Apheresis Collection Research Articles

Gene Therapy in Transfusion-Dependent Non-β0/β0 Genotype β-Thalassemia: First Real-World Experience of Beti-cel

Gene addition and editing strategies for transfusion-dependent β-thalassemia have gained momentum as potentially curative treatment options, with studies showcasing their efficacy and safety. We report the first real-world application of betibeglogene autotemcel (beti-cel; ZYNTEGLO™) during its period of active license in Europe from January 2020 to March 2022 for patients aged ≥ 12 years without a β0/β0 genotype and without a human leukocyte antigen (HLA)-matched sibling donor, before beti-cel marketing authorization was withdrawn by its holder due to non-safety reasons. Among 15 screened patients, 4 opted out for fertility and safety concerns, 2 were excluded because of marked hepatic siderosis, and 1 had apheresis collection failure. Eight patients received beti-cel post busulfan myeloablative conditioning, all achieving transfusion independence within 8 to 59 days with posttreatment hemoglobin levels ranging from 11.3 to 19.3 g/dL. No deaths occurred, but acute toxicity mirrored busulfan's known effects. Posttreatment platelet management faced challenges due to HLA-antibodies in 3 patients. Monitoring up to Month 24 revealed pituitary-gonadal endocrine dysfunction in all 3 female and in 2 of 5 male patients. Additionally, we observed unexpected posttreatment sequelae: 1 patient developed polycythemia that could not be explained by known genetic or acquired mechanisms, 1 patient developed posttreatment depression and anxiety prohibiting her from returning to work, and 1 patient developed fatigue severely compromising both quality of life and work capacity. This real-world experience corroborates beti-cel's efficacy and safety and provides information on adverse events observed during real-world use of the therapy.

Development of a Uniform Apheresis Case Report Form forStandardized Collection of Apheresis Data.

Apheresis is performed worldwide for an increasing number of indications. The development of common data elements (CDE) for apheresis related areas may facilitate conduct of new research, enhance quality initiatives including benchmarking, and improve patient care. This report describes the systematic development of the Uniform Apheresis Case Report Form (UACRF) as part of the Apheresis in the United States (ApheresUS) program. A consensus panel of 17 diverse experts in apheresis, related specialties, and electronic case report form (eCRF), and database development was assembled. The panel met via online conferencing from November 17, 2020 to December 1, 2021. A draft document was posted online for public comment from October 11, 2021 to November 10, 2021. Feedback was collected using an online survey tool. The consensus panel revised the UACRF. This version was converted to an eCRF with additional changes made to improve usability in this format. The final version of the UACRF was created on August 24, 2023. The UACRF contains 16 modules: procedure and subject eligibility, patient demographics, general procedure information, laboratory parameters, vascular access, common procedure elements, eight procedure specific modules (mononuclear cell collection and seven therapeutic modalities), outcomes, and site information. A total of 137 data elements were created, including 57 with one or more subelements. The UACRF is the first systematic attempt to develop CDE for therapeutic apheresis and white blood cell collections. Further validation of the UACRF is necessary to confirm the tool's ability to collect the relevant data elements and determine the usability of the form.

Preemptive Approach to Plerixafor Use Is Optimal in Patients With Relapsed/Refractory Germ Cell Tumors Undergoing Peripheral Blood Hematopoietic Stem Cell Collection: Effect on Collection Days, Yields, and Cost.

Evidence describing the use of plerixafor in the off-label population of relapsed/refractory germ cell tumors (GCT) is limited. Weaim to describe the effect of rescue versus preemptive plerixafor use on apheresis collection days, collection yields, and cost. We retrospectively collected data on 77 consecutive patients (at least 15 years of age) with GCT who underwent peripheral blood stem cell (PBSC) collection for autologous stem cell transplant between January 1, 2020 and May 1, 2022. Depending on insurance approval, plerixafor was given either as "rescue" (after a first apheresis collection of < 5 × 106 CD34+ cells/kg) or as "preemptive" on Day 4 of granulocyte-colony stimulating factor (G-CSF) prior to the first apheresis collection, if the Day 4 peripheral blood CD34+ count was < 40 cells/μL. A total of 66% of patients who received preemptive plerixafor completed collection in 1 day, similar to good mobilizers who only needed G-CSF (71%, p = 0.366). In contrast, all poor mobilizers in the rescue group required at least 2 days of collection and had lower CD34+ cell yields than the preemptive group (7.15 vs. 9.81 × 106/kg, p = 0.0055). A cost analysis revealed that preemptive plerixafor may save approximately $7000 per patient compared with a rescue approach. Preemptive plerixafor in GCT patients undergoing PBSC collection allows relatively poor mobilizers to collect in fewer days and with lower overall cost. Fewer apheresis procedures result in less risk to the patient, increased patient satisfaction, and the ability to schedule more patients within the constraints of staffing.

