Total Cell Dose Research Articles

In vivo effects of cell seeding technique in an ex vivo regional gene therapy model for bone regeneration.

When delivering cells on a scaffold to treat a bone defect, the cell seeding technique determines the number and distribution of cells within a scaffold, however the optimal technique has not been established. This study investigated if human adipose-derived stem cells (ASCs) transduced with a lentiviral vector to overexpress bone morphogenetic protein 2 (BMP-2) and loaded on a scaffold using dynamic orbital shaker could reduce the total cell dose required to heal a critical sized bone defect when compared with static seeding. Human ASCs were loaded onto a collagen/biphasic ceramic scaffold using static loading and dynamic orbital shaker techniques, compared with our labs standard loading technique, and implanted into femoral defects of nude rats. Both a low dose and standard dose of transduced cells were evaluated. Outcomes investigated included BMP-2 production, radiographic healing, micro-computerized tomography, histologic assessment, and biomechanical torsional testing. BMP-2 production was higher in the orbital shaker cohort compared with the static seeding cohort. No statistically significant differences were noted in radiographic, histomorphometric, and biomechanical outcomes between the low-dose static and dynamic seeding groups, however the standard-dose static seeding cohort had superior biomechanical properties. The standard-dose 5 million cell dose standard loading cohort had superior maximum torque and torsional stiffness on biomechanical testing. The use of orbital shaker technique was labor intensive and did not provide equivalent biomechanical results with the use of fewer cells.

Comparison of Various Stem Cell Mobilization Regimens for Multiple Myeloma - a 15-Year Retrospective Institutional Analysis from India

Introduction For multiple myeloma (MM), Cyclophosphamide (Cy) based chemo-mobilization offers advantage in term of higher stem cell yield and can overcome adverse impact of prior lenalidomide therapy on stem cell harvest. However, considering need for hospital stay and risk of febrile neutropenia, steady state mobilization with G-CSF and pre-emptive plerixafor (PEF) is becoming increasingly popular. Until few years back, cost of PEF was prohibitive thereby limiting its use and prompting a search for alternative cheaper drugs. Mouse models have shown that Proteasome Inhibitors aid in hematopoietic stem-cell (HSC) mobilization by down-regulating very-late antigen-4 (VLA-4) on bone marrow stromal cells which interacts with vascular cell adhesion molecule (VCAM-1) on HSC. We previously reported that bortezomib (Bort) in combination with Cy-G-CSF showed a trend towards higher CD34 yield and reduced need for apheresis sessions, without additional toxicity. There is limited data comparing mobilization regimens, especially from low-middle income countries where HSC collection may not be done after 4-6 cycles of first-line therapy. We hereby report our 15-year experience of various mobilization regimens for MM. Methods All patients with MM who underwent HSC mobilization from September 2007 - December 2022 were included in this analysis. Various mobilization regimens [Group 1 - G-CSF + Bort (August 2018 - December 2022); Group 2 - G-CSF + plerixafor (August 2018 - December 2022); Group 3 - Bort-Cy-GCSF (April 2016 - July 2018); Group 4 - Cy + G-CSF (September 2007 - March 2016)] were used as per respective time-periods (Table 1). Our aim was to collect CD34 cell dose of 5million/kg to suffice for 2 transplants. G-CSF (5 mcg/kg subcutaneously BD) was given on days 1-5 (Groups 1 and 2). Bort (1.3mg/m 2 iv / sc) (Group 1) or PEF (Group 2) were given on day 4 twelve hours prior to planned apheresis on day 5. Apheresis was done on day 5, and additionally on day 6, if first harvest was inadequate for 2 transplants. In group 3, Bort (1.3mg/m 2 iv) was given on days-1,4,8,11, with Cy (1gm/m 2 iv) on days 8 and 9, followed by G-CSF from day 11 onwards. Cy (1gm/m 2 iv) was given on day 1 and 2 (Group 4), followed by G-CSF from day 4 onwards. Stem cell harvest was done in groups 3 and 4 once peripheral blood CD34 (PB CD34) was >20/µL. In groups 3 and 4, if PB CD34 was <20/µL for 2 subsequent days, PEF was used according to physician's discretion, if available. Similarly in Group 1, if PB CD34 was between 10-20/µl on day 4, PEF was used according to physician's discretion. PEF was used in any group after the first harvest if the cell dose was deemed inadequate, at physician's discretion. Primary objective was to determine proportion of patients with CD34 + dose ≥5x10 6/kg in first apheresis in various groups. Secondary objectives were to determine total median CD34 + dose (x10 6/kg) collected, median CD34 + (x10 6/kg) in first apheresis, and frequency of mobilization failure. Mobilization failure was defined as total collected CD34 + dose of <2x10 6/kg or abandoned harvest attempt anticipating a poor collection.Analysis was done as per planned regimen (intention to treat analysis). Results Total 205 patients were analyzed. Median age was 48 years (27-65 years) and 143 (70%) were males (Table 1). Median day of first collection in groups 3 and 4 was day10 from the first dose of cyclophosphamide. Percentage of patients who collected ≥5x10 6 CD34 + cells/kg in first apheresis were 26%, 53%, 69% and 63%, respectively in Groups 1-4 (p=0.0001) (Table 1). CD34+ cell-dose in first apheresis and total dose collected are shown in Table 1. In comparison to groups 1 and 2, both chemo-mobilization groups (group 3 and 4) had a significantly higher total cell dose (Figure 1). In comparison to group 1, both Cy chemo-mobilization groups had a significantly higher cell dose in first harvest, but there was no significant difference between group 2 and group 4 (p=0.23) (Figure 1). PEF was required additionally in 47%, 22%, and 7% of patients in groups 1, 3 and 4 respectively (P<0.00001). Amongst transplanted patients, there was no difference in neutrophil or platelet engraftment and the frequency of engraftment syndrome in various groups. Conclusion Cy-based chemo-mobilization regimens have advantage of higher total stem cell yield, while they are equivalent to G-CSF + Plerixafor for harvest in single apheresis. Addition of Bortezomib to Cyclophosphamide may help to increase stem cell yield.

