Leukapheresis Collections Research Articles

INTEGRICELLTM: Accelerating cellular therapy, de-risking leukapheresis collection, cryopreservation, and integrating global logistics

INTEGRICELLTM: Accelerating cellular therapy, de-risking leukapheresis collection, cryopreservation, and integrating global logistics

Delivery of Obecabtagene Autoleucel (obe-cel, AUTO1) for the FELIX Pivotal Study Demonstrating Robust Cell Processing, Robust Release Testing, and Reliable Logistics, Together with Readiness for Sustainable Patient (pt) Care

Background: Obe-cel is an autologous chimeric antigen receptor (CAR) T cell product whose CD19 binding domain has a unique fast off-rate and was designed for improved safety and persistence over existing CD19 CAR T therapies. Successful CAR T therapy relies on a rapid and effective end-to-end process. The challenge for product manufacturing is twofold: first, pts with high tumor burden can have T cells that are highly differentiated and exhausted; second, pts with leukemic cells in circulation have apheresis containing a substantial proportion of leukemic cells that require removal before manufacture can start. Having robust, reliable manufacturing, testing, logistics processes, and teams that are able to provide a consistent CAR T product is key to achieving timely vein-to-certification/vein-to-delivery (V2C/V2D) targets. This abstract reports on the manufacturing, quality control (QC) and logistics processes set up for the FELIX study, and the impact of on-study optimization for scale up of production. Methods: The FELIX clinical study design required manufacturing, testing and logistics processes that could support a global, multicenter study with a target infusion of ~90 pts. The key elements were: 1) manufacturing and testing location: obe-cel was manufactured in Stevenage, UK, chosen because of proximity to a major international airport (London Heathrow) that provides logistics access to Europe and the US, and the ability to leverage existing knowledge and talent; 2) manufacturing process: manufacturing was carried out 7 days/week, was highly automated with mainly closed-system processing, using a Miltenyi Biotec Prodigy ® (https://www.miltenyibiotec.com), 3) analytical procedures: QC procedures, including measuring biological activity and sterility testing, were carried out in-house to reduce turnaround time (TAT); 4) logistics: TAT was minimized and reliability improved by establishing primary and secondary routes plus turnkey contingency plans with courier and charter flight options 5) targets: V2C (time from leukapheresis to quality release) and V2D (time from leukapheresis to delivery of product to the hospital) of ~23 and ~25 days, respectively, for the FELIX study. Results: Between Jul 2021 and Dec 2022 (FELIX Phase II Cohort A), the 32 clinical sites for FELIX in the US, UK and Spain were all supplied obe-cel from the UK. Median V2C and V2D times were 21 and 24 days, respectively. Despite the median leukemic B-cell content in apheresis being 21% (range 0-97%) and the median CD3+ T cell content being 13% (range 1-91%), 96% of manufactured obe-cel batches reached their target dose of 410 x 10 6 CAR T cells. From apheresis to product the median (range) percentage of naïve T cells increased from 20% (1-73%) to 31% (2-74%) while the medians for T CM and T EM remained unchanged at 13% to 13% and 19% to 17%, respectively, and the median TEMRA content decreased from 37% (range 4-93%) to 31% (range 5-90%), indicating that the manufacturing process managed to drive towards a less differentiated T cell phenotype in the final drug product. Transduction rates were consistently high with a median of 70% (range 14-87%). In the morphological Phase II Cohort A of the FELIX study, 112 pts were leukapheresed and 94 (84%) were dosed. All leukapheresis collections and cell product deliveries were executed successfully and with the expected quality, despite the challenges posed by the COVID-19 pandemic. In addition, US international airline flights decreased by 43 % (US Dept of Transportation, Bureau of Transportation Statistics; July 2023) compared with pre COVID-19 pandemic, but sample collection and product delivery were successfully maintained, with no batches impacted. Maintaining consistently short QC and release times were driven by streamlined analytical strategy and batch records. Conclusions: The FELIX study successfully demonstrated the robust operability of obe-cel manufacturing, QC and logistics processes, meeting target V2C and V2D. All apheresis starting material successfully processed despite the multitude of constraints posed by the COVID-19 pandemic. Further optimization and improvements made during the study increased reliability, consistency and precision of the manufacturing process, and supported the development of a new obe-cel manufacturing facility with greater production capacity that aims to achieve a ≥95% manufacturing success rate with ≤15-day V2C times.

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 <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.

Feasibility of manufacture of chimeric antigen receptor-regulatory T cells from patients with end-stage renal disease

BackgroundGene-modified cell therapy with regulatory T cells (Tregs) is a promising approach to prevent graft rejection and induce immunological tolerance in organ transplantation. We are developing a cell therapy comprising autologous naïve Tregs that are isolated from leukapheresate, transduced with lentiviral vector encoding a chimeric antigen receptor (CAR) recognising human leukocyte antigen class I molecule A*02 (HLA-A*02), and expanded ex vivo before cryopreservation as resultant drug product (TX200-TR101). In an ongoing first-in-human study (NCT04817774), kidney transplant recipients will receive a single infusion of TX200-TR101 2–3 months after transplantation. The phase 0 study described here evaluated the feasibility of manufacture of TX200-TR101 for the target population, i.e., end-stage renal disease (ESRD) necessitating kidney transplantation. Participants in this study did not receive an infusion of drug product.MethodsFour patients with ESRD and HLA-A*02 negative typing underwent leukapheresis to collect starting material for manufacture of TX200-TR101. Manufacturing success criteria were predefined as a batch of CAR-Tregs with cell quantity in each batch ≥ 104 cells/kg body weight, cell viability ≥ 70%, transduction efficiency ≥ 20% and hypomethylation of the FoxP3 gene (Treg-specific demethylated region [TSDR]) ≥ 80%. Other manufacturing variables included Treg identity and maturation by phenotyping, residual bead count, vector copy number, endotoxin level, sterility, and presence of mycoplasma. The characteristics of leukapheresate starting material and drug product from patients with ESRD were compared with those from commercially purchased leukapheresate from 10 healthy donors.ResultsNo safety issues were identified during leukapheresis collections. Batches of drug product were manufactured from all 4 patients with ESRD and met the predefined success criteria. There was some variability in leukapheresate starting material in terms of volume of apheresis and total leukocyte counts between patients with ESRD and healthy donors, but percentage differential white blood cell counts were comparable. The quality, quantity and functional activity of manufactured CAR-Tregs were similar between ESRD patients and healthy donors. CAR-Treg drug product from one patient with pre-existing lymphopenia had similar high quality but reduced cell quantity compared with batches from the other patients with ESRD, although yield was still above the predefined target minimum number of cells.ConclusionsManufacture of high-quality naïve CAR-Tregs from patients with ESRD is safe and feasible.