Optimizing autologous stem cell collections for patients with multiple myeloma receiving G-CSF and Plerixafor: A single center project.

Increasing indications for cellular therapy collections have stressed our healthcare system, with autologous collections having a longer than desired wait time until apheresis collection. This quality improvement initiative was undertaken to accommodate more patients within existing resources. Patients with multiple myeloma who underwent autologous peripheral blood stem cell collection from October 2022 to April 2023 were included. Demographic, mobilization, laboratory, and apheresis data were retrospectively collected from the medical record. This cohort included 120 patients (49.2% male), with a median age of 60 years. All received G-CSF and 95% received pre-emptive Plerixafor approximately 18 hours pre-collection. Most (79%) had collection goals of at least 8 × 106/kg CD34 cells, with 63% over 70 years old having this high collection goal (despite 20 years of institutional data showing <1% over 70 years old have a second transplant). With collection efficiencies of 55.9%, 44% of patients achieved their collection goal in a single day apheresis collection. A platelet count <150 × 103/μL on the day of collection was a predictor for poor mobilization; among 27 patients with a low baseline platelet count, 17 did not achieve the collection goal and 2 failed to collect a transplantable dose. With minor collection goal adjustments, 15% of all collection appointments could have been avoided over this 6-month period. Other strategies to accommodate more patients include mobilization modifications (Plerixafor timing or substituting a longer acting drug), utilizing platelet counts to predict mobilization, and modifying apheresis collection volumes or schedule templates.

Bacterial Contamination of Platelet Products.

Transfusion of bacterially contaminated platelets, although rare, is still a major cause of mortality and morbidity despite the introduction of many methods to limit this over the past 20 years. The methods used include improved donor skin disinfection, diversion of the first part of donations, use of apheresis platelet units rather than whole-blood derived pools, primary and secondary testing by culture or rapid test, and use of pathogen reduction. Primary culture has been in use the US since 2004, using culture 24 h after collection of volumes of 4-8 mL from apheresis collections and whole-blood derived pools inoculated into aerobic culture bottles, with limited use of secondary testing by culture or rapid test to extend shelf-life from 5 to 7 days. Primary culture was introduced in the UK in 2011 using a "large-volume, delayed sampling" (LVDS) protocol requiring culture 36-48 h after collection of volumes of 16 mL from split apheresis units and whole-blood derived pools, inoculated into aerobic and anaerobic culture bottles (8 mL each), with a shelf-life of 7 days. Pathogen reduction using amotosalen has been in use in Europe since 2002, and was approved for use in the US in 2014. In the US, recent FDA guidance, effective October 2021, recommended several strategies to limit bacterial contamination of platelet products, including pathogen reduction, variants of the UK LVDS method and several two-step strategies, with shelf-life ranging from 3 to 7 days. The issues associated with bacterial contamination and these strategies are discussed in this review.

Pre-Apheresis Prediction of CD34 + Cell Yield from Collecting Efficiency Is Not Reliable in Peripheral Blood Stem Cell Collection