An evidence-based and risk-adapted GSF versus GSF plus plerixafor mobilization strategy to obtain a sufficient CD34+ cell yield in the harvest for autologous stem cell transplants

BackgroundPlerixafor is a bicyclam molecule with the ability to reversibly bind to receptor CXCR-4 thus leading to an increased release of stem cells (SC) into the circulation. This study aims to evaluate the efficacy of G-CSF plus plerixafor versus G-CSF alone mobilizing regimens on the basis of CD34+ cell yield and engraftment kinetics following hematopoietic SC transplants. MethodsThe study incorporated 173 patients with plasma cell neoplasms (PCN), Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), undergoing mobilization and following autologous SC-transplant. For patients with mobilization failure and those predicted to be at risk of harvesting inadequate CD34+ yields (poor-responders), plerixafor was administered. Data was collected and compared in relation to the harvesting protocols used, cell quantification, cell-engraftment potential and overall clinical outcome. ResultsA total of 101 patients received plerixafor (58.4 %) and the median CD34+increase was 312 %. Chemotherapy-mobilized PCN-patients required less plerixafor administration (p = 0.01), no difference was observed in lymphoma groups (p = 0.46). The median CD34+cell yield was 7.8 × 106/kg bm. Patients requiring plerixafor achieved lower, but still comparable cell yields. Total cell dose infused was in correlation with engraftment kinetics. Patients requiring plerixafor had delayed platelet engraftment (p = 0.029). ConclusionsAdequately selected plerixafor administration reduces "mobilization-related-failure" rate and assure a high-level cell dose for SC transplants, with superior "therapeutic-potential" and safety profile. The mobilization strategy that incorporates "just-in-time" plerixafor administration, also leads to a reduction of hospitalization days and healthcare resource utilization. For definitive conclusions, further controlled/larger clinical trials concerning correlation of CD34+ cell count/yield, with hematopoietic reconstitution are required.

γ-Secretase inhibitor in combination with BCMA chimeric antigen receptor T-cell immunotherapy for individuals with relapsed or refractory multiple myeloma: a phase 1, first-in-human trial

γ-Secretase inhibitors (GSIs) increase B cell maturation antigen (BCMA) density on malignant plasma cells and enhance antitumour activity of BCMA chimeric antigen receptor (CAR) T cells in preclinical models. We aimed to evaluate the safety and identify the recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat (LY3039478) for individuals with relapsed or refractory multiple myeloma. We conducted a phase 1, first-in-human trial combining crenigacestat with BCMA CAR T-cells at a single cancer centre in Seattle, WA, USA. We included individuals aged 21 years or older with relapsed or refractory multiple myeloma, previous autologous stem-cell transplant or persistent disease after more than four cycles of induction therapy, and Eastern Cooperative Oncology Group performance status of 0-2, regardless of previous BCMA-targeted therapy. To assess the effect of the GSI on BCMA surface density on bone marrow plasma cells, participants received GSI during a pretreatment run-in, consisting of three doses administered 48 h apart. BCMA CAR T cells were infused at doses of 50 × 106 CAR T cells, 150 × 106 CAR T cells, 300 × 106 CAR T cells, and 450 × 106 CAR T cells (total cell dose), in combination with the 25 mg crenigacestat dosed three times a week for up to nine doses. The primary endpoints were the safety and recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat, an oral GSI. This study is registered with ClinicalTrials.gov, NCT03502577, and has met accrual goals. 19 participants were enrolled between June 1, 2018, and March 1, 2021, and one participant did not proceed with BCMA CAR T-cell infusion. 18 participants (eight [44%] men and ten [56%] women) with multiple myeloma received treatment between July 11, 2018, and April 14, 2021, with a median follow up of 36 months (95% CI 26 to not reached). The most common non-haematological adverse events of grade 3 or higher were hypophosphataemia in 14 (78%) participants, fatigue in 11 (61%), hypocalcaemia in nine (50%), and hypertension in seven (39%). Two deaths reported outside of the 28-day adverse event collection window were related to treatment. Participants were treated at doses up to 450 × 106 CAR+ cells, and the recommended phase 2 dose was not reached. Combining a GSI with BCMA CAR T cells appears to be well tolerated, and crenigacestat increases target antigen density. Deep responses were observed among heavily pretreated participants with multiple myeloma who had previously received BCMA-targeted therapy and those who were naive to previous BCMA-targeted therapy. Further study of GSIs given with BCMA-targeted therapeutics is warranted in clinical trials. Juno Therapeutics-a Bristol Myers Squibb company and the National Institutes of Health.

