patient underwent high-dose melphalan treatment, 24 hours following which, the autologous stem cells were infused. In the cryopreserved group, the cells were cryopreserved for variable lengths of time until their infusion 48 hours after high-dose melphalan therapy. RESULTS: Thirty-two (32) patients received fresh product and 32 received cryopreserved product. Baseline characteristics were similar between the groups and summarized in Table 1. Median CD34 count at infusion was 3.90 (range 2.83 to 5.68) million/kg in the fresh group and 4.32 (range 2.46 to 7.41) million/kg in the cryopreserved group, which was not statistically different (p= 0.07). Thirty patients in the fresh group and 27 in the cryo group received plerixafor. All the patients engrafted. The median time to neutrophil engraftment was 11 days (range 10-13 days) in the fresh group compared to 12 days (range 10-18 days) in the cryopreserved group (10.71±0.77 vs 12.18±1.71 days. P= 0.90). ANC &amp;lt;500 developed earlier in the fresh group vs cryopreserved group at median of 5 vs 8 days respectively (5.46±0.8 vs 7.78±1.07 days. P = &amp;lt;0.0001) and the duration of neutropenia was not significantly longer (6.25±1.04 vs 6.4±1.96 days. P = 0.70). The median time to platelet engraftment was 18 days (range 14-21 days) in the fresh group versus 23 days (range 16-50 days) in the cryopreserved group (16.25±4.72 vs 23±6.53 days. P &amp;lt; 0.0001). The median LOS was 13 days (range 10-21 days) in the fresh group versus 17 days (range 13-30) in the cryopreserved group (13.25±2.83 vs 16.59±4.18 days. P = 0.0004). CONCLUSIONS: Time to platelet engraftment and length of stay in the hospital were significantly shorter with use of fresh autologous stem cells compared to cryopreserved cell for transplantation for multiple myeloma. Despite earlier development of neutropenia in the fresh group, time to engraftment and duration of neutropenia were not significantly longer. CD34 cell dose was actually lower in the fresh group, with the difference approaching statistical significance. The use of fresh autologous product is both feasible and safe. Early hematologic recovery, particularly in terms of platelet engraftment with use of fresh product has further helped with early discharge from the transplant clinic to the referring oncologists. It has also led to subsequent utilization of this approach in performing outpatient autologous transplants at our center. Table Disclosures Randolph: Bristol Myers Squibb, Celgene: Speakers Bureau. Khaled:Celgene, Seattle Genetics, Jazz, Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Translating the Rapidly Evolving CAR-TCR Research Portfolio to Clinical Practice, a Single Institution Experience at an Academic Medical Center

Topic Significance/Background This is an unparalleled time in cancer research, particularly with the use of cellular therapeutics to target both solid and liquid tumors. This science is challenging the boundaries of clinical care to update guidelines, streamline processes, and integrate clinical and research teams. The University of Kansas Cancer Center was launched into the cell therapy space when we enrolled the first adult patient on Novartis Juliet CAR-T trial for DLBCL. Our team learned that inpatient and outpatient infrastructure and multidisciplinary collaboration are essential to promoting quality clinical care to complement this innovative new field of cellular immunotherapy. As of September 2019, our center has 19 active cell therapy trials, 10 studies in start-up, and 27 in the research pipeline. We have had the opportunity to participate in clinical trials assessing safety and efficacy of TILs, CAR-T and TCR in the cell therapy space, along with expanded umbilical, placental, and mesenchymal stem cell products. In order to optimally contribute in this dynamic and vastly changing landscape, it was essential for our teams, both research and clinical, to come together and develop a forum in which all new cell therapies are discussed and vetted. Intervention In order to facilitate this multi-disciplinary cell therapy forum, we instituted the Immune Effector Cell (IEC) Committee. The IEC committee started as a pre-start-up review panel that focused on feasibility of trial conductivity and ensuring appropriate resources and processes were in place for each trial. The IEC committee has now evolved into a formal Disease Working Group (DWG) responsible for the review of all open trials in the cell therapy space, deviation review and reporting, enrollment and accrual management, and patient safety in addition to feasibility and care algorithm management. The IEC DWG meets monthly. Members include clinicians, dedicated cell therapy research staff, pharmacists, laboratory personnel and cancer center leadership. Discussion/Implications The new era of cellular immunotherapy is challenging current clinical trial oversight and workflow development to ensure novel therapies are transitioned seamlessly into clinical care. Developing a structured, multidisciplinary IEC DWG with supervision over all cell therapy clinical research serves as a foundation for innovative, quality care. This forum's assessment of upfront workload promotes synergy with all teams supporting cell therapy including research, apheresis, cell processing, inpatient and outpatient clinical programs. The IEC DWG infrastructure is a supportive environment for solid tumor cell therapy to integrate into the existing clinical framework. Future work at our institution includes a standard site readiness tool to facilitate communication and integration.