Introduction Procurement of enough CD34 + cells (CD34 +) by apheresis is essential for the successful engraftment of peripheral blood stem cell transplantation (PBSCT). To determine the processed blood volume of apheresis to obtain a certain number of CD34 +, collecting efficiency 2 (CE2) is generally used, which is calculated from the total amount of CD34 + in the product (PBSC-CD34 +) divided by pre-apheresis CD34 + in the peripheral blood (PB-CD34 +) and by processed blood volume, and CE2 is determined beforehand from the accumulated data in each apheresis site. However, we sometimes experience cases in which collected amount of CD34 + are far from our expected values. It is probably because of the introduction of various CD34 + mobilization methods, new automated blood cell separators and protocols. Hence, we investigated thoroughly the kinetics of CD34 + in PBSC collection. Patients and Methods PBSC collections in 55 subjects, including 46 patients undergoing autologous PBSCT and 9 healthy allogeneic PBSCT donors, were analyzed prospectively. All patients were administered G-CSF 10 μg/day sc for 4 days and plerixafor 0.24 mg/kg sc at 22:30 on day 4, and PBSC collection was started from 9 am on day 5. Donors were given G-CSF 10 μg/day for 4 to 5 days until 2x10 6/kg (pt's body weight) CD34 + were collected. Apheresis was performed using Spectra Optia ® in CMNC mode. The apheresis time was adjusted depending on the CD34 + yield. CD34 + was analyzed by flow cytometry according to the ISHAGE protocol. Peripheral blood samples were obtained before, and every 1-1.5 hour after the start until the end of apheresis. Samples were also taken from the apheresis collection bag at the same timings as PB. Collecting efficiencies were calculated as follows: CE1(%) = [collected CD34 +]x100/([prePB-CD34 +]+[postPB-CD34 +])/2 x [processed blood volume]). CE0(%) = [collected CD34 +]x100/sum of ([prePB-CD34 +]+[postPB-CD34 +])/2 x [processed blood volume]) in each section (*CE0 was defined in this particular study). This study was conducted as a part of the prospective study, which was approved by the Ethics Committee of Keio University School of Medicine. Results More than 2x10 6/kg CD34 + were collected in the first day in 48 subjects (87%). The median processed blood volume and time was 5575 (range 3873-10903) mL and 185 (range 120-292) min, respectively. CE2 was calculated as a median 53% (range 20-88) from this cohort, but it varied markedly; CE2&amp;gt;70% in 6 cases, and &amp;lt;30% in 4. On the other hand, CE1 was median 65.5% (range 26.2-106.4%), and it also varied markedly; CE1&amp;gt;80% in 6 cases, and &amp;lt;40% in 7. CE1 and CE2 correlated strongly with each other (r=0.93), but CE1 was higher than CE2 in all cases but one; median 124(range 95-149) % (Fig.1). The main reason why CE1 was always higher than CE2 was probably because PB-CD34 + concentration was always declining during apheresis, which might be more influenced in plerixafor mobilization where CD34 + concentration changed in an hour-depending manner. In some apheresis cases with extremely low CE, it was revealed that cell aggregates containing myriads of platelets might have blocked the outlet of the collection kit, which was difficult to be recognized during apheresis. CE1 in each apheresis section also changed markedly during each apheresis and CE2 was not helpful for predicting optimal apheresis time (Fig. 2). In the first 10-20 minutes until the interphase formation, a real collection was not yet started so that CE1 in the first 1 hour became low. In the following sections, collection was supposed to become relatively steady, where CE would be influenced largely by interphase stability. In the last part of collection, CE tended to be relatively high for unknown reasons. CEs varied markedly during the whole collection periods. Conclusions Because multiple factors are associated with CE variability, CD34 + yields were sometimes far from the expected values calculated from prePB-CD34 + and CE2. Therefore, enumeration of CD34 + in the product during apheresis is recommended for secure and effective collections.

The Impact of Leukapheresis Collection on Patients Undergoing Chimeric Antigen T-Cell Receptor Therapy with Tisagenlecleucel

Introduction Autologous chimeric antigen receptor T-cell therapy (CAR-T) has revolutionized the treatment of acute lymphoblastic leukemia (ALL) in children and young adolescents, with an impressive overall remission rate of 81% in patients with relapsed or refractory (r/r) disease 1. Approved by the US FDA, tisagenlecleucel (Kymriah ®) is now approved for patients up to 25 years of age with pre B -cell ALL that is refractory or in second or later relapse as well as adult patients with r/r non-Hodgkin lymphoma (NHL) including diffuse large B cell lymphoma (DLBCL) after two or more lines of systemic therapy 2. Apheresis is the first step of CAR-T cell manufacturing with the recommended collection goal of ≥1.0x10 9 CD3 cells or ≥2.0x10 9 total nucleated cells (TNC) 3. However, this crucial process can be affected by technical and patient specific factors, including heavily pre-treated patients, resulting in CAR T-cell manufacture failure rates of 1-14% in clinical trials 4, 5, 6, 7. The clinical impact of CD3+ count in peripheral blood at the time of leukapheresis and optimal apheresis collection parameters remain to be established. Methods: This is a retrospective study of patients 0-88 years with r/r DLBCL or ALL who underwent leukapheresis between August 31 st 2017 to August 1 st, 2022 for tisagenlecleucel therapy. Spearman correlation was conducted to evaluate the association between absolute lymphocyte count (ALC) and cell counts prior to leukapheresis. The Kaplan-Meier method was employed to evaluate overall survival (OS) and event-free survival (EFS) from date of CAR-T. Cox proportional hazards regression analysis was utilized to examine the associations between OS or EFS and measures of interest, including white blood cell count (WBC), ALC, CD3+ cells from leukapheresis, CD3+%, cell viability, viable T cells %, total cell count, IFNγ CD19, maximum cytokine release syndrome (CRS) score (Max CRS), maximum immune effector cell-associated neurotoxicity syndrome (ICANS) score (Max ICANS), duration of CRS, duration of ICANS, and post-CART transplantation. Results: Forty patients were included, 22 (55%) patients were male, and the median age at apheresis was 50 years (5-88 years). There were significant positive correlations between ALC and the CD3+ cells from leukapheresis (Correlation: 0.62 [0.33 - 0.83], p-value &amp;lt;0.001) and CD3+ % (Correlation: 0.46 [0.001 - 0.78], p-value 0.020). For the 39 patients with data available, the median OS was 94.2 months (95% CI: 55.3 - 129.1). For EFS from date of CAR-T, the median survival from the date of diagnosis until the date of first recurrence event (relapse, secondary malignancy, and death) or the last follow-up date was 75.1 months (95% CI: 34.7 - 104.5). Based on univariate survival analysis results, there was no significant association observed between OS or EFS and the variables of interest. The median IFNγ reported on certificate of analysis from manufacturer was 162 (13-818) with 14 (36%) patients maximum grade CRS equal to 0, and 27 (69%) maximum grade ICANS equal to 0. For those who experienced any grade of CRS, there was a significant correlation with CD3+ cells (p-value 0.007) but not significant when compared to IFNγ (p-value 0.51). There was no significant correlation between those who experienced ICANS compared to CD3+ cells (p-value 0.17) or IFNγ (p-value 0.1). There was no significant positive correlations between IFNγ and the duration CRS (Correlation: 00.292 [-0.045, 0.554], p-value 0.07) or duration of ICANS (Correlation: 0-0.241 [-0.531, 0.064], p-value 0.16). Of note, we had one patient who had a manufacturing failure and unfortunately passed away before second round of apheresis. For this patient, WBC on apheresis was 4.3k/microL, ALC was 560 and CD3+ cells was 84.4%. Conclusion: A significant positive correlation was observed between ALC and the CD3+ cells as well as its percentage from leukapheresis for pediatric and adult patients undergoing tisagenlecleucel CAR-T. However, no significant effects were found when detecting the association between ALC with OS or EFS. In addition, there were no significant findings between IFNγ and the duration of CRS or ICANS. Funding: This work was supported in part by the NIH/NCI Cancer Center Support Grant (award number P30 CA016672) and used the Biostatistics Resource Group.