A Phase I Clinical Trial of Point-of-Care Manufactured Fresh Anti-CD19/20/22 Chimeric Antigen Receptor T Cells for Treatment of Relapsed or Refractory Lymphoid Malignancies (Non-Hodgkin Lymphoma, Acute Lymphoblastic Leukemia, Chronic Lymphocytic Leukemia, B Prolymphocytic Leukemia)

Background: CD19-targeted CAR-T cells have transformed the treatment of relapsed/refractory B-cell malignancies. However, relapse occurs in over 50% of patients treated with commercial CD19 directed CAR-T due to antigen escape from selective immune pressure either on the target antigen or due to exhaustion and lack of persistence of CAR-T cells. Hence there is an unmet need for next-generation CAR-Ts that have broad antigen recognition and novel costimulatory domains to promote survival and decrease exhaustion of CAR-Ts. Targeting three B-cell antigens simultaneously is a novel approach to prevent antigen-negative relapse while inclusion of the OX40 domain will promote CAR-Ts persistence and expansion. Whether targeting three commonly expressed antigens is feasible or safe and will result in durable remissions is not known. We hypothesize that this trispecific targeting of CD19/20/22 CAR-T construct will be safe and effective in patients with relapsed, refractory B-cell malignancies. Study Design and Methods: This parallel group phase 1 study follows a 3+3 phase 1 design to determine the safety of the infusion of point-of-care manufactured fresh CAR-T cells targeting CD19/20/22 and to find the recommended phase II dose for this cellular therapy. CAR-T cells will be manufactured over 6 days. Patients will be enrolled in one of the two cohorts: Cohort A (single infusion): Non-Hodgkin's Lymphoma (NHL) with lesions <= 5 cm; Indolent lymphomas; B-PLL (B-Pro lymphocytic leukemia); Chronic Lymphocytic Leukemia -CLL (without Richter's transformation), Cohort B (split infusion): B-ALL (B-Acute lymphoblastic leukemia); Lymphoid blast crisis of Chronic myeloid leukemia; CLL (with Richter's transformation); NHL with lesions > 5 cm and/or lymphoblastic lymphoma, or NHL with circulating lymphoma cells. Patients who are CAR-naïve will be enrolled in this study. The study involves infusion of anti-CD19/20/22 CAR-T cells administered after an immunosuppressive conditioning regimen of fludarabine and cyclophosphamide. Cyclophosphamide 60 mg/kg will be infused intravenously over 60 minutes on day -6. Fludarabine 25 mg/m2 will be infused intravenously daily over 30 minutes on days -5 to -3. In each group, patients will be enrolled sequentially to each dose level, starting with dose level 1. Three CAR-T dose-levels: 5x105, 1x106 & 2x106 cells/kg/dose will be explored to determine MTD. For cohort B patients, the total CAR-T cell dose infused, will be divided in two doses, dose 1 (day 0) is 40% of the total dose and dose 2 (day 7) is 60% of total dose. The minimum number of subjects enrolled in the complete study will be 18 (9 per cohort) and the maximum number in dose escalation will be 36 (18 per cohort). Patients in each cohort will be monitored for 30 days before enrollment of next patientTwo patients have been enrolled in the trial and received fresh infusion of Anti CD19/20/22 CAR-T cells and accrual is ongoing. Updates on the initial cohort of patients will be presented at the meeting. ClinicalTrials.gov NCT: NCT05418088 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Incidence and risk factors for graft failure in the modern era of cord blood transplantation.

Graft failure (GF) after cord blood transplant (CBT) has decreased with improved supportive care and cord selection strategies. We aimed to evaluate cord blood selection and factors associated with retransplantation on the incidence of GF, determine risk factors for GF including host antibodies to Kell antigen and evaluate survival after GF. We retrospectively reviewed 84 patients who underwent CBT at the University of Oklahoma between 2000 and 2016 and compared outcomes in patients with/without engraftment by Day 28. The nonengraftment cohort was further divided into patients who underwent retransplantation. Kaplan-Meier curves with log-rank tests were calculated to assess the association between mortality and engraftment. Engraftment following CBT was high at 81%, with 52% engrafting by Day 28 and an additional 29% engrafting by a median of 36 days. Retransplantation led to 88% engraftment at a median of 53 days. Overall, 75% of the 40 patients who did not engraft by Day 28 died. Female sex and total nucleated cell count < 3.5/kg were significantly associated with lack of engraftment and higher mortality. Antibodies to Kell fetal antigen were not identified. Retransplantation by Day 28 for primary GF conferred a survival advantage. This study demonstrates that failure to engraft by 28 days was associated with increased mortality, and risk was mitigated with early retransplantation. Female sex and low total cell dose were associated with increased mortality. Early identification of GF coupled with early retransplantation can reduce mortality in CBT.

Large-scale manufacturing and characterization of CMV-CD19CAR T cells

BackgroundAdoptive transfer of CD19-specific chimeric antigen receptor (CD19CAR) T cells can induce dramatic disease regression in patients with B cell malignancies. CD19CAR T cell therapy may be limited by insufficient...

CD11c+ dendritic cells mediate antigen-specific suppression in extracorporeal photopheresis.

Extracorporeal photopheresis (ECP) represents one of the most widespread and effective cell therapies for graft-versus-host disease and other T cell-mediated disorders. However, the key factors affecting the therapeutic efficacy of ECP remain unclear. We hypothesized that therapeutic effects are mediated by ECP-treated antigen-presenting dendritic cells (DC). To test this hypothesis, we used the experimental model of contact hypersensitivity (CHS). The ECP's therapeutic activity improved when the total cell dose of the ECP-treated cells was increased. We used different haptens during sensitization to demonstrate that the anti-inflammatory activity of ECP is antigen-specific. This confirmed the hypothesis that professional antigen-presenting cells are involved in the mode of action. Also, the ECP's therapeutic activity was abrogated by the depletion of CD11c+ DC, which represents fewer than 1% of all the ECP-exposed cells. Finally, we confirm the critical importance of CD11c+ DC for ECP activity by showing that only a few purified CD11c+ DC are sufficient to mediate its therapeutic effect. The finding that ECP-treated, physiological antigen-presenting DC alone mediate antigen-specific modulation of a pathological immune response may result in better-targeted interventions when treating patients.