Results of point-of-care manufacturing of bispecific chimeric antigen receptor (CAR) LV20.19 CAR-TCELLS in a phase i study for relapsed/refractory (R/R), non-hodgkin lymphoma (NHL)

Background & Aim Current approved CAR-T cell manufacturing employs off-site production, a labor-intensive process that requires several weeks and involves shipping back and forth to the production site. This production model may limit availability and accessibility to CAR-T cells and delay treatment for patient (pt) s with rapidly progressive disease. To address these limitations, we utilized the Miltenyi CliniMACS Prodigy, a GMP-compliant device, to produce CAR-T cells on-site for a first-in-human bi-specific CD19/20 (LV20.19) CAR-T Phase I trial. Methods, Results & Conclusion CAR-T cells were manufactured within our Cell Therapy Laboratory, an ISO7 facility. Pt's mononuclear cell apheresis was collected and loaded onto the Prodigy to isolate CD4/CD8 T cells. The T cells were suspended in TexMACS supplemented with human AB serum and IL-2, activated with TransACT, and then transduced by LV20.19 the next day. Culture wash and feedings were done during the production process, and the final cell product was harvested at day 14. The process was automated with limited machine/operator interactions. CAR-T expression was assessed by protein L staining. CAR-T cells were either administered fresh or cryopreserved for later infusion. Dose was escalated in a 3+3 manner from 2.5e+5 cells/kg to a target dose of 2.5e+6 cells/kg. CAR-T cells have been successfully generated for 11/11 enrolled pts thus far with no manufacture failures, and all products met release criteria. Median T cell recovery from the apheresis products was 59.5% with a median CD3+ purity of 97.2% (Figure 1). Final product CAR expression was at a median of 17.6%. A median of 4.7e+8 CAR T cells was obtained at harvest, exceeding the required target dose. CAR-T cells contained both CD4 and CD8 T cells with higher CD4 content. The majority of CAR-T cells showed an effector-memory phenotype. CAR-T cells were able to kill CD19+/20+ target cells and produced IFN-gin response to antigens. Among the 11 treated pts 8 received fresh CAR-T cells and 3 received CAR-T cells after cryopreservation. As no DLTs were observed at the 3 dose levels, a dose of 2.5e+6 cells/kg has been selected for further expansion. Clinical response shows 6 pts have achieved a CR at Day +28 with a duration of response between 2 to 14 months. In summary, initial results demonstrated feasibility for point-of-care manufacturing of bispecific LV20.19CAR-T cells with safe and promising efficacy in treating heavily pretreated pts with R/R B-cell NHL, which warrants further investigations.