Early Collected Leukapheresis Material Improves Tisagenlecleucel Manufacturing Success and Time from Order to Pickup of the Leukapheresis Product: A Real-World Experience

Background Patient leukapheresis material collected for tisagenlecleucel manufacture can be cryopreserved for up to 30 months prior to use in manufacturing, allowing potential benefits in scheduling, shipping, and patient management (Qayed et al, Cytotherapy 2022). Recent data have reported leukapheresis in patients with DLBCL after failure to first line of therapy and the positive impacts of collecting these patients early on the quality of the leukapheresis material (Grisariu et al, EBMT 2022; Farina et al, EBMT 2022). Here, we evaluated the prevalence of tisagenlecleucel orders manufactured from leukapheresis material collected prior to order submission (historical collections), assessed whether this prevalence was similar across countries and indications, and reported the impacts of using historical collections on tisagenlecleucel manufacturing outcomes. Methods Between Jan-2020 and Jun-2022, over 5000 patients with ALL or DLBCL globally had cryopreserved leukapheresis material available for commercial tisagenlecleucel manufacture. Date of order submission for tisagenlecleucel manufacture, leukapheresis collection, and pickup of the cryopreserved leukapheresis product from the treatment center was captured through CellChain™, the Novartis online portal for order management. Leukapheresis material was defined as a historical collection if the date of leukapheresis occurred before the date of order submission. Prevalence of historical collections between the US, Europe, and other countries where tisagenlecleucel is commercially available was analyzed. Manufacturing success rates (MSR) and time between order submission to pickup of the cryopreserved leukapheresis material were evaluated. Results Treatment center practices on when leukapheresis was performed relative to order submission were significantly different between countries and indication. Amongst patients with DLBCL, the percentage of patients where leukapheresis occurred prior to order submission was 41% in Europe, 5% in the US, and 22% in other countries. Further, the percentage of patients with DLBCL collected more than 60 days prior to order submission was 6% in Europe, less than 0.5% in the US, and 1% in other countries (Fig. 1A). For patients with ALL, differences in percentage of historical collections were less striking: 42% in Europe, 32% in the US, and 23% in other countries. Interestingly, the percentage of patients with ALL collected more than 60 days prior to order submission was the highest in the US at 4%, vs. 2% in Europe or other countries (Fig. 1B). Compared to batches manufactured from collections performed after order submission, batches manufactured from historical collections showed a reduced median time between order submission and pickup of the cryopreserved leukapheresis material from 12 days to 5 days. Compared to products manufactured from leukapheresis material collected after order submission, a marginally higher tisagenlecleucel MSR was observed when the leukapheresis material was collected more than 60 days prior to order submission (96% MSR vs. 93% MSR), with no manufacturing terminations reported (Fig. 2). Conclusions This report describes the real-world experience of when patients are collected compared to when the order for tisagenlecleucel manufacture is submitted. Having a leukapheresis material already collected and cryopreserved at time of order submission, decreased the median time between order submission and pickup of the leukapheresis product from 12 to 5 days, potentially leading to faster patient access to tisagenlecleucel. A small but significant number of patients intended for tisagenlecleucel treatment were collected more than 60 days prior order submission, suggesting that these patients may have been collected prior to their eligibility for CAR-T. However, this practice to collect patients pre-emptively, especially more than 60 days prior order submission, was highly variable and was most prevalent in Europe for patients with DLBCL at 6% of patients and most prevalent in the US for patients with ALL at 4% of patients. A marginally higher MSR when using a leukapheresis material collected more than 60 days prior order submission was observed. However, the numbers were small and thus the benefits of early collected leukapheresis material on the manufacturing outcomes remain to be confirmed in larger sample size. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Considerations for immune effector cell therapy collections: a white paper from the American Society for Apheresis

Background aimsThis white paper was developed to provide leukapheresis guidance for the collection of mononuclear cells from adult and pediatric patients who are destined for immune effector cell (IEC) therapies for commercial and research applications. Currently, there is considerable variability in leukapheresis processes and limited published information regarding best practices relevant to new cellular therapies, especially IECs. Herein the authors address critical leukapheresis questions in five domains to help guide consistent collection processes and ensure high-quality products. The first four domains are onboarding, pre-collection, collection and post-collection, with protocol feasibility, preparation, care and follow-up of the patient/donor at each step, respectively, and technical considerations during collection. The fifth domain of quality assurance focuses on ensuring product potency, purity, safety and auditing. MethodsThe American Society for Apheresis (ASFA) Clinical Applications Committee (IEC Therapy Subcommittee) was charged by the society's board of directors with working collaboratively with other ASFA committees and organizations, including the Foundation for the Accreditation of Cellular Therapy, Association for the Advancement of Blood and Biotherapies, American Society for Transplantation and Cellular Therapy, National Marrow Donor Program and International Society for Cell & Gene Therapy, to develop guidelines regarding leukapheresis collection of cells destined for the manufacture of IEC therapies. After a review of the literature and discussion with members of the involved committees and various institutions, a draft guidance was created and circulated for comment and revision. ResultsCritical aspects of apheresis that could affect the quality and quantity of the leukapheresis product were identified. These areas were then discussed and reviewed. After consensus, the best practice guidelines were proposed and accepted. ConclusionsIn the current era of rapid growth of IEC therapies, it is important to address critical leukapheresis steps to provide high-quality products and more consistent practices and to eliminate redundant efforts.