Peripheral Blood to Marrow Collection White Cell Count Ratio As an Indicator of Harvest Quality and Transplant Outcomes in G-CSF-Primed Marrow Grafts

Background: G-CSF-primed bone marrow (G-BM) is used in some centers as the product of choice in related donor transplants because of lower rates of GVHD and fast engraftment (Morton 2001; Deotare et al. 2015). Marrow collections substantially diluted with peripheral blood may, however, compromise the GVHD-sparing effect of G-BM. Concerns over the variability in the quality of bone marrow harvest have been reported (Spitzer et al. 1994; Prokopishyn et al. 2019). We explored the role of a simple novel quality indicator, i.e peripheral blood to marrow harvest WBC ratio, as a predictor of GVHD risk. Materials and methods: We analysed 156 successive bone marrow harvests for patients suffering from severe thalassemia using G-BM donated by fully matched (10/10 or 12/12) related donors (143 siblings and 13 mothers) at Sankalp-People Tree Centre for Paediatric BMT - Bangalore (80 patients) and Sankalp-CIMS Centre for Paediatric BMT - Ahmedabad (76 patients). G-CSF priming was done for 5 days before the transplant with 10 mcg/kg/day in 2 divided subcutaneous doses and peripheral counts were obtained within 24 hours of collection. G-BM was collected in bags with 20 ml 50 U/ml heparin for the first 200 ml and further 10ml/100ml of marrow. We defined quality of marrow as follows: Quality of Marrow = WBC count of G-CSF primed BM / Peripheral WBC count of donor Quality of Marrow &lt; 0.9 was categorised as poor as it signifies that the concentration of WBCs in bone marrow is same or lesser than the pre-harvest peripheral counts. EBMT criteria was used for grading GVHD. We calculated the odds of aGVHD based upon the quality of marrow. Beside marrow quality, the sex of the recipient seemed to impact GVHD rates and thus regression analysis was used to study the impact of G-BM quality independent of other factors. Statistics were performed on R software (version 3.5.x). Mann Whitney and Fisher's exact tests were used to compare numeric and categorical data respectively. Results: Of the 156 harvests, 18 had poor marrow quality; 33% (6 of 18) of these developed acute GVHD grade II-IV as opposed to 9% (13 of 138) (see Figure 1). Between those who developed GVHD and those who did not, marrow quality (p=0.012), patient sex (p=0.006) and sex mismatch status (p=0.014) were significantly different. Differences in total cell dose (p=0.65), transplant centre (p=0.62), bone marrow cell count (p=0.14) and peripheral cell count before harvest (p=0.72), donor age at BMT (p=0.42), relationship with the donor (p=1.0) and average volume of marrow per draw of syringe (p=0.23) showed no statistical significance. Variables with significant difference (sex mismatch and bone marrow quality) were entered in the Cox's proportional hazards model for acute GVHD II-IV which showed that poor marrow quality is the most significant independent risk associated with a hazard ratio of 3.6 (p=0.009), followed by male to male transplants which were associated with a hazard ratio of 10.6 (p=0.03) See Table 1. Further, poor quality marrow was associated with lower cell count in marrow (p&lt;0.001), higher peripheral counts (p&lt;0.001), higher collection volume (p=0.035) and was seen more in one centre compared to the other (p&lt;0.001). Donor age (p=0.81), donor's relationship (p=1.0), donor sex (0.62), donor's carrier status (p=0.64), donor weight (p=0.85) and average volume of marrow per draw of syringe (p= 0.76) were not statistically significant. Discussion: The role of tolerance induced by mesenchymal stem cells in reduction (Aggarwal 2005) and even the treatment of GVHD (Le Blanc et al. 2004; Baron and Storb 2012) has been reported elsewhere and supports our findings though this is yet to be established (Fisher et al. 2019). Simple quality control of BM could be highly beneficial in all settings in minimising acute GVHD. This is particularly important in low- and middle-income countries where GVHD can be very challenging (Saleem et al. 2018) and is associated with high costs of management and prolonged hospitalisation (Grubb et al. 2016). Our indicator is accurately measurable, inexpensive, precise and timely; further it does not involve any sophisticated equipment (flow cytometry for CD counts), thus may be widely applicable and usable. Conclusion: Quality of BM harvest, as indicated by PB to BM WBC ratio, is a simple, effective and inexpensive informative indicator in the context of G-BM. Poorer quality of marrow collection may be contributing to increased risk of acute GVHD. Disclosures No relevant conflicts of interest to declare.

A Retrospective Analysis of Safety and Efficacy of Wharton's Jelly Stem Cell Administration in Children with Spina Bifida.

The aim of this paper was to describe the outcome of therapeutic administration of mesenchymal stem cells (MSC) obtained from Wharton's jelly (WJ-MSCs) in paediatric patients with spina bifida (SB) during a medical therapeutic experiment. We retrospectively analysed the records of twenty-eight patients aged 1-18years (median age 4years) recruited in daily clinical practice. Each patient received 0.9-5.0 × 106 WJ-MSCs/kg (median 2.6 × 106 WJ-MSCs/kg) administered in 1-5 injections as an experimental treatment for SB (allogenic administration). All the patients were examined by the same neurologist (study investigator, SI) on the day of each infusion. Based on the neurological examination, the SI used a six-point Likert scale to assess the quality of life and self-service of each patient. Twenty-six follow-up observations after MSC administration were analysed retrospectively. In addition, the assessments of the parents and other healthcare professionals were obtained for 5 patients and compared with the SI's assessment. Twenty-one of 26 patients (81%) experienced some improvement in their health status. Twenty-one (81%) patients experienced increased quality of life (median 2.0) and 10 patients (38%) achieved a slight increase in their self-service level (median 1). Improvement was achieved in 12 out of 17 areas. Five were significant in low-power sign test: muscle tension, muscle strength, gross motor development, micturition/defecation control, and cognitive functions. Adverse events were mild and temporary. Age, body mass, single dose or poor response after the first administration were not significant predictors of later response to treatment in contrast to the total cell dose per one kg in the whole treatment course. WJ-MSC administration is a safe and effective procedure that improves motor functions, micturition/defecation control, and cognitive functions, and improves the quality of life in children with SB.