Ksd-101 in Patients with EBV-Associated Hematologic Neoplasms: Results from an Ongoing Phase I Clinical Study

Background: The Epstein-Barr Virus (EBV), which has been confirmed by WHO as the first human tumor-associated virus, is a lifelong carrier once infected. EBV-associated Hematologic Neoplasms seriously threats human health, with rapid disease progression, poor prognosis, easy to relapse, and high mortality rate. KSD-101 is a First-in-class autologous dendritic cell (DC) vaccine which is developed to control EBV-associated hematological diseases. Autologous DCs were loaded with the EBV-transformed B-LCL (B-Lymphoblastoid Cell Line) lysates which contains a broad-spectrum EBV antigen to prepare KSD-101 for the treatment of EBV-associated diseases. Methods: In this study, patients (pts) with EBV-associated Hematologic Neoplasms who fail to respond to or relapse after conventional treatment will be recruited and undergo leukapheresis to collect autologous peripheral blood mononuclear cell (PBMC). The enriched monocytes will be induced to differentiate into DCs and prepared as KSD-101 after loading with EBV-transformed B-LCL. Pts will undergo monotherapy with KSD-101, without the need for pre-treatment of lymphodepletion or prophylactic medication. Administration route of KSD-101 is subcutaneous injection, once every 2 weeks for 3-5 vaccinations in total. The study used a 3+3 dose-escalation design. Safety assessment is conducted according to NCI-CTCAE 5.0 grading criteria. The primary objective is to assess the tolerability and safety of KSD-101 in the treatment of EBV-associated Hematologic Neoplasms, explore the dose-limiting toxicities (DLT) and the maximum tolerated dose (MTD). The secondary objective is to explore the clinical efficacy and immune responses of KSD-101 in the patients with EBV-associated Hematologic Neoplasms. Results: As of July 2023, 9pts were enrolled in the study and 8 pts DC vaccine preparations were completed. Three pts were injected 5.0 × 10 6 cells/dose without exploring any DLT. The first two pts in the second dose level were injected 7.5 × 10 6 cells/dose. However, the third pt in second dose level was injected 5.0 × 10 6 cells/dose due to insufficient apheresis collection. Based on the safety and preliminary efficacy data, we decided not to proceed with dose-escalation to the third dose group. In the expansion phase, all pts were injected with a dose of 5.0 × 10 6 cells. Currently observed KSD-101-related toxicities were pyrexia (grade ≤ 2), injection site reactions (grade 1), and lymphadenopathy (grade 1). In all the pts who were injected, neither DLT nor MTD were explored. Based on the comprehensive considerations of safety, efficacy and easibility, we propose that the most appropriate dose is 5.0 × 10 6 cells. Of all pts enrolled, one was excluded due to hemophagocytic lymphohistiocytosis (HLH), among whom 4 pts completed the 12-week follow-up after the first injection for clinical and laboratory assessments. The results showed an overall response rate (ORR):100%, complete response (CR):100%, including 1 case of AITL (EBV infected T, B, NK),1case of CAEBV (EBV infected T, NK),2cases of NK-T cell lymphoma (1 EBV infected B, NK, and the other EBV infected NK). After vaccinations, the total number of lymphocytes of these four pts increased by 1.5 to3.4 times, including 1.6 to 4.2 times of T cells, 2.1 to 6.5 times of activated T cells (CD3 + HLA -DR +), 1.4 to 5.0 times of CD8 + T cells, 1.2 to 3.7 times of CD4 + T cells, 1.7 to 2.0 times of NK cells, and 1.2 to 32.4 times of B cells; meanwhile, the proportion of EBV-specific CTLs increased significantly (ranging from a 2.51% to 4.16% increase), and the proportion of Tregs significantly decreased compared with the baseline (ranging from a 32.49% to 73.81% reduction). Conclusions：Our current study shows that KSD-101 has excellent safety and good preliminary efficacy against EBV-associated Hematologic Neoplasms with efficient induction of immune responses. The results indicate that KSD-101 is a promising therapeutic reagent for the treatment of EBV-associated Hematologic Neoplasms. Larger prospective studies and longer follow-up time is needed to be conducted.