Impact of Autologous Hematopoietic Cell Transplant (HCT) Followed By Dendritic Cell/Myeloma Fusion Vaccine with Lenalidomide Maintenance in Increasing Multiple Myeloma (MM) Immunity (BMT CTN 1401)

Impact of Autologous Hematopoietic Cell Transplant (HCT) Followed By Dendritic Cell/Myeloma Fusion Vaccine with Lenalidomide Maintenance in Increasing Multiple Myeloma (MM) Immunity (BMT CTN 1401)

Single institution retrospective review of CD34+ apheresis collection based on mobilization regimen and primary diagnosis

Abstract Introduction/Objective Plerixafor has been increasingly used in conjunction with G-CSF to increase mobilization of CD34+ stem cells in patients undergoing leukapheresis collection for autologous stem cell transplantation. While G- CSF and plerixafor have been shown to increase CD34+ counts, studies examining patients’ ability to meet collection goals for patients with multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and non-Hodgkin lymphoma (NHL) have not been well documented. Methods/Case Report All autologous stem cell collection patients between 2017-2021 were retrospectively reviewed and categorized by primary diagnosis. Patients received a mobilization regimen of G-CSF alone, G-CSF and plerixafor, G-CSF and chemotherapy, or G-CSF, plerixafor, and chemotherapy. CD34+ stem cell collection results were recorded. All units of CD34+ stem cells were processed according to protocol and viability was assessed by Trypan blue method on a thawed aliquot 2 weeks after processing. Results (if a Case Study enter NA) 385 patients (271 MM, 41 DLBCL, 30 HL, and 43 NHL patients; male: 242; female: 143; age range: 16-78) were identified. Binomial logistic regression demonstrated that use of plerixafor with G- CSF was negatively associated with meeting CD34+ goal collection (OR= -1.57, p= 0.003) compared to G-CSF alone. This result was particularly true for MM patients (OR= -1.62, p= 0.014). A pairwise t-test indicated that patients receiving G-CSF and plerixafor had lower CD34+ cell viabilities (t= 2.21, p = 0.028); after Bonferroni correction for multiple comparisons, MM samples also had significantly lower percentage viability than DLBCL (p= 0.003) or HL (p= 0.022) samples. Conclusion A higher percentage of patients mobilized on G-CSF alone were able to meet collection goal versus patients who were mobilized on G-CSF and plerixafor. Patients who received G-CSF and plerixafor had significantly lower viability than those who received G-CSF alone. We hypothesize these findings to be due to lower baseline mobilization for patients on G-CSF and plerixafor.

The Post-Thaw Recovery of Cryopreserved Chimeric Antigen Receptor (CAR) T-Cells during Manufacture Is Better Than That of Cryopreserved Peripheral Blood CD3+ Cells

As clinical applications for Chimeric Antigen Receptor (CAR) T-cell therapy extend beyond early phase trials, larger scale cell manufacturing processes incorporating cryopreservation steps become a logistical necessity. Mononuclear cells (MNC) collected by leukapheresis, which serve as the pre-culture “starting” fraction (PeC) for CAR T-cell production, may need to be cryopreserved prior to cell transportation to a centralized manufacturing facility. PeC aliquots may be cryopreserved for repeat cultures in cases of primary culture failure or for patient re-dosing. Further, cryopreservation of the post-culture (PoC) CAR T-cell product is essential in order to ease post-manufacture transportation, ensure completion of regulatory testing prior to product release and avoid product wastage due to unanticipated patient-related delays. The effect of cryopreservation on CAR T-cell viability, expansion, phenotype and transduction efficiency merit evaluation prior to routine use.From 2012 to 2017, data was obtained on 160 consecutive autologous CAR T-cell cultures initiated for patient infusion on 6 clinical trials of different CAR T-cell types (1. Anti-CD19-pediatric trial (n=58), 2.Anti-GD2 (n=14), 3.Anti-BCMA (n=21), 4.Anti-CD22 (n=43), 5. Anti-CD19-adult trial (n=22), 6. Anti-CD30 (n=2)).Following autologous leukapheresis collection, PeC was either cryopreserved-thawed (PeC-C) or used fresh (PeC-F) for CAR T culture initiation, depending on patient needs. Thereafter, cell selection, transduction-expansion and harvest were performed according to trial protocol specific instructions. The harvested PoC was again cryopreserved (PoC-C) or infused fresh (PoC-F). Data on patient demographics, number, timing and duration of cryopreservation, Total Nucleated Cell (TNC) count, viability, fold expansion (FE), transduction efficiency (TE), CD3% and CD4/CD8 ratios was collected. Post thaw TNC recovery (PTR) % was calculated as TNC*viability at thaw/TNC* viability at cryopreservation.Afterdiscarding 13 products due to manufacturing failure (MF) during culture, 147CAR T infusates were prepared for pediatric or adult patients with hematologic malignancies (135, 91.8%) or solid tumors (12, 8.2%). Mean age was 27.7 years (±19.9), weight 60.7 kg (±26.3), median CAR T-cell dose of 1x106 CAR-transduced viable CD3+ cells/kg (Range: 1x105 - 1x107). Gamma-retroviral or lentiviral vectors were used for CAR delivery in 88 (59.9%) and 59(40.1%) infusates, respectively. Pre-culture lymphocyte concentration included ficoll alone (42, 28.6%), flask adherence and/or elutriation (14, 9.5%), CD3.CD28 and/or CD4/CD8 selection (68, 46.3%). Of the 147 infusates, PeC-C and PoC-C were 69 and 78, respectively. Median duration of PeC-C and PoC-C was 6 days (Range: 3-868) and 9 days (Range: 1-408), respectively. TNC and CD3 PTR were lower in PeC-C than in PoC-C (Figure). At harvest (PoC), Mean FE, TE, CD3% and CD4/CD8 ratio were 12.8 (±12.5), 54.3 (±21.9), 97.6 (±4.4) and 1.9 (±2.0) respectively. In multivariate analyses, FE, TE, CD3% and CD4/CD8 ratios at harvest were unaffected by PeC-C status or duration. TE differed by trial. Twelve out of 13 (92%) MFs were due to failure of PeC transduction and/or expansion. Of the 12 MFs, 6 products were PeC-C (50%); 6 were PeC-F (50%). Among the 6 PeC-C MFs, none of the failures could be attributed to cryopreservation. In fact, 4 of the 6 PeC-C cultures were re-initiated with an additional PeC-C and all 4 yielded a successful product.Pre-culturecryopreservation impacts viable TNC and CD3% recovery during CAR T-cell manufacture compared to post-culture cryopreservation. Within this PeC fraction, CD3+ cells appear to have better viable recovery than the whole TNC fraction. This is likely due to a higher (differential) loss of monocytes and NK cells within TNCs. Despite these findings, most products manufactured after initial cryopreservation met clinical dose requirements. FE, TE, CD3% and CD4/CD8 ratios were unaffected by initial cryopreservation. The post thaw viability of CAR T-cells cryopreserved post culture was greater than cryopreserved peripheral blood CD3+ cells. Cryopreservation at either end of CAR T-cell manufacture is a viable strategy and may ease logistical constraints associated with large scale cell production. If optional, CAR T-cells cryopreserved post manufacture may be preferable to cryopreserving the starting fraction. [Display omitted] DisclosuresKochenderfer:Bluebird bio: Research Funding; Kite Pharma: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.