Efficiency of CAR-T Therapy for Treatment of Solid Tumor in Clinical Trials: A Meta-Analysis.

Background Chimeric antigen receptor T (CAR-T) cell therapy has achieved unprecedented success among hematologic tumors, but its role in treating solid tumors is still unclear. Methods A comprehensive search of electronic databases up to June 1, 2018, was carried out by two independent reviewers. We included studies which focused on the association between CAR-T cell therapy and patient response rate and survival time in solid tumors. Results 22 studies with 262 patients were included in our meta-analysis. The overall pooled response rate of CAR-T cell therapy was 9% (95% confidence interval (CI): 4-16%). Subgroup analysis (analyses) demonstrated that CAR-T therapy could perform its best therapeutic effect on neuroblastoma, while barely works among gastrointestinal malignancies. Moreover, the treatment efficacy was not significantly impacted by different treatment strategies (lymphodepletion before T cell infusion, transfection method, cell culture duration, persistence of CAR-T cells, transfection efficacy, total cell dose, and administration of IL-2). Only T cell culture duration was associated with better clinical prognosis. Conclusions Although CAR-T cell therapy did not have satisfactory responses in solid tumors, researchers were still holding an optimistic attitude towards its future efficacy with more modifications of its structure.

Fully Human Bcma Targeted Chimeric Antigen Receptor T Cells Administered in a Defined Composition Demonstrate Potency at Low Doses in Advanced Stage High Risk Multiple Myeloma

Fully Human Bcma Targeted Chimeric Antigen Receptor T Cells Administered in a Defined Composition Demonstrate Potency at Low Doses in Advanced Stage High Risk Multiple Myeloma

The severe cytokine release syndrome in phase I trials of CD19-CAR-T cell therapy: a systematic review.

CD19 chimeric antigen receptor (CAR) T cell therapy has shown impressive results in treating acute lymphoblastic leukemia (B-ALL), chronic lymphoblastic leukemia (B-CLL), and B-cell non-Hodgkin lymphoma (B-NHL) over the past few years. Meanwhile, the cytokine release syndrome (CRS), which could be moderate or even life-threatening, has emerged as the most significant adverse effect in the clinical course of this novel targeting immunotherapy. In this systematic review, we analyzed the incidence of severe CRS in 19 clinical trials selected from studies published between 2010 and 2017. The pooled severe CRS proportion was 29.3% (95% confidence interval [CI] 12.3-49.1%) in B-ALL, 38.8% (95%CI 12.9-67.6%) in B-CLL, and 19.8% (95%CI 4.2-40.8%) in B-NHL. In the univariate meta regression analysis, the proliferation of CD19-CAR-T cell in vivo was correlated with the severe CRS. Specifically, total infusion cell dose contributed to the severe CRS occurring in B-ALL patients but not in B-CLL or B-NHL patients. Tumor burden was strongly associated with the severity of CRS in B-ALL. Besides, post-HSCT CD19 CAR-T cell infusion represented lower severe CRS incidence. Further investigations into the risk factors of CRS in B-CLL and B-NHL are needed.

Concise Review: Quantitative Detection and Modeling the In Vivo Kinetics of Therapeutic Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) present a promising tool in cell‐based therapy for treatment of various diseases. Currently, optimization of treatment protocols in clinical studies is complicated by the variations in cell dosing, diverse methods used to deliver MSCs, and the variety of methods used for tracking MSCs in vivo. Most studies use a dose escalation approach, and attempt to correlate efficacy with total cell dose. Optimization could be accelerated through specific understanding of MSC distribution in vivo, long‐term viability, as well as their biological fate. While it is not possible to quantitatively detect MSCs in most targeted organs over long time periods after systemic administration in clinical trials, it is increasingly possible to apply pharmacokinetic modeling to predict their distribution and persistence. This Review outlines current understanding of the in vivo kinetics of exogenously administered MSCs, provides a critical analysis of the methods used for quantitative MSC detection in these studies, and discusses the application of pharmacokinetic modeling to these data. Finally, we provide insights on and perspectives for future development of effective therapeutic strategies using pharmacokinetic modeling to maximize MSC therapy and minimize potential side effects. Stem Cells Translational Medicine 2018;7:78–86

Engineering cord blood to improve engraftment after cord blood transplant.

Umbilical cord blood transplant (CBT) has traditionally been associated with slower engraftment of neutrophils, delayed immune reconstitution and consequently higher risk of infections as compared with peripheral blood progenitor cell (PBPC) or bone marrow (BM) transplants. This is primarily due to low numbers of total nucleated cells (TNCs) and the naive nature of CB immune cells. The use of double unit CB transplant (DCBT) increases the total cell dose in the graft, but it still does not produce as rapid engraftment as seen with PBPC or even BM transplants. Herein, we discuss strategies to improve engraftment after CBT. We describe methods of (I) expansion of CB graft ex vivo to increase the total cell dose; and (II) enhancement of BM homing capability of CB progenitor cells; (III) ex vivo expansion of CB derived T cells for improving T cell function against viruses, tumors and protection from graft versus host disease (GVHD). With these novel approaches, engraftment after CBT is now reaching levels comparable to that of other graft types.

Equine Airway Mast Cells are Sensitive to Cell Death Induced by Lysosomotropic Agents.