PB0695 Comparison of Platelet Quality and Hemostatic Function Across Apheresis Collection Platforms

PB0695 Comparison of Platelet Quality and Hemostatic Function Across Apheresis Collection Platforms

A donor safety evidence literature review of the short- and long-term effects of plasmapheresis.

Many blood establishments are expanding plasmapheresis collection capacity to achieve increasing plasma for fractionation volume targets, driven by immunoglobulin product demand. Some adverse events occur in both apheresis and whole blood collection, such as venepuncture-related trauma and vasovagal reactions. Others are specifically related to the apheresis procedure, such as citrate reactions, haemolysis, infiltration and air embolism. Whilst plasmapheresis procedures are generally well tolerated, theoretical longer term donor health considerations, such as the effects on donor plasma protein levels, bone mineral density, iron deficiency and malignancy also require consideration. An evidence-based framework that supports a safe and sustainable increase in the collection of plasma is essential. Our review demonstrates a lack of high-quality evidence on risks and outcomes specifically in plasmapheresis. Whilst conservative procedural controls and donor harm minimization policies will mitigate risk, high-quality evidence is needed to facilitate practice change that is safe and sustainable and maximizes the potential of individual donor differences.

Apheresis collection of mononuclear cells for chimeric-antigen receptor therapies.

Collections of lymphocytes to be genetically modified to treat hematologic malignancies have seen a dramatic increase over the last few years as commercial products have been approved. Reports of new products in development that can possibly treat solid organ malignancies represent a massive change in the field. Apheresis is at the center of the collection of cells for the manufacture of these chimeric-antigen receptor therapy products. The expansion of these collections represents one of the areas of apheresis procedures growth. This review will summarize concepts important to this type of collection and variables that need to be optimized to obtain desired cell yields while increasing patients' safety.

A machine-learning model that incorporates CD45 surface expression predicts hematopoietic progenitor cell recovery after freeze–thaw

Background aimsSufficient doses of viable CD34+ (vCD34) hematopoietic progenitor cells (HPCs) are crucial for engraftment. Additional-day apheresis collections can compensate for potential loss during cryopreservation but incur high cost and additional risk. To aid predicting such losses for clinical decision support, we developed a machine-learning model using variables obtainable on the day of collection. MethodsIn total, 370 consecutive autologous HPCs, apheresis-collected since 2014 at the Children's Hospital of Philadelphia, were retrospectively reviewed. Flow cytometry was used to assess vCD34% on fresh products and thawed quality control vials. The ratio of vCD34% thawed to fresh, which we call “post-thaw index,” was used as an outcome measure, with a “poor” post-thaw index defined as <70%. HPC CD45 normalized mean fluorescence intensity (MFI) was calculated by dividing CD45 MFI of HPCs to the CD45 MFI of lymphocytes in the same sample. We trained XGBoost, k-nearest neighbor and random forest models for the prediction and calibrated the best model to minimize falsely-reassuring predictions. ResultsIn total, 63 of 370 (17%) products had a poor post-thaw index. The best model was XGBoost, with an area under the receiver operator curve of 0.83 evaluated on an independent test data set. The most important predictor for a poor post-thaw index was the HPC CD45 normalized MFI. Transplants after 2015, based on the lower of the two vCD34% values, showed faster engraftment than older transplants, which were based on fresh vCD34% only (average 10.6 vs 11.7 days, P = 0.0006). ConclusionsTransplants taking into account post-thaw vCD34% improved engraftment time in our patients; however, it came at the cost of unnecessary multi-day collections. The results from applying our predictive algorithm retrospectively to our data suggest that more than one-third of additional-day collections could have been avoided. Our investigation also identified CD45 nMFI as a novel marker for assessing HPC health post-thaw.

Primed for change: The effect of a blood prime on peripheral blood stem cell collection and accuracy of a prediction tool in pediatric patients.