Factors Affecting Lymphocyte Collection Efficiency and Manufactured Product Specification during Leukapheresis for Diffuse Large B Cell Lymphoma Patients Treated with Commercial Tisagenlecleucel

Introduction: Leukapheresis and adequate cell collection of patient derived T cells is critical in the successful manufacturing of chimeric antigen receptor T cell (CAR-T) therapy. There are limited published data on parameters that might affect quality of leukapheresis collection or collection efficiency (CE). The aim of this study is to identify patient characteristics or collection parameters that might affect leukapheresis CE and any potential impact on manufactured product characteristics. Methods: We retrospectively reviewed collection data from 23 consecutive patients with relapsed/refractory diffuse large B cell lymphoma (DLBCL) who underwent leukapheresis for Kymriah® (tisagenlecleucel) at our institution from May 1st, 2018 to July 1st, 2020. The study was approved by the Institutional Review Board. Non-stimulated mononuclear cell (MNC) collections were performed on the Spectra Optia, Terumo BCT, using the continuous MNC collection protocol. Acid citrate dextrose A (ACD-A) was used for anticoagulant. Inlet/AC ratio maintained at 15:1. Collection flow rates were targeted for 60-85 mL/min, with goal of processing 2-4 times the patient's total blood volume (TBV). Patients underwent peripheral collection if they had adequate venous access, otherwise central venous access was obtained. Peripheral blood (PB) flow cytometry was obtained prior to leukapheresis. Collection efficiency (CE) was calculated using the following formula: CE = Product CD3+ x106/ (PB CD3+ cells/μL x Total Volume Processed/ 1000). Descriptive summary statistics included median (range) for continuous variables and number (percentage) for categorical variables. Routine methods of continuous and categorical data analysis were performed using Statistica (version 7). Graph Pad Prism 8 was used to look for possible relations between variables. Results: Table 1 summarizes patient baseline characteristics (at time of leukapheresis) and pre-collection laboratory results. Table 2 summarizes the apheresis product. The median CE was 73% (with SD=13%). Baseline characteristics did not correlate significantly with collection efficiency. Collection by tunneled catheter or peripheral vein access did not significantly impact collection efficiency. PB absolute lymphocyte count (ALC) strongly correlated with PB CD3 cell and apheresis product CD3 counts (p<0.0001). PB CD3 cell count significantly correlated with apheresis product CD3 count (p<0.03; Figure 1). Notably, one patient with PB ALC of 0.0 K/uL and PB CD3+ of 0.204 K/uL was adequately collected, with successful product manufactured. All patients were able to receive manufactured product. Table 3 summarizes the tisagenlecleucel product information. Nine patients received out of specification (OOS) product due to low cell viability. All OOS products were due to cell viability below 80% threshold. When OOS products were compared to in specification products, OOS products were significantly associated with lower patient weight, less total volume processed, shorter collection run times, and smaller final collection product volumes. TBV processed was similar between groups. Conclusions: High CE is feasible in heavily pre-treated DLBCL patients, even with very low peripheral blood lymphocyte counts (ALC < 100). Our study confirms that pre-collection ALC and PB CD3 are strong indicators of CD3 yield in the leukapheresis collection product. OOS products were significantly associated with smaller volume processed, shorter run times, and smaller product volumes. This warrants further investigation. Disclosures Shah: NCCN: Vice-Chair, Acute Lymphoblastic Leukemia Working Group: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead, Precision Biosciences, Novartis, AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Kite/Gilead, Celgene/Juno/BMS, Novartis, Pfizer, Amgen, Spectrum/Acrotech, Precision Biosciences, Beigene, AstraZeneca, Pharmacyclics/Jansen, Adaptive: Honoraria; Moffitt Cancer Center: Current Employment; Kite/Gilead, Jazz, Incyte: Research Funding. Nishihori:Karyopharm: Other: Research support to institution; Novartis: Other: Research support to institution. Locke:Calibr: Consultancy; Allogene: Consultancy; Cellular Biomedicine Group: Other: Consultancy with grant options; Celgene/Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Wugen: Consultancy; GammaDelta Therapeutics: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding. Jain:Kite/Gilead: Consultancy; Novartis: Consultancy.

