Leukapheresis Products Research Articles

Evaluation of a flow cytometry-based method for determination of T-lymphocyte subtypes for quality assessment of cell therapy products

Chimeric antigen receptor-T (CAR-T) cell therapy is currently the best-known type of immune effector cells therapy. For CAR T-cell therapy, the determination of CD3+ T cells is necessary for the quality control of fresh leukapheresis product as starting material. The aim was to validate analytical method for quantification of percentage and absolute count of T lymphocyte subtypes (CD3+, CD4+ and CD8+ cells) in fresh apheresis products using single-platform method on flow cytometer BD FACS Canto II. Validation study included determination of precision, trueness (bias), assessment of linearity, carryover, comparison of results obtained with two different protocols on flow cytometer for CD3+ cells determination and stability study. For between-run precision coefficients of variation (CVs) were <20%, as well as bias for all T-lymphocyte subtypes. For within-run precision, CVs were <10%, except for low CD8+ cell (percentage 10.51% and viable absolute count 12.37%). Comparison of results obtained with two different protocols for CD3+ cells determination shows no statistically significant difference. Statistically significant differences between results of the analysis of CD4+ cells in fresh samples and results obtained after storage at 4 °C (p = .004) and at room temperature (p = .018) were found. In conclusion, method for enumeration of T-lymphocyte subtypes can be used in routine work on BD FACS Canto II instrument for quality assessment of fresh cell products collected by leukapheresis procedure.

Choosing T-cell sources determines CAR-T cell activity in neuroblastoma.

The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.

Monocytes in leukapheresis products affect the outcome of CD19–targeted CAR T-cell therapy in patients with lymphoma

AbstractCD19–directed chimeric antigen receptor (CAR) T cells can induce durable remissions in relapsed/refractory large B-cell lymphomas (R/R LBCLs), but 60% of patients do not respond or relapse. Biological mechanisms explaining lack of response are emerging, but they are largely unsuccessful in predicting disease response at the patient level. Additionally, to maximize the cost-effectiveness of CAR T-cell therapy, biomarkers able to predict response and survival before CAR T-cell manufacturing would be desirable. We performed transcriptomic and functional evaluations of leukapheresis products in 95 patients with R/R LBCL enrolled in a prospective observational study, to identify correlates of response and survival to tisagenlecleucel and axicabtagene ciloleucel. A signature composed of 4 myeloid genes expressed by T cells isolated from leukapheresis products is able to identify patients with a very short progression-free survival (PFS), highlighting the impact of monocytes in CAR T-cell therapy response. Accordingly, response and PFS were also negatively influenced by high circulating absolute monocyte counts at the time of leukapheresis. The combined evaluation of peripheral blood monocytes at the time of leukapheresis and the 4-gene signature represents a novel tool to identify patients with R/R LBCL at very high risk of progression after CAR T-cell therapy and could be used to plan trials evaluating CAR T cells vs other novel treatments or allogeneic CAR T cells. However, it also highlights the need to incorporate monocyte depletion strategies for better CAR T production.

Flow-based immunomagnetic enrichment of circulating tumor cells from diagnostic leukapheresis product.