Expandable Bioreactor for the culture of immune cells

Background & Aim With the recent successes of autologous immunotherapy, with 2 therapeutic products approved by the Food & Drugs Administration (FDA) since 2017 and more in the clinical pipeline, there is a need for cell and gene therapy companies to transition from lab scale production to mass manufacture. Traditionally, bioreactors have been routinely used in process development and scale up manufacturing of biologics. However, such bioreactor systems are not suitable for immunotherapy production as, beyond standard external factors such as shear stresses, immune cells are also sensitive to factors such as density and require cell-to-cell contact to proliferate well. Methods, Results & Conclusion A novel single use bioreactor for non-adherent cell culture has been developed in SIMTech (Patent Application WO2018/182533 A1) that allows for culturing cells at constant density while increasing volume of culture media to support cell proliferation. The bioreactor features two different chambers, one for media reserves and the other for cells. The two chambers are separated by a membrane that allow for fluidic exchange between these chambers. The cell chamber has a built-in expansion mechanism that can be adjusted as cells proliferate, thus allowing the user to adjust at any time point and maintain consistent culture density. The bioreactor has been validated with Jurkat cell line and T cells derived from commercial PBMCs with promising results; it was observed that the cultured cells exhibited high viability and selectivity. Jurkat cells had a 240-fold increase in cell number after 14 days in culture while T cells (from commercial PBMCs), had a 47-fold increase in cell number after 21 days in culture. A direct comparison study (1 bioreactor vs multiple 24-well plates for the same increase in cell number) also demonstrated a significant reduction in protocol steps and culture handling time. The bioreactor is currently being tested on donor PBMCs and other cell types, in collaborations with clinicians and cell therapy laboratories, in order to develop a GMP workflow for clinical use.

A Phase I Study to Assess the Safety and Cancer-Homing Ability of Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells in Men with Localized Prostate Cancer

Animal models show that systemically administered bone marrow‐derived mesenchymal stem cells (MSCs) home to sites of primary and metastatic prostate cancer (PC)—making them candidates to selectively deliver cytotoxic agents. To further assess this potential as a cell‐based therapeutic vehicle, a phase I study testing homing of systemically infused allogeneic MSCs preprostatectomy was conducted. The primary objective was to assess safety and feasibility and to determine if MSCs accumulate within primary PC tissue. MSCs were quantified using beads, emulsion, amplification, magnetics digital polymerase chain reaction (limit of detection: ≥0.01% MSCs) to measure allogeneic MSC DNA relative to recipient DNA. MSCs were harvested from healthy donors and expanded ex vivo using standard protocols by the Johns Hopkins Cell Therapy Laboratory. PC patients planning to undergo prostatectomy were eligible for MSC infusion. Enrolled subjects received a single intravenous infusion 4–6 days prior to prostatectomy. The first three subjects received 1 x 106 cells per kilogram (maximum 1 x 108 cells), and subsequent four patients received 2 x 106 cells per kilogram (maximum 2 x 108 cells). No dose‐limiting toxicities were observed and all patients underwent prostatectomy without delay. Pathologic assessment of prostate cores revealed ≥70% tumor involvement in cores from four subjects, with benign tissue in the others. MSCs were undetectable in all subjects, and the study was stopped early for futility. MSC infusions appear safe in PC patients. Although intended for eventual use in metastatic PC patients, in this study, MSCs did not home primary tumors in sufficient levels to warrant further development as a cell‐based therapeutic delivery strategy using standard ex vivo expansion protocols. stem cells translational medicine 2019;8:441–449

Automated Manufacturing of CD20.19 Bi-Specific Chimeric Antigen Receptor T (CAR-T) Cells at an Academic Center for a Phase I Clinical Trial in Relapsed, Refractory NHL

Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, but limitations exist with off-site commercial CAR-T production: (1) manufacturing can take several weeks and requires shipping patient cells; (2) off-site manufacturing limits treatment options for patients with rapidly progressive disease; (3) high cost of the products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy GMP-compliant device to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR). Using reagents from Miltenyi Biotec, production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory. Manufacturing time was 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) collected from patients by apheresis were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. The T cells were cultured in 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. Next day, lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3z and 4-1BB stimulatory domains was added to the cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on day 14. Eligible patients either received fresh CAR-T cells, or cells that had been cryopreserved and administered on a later date. CAR-T cell products have been successfully generated for all 8 patients enrolled thus far on the Phase 1 clinical trial with no production failures, and all products passed release criteria for infusion. Three patients received cryopreserved product and 5 patients received fresh product. Average T cell recovery from the apheresis cell products was 66.0% (56.0-76.0%), and the enriched T cells were 94.3% CD3+ (87.8-97.4%) (Table 1). Protein L staining of the final cell products indicated 19.6% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFNg upon antigen stimulation. An average yield of 5.27e+8 CAR T cells was obtained at harvest, exceeding the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with greater percentages of CD4 T cells. The majority of T cells (avg. 83.2%) had an effector-memory phenotype. In summary, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. A major clinical advantage of CAR-T cells generated on-site is flexibility in treatment, with either immediate (i.e., fresh) or later (cryopreserved/thaw) infusion, dependent on patient need.

Point-of-Care Manufacturing of CD20.19 Bi-Specific Chimeric Antigen Receptor T (CAR-T) Cells in a Standard Academic Cell Processing Facility for a Phase I Clinical Trial in Relapsed, Refractory NHL

Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, which has led to FDA approvals for two CAR-T products. However, limitations exist with commercial CAR-T centralized production: (1) off-site manufacturing can take several weeks and requires shipping from and to the treating facility; (2) off-site manufacturing limits treatment options for progressing patients; (3) high cost of the commercial products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy device, a GMP-compliant closed system, to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR).CAR-T manufacturing was performed exclusively using the CliniMACS Prodigy device and reagents obtained from Miltenyi Biotec. Production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory, an ISO7 air handling environment. Manufacturing was set at 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) were collected by apheresis, with a collection goal of 4 blood volumes to eliminate risk of a low CD3 yield in heavily pre-treated patients. Next, MNC were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. To start the culture process, enriched T cells were suspended in TexMACS medium supplemented with 3% human AB serum and 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. The following day (day 1), lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3ζ and 4-1BB stimulatory domains was added to the stimulated cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on Day 14. Protein L staining was used to detect expression of CD20.19 CAR on the T cells. On Day 14, eligible patients received fresh CAR-T cells, while for others the product was cryopreserved and administered on a later date.To date, the MCW Cell Therapy Laboratory has successfully generated CAR-T cell products for all 6 patients enrolled thus far on the Phase 1 clinical trial with no production failures (Table 1). Three patients received cryopreserved product and 3 patients received fresh product. The enriched T cells were 94.3% CD3+ (87.8-97.4%), and average T cell recovery from the apheresis cell products was 65.2% (54.2-80.0%). Protein L staining indicated 20.8% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFN-gamma in response to the same target cells. An average yield of 5.9e+8 (4.3-7.9e+8) CAR T cells was obtained at harvest, which exceeded the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with higher expression on CD4 T cells; average CAR-T CD4:CD8 ratio on the final products was 2.8. The majority of T cells (average of 81.5%) had an effector-memory phenotype. In-process testing performed on Day 8 of manufacturing demonstrated sufficient numbers of CAR-T cells needed for patient infusions were already present, and that the CAR-T cells only expanded an additional 1.9 to 3.5-fold between Days 8 and 14.In conclusion, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. Time to production was efficient (14 days), and patient-derived CAR-T cell products were reproducibly generated in a standard cell processing laboratory within an academic medical center. A major clinical advantage of CAR-T cells generated on-site is the flexibility in treatment. Patients can receive cells either immediately (i.e., fresh) or the cells can be cryopreserved for later infusion if the patient is not able to receive fresh cells. Based on our results, we intend to decrease the cell processing time to 10 days. [Display omitted] DisclosuresZhu:Lentigen Technology Inc., A Miltenyi Biotec Company: Research Funding. Shah:Juno Pharmaceuticals: Honoraria; Oncosec: Equity Ownership; Geron: Equity Ownership; Exelexis: Equity Ownership; Miltenyi: Other: Travel funding, Research Funding; Lentigen Technology: Research Funding. Schneider:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Keever-Taylor:Medical College of Wisconsin: Research Funding. Dropulic:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Hari:Bristol-Myers Squibb: Consultancy, Research Funding; Amgen Inc.: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Kite Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding. Johnson:Miltenyi: Research Funding.