A Pilot Study of Acalabrutinib with Bendamustine/Rituximab Followed By Cytarabine/Rituximab (R-ABC) for Untreated Mantle Cell Lymphoma

A Pilot Study of Acalabrutinib with Bendamustine/Rituximab Followed By Cytarabine/Rituximab (R-ABC) for Untreated Mantle Cell Lymphoma

Characterization of Peripheral Blood Mononuclear Cells Addback Following CD34 Enrichment, Engraftment and T and NK Cells Immune Reconstitution in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Background: CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998) are designed to deplete T cells and reduce AGVHD following allogeneic stem cell transplantation (AlloSCT). These approaches achieved low rates of AGVHD, but the grafts had few T and B cells. To improve immune reconstitution we undertook an alternative approach to addback small numbers and percentages of immune cells in the final HSCT product. We previously reported a very low incidence of AGVHD in pediatric recipients receiving CD34 enriched HPC products with peripheral blood mononuclear cells (PBMNC) addback containing a fixed dose of 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Recently we demonstrated that despite a 5 log depletion of T cells, PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid hematopoietic engraftment, high levels of donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. Patients had a 1 yr OS of 90% following familial haploidentical (FHI) CD34 Enriched Stem Cell Transplantation in patients with SCD (Cairo, JAMA Pediatr, 2020). Objective: To determine the final immune cell concentration following CD34 enrichment and PBMNC (2 x 105 CD3/kg) addback and determine the effect on engraftment and T and NK cell immune reconstitution. Methods: Patients and/or their guardians signed written informed consents and/or assents (NCT NCT02675959). CD34+ enrichment was performed using a CD34+ reagent system (CliniMACS; Miltenyi Biotec). Mononuclear cells (2 × 105 CD3 cells/kg of recipient body weight) were removed from the leukapheresis collection prior to CD34+ enrichment and were cryopreserved as a source of MNC addback (T cells). The addback products were analyzed for CD3+CD56- T cells, CD3-CD56+ NK cells, CD3+CD56+ NKT cells, Lin-CD123+ HLA-DR+ DC cells and Lin-CD11c+ HLA-DR+ DC cells by multicolor flow cytometry analysis. Th1/Th2 cytokines were measured by multiplex assays. T cell activity was measured by viral T cells IFN-g and plasma cytokines. NK function was measured by NK receptor expression by flow cytometry analysis and in vitro cytotoxicity. Results: We identified in the PBMNC addback, mean+SEM white blood cell (WBC) percentage of: CD3+ CD56- T cells = 56.4±5%; CD3- CD56+ NK cells = 4.6±1%; CD3+ CD56+ NKT cells = 5.1±0.6%; CD19+ B cells = 29.9±3.5%. Lin- WBC consisted of: CD123+ HLA-DR+ DC cells = 18.4±8.2%; CD11c+ HLA-DR+ DC cells = 6.0±3.0%. There were 20.0+9.1e6 T cells, 1.1+0.3e6 NK cells, 1.6+0.7 e6 NKT cells, 8.6+2.5e6 B cells, 1.2+0.6e6 CD123+DC and 0.8+0.5e6 CD11c DC in the final infused products (Fig.1). We found that percentages of IFN-g+ in CD4 cells in response to CMV (pp65), ADV (hexon) and EBV (BZLF1), ranged from 0.2%+0.1% to 0.5%+0.1%, while percentages of IFN-g+ in CD8 cells in response to the antigens ranged from 0.7%+0.3% to 3.7%+1.8% when examined at days 180, 270 and 365. NK (CD3- CD56+) reconstitution was extremely rapid and occurred as early as day 30 (35.5±8.6%, 2710+1624.4 cells/ul total cells; p<0.01 vs pre-t). There were no significant differences pre-HSCT vs day 365 in plasma cytokines (Th1 and Th2) and growth factors released including IFN-g, TNF-a, IL-18, IL-4, IL-5, IL-6, IL-10, G-CSF, MCP-1 and MIP1a. There was also robust expression of NK receptor expression including NK cytotoxicity receptors, NK KIR receptors, and C-type lectin-like receptors at day 30 as compared to pre-HSCT. NK cytotoxicity, as measured using PBMC cells from recipients at different time points against K562 (E:T=10:1), was also significantly increased at day 30 (26.2±2.8%) and day 180 (28.3±3%) vs pre-HSCT (16.1±2.1%) (p<0.01). As a NK cell activation marker, CD107a expression and granzyme B levels in gated NK cells peaked at day 30. Conclusion: PBMNC addback to CD34 enriched HPC products, with a final dose of 2 × 105 CD3 cells/kg, led to stem cell products with a diverse mixture of T, NK, NKT, DC1, and DC2 cells. Immune reconstitution following PBMNC addback to CD34 enriched cells resulted in excellent CD4 and CD8 responses to CMV, ADV and EBV, and rapid functional NK cell reconstitution (Supported by FDA R01FD004090 (MSC)). Disclosures Baxter-Lowe: CHLA: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Patents related to HLA typing, Research Funding. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Evolving the supply chain to counter key challenges in apheresis and leukapheresis collection

Evolving the supply chain to counter key challenges in apheresis and leukapheresis collection

An Innovative Standard Operation Procedure for Isolating GMP-Grade CD4+CD25+ T Cells from Non-Mobilized Leukapheresis.

This SOP describes a closed system for isolating GMP-grade CD4+CD25+ T cells from non-mobilized leukapheresis collections (nMLCs), independent of a clean room in a certified GMP premises, by using CliniMACS format GMP grade reagents (CD25-labeled magnetic beads with/without pre-depletion of CD8+ T cells and CD19+ B cells), a GMP grade-A laminar hood and CliniMACS cell processing system.

Phase II Study Evaluating the Safety and Efficacy of BL-8040 for the Mobilization of Donor Hematopoietic Stem and Progenitor Cells for Allogeneic Hematopoietic Cell Transplantation and Phenotypic Characterization of the Leukapheresis Product