Mast cells are known for their detrimental effects in various inflammatory conditions. Regimens that induce selective mast cell apoptosis may therefore be of therapeutic significance. Earlier studies have demonstrated that murine- and human-cultured mast cells are highly sensitive to apoptosis induced by the lysosomotropic agent LeuLeuOMe (LLME). However, the efficacy of lysosomotropic agents for inducing apoptosis of invivo-derived airway mast cells and the impact on mast cells in other species have not been assessed. Here we addressed whether lysosomotropic agents can induce cell death of equine invivo-derived mast cells. Bronchoalveolar lavage (BAL) fluids from horses were incubated with LLME at 15-100μm for up to 48h. The overall cell viability was unaffected by 15μm LLME up to 48h, whereas a relatively modest drop in total cell counts (~30%) was seen at the highest LLME dose used. In contrast to the relatively low effect on total cell counts, LLME efficiently and dose dependently reduced the number of mast cells in BAL fluids, with an almost complete depletion (96%) of mast cells after 24h of incubation with 100μm LLME. A significant but less dramatic reduction (up to ~45%) of lymphocytes was also seen, whereas macrophages and neutrophils were essentially resistant. The appearance of apoptotic bodies suggested a mechanism involving apoptosis rather than necrosis. These findings suggest that equine airway mast cells are highly sensitive to lysosomotropic agents. Possibly, lysosomotropic agents could be of therapeutic value to treat disorders involving harmful accumulation of mast cells in the airways.

Sustained Remissions Following Chimeric Antigen Receptor Modified T Cells Directed Against CD19 (CTL019) in Patients with Relapsed or Refractory CD19+ Lymphomas

BACKGROUND: Autologous T cells genetically modified to express a chimeric antigen receptor consisting of an external anti-CD19 single chain antibody domain with CD3ζ and 4-1BB signaling domains (CTL019 cells) can mediate potent anti-tumor effects in patients (pts) with relapsed or refractory chronic lymphocytic and acute lymphoblastic leukemias. We are conducting a phase IIa clinical trial to evaluate the safety and efficacy of CTL019 cells in pts with relapsed or refractory CD19+ non-Hodgkin lymphomas (NHL).METHODS: Pts with CD19+ diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), or mantle cell lymphoma (MCL) with no available curative treatment options, a limited prognosis (<2 years anticipated survival), and responsive or stable disease with most recent therapy are eligible. Pts with DLBCL have residual disease after primary and salvage therapies and are not eligible for autologous stem cell transplant (ASCT) or have relapsed or residual disease after ASCT; FL pts have progression of lymphoma <2 years after second or higher line of therapy (not including single agent monoclonal antibody therapy); MCL pts have relapsed, residual, or progressive disease after rituximab-chemotherapy combination therapy and are not appropriate for transplant or have relapsed after transplant. After steady state apheresis to collect peripheral blood leukocytes, pts receive lymphodepleting chemotherapy based on disease burden, histology, and past therapies. One to 4 days after chemotherapy, pts receive a single dose of CTL019 cells by intravenous infusion. Peripheral blood and marrow samples are collected for immunophenotypic, cytokine, and molecular studies at pre-specified times after T cell infusion. Initial tumor response assessment is performed 3 months after T cell infusion using International Working Group response criteria. Enrollment started in February 2014; data reported here are through July 26, 2015.RESULTS: To date, 38 pts have enrolled (DLBCL 21; FL 14; MCL 3). The median age is 56 years (range: 25-77), male: female ratio is 22:16, median number of prior therapies is 4 (range: 1-10), and number of pts with prior transplant is 12 (32%; 11 ASCT, 1 allotransplant). Ann Arbor stages at enrollment are: Stage IV 23 pts (61%), Stage III 7 pts (18%), Stage II 6 pts (16%), Stage 1E 2 pts (5%); 11 pts (29%) had bone marrow involvement. LDH was increased in 28 pts (74%). ECOG PS was 0 in 16 pts (42%) and 1 in 22 pts (58%). As of July 26, 2015, 24 patients have received the protocol-specified dose of CTL019 cells (13 DLBCL; 9 FL; 2 MCL). Lymphodepleting chemotherapy regimens were bendamustine (6 pts), cyclophosphamide (11 pts), cyclophosphamide-fludarabine (1 pt), modified EPOCH (3 pts), and radiation-cyclophosphamide (3 pts). Median total CTL019 cell dose is 5.00e8 (range: 1.79e8 - 5.00e8); median CTL019 cell dose/kg is 5.84e6 (range: 3.08e6-8.87e6). Median peak CTL019 cell expansion in blood occurred 7 days after infusion (range: 2-14 days); there was no difference in peak expansion between responders and non-responders. Cytokine release syndrome (CRS) occurred in 16 pts (14 grade 2; 1 grade 3; 1 grade 4) and did not predict response. Neurologic toxicity occurred in 3 pts: 2 episodes of delirium (1 grade 2, 1 grade 3) and one possibly related grade 5 encephalitis. 22 pts are evaluable for response (DLBCL 13, FL 7, MCL 2). Overall response rate (ORR) at 3 months is 68% (15/22): DLBCL 54% (7/13); FL 100% (7/7); MCL 50% (1/2). At the median follow-up 11.7 months, progression-free survival (PFS) from CTL019 infusion is 62% (DLBCL 43%; FL 100%). For responders at median follow up, response duration is 83% for DLBCL and 100% for FL.CONCLUSIONS: These results demonstrate that CTL019 cells can be prepared from extensively pretreated pts with active NHL and can induce durable responses with toxicity that is less than reported for chronic lymphocytic and acute lymphoblastic leukemias. [Display omitted] DisclosuresSchuster:Phamacyclics: Consultancy, Research Funding; Novartis: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Genentech: Consultancy; Hoffman-LaRoche: Research Funding. Svoboda:Seattle Genetics: Research Funding; Celgene: Research Funding; Celldex: Research Funding; Immunomedics: Research Funding. Dwivedy Nasta:BMS: Research Funding; Millenium Takeda: Research Funding. Porter:Genentech: Other: Spouse Employment; Novartis: Patents & Royalties, Research Funding. Mato:Celgene Corportation: Consultancy, Research Funding; Genentech: Consultancy; Pharmacylics: Consultancy, Research Funding; Pronai Pharmaceuticals: Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy; TG Therapeutics: Research Funding; Gilead: Consultancy, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Chew:Novartis: Patents & Royalties. Hasskarl:Novartis: Employment, Equity Ownership. Shah:Novartis: Employment, Equity Ownership. Wasik:Janseen and Novartis: Research Funding. Zheng:Novartis: Patents & Royalties. Levine:Novartis: Patents & Royalties, Research Funding. June:Novartis: Patents & Royalties, Research Funding.