Pediatric apheresis collection of peripheral blood stem cells for autologous transplantation often requires use of a blood prime. We evaluated the relationship between pre-apheresis blood CD34+ counts and final CD34+ yield with use of a blood prime. Forty patients underwent apheresis stem cell collection in a 5 year period in our hospital, of which 27 required blood priming of the apheresis machine. Despite the blood prime group having significantly higher pre-apheresis CD34+ cell counts, this group processed a relatively higher volume of blood due to a higher dilutional effect and collected significantly less than predicted CD34+ cell yield. Use of weight-specific collection efficiencies and dilution-adjusted pre-apheresis CD34+ counts will help in accurately estimating the whole blood volume to process for PBSC collection and therefore increase efficiency and decrease the overall cost of collection.

Comparison of Platelet Quality and Function Across Apheresis Collection Platforms.

Platelet concentrates (PLT) can be manufactured using a combination of apheresis collection devices and suspension media (plasma or platelet additive solution (PAS)). It is unclear how platelet quality and hemostatic function differ across the current in-use manufacturing methods in the United States. The objective of this study was therefore to compare baseline function of PLT collected using different apheresis collection platforms and storage media. PLT were collected at two sites with identical protocols(N=5 per site, N=10 total per group) on the MCS®+ 9000 (Haemonetics; "MCS"), the Trima Accel® 7 (Terumo; "Trima"), and the Amicus Cell Separator (Fresenius Kabi, "Amicus"). MCS PLT were collected into plasma while Trima and Amicus PLT were collected into plasma or PAS (Trima into Isoplate and Amicus into InterSol; yielding groups "TP", "TI" and "AP", "AI", respectively). PLT units were sampled 1 hour after collection and assayed to compare cellular counts, biochemistry, and hemostatic function. Differences in biochemistry were most evident between plasma and PAS groups, as anticipated. MCS and TP had the highest clot strength as assessed by viscoelastometry. AI had the lowest thrombin generation capacity. Both TP and TI had the highest responses on platelet aggregometry. AI had the greatest number of microparticles. Platelet quality and function differs among collection platforms at baseline. MCS and Trima platelets overall appear to trend towards higher hemostatic function. Future investigations will assess how these differences change throughout storage, and if these in vitro measures are clinically relevant. This article is protected by copyright. All rights reserved.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

A guide to the collection of T-cells by apheresis for ATMP manufacturing-recommendations of the GoCART coalition apheresis working group.

Autologous chimeric antigen receptor-modified T-cells (CAR-T) provide meaningful benefit for otherwise refractory malignancies. As clinical indications for CAR-T cells are expanding, hospitals hitherto not active in the field of immune effector cell therapy will need to build capacity and expertise. The GoCART Coalition seeks to disseminate knowledge and skills to facilitate the introduction of CAR-T cells and to standardize management and documentation of CAR-T cell recipients, in order to optimize outcomes and to be able to benchmark clinical results against other centers. Apheresis generates the starting material for CAR-T cell manufacturing. This guide provides some initial suggestions for patient's apheresis readiness and performance to collect starting material and should thus facilitate the implementation of a CAR-T-starting material apheresis facility. It cannot replace, of course, the extensive training needed to perform qualitative apheresis collections in compliance with national and international regulations and assess their cellular composition and biological safety.

Altered Cellular Sedimentation during Apheresis Impacts Outcomes of Peripheral Blood Stem Cell Collection Efficiency in Patients with Sickle Cell Disease