▪Background. Mobilized hematopoietic stem/progenitor cells (HSPCs) are the most commonly used graft source for hematopoietic cell transplantation (HCT). Current practices for harvesting HSPCs with G-CSF and/or plerixafor are costly, involve a multi-day procedure with suboptimal HSPC yields in up to 30% of patients and are associated with some morbidity and mortality. Here, we sought to test BL-8040, a high affinity CXCR4 antagonist with rapid mobilizing kinetics and long receptor occupancy, in donors for allogeneic HCT (alloHCT).Methods. The primary endpoint was to assess the efficacy of a single injection of BL-8040 to mobilize ≥ 2 x 106 CD34+ cells/kg of recipient weight after no more than two leukapheresis (LP) collections. Donors received BL-8040 by subcutaneous (SC) injection 3 hours prior to LP on day 1. The study was conducted in 2 parts. Part 1 enrolled HLA-identical pairs and 14 donors were treated with 1 mg/kg BL-8040. Part 2 enrolled HLA-identical and haploidentical pairs and treated 11 donors with 1.25 mg/kg BL-8040. When < 5x106 CD34+ cells/kg recipient weight were collected, a second injection of BL-8040 and LP collection was performed the next day. Immunophenotyping of lymphoid, myeloid, and HSPC subsets in the blood and LPs was performed by flow cytometry.Results. 25 donor-recipient pairs were enrolled in the study. The median age of donors was 55 years (range 20-69); 18 were fully HLA-matched siblings, and 7 were haploidentical donors. One donor was replaced per protocol because of problems with vascular access. All 11 donors treated at the 1.25 mg/kg dose and 22/24 (92%) patients overall collected the minimum goal of 2 x 106 CD34+ cells/kg recipient weight in 2 LPs. 9/11 donors treated with 1.25 mg/kg BL-8040 collected the target of 5 x 106 CD34+ cells/kg recipient weight. The median CD34+ cell count was 15 cells/µL blood at 3-4 hours after BL-8040 and was maintained for > 24 hours. BL-8040-adverse related events consisted primarily of grade 1-2 injection site reactions and transient systemic reactions (hives). Recipients were a median of 58 years of age (26-71) with an ECOG PS of 0-2 undergoing alloHCT for AML (n=16, 64%), ALL (n=4, 16%), MDS (n=3, 12%), MPN (n=1, 4%) or Hodgkin lymphoma (n=1, 4%). 22 recipients were transplanted with BL-8040-mobilized grafts. Time to neutrophil engraftment occurred at a median of 13 days (n=22, range 11-26) and platelet engraftment occurred at a median of 20 days (n=20, range 15-41; no platelet nadir for 1 pt and no engraftment in 1 pt). With a median follow-up of 258 days, 5 deaths have been observed on study. These deaths were due to sepsis (Day +84), complications of sepsis in setting of grade 3 GVHD (Day+67), AML relapse (Day+143,+166), and complications of intrabdominal infection (Day+252). Grade II acute GVHD was observed in 2/22 recipients (9%) both of which resolved. Grade III-IV GVHD was observed in 5 out of 22 recipients (23%), of which 3 resolved. Flow cytometric evaluation of HSPCs purified from LPs by CD34 immunoselection revealed that BL-8040 preferentially mobilized CD34+ plasmacytoid dendritic cell (pDC) precursors (pre-pDCs) that express high levels of CXCR4 (Fig. 1A). These pre-pDCs are the immediate progenitors of pDCs; a distinct DC population capable of producing large amounts of type 1 interferons in response to viral infections. In contrast to plerixafor, BL-8040 remained bound (as determined by inhibition of anti-CXCR4 binding) to CXCR4 on all lymphoid, myeloid, and HSPCs after LP (Fig. 1B and data not shown). BL-8040 induced pan-mobilization of all major myeloid and lymphoid subsets, with maximum relative changes in the absolute numbers of circulating pDCs, B cells, basophils, myeloid DCs (mDCs), CD8+ T cells and CD14+HLA-DR+CD16lo classical monocytes (Fig. 1C). In general, the magnitude of mobilization of each subset correlated with the level of surface CXCR4 at baseline (Fig 1C, gray bars, R2 = 0.4; P = 0.02). Although there was pan mobilization of all CD8+ and CD4+ T cell subsets, BL-8040 preferentially mobilized naïve and central memory CD8+ T cells relative to the other T cell subsets (Fig. 1D).Conclusions. Treatment of normal donors with a single dose of BL-8040 results in rapid and sustained mobilization of HSPCs for use in allogeneic transplant. BL-8040 mobilized grafts result in rapid and reliable engraftment in transplant recipients. Additional data is required to assess the impact of BL-8040-mobilized grafts on rates of GVHD and relapse. [Display omitted] DisclosuresRettig:Novimmune: Research Funding; Amphivena Therapeutics: Research Funding. Uy:Curis: Consultancy; GlycoMimetics: Consultancy. Devine:Kiadis Pharma: Consultancy. Jaglowski:Juno: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding. Vainstein:BioLineRx Ltd.: Employment. Sorani:BioLineRx Ltd.,: Employment. Chen:BioLineRx Ltd.,: Employment. Bohana-Kashtan:BioLineRx Ltd.,: Employment, Equity Ownership; Cell Cure Neurosciences: Equity Ownership. Shaw:BioLineRx Ltd.: Employment, Equity Ownership.

Risks of leukapheresis and how to manage them—A non-systematic review

Leukapheresis is like any other preparative apheresis, except it isn’t: Leukapheresis typically takes much longer, larger blood volumes are processed and, consequently, larger ACD-A volumes are administered. Blood component donors and leukapheresis subjects are also quite different populations. Allogeneic donors tend to be younger and many are first-time donors, both of which are risk factors for adverse reactions during blood donation. Moreover, more than half of all leukapheresis collections are performed in patients. Here it is the age distribution, including patients at the extremes of age, as well as the underlying disease and co-morbidities which may expose them to higher, or different, risks compared to donors. Both groups thus have good reasons why adverse effects to leukapheresis might be more frequent, more severe, or even different in nature altogether. Compared to other preparative apheresis types like platelet or plasma apheresis, the risks of leukapheresis have been studied less extensively, as it is in comparison a relatively low-frequency intervention. Often leukapheresis remains a domain of hematologists who have a different sense of procedural safety than transfusionists. Furthermore, G-CSF mobilized “stem cell” aphereses by a wide margin outnumber unmobilized aphereses, so that the very strong signal from adverse reactions to G-CSF all but drowns out signals from the apheresis proper. This focused review assesses observations from leukapheresis as well as extrapolation of observations from other forms of preparative apheresis in an attempt to gauge the safety of leukapheresis and identify potential approaches to its further improvement. In short, the overall impression is one of a very satisfactory safety record of leukapheresis, with occasional issues with venous access or vasovagal problems, and frequent, but highly responsive and rarely limiting ACD-A toxicity.