Autologous Cord Blood Infusion for the Treatment of Brain Injury in Children with Cerebral Palsy

Background: Cerebral palsy (CP) results from in utero or perinatal injury to the developing brain, often through stroke, hypoxic insult, or hemorrhage. Current treatments are supportive, focusing on managing sequelae with physical therapies, medications, and surgery. However, there are no therapies to address the underlying brain injury. Umbilical cord blood (CB) has been shown to prevent neurologic damage in children with leukodystrophiesand to improve motor function in animal models of brain injury and CP. Previously, we demonstrated safety of intravenous autologous CB infusion in young children with brain injuries. The Cerebral Palsy-Autologous Cord Blood (CP-AC) study was designed to assess the efficacy of a single intravenous infusion of autologous CB in young children with spastic CP.Methods: The CP-AC study is a prospective, randomized, double blind, placebo controlled crossover study of a single intravenous infusion of autologous CB in children ages 1-6 years with spastic CP. The Gross Motor Classification System (GMFCS) was utilized to classify the level of motor function at study entry and follow-up. Children were eligible if they were (1) GMFCS level 2-4 or (2) GMFCS level 1 with hemiplegia if they used their affected hand as an assist only. Children with known genetic conditions, intractable seizures, or severe microcephaly were ineligible. Autologous CB units had to have a documented pre-cyropreservation total cell dose of ≥1x107/kg, negative sterility culture, negative maternal infectious disease screening, and confirmed identity through HLA typing of the subject and a segment attached to the CB unit. Subjects were evaluated at baseline, one year, and two years with functional evaluations (Gross Motor Function Measure-66 (GMGM-66), Peabody Developmental Motor Scales-2, Assisting Hand Assessment, Bayley Scales of Infant Development) and brain MRI. They were randomized to the order in which they received CB and placebo infusions (given one year apart). The primary endpoint was change in GMFM-66 score at one year after the initial infusion (CB or placebo). Cryopreserved CB units were shipped to and stored at Duke until the day of treatment when they were thawed and washed in dextran 40 +5% human serum albumin (DA). Infusions, dosed at 1-5x107/kg based on the pre-cryopreservation total nucleated cell count (TNCC) and diluted in 1.25mL/kg of DA, were administered intravenously over 5-10 minutes in the outpatient setting after premedication with oral acetaminophen, IV Benadryl, and IV Solumedrol. Subjects received IV fluids and were monitored for 2-4 hours post-infusion.Results: Sixty-three children were enrolled with a median age of 2 years (range 1- 6) at baseline. Median TNCC of CB infusion was 2x107/kg (range 0.4-5) with a median CD34 dose of 0.5x105/kg (range 0.05-4). Despite negative pre-cryopreservation cultures, one CB unit grew β-hemolytic strepfrom a sample of the thawed CB unit. There were no clinical infections in this or any other study patient. One subject had transient infusion reactions consisting of hives +/- low-grade fever after each infusion; an additional dose of Benadryl was administered after the first reaction. Preliminary analysis of the 63 patients at one year showed no statistically significant overall difference in GMFM-66 change scores between placebo and treated groups (6.9 vs. 7.5, p =0.72). However, treated subjects who received pre-cryopreservation cell doses of >2.5x107/kg demonstrated statistically significant improvement in GMFM-66 change scores compared to subjects who received lower cell doses (p <0.01). A similar result was observed comparing subjects who received above (or below) the median infused cell dose of 1.98x107/kg (p =0.05) (Figure 1). The infused cell dose was not related to type of CP (p =0.32) or severity (p=0.46).Conclusions: When adequately dosed, cord blood may improve motor function in young children with spastic CP. We observed improvement at pre-cryopreservation cell doses (>2.5x107/kg) that correspond with the minimum cell dose used for hematopoietic reconstitution in patients undergoing allogeneic CB transplantation. In order to extend this therapy to children who do not have adequate autologous CB units available, safety and efficacy of allogeneic CB products should be investigated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Point-of-Care Platform for Hematopoietic Stem Cell Gene Therapy