Recent reports have demonstrated the potential for gene therapy as a therapeutic option to achieve a durable cure in patients with sickle cell disease (SCD). However, for these emerging cell therapies, relatively large numbers of peripheral blood hematopoietic stem cells (PB HSCs) are required to generate a therapeutic dose. Outcomes of stem cell collections in patients with SCD are highly variable, at times yielding far fewer numbers of HSCs in the product than predicted by the number of PB CD34+ cells. While the CXCR4 antagonist plerixafor has emerged as a safe and reliable drug to mobilize CD34+ cells in patients with SCD, collection failures continue to occur requiring additional cycles or failure to generate a gene therapy product. We sought to understand the mechanics affecting stem cell sedimentation in real time and posited that SCD disease associated changes to red cells results in altered HSC sedimentation during apheresis, negatively impacting collection. Methods Autologous PB stem cell collections were performed in patients with SCD following mobilization with 240ug/kg plerixafor. To uncover the mechanisms impacting apheresis efficiency for collecting PB HSCs, we devised a method to interrogate the sedimentation of cells in peripheral blood in real time, by interrupting the return collection circuit line. Cellular composition analyses of the collecting interfaces were performed including CD34+ enumeration by flow cytometry, CBC and differential, reticulocyte counts, and surface CD47 expression of red blood cells (RBCs). Results Patients were mobilized with 240ug/kg of plerixafor up to 6 hours before apheresis collection. We collected fewer cells than predicted (3.78 million cells/kg) in these patients with an overall average apheresis collection efficiency of 38%, and a strikingly wide range of 2%-107%. We determined that the lower collection efficiency outcomes were primarily derived when using standard apheresis interface settings. The pre-apheresis PB CD34+ counts were less predictive of total CD34+ cells collected (p=0.012) in patients with SCD (r2=0.2557, p=0.0023) when compared to historical control patients without SCD (r2=0.7638, p<0.0001). Real time measurement of cell counts at standard (Hematocrit 3%, S) and deep (Hematocrit 5%, D) collecting interfaces of the apheresis machine showed a significant decrease in lymphocytes (p=0.0146), increase in monocytes (p=0.0496), and increase enrichment in CD34+ cells at the D interface (Panel A). Enrichment of CD34+ cells correlated with monocyte enrichment and (n=21, r2=0.6706, p<0.0001) and an increased fold change in CD34+ cells between the S and D interfaces correlated with higher PB reticulocyte percentage (r2=0.5295, p=0.0111). Further analysis of the individual collection interfaces revealed that most RBCs were HbS containing despite a PB HbS concentration <30%, consistent with an enrichment for shorter lived sickle red cells. CD47 expression has been previously reported in higher concentrations on the surface of younger RBCs during erythropoiesis. Surface expression of CD47 was found to be significantly higher at both the S and D interfaces compared to the PB of patients, indicating that the accumulation of a younger population of RBCs at the collecting interfaces. As microcytic RBCs have been associated with poor collection efficiencies, the SCD red cell hemoglobin concentration in the PB was used as a marker for size and a significant correlation was determined (r2=0.6086, p=0.0001). Patients with SCD can mobilize PB HSC in response to plerixafor but demonstrate specific characteristics that impede the collection of stem cells. During apheresis, CD34+ stem cells sediment at a deeper hematocrit interface in the buffy coat as a result of displacement by the younger patient-derived population of RBCs. The relative shift in erythrocyte sedimentation requires a corresponding alteration in collection strategies to improve the collection efficiency. In addition to red cell age, other factors that affect red cell buoyancy including iron status or additional globin gene mutations negatively impact apheresis stem cell collections. Full assessment of red cell parameters can identify patients that are at risk for poor stem cell collection outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

G-CSF Mobilized Apheresis As an Alternative Source of CAR T-Cells

Background Cellular therapies are traditionally manufactured from an apheresis product collected at 'steady state' i.e. without any cytokine administered prior. We sought to evaluate the feasibility of utilising the CD34-negative fraction of recombinant human granulocyte colony stimulating factor (rhG-CSF) mobilised apheresis products for the manufacture of CAR T-cells. The effect of rhG-CSF on CAR T-cell function is largely unknown. We conducted an extensive comparison of phenotypic and functional attributes of CAR T-cells manufactured after rhG-CSF exposure (mobCAR) to those manufactured from a steady state apheresis (ssCAR), in order to test the hypothesis that rhG-CSF mobilised CAR T-cells are functionally equivalent to steady state CAR T-cells. Methods Untransduced T-cells (UTD) and CAR T-cells targeting CD33 (CART33) were manufactured using paired steady state (ss) cells and cells collected after rhG-CSF treatment (mob). On a protocol approved by the institutional review board, 4 healthy volunteers had ss cells collected from peripheral blood and cryopreserved, and later underwent an apheresis collection after 4 days of G-CSF (10ug/kg/daily) to collect and cryopreserve mob cells. CART33 were then manufactured from thawed ss and mob cells in paired expansions. Cells were activated using CD3/CD28 beads, transduced with lentiviral vector and expanded using Ex-Vivo based cell culture media (fortified with IL-7/IL-15 and human serum), and cryopreserved when mean cell volume was less than 300fL. Extensive comparisons between ssCART33 and mobCART33 were completed, including analysis by flow cytometry, CyTOF, single cell RNA sequencing (scRNAseq) and metabolomics by mass spectrometry. In vitro assays of degranulation, cytokine production and killing were performed, and in vivo function was assessed using an immunodeficient murine model after engraftment with a CD33 expressing cell line. Results Paired effector T-cells were manufactured with lower fold expansion of mobCAR [4 vs. 7 population doublings, p<0.01] though a sufficient number of mob-CAR were able to be manufactured. Mean CAR expression was higher in ssCAR though this was not statistically significant [ssCAR 78.6% (55-79%) vs. 64.7% (59-71%), p>0.05], and no significant differences in CD4/CD8 ratio were observed. In vitro degranulation, cytokine production and tumour cytolysis were comparable between ssCAR and mobCAR. Functional equivalence was also seen between ssCAR and mobCAR in mouse xenografts of an acute myeloid leukemia (AML) model, with no differences in body weight, toxicity, or mouse survival observed (Figure 1). While significant inter-donor differences in T-cell subsets by CyTOF were observed, minimal differences were seen between the ss and mob products for a given donor, and integrated analysis of scRNASeq data showed a similar transcriptomic profile for ssCAR and mobCAR cells (Figure 2). Metabolomics analysis is ongoing. Conclusions and future directions We were able to successfully manufacture CAR T-cell products from G-CSF mobilized cryopreserved apheresis products, and found comparable in vivo anti-tumor efficacy and toxicity, with no significant differences in the G-CSF exposed product by multi-omic analyses. Successful completion of this work may support the use of cryopreserved G-CSF stimulated apheresis units for cell therapy applications. This may allow for an adjunct cell therapy to hematopoietic stem-cell transplant to be manufactured from the same mobilized collection, or provide a means by which previously cryopreserved mobilized units may be utilised, noting frequently lower cumulative chemotherapy exposure in historically cryopreserved T-cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Peripheral Blast Count at Apheresis Acts Independent of Disease Burden As a Risk Factor for Survival Following Tisagenlecleucel in Children and Young Adults