Production of Anti-CD19 CAR T Cells for ZUMA-3 and -4: Phase 1/2 Multicenter Studies Evaluating KTE-C19 in Patients With Relapsed/Refractory B-Precursor Acute Lymphoblastic Leukemia (R/R ALL)

Introduction: ZUMA-3 and -4 (NCT02614066 and NCT02625480) are ongoing phase 1-2 multicenter trials evaluating KTE-C19, a CD28/CD3ζ anti-CD19 chimeric antigen receptor (CAR) T cell therapy, in adult and pediatric patients respectively with R/R ALL. The manufacturing process developed to support the clinical trials aimed to accommodate variability in the incoming apheresis material, maximize manufacturing success rate, and minimize timing between leukapheresis collection and KTE-C19 administration at bedside.Methods: Once eligibility criteria were satisfied, patients underwent leukapheresis to process 12-15 L of blood (2-5 blood volumes for pediatric patients) to target collection of 10 × 109 mononuclear cells (MNCs). After collection, leukapheresis material was shipped to the central manufacturing site and processed to enrich T cells using an automated and closed system. The T cell enrichment step was implemented to remove cellular impurities such as leukemic blasts and myeloid cells that may negatively impact T cell activation and expansion. Enriched T cells were activated using an antigen presenting cell (APC)-free and bead-free method and cultured in serum-free medium containing 300 IU/mL of interleukin-2. Activated T cells were transduced with a gamma retroviral vector that encodes the CAR gene and further expanded to achieve target dose of 2 × 106 CAR-positive T cells/kg body weight (minimum of 1 × 106). Overall target duration of the manufacturing process was 8 days with an allowed range for harvest set between process days 6 and 10. Final KTE-C19 product was washed, formulated, cryopreserved and tested for identity, potency, and adventitious agents. After acceptance criteria were met, KTE-C19 was shipped to the clinical site using a validated cryoshipper.Results: As of July 2016, 5 of the 6 patients enrolled in the clinical trials received the target dose of 2 × 106 CAR-positive cells/kg body weight. The assigned dose could not be generated for one patient enrolled in the ZUMA-4 trial. In this patient, analysis performed on the leukapheresis material revealed >99% leukemic blasts. For the other 5 patients, the target dose was achieved within 6 - 7 process days notwithstanding a wide range in T cell composition of the incoming leukapheresis material (range, 4.7% to 81%), and wide variation in percent leukemic blasts (range, 11% to 92%). Median frequency of CAR expression in the KTE-C19 final product was consistent across product lots. The manufacturing process led to final products highly enriched for T cells with a median CD3+ cell content of >99%. Expansion of T cells was consistent among the 5 product lots with a mean 7-fold expansion from transduction to harvest. Median time from leukapheresis collection to final product release was 14 days.Conclusions: Reproducible manufacturing of high-quality, clinical-grade autologous CAR T cell products may be challenged by the inherent variability of starting material in patients with high content of leukemic blasts in peripheral blood. The bead- and serum-free manufacturing process developed to support the ZUMA-3 and -4 clinical trials was able to generate products within 6 -7 days with a low failure rate (including from patients with high percentages of leukemic blasts), and most importantly, a short duration from leukapheresis collection to shipment of KTE-C19 final product back to the clinical center for patient administration. DisclosuresSabatino:Kite: Employment, Equity Ownership. Choi:Kite Pharma: Employment, Equity Ownership. Chiruvolu:Kite Pharma: Employment, Equity Ownership. Better:Kite Pharma: Employment, Equity Ownership.

Abstract LB-083: Phase I study of T cells redirected to EGFRvIII with a chimeric antigen receptor in patients with EGFRvIII+ glioblastoma

Abstract We initiated a Phase I study of intravenous delivery of autologous T cells re-directed to the EGFR variant III mutation by means of a lentiviral vector encoding a chimeric antigen receptor (CAR). Patients with recurrent glioblastoma (GBM) have their tumors screened for the presence of the EGFRvIII mutation by a next-generation sequencing based assay. Eligible patients undergo leukapheresis for collection of autologous T cells, which are genetically modified ex vivo to express the EGFRvIII CAR, expanded, and then cryopreserved for infusion. We report results on the nine patients we have treated. We targeted a challenging subset of GBM patients, with multi-focal residual, progressive disease refractory to standard and experimental therapies, including temozolomide, bevacizumab, and radiation. To date, we have found that infusion of 1-5×10⁁8 CART-EGFRvIII cells is feasible to manufacture and infusion is safe, without evidence of off-tumor toxicity or cross-reactivity to wild type EGFR. No clinical or laboratory signs of systemic cytokine release syndrome have been observed, though elevations in serum IL-6 occur concurrently with CART-EGFRvIII expansion in the peripheral blood. One patient developed non-convulsive status epilepticus nine days after CART-EGFRvIII infusion, which resolved with standard treatment and anti-cytokine therapy. All patients have had significant expansion of CART-EGFRvIII cells between 7-10 days post-infusion, as measured by flow cytometry and quantitative PCR in peripheral blood samples. Five patients have undergone surgical resection of tumor between 6-120 days after infusion, and pathologic evaluation has demonstrated infiltration of activated CAR T cells, recruitment of new T cells (as assessed by next generation T cell receptor deep sequencing), and specific EGFRvIII target antigen loss in GBM cells in some cases. These findings provide evidence that CART-EGFRvIII cells are safe, without evidence of off-target toxicity or cytokine release syndrome, and are immunologically active. The data also suggest a mechanism of antigen editing by activated CART-EGFRvIII cells that track to specific EGFRvIII GBM cells. Citation Format: Donald M. O’Rourke, MacLean P. Nasrallah, Jennifer Morrissette, Jan J. Melenhorst, Simon F. Lacey, Keith Mansfield, Maria Martinez-Lage, Arati Desai, Steven Brem, Eileen Maloney, Suyash Mohan, Sumei Wang, Gaurav Verma, Jean-Marc Navenot, Angela Shen, Zhaohui Zheng, Bruce L. Levine, Hideho Okada, Carl H. June, Marcela V. Maus. Phase I study of T cells redirected to EGFRvIII with a chimeric antigen receptor in patients with EGFRvIII+ glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-083.