Hematopoietic stem and progenitor cell (HSPC) gene therapy holds great promise but requires highly technical and dedicated facilities. Current state-of-the-art requires ex vivo HSPC gene transfer in dedicated Good Manufacturing Practices (GMP) facility infrastructure, limiting treatment to highly developed countries. A patient point-of-care strategy would therefore make HSPC gene therapy available to patients worldwide. We developed a short, semi-automated, mostly-closed platform for ex vivo isolation and lentivirus (LV) gene modification of CD34+ HSPCs from either bone marrow or mobilized peripheral blood sources using the CliniMACS Prodigy® (Figure 1). Experiments were performed with a biosafety cabinet and personal protective equipment to simulate anticipated conditions in clinical facilities of underdeveloped countries. A total of 7 custom programs were developed for bone marrow or mobilized peripheral blood CD34+ cell isolation and LV transduction. Addition of a pyrimidoindole derivative, UM 729, permitted efficient transduction of CD34+ HSPCs in <18 hours. Complete semi-automated production took <34 hours from collection to infusion of the gene modified cell product, requiring very little operator hands-on time. Autologous, LV gene-modified CD34+ HSPCs from two nonhuman primates produced using this platform engrafted and supported multilineage hematopoietic repopulation after myeloablative total body irradiation at 1020 cGy. Total cell doses achieved were 27 x 106 and 5.4 x 106 CD34+ cells/kg body weight, respectively. We observed stable, persistent and polyclonal gene marking in peripheral blood granulocytes up to 40% and lymphocytes up to 15% within 4 months after infusion. Neither animal displayed evidence for increased toxicity, including potential contamination from the cell products. We then validated processing of human bone marrow and mobilized apheresis products from healthy adult donors. We demonstrate efficient isolation of human CD34+ HSPCs and up to 60% transduction efficiency with a clinical-grade LV currently being tested in a phase I clinical trial for treatment of HIV-associated lymphoma. Products tested met current FDA approved specifications for infusion. Xenotransplantation of these products into immunodeficient mice resulted in polyclonal engraftment over 12 weeks. These data demonstrate preclinical safety and feasibility of a patient point-of-care strategy for ex vivo LV gene transfer into HSPCs. This platform represents a major advance in global portability of LV mediated HSPC gene therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Unrelated Cord Blood Transplantation (UCBT) In Adults In The UK: Evolution, Experience and Outcomes. A Retrospective Analysis On Behalf Of The British Society of Blood and Marrow Transplantation (BSBMT) and Eurocord

In the UK, unrelated cord blood transplantation (UCBT) has been used increasingly in adults since 2000. National guidelines were published in 2009 (Shaw et al, 2009) and two national prospective clinical trials have been established (EUDRACT registrations; RIC 2004-003845-41 and MAC 2009-011818-21). However, national trends and outcomes have never been comprehensively appraised. We have therefore analysed the demographic data and outcomes in adults (>18 years) undergoing UCBT in 23 UK transplant centres from 2000-2012 using the BSBMT and Eurocord databases.From the first adult UCBT in 2000 to the end of 2102 there were a total of 176 centre UCBT registrations with corresponding cord blood bank data, including 28 patients in the national prospective clinical trials which have been excluded from any further analysis. Outcomes were analysed for 148 patients with a median age of 40.8 (range 18-72) years, with acute leukaemia (n=80), myeloproliferative disorders/myelodysplastic syndrome (MDS) (n=43), lymphoproliferative diseases (n=20) or bone marrow failure (n=5). Half the activity was between 2000-2008 and half between 2009-2012, reflecting a greater than doubling of activity in recent years. Various conditioning regimens were used, with the majority receiving a reduced intensity conditioning regimen. Most patients (72%) received double cord blood units (dCBU) and the remainder a single CBU. Recorded median total cell dose infused was 3.61 x107/kg (range 0.41-34.35) for total nucleated cell count (TNC) and 1.69 x105/kg (range 0.13-14.97) for CD34+ cells. Engraftment of neutrophils to >0.5 x109/L occurred at a median of 22 (range 3-52) days and platelets to >20 x109/L at a median of 39 (range 10-117) days. Overall survival at 1 year was 46.4% (CI 38.8-55.5%) and 2 years was 40% (CI 32-49%), with an overall median survival of 27.1 (range 3.2-83.7) months. The incidence of grade II to IV acute graft-versus-host disease (GVHD) was 17.6% (CI 11.5-24.8%) and chronic GVHD at 1 year was 11.4% (CI 6.7-17.6%). In patients treated for malignant disease with remission status available (n=137), cumulative incidence of relapse was 11.0% (CI 6.4-16.9%) at 100 days and 24.6% (CI 17.5-32.4%) at 1 year, and treatment related mortality was 22.6% (CI 16.0-30.0%) at 100 days and 34.6% (CI 26.5-42.7%) at 1 year. In univariate analysis, overall survival (OS) at 2 years was strongly related to stage of disease; 54% for early (CR1, chronic phase, MDS subtype-RA, good remission) versus 47% for intermediate (CR2, accelerated phase, MDS transformation, PR) versus 21% for advanced (non-remission, other subtypes of MDS) (p=0.0001). There was no impact of gender, age, diagnosis, intensity of conditioning regimen, use of serotherapy in the conditioning regimen, CBU number, TNC or CD34+ dose, HLA or ABO matching, or year of UCBT (2000-08 versus 2009-12) on OS. In a subgroup analysis of acute leukaemia, the relationship between 2 year OS and disease status was stronger; 60% for early versus 43% for intermediate versus 0% for advanced (p=0.000008), with improved survival outcomes with the use of dCBU over single unit UCBT (50% vs 34%), although this did not achieve significance (p=0.15).This retrospective national analysis supports the evolution of UCBT as an effective treatment in adults without an otherwise available donor. Outcomes are comparable to similar patient groups treated with unrelated blood and marrow transplantation. The best outcomes are achieved in early and intermediate risk disease. The benefit of national guidelines is supported by more than doubling of activity since publication in 2009 without deterioration in outcomes. Whether the national prospective clinical trials will deliver improved UK outcomes will be determined following initial analysis in 2014. ReferenceShaw BE, et al. Recommendations for a standard UK approach to incorporating umbilical cord blood into clinical transplantation practice: conditioning protocols and donor selection algorithms. Bone Marrow Transplantation 2009;44:7-12 Disclosures:Gluckman:Cord use: Honoraria; gamida: Honoraria.