The clinical utility of circulating tumor cells (CTCs) is hampered by the low number of cells detected. Diagnostic leukapheresis (DLA) offers a solution but, due to the observed non-specific binding and clumping, processing of DLA samples using the CellSearch system only allows for the processing of aliquots consisting of ~ 2% of the total DLA sample per test. Here, we introduce a flow enrichment target capture Halbach-array (FETCH)-based separation method in combination with a DNase preprocessing step to capture CTCs from larger fractions of DLA products without clumping. To evaluate the FETCH method, we processed peripheral blood samples from 19 metastatic castration-naïve prostate cancer (mCNPC) patients with CellSearch, and processed 2% aliquots of leukapheresis samples from the same patients with CellSearch as well as FETCH with or without DNase preprocessing. Using 2% aliquots from six patients, the use of FETCH with fewer immunomagnetic epithelial cellular adhesion molecule (EpCAM) conjugated ferrofluids was tested, whereas 20% aliquots from four patients were used to evaluate the processing of 10-fold larger DLA samples using FETCH. Results show that the cell clumping normally seen after immunomagnetic enrichment of DLA material was greatly reduced with the use of DNase pretreatment, while the number of CTCs detected was not affected. The number of CTCs detected in 2% aliquots of DLA using FETCH was unchanged compared to CellSearch and did not decrease when using down to 10% of the volume of immunomagnetic anti-EpCAM ferrofluids normally used in a CellSearch test, whereas the number of co-enriched white blood cells reduced a median 3.2-fold. Processing of a 20% aliquot of DLA with FETCH resulted in a 14-fold increase in CTCs compared to the processing of 2% aliquots of DLA using CellSearch and a total 42-fold median increase in CTCs compared to peripheral-blood CellSearch.

Hematopoietic Progenitor Cell Counts of the Leukapheresis Product Determined Using Sysmex XN Analyzers Predict a Sufficient Number of CD34+ Stem Cells in a Peripheral Blood Stem Cell Harvest for Autologous Transplantation.

Objective Several institutions outsource CD34+ cell counting of leukapheresis products, limiting rapid measurements, as results are obtained the next day. This problem is compounded with plerixafor use, a stem cell-mobilizing drug that increases leukapheresis efficiency but requires administration the day before leukapheresis. Use of this drug for a second leukapheresis procedure before the first-day leukapheresis CD34+ count results are confirmed causes unnecessary leukapheresis and expensive plerixafor administration. We investigated whether or not measuring hematopoietic progenitor cells in leukapheresis products (AP-HPCs) using a Sysmex XN-series analyzer could resolve this problem. Methods We retrospectively compared the absolute AP-HPC value per body weight with the CD34+ (AP-CD34+) count in 96 first-day leukapheresis product samples obtained between September 2013 and January 2021. Comparisons were also conducted according to regimen: granulocyte colony-stimulating factor (G-CSF) monotherapy, chemotherapy plus G-CSF, or plerixafor mobilization. Results AP-CD34+ and AP-HPC counts correlated strongly (rs=0.846) overall and, in particular, under chemotherapy plus G-CSF (rs=0.92) but correlated mildly under G-CSF monotherapy (rs=0.655). AP-HPCs could not completely be dichotomized based on an AP-CD34+ threshold of 2×106/kg for any stimulation procedure. In most cases with AP-HPCs >6×106/kg, the AP-CD34+ count exceeded 2.0×106/kg, but in 5.7% of these cases, the AP-CD34+ count was <2.0×106/kg. A cut-off of AP-HPCs >4.843×106/kg yielded a sensitivity of 71% and specificity of 96% for predicting AP-CD34+≥2×106/kg. Conclusion AP-HPCs can identify cases in which sufficient stem cells have been collected.

A workflow for the enrichment, the identification, and the isolation of non-apoptotic single circulating tumor cells for RNA sequencing analysis.

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells-we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients-were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.

A critical time window for leukapheresis product transportation to manufacture clinical-grade dendritic cells with optimal anti-tumor activities