Standard Processing of Apheresis Products for HSCT Provides Significant Cost Savings over Automated Processing without Impact on Time or Product Quality

Introduction: Automated cell manufacturing is becoming increasingly common in advanced manufacturing protocols in order to provide more linearity and quality control. However, most of the manufacturing performed within full-service cell therapy laboratories consists of routine processing for HSCT. Automated systems are also employed for basic cell processing although the value of these systems is not routinely assessed. We compared our standard method for cell washing and concentration using the Terumo 2991® to manual centrifuge-based processing autologous apheresis products, testing the hypothesis that manual centrifuge-based processing would decrease cost and staff time for processing of apheresis HSCT products.Methods: A total of 10 products were compared (9 myeloma and 1NHL). Using the Terumo 2991®, the collection bag was rinsed x2 with a Plamalyte A®/Sodium Citrate prior to automated centrifugation and the centrifuge module was rinsed in a similar manner following concentration and expression of the cell product. For centrifuge-based manual processing, the product was transferred from the collection bag to a transfer bag, centrifuged, and excess plasma expressed. Each product was diluted to 10% within 5 x 108WBC/ml with autologous plasma. Samples were obtained for cell counting, viability, and CD34 enumeration by flow cytometry at the beginning and end of processing and following cryopreservation/thawing and 7d storage. Hands-on time was assessed by an observer and materials cost was based on standard pricing for UAB Hospital. Statistical determinations consisted of mean, SD, and student's t-test.Results: The WBC/kg dose recovery was not significantly different between automated processing (x̅=0.894±0659) and manual centrifuge-based processing (x̅=0.938±0.0696, p=0.163). The CD34+/kg dose recovery also did not show a significant difference between automated (x̅=0.875±0.0920) and centrifuge-based processing (x̅=0.937±0.0897, p=0.302). Post processing total viability, however, was significantly improved for the manual centrifuge-based processing (x̅=0.940±0.0260 vs. x̅=0.890±0.0390 p=0.031). Post thaw and wash CD34+ viability showed no difference between automated processing (x̅=0.8260, SD=0.0840) and manual centrifuge-based processing (x̅=0.810, SD=0.0260, p=0.592). Patients that were transplanted the manual centrifuge-based processed products engrafted between days 11 and 13. The institution engraftment benchmark is 15 days with an average of 11.3 days. Hands-on time for processing was also not significantly different between the two methods (x̅=27.89±7.897min for automated vs x̅=34.88±9.975min for standard, p=0.169). The combined cost for labor and supplies for automated Terumo® 2991 is $174.01 per product processed and $63.58 for manual centrifuge-based product processed. This produces a cost saving of $110.43 per product processed.Conclusions: Taken together, these findings show that manual centrifuge-based processing creates significant cost savings and improved product viability for the same hands-on processing time and with no significant total or CD34+ cell loss. With the current financial trends in healthcare, significant savings in standard care is important for the laboratory and hospital providing care. DisclosuresLamb:Incysus Therapeutics Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Automation: what will the cell therapy laboratory of the future look like?

Automation: what will the cell therapy laboratory of the future look like?

226 - Developing a robust competency program for the GMP novel cell therapy laboratory in the cell manipulation core facility laboratory at Dana-Farber Cancer Institute, Boston

226 - Developing a robust competency program for the GMP novel cell therapy laboratory in the cell manipulation core facility laboratory at Dana-Farber Cancer Institute, Boston