Introduction: Chimeric antigen receptor (CAR) T-cells have provided a therapeutic option for patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) who have failed standard chemotherapy approaches. Despite initial responses, relapse rates after tisagenlecleucel approach 40% for pediatric patients necessitating a need to improve outcomes with this therapy. High pre-infusion disease burden (defined as ≥5% blasts on marrow or extramedullary disease) has been associated with inferior overall survival (OS) and event free survival (EFS) (Schultz 2021). However, it remains unclear if debulking disease will improve outcomes. Additionally, bridging chemotherapy used to debulk may introduce toxicity, without established benefit. Peripheral blast count represents high burden disease in the bone marrow. No current clinical standard exists guiding peripheral blast thresholds prior to apheresis. It remains unclear if having peripheral blasts at the time of apheresis is a surrogate marker for disease burden, or if peripheral blasts at time of apheresis collection has inherent independent predictive value. Methods: We performed a retrospective analysis from the Pediatric Real-World CAR Consortium (PRWCC) consisting of 15 US institutions. All patients with data available regarding their peripheral blast count at time of apheresis were included. Primary outcomes were day 28 response, EFS (events; second malignancy, relapse, or death) and OS. Baseline factors of interest included age at diagnosis, sex, pre-infusion disease burden, prior lines of therapy, prior CD19 therapy, time from diagnosis to CAR infusion, CAR dose, prior stem cell transplantation (SCT), and timing of 1st relapse post SCT (Schultz 2021). The impact of peripheral blast count at apheresis was assessed using Cox models adjusting for all baseline factors of interest. Kaplan Meier (KM) curves were reported for time-to-death and time-to-first event. Results: Of 185 patients who were infused with tisagenlecleucel in the PRWCC database, 162 patients had peripheral blast data at time of apheresis available for analysis: 120 (74%) had no peripheral blasts on CBC and 42 (26%) had >0 blasts at apheresis. A greater proportion of patients with >0 blasts had high disease burden (79%) compared to those with no peripheral blasts (44%) (table 1). The median (interquartile range, IQR) from apheresis to CAR T infusion is 7 weeks (wks) (5.2-10 wks) and there was no difference in time to infusion for patients with no blasts (n=120): 7 wks (6-10) compared to those with >0 blasts (n=42): 6 wks (5-8.8). Day 28 response did not differ between those with no blasts (87% CR) and those with >0 blasts (83% CR) (p=0.83). With a median follow-up of 334 (143-536) days (>0 peripheral blasts; 248 (92, 520), no blasts; 353 (186, 549)), death occurred more often in patients with >0 blasts (43%) when compared to those with no blasts (22%). Patients who had >0 blasts had significantly inferior event-free survival (p=0.008) and overall survival (p=0.004) compared to those with no peripheral blasts at apheresis. Univariate analysis showed that for every 10% increase in blast percentage, the hazard of death increased 1.19-fold (95% confidence interval (CI) 1.05-1.35;p=0.006) and the hazard of events increased 1.10-fold (95% CI 0.99-1.23; p=0.077). High pre-infusion disease burden had a significantly higher hazard of death (p=0.002) and of event (p=0.002), compared to no detectable disease. After adjustment for baseline factors including high vs no vs low disease burden, multivariable analysis demonstrated higher blasts remained significantly associated with higher hazard of death (p=0.004) and event (p=0.04). KM curves were stratified by pre-infusion disease burden (high versus low/no) and peripheral blast count at apheresis (no blasts versus >0 blasts) (figure 1). Patients with low/no disease burden and no blasts at apheresis had the best EFS when compared to those with high disease burden (p<0.001). Although not statistically significantly, amongst patients with high pre-infusion disease burden, those with >0 blasts had slightly worse OS (p=0.13) and EFS (p=0.47) compared to those with 0 blasts. Conclusion: Higher peripheral blasts at apheresis associates with inferior outcomes after tisagenlecleucel, even after multivariable analysis. Further study is needed to determine the mechanism of this association and if it is modifiable. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

National Blood Foundation 2021 Research and Development summit: Discovery, innovation, and challenges in advancing blood and biotherapies.

National Blood Foundation 2021 Research and Development summit: Discovery, innovation, and challenges in advancing blood and biotherapies.