276. Long-Term Relapse-Free Survival of Patients with Acute Myeloid Leukemia (AML) Receiving a Telomerase-Engineered Dendritic Cell Immunotherapy

There are few treatment options for patients with intermediate and high risk AML, and remission and relapse rates are dismal especially in patients ≥60 years old. A Phase 2 clinical trial was conducted in subjects with AML to assess a dendritic cell immunotherapy (AST-VAC1) engineered to express a modified form of telomerase that is processed through both the MHC Class I and II antigen presentation pathways. AST-VAC1 was prepared from leukapheresis collections from 33 subjects and were transfected with an mRNA encoding near full length telomerase (hTERT) modified with a lysosomal sorting signal, LAMP-1, which enhances presentation to both CD4+ helper and CD8+ cytotoxic T cells. hTERT is essential for maintaining the extended proliferative lifespan of tumor cells. AML patients were enrolled if they were in complete remission (CR1 or CR2) with intermediate or high risk cytogenetics. AST-VAC1 was prepared after induction therapy and before or after completion of consolidation cycles. AST-VAC1 containing 1 × 107 cells was administered as 6 weekly followed by 6 biweekly intradermal injections. If AST-VAC1 doses were available, patients were eligible to receive additional monthly boosts. Twenty one patients (median age: 55) in complete remission (16 CR1 and 3 CR2) or early relapse (2) received at least 3 injections of AST-VAC1. Only one Grade 3 or 4 adverse event, (idiopathic thrombocytopenia), possibly related to the immunotherapy was observed. The majority of adverse events were transient including headache, fatigue, erythema and rash. The two patients who were vaccinated during early relapse progressed rapidly and did not receive the full dosing regimen of AST-VAC1. Of the 19 patients that were in CR, 13 received all 12 doses of AST-VAC1. Fifty-eight percent (11/19) developed T cell immune responses to hTERT as assessed by ELISpot analysis. Eleven of 19 patients (median follow-up 52 mos.) were in remission as of last follow-up; seven developed detectable cellular immune responses to hTERT. Of the 19 CR patients, 7 were ≥ 60 years old at the time of AST-VAC1 immunotherapy. Four of 7 patients ≥ 60 years old remained relapse free 52-59 months post AST-VAC1 immunotherapy with all four developing immune responses to hTERT. The three patients that received AST-VAC1 while in CR2 were in remission as of their last follow-up of 24, 50 and 59 months with two having hTERT immune responses. The median duration of complete remission was greater than that observed in historical controls especially for patients ≥ 60 years old where relapse-free survival at 4 years is typically 5-20%. The results suggest that immunotherapy with AST-VAC1 is safe, can stimulate immune responses to telomerase, and may extend relapse-free survival even in patients with high risk AML.

Blockade of PD-1 in Combination with Dendritic Cell/Myeloma Fusion Cell Vaccination Following Autologous Stem Cell Transplantation Is Well Tolerated, Induces Anti-Tumor Immunity and May Lead to Eradication of Measureable Disease

Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to incorporate immunotherapy in an effort to target residual disease. Our group has developed a cancer vaccine in which dendritic cells (DCs) are fused to autologous tumor cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Up-regulation of the PD-1/PDL1 pathway represents a key element contributing to tumor-mediated tolerance, and potentially muting response to vaccination. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (Pidilizumab, MDV9300) in combination with a dendritic cell/myeloma fusion cell vaccine following autologous transplantation. 22 patients have been treated with post-transplant immunotherapy. Mean age was 64. MM cells were isolated from bone marrow and were identified by expression of CD38 or CD138. Mean tumor cell yield was 118x106 cells. Adherent mononuclear cells were isolated from leukapheresis collections and cultured with GM-CSF and IL-4 for 5-7 days, then exposed to TNFα for 48-72 hours to generate mature DCs. DCs expressed co-stimulatory (mean CD86 75%) and maturation markers (mean CD83 50%). DC and MM cells were co-cultured with PEG and fusion cells were quantified by determining the percentage of cells that co-express unique DC and myeloma antigens. Mean fusion efficiency was 41% and the mean cell dose generated was 4 x 106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 92%, 89%, and 85%, respectively. As a measure of their potency as antigen presenting cells, DC/MM fusions potently stimulate allogeneic T cell proliferation ex-vivo (Mean stimulation index of 1.9, 9.2 and 7.1 for tumor, DC and DC/myeloma fusions respectively, n=21) Post-transplant immunotherapy was initiated after recovery from transplant-related toxicities. Median time from transplant to initiation of post-transplant immunotherapy was 80 days. Patients received 3 doses of Pidilizumab at 6-week intervals. DC/myeloma fusion cells vaccination is administered 1 week before each dose of Pidilizumab. To date, 22 patients have completed vaccinations and Pidilizumab. Adverse events judged to be potentially treatment related included grade 1-2 diarrhea, arthralgias, myalgias, fatigue, headache, nausea, chills, transaminitis, cytopenia, elevated TSH, and vaccine site reactions. A significant increase in circulatingtumor reactive lymphocytes was noted following post-transplant immunotherapy, as determined by T cell expressionof IFN-γ by CD8 cells following ex-vivo co-culture withautologous myeloma cell lysate. Mean percentage of tumor reactiveCD8 cells increased from 1.8% post-transplant to a peak of 9.16% following immunotherapy. In the post-transplant period, regulatory T cells fell to minimal levels and remained low throughout the period of immunotherapy. 6 patients achieved a best response of VGPR, 6 patients have achieved a nCR/CR, including 3 who converted to CR following immunotherapy. Median PFS from transplant is 19 months with ongoing follow up. In summary, DC/MM fusion cell vaccination in conjunction with PD1 blockade following ASCT was well tolerated, potently induced anti-tumor immunity, and in a subset of patients, resulted in the eradication of post-transplant measurable disease. DisclosuresRichardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.