Background aimsDendritic cell (DC)-based immunotherapy is a promising approach to treat cancer. However, key aspects governing the reproducible manufacturing of high-quality DC remain incompletely defined. Here, we show that the time window between leukapheresis and DC manufacturing is critical. MethodsTranscriptomic profiling by RNA-seq was used to unbiasedly characterize cellular states during each step of DC manufacturing process, and functional assays were used to determine the anti-tumor activities of DC. ResultsDuring preclinical development of a DC-based cytotherapy platform, CUD-002 (NCT05270720), we found that DC quality varied among different batches, even though commonly used DC maturation markers CD80, CD83 and CD86 were indistinguishable. Multivariate analysis indicated that DC quality was negatively associated with the shipping time from the leukapheresis site to the manufacturing center. To investigate the potential effect of shipping time, we stored leukapheresis materials from three donors for 0, 1, 2 or 3 days before DC manufacturing. For each step, we carried out RNA-seq analysis to unbiasedly characterize cellular states. Integrated bioinformatic analyses indicated that longer storage time reduced the expression of several transcription factors to attenuate interferon pathways. ConclusionsConsistently, we found that 3-day storage of leukapheresis materials significantly lowered the efficiency to generate DC but also impaired DC responses to inflammatory signals, resulting in inferior antigen-presentation and cytotoxic T-cell activities. Thus, we recommend using leukapheresis materials within 48 h to manufacture therapeutic DCs.

CAR-T Phenotype and Efficacy Is Impaired By Lymphoma-Associated CD39 + T Cells

Background: Autologous chimeric antigen receptor (CAR) T-cell therapy is standard of care for patients with relapsed or refractory Large B cell Lymphoma (R/R LBCL). Each patient's CAR T product is unique, and the patient-specific determinants of CAR T-cell quality are poorly understood. CD39 is an ectonucleosidase that participates in the conversion of ATP and ADP and is upregulated in cancer. On T-cells, CD39 is expressed on CD4+ Tregs and on exhausted CD8+ T-cells that are prone to apoptosis. Methods: This is a retrospective single-center cohort study (N=72) of patients who received CD19 CAR T-cell therapy for LBCL (axi-cel n=52; tisa-cel n=20). Leukapheresis material was obtained from n=51 patients and flow cytometry was performed to phenotype CD4+ and CD8+ stem-central memory, memory, and effector T-cell subsets (using CCR7 and CD45RO), and for immune checkpoint expression (PD-1, LAG3, TIGIT, and CD39). 10x multiome (ATAC and RNA) single cell sequencing (scRNA-seq) was performed on n=8 leukapheresis products for deeper characterization. Serum metabolomics was performed on paired samples from n=21 patients. 10X Genomics Chromium scRNAseq was also performed on n=57 CAR T-cell infusion products, including n=36 with paired leukapheresis material previously phenotyped by flow cytometry. In vitro CAR T-cells were manufactured from starting patient T-cells and analyzed for CAR T-cell phenotype and function. Results: In the leukapheresis material of patients that did not achieve long-term remission after CAR T-cell therapy, and in those with high tumor burden, we found higher numbers of CD4+CD39+ and CD8+CD39+ T-cells ( Figs. 1 and 2). 10X multiome sequencing confirmed that CD39+ T-cells exhibit characteristics of exhaustion. Recent platinum-based chemotherapy did not affect CD39+ T-cell levels. In n=21 patients with paired serum analyzed by metabolomics, high CD8+CD39+ T-cells associated with reduced metabolites in the inosine-hypoxanthine pathway, which are downstream of the ectonucleotidase function of CD39 (inosine; P=0.006; hypoxanthine P=0.07). Increased numbers of CD39+ T-cells in the starting leukapheresis material translated into manufactured CAR T-cells having less favorable product characteristics, including higher CD39 expression and fewer memory cells in both paired patient samples and in experiments of in vitro CAR T-cell manufacturing. ScRNA-seq of the infused CAR T-cell product revealed differential gene signatures, both globally and in association with response, between axi-cel and tisa-cel CAR T-cell products, highlighting the profound product-specific differences. Nonetheless, both products were adversely affected by high CD39+ T-cells in the leukapheresis material used for manufacture. Conclusions: CD39+ T-cells, found in patients with high lymphoma tumor burden and an unfavourable immunometabolic environment, associate with poor CAR T-cell quality and adverse patient outcomes in R/R LBCL. Strategies are needed to improve patient T-cell quality prior to leukapheresis and/or improve CAR T-cell manufacturing from patients with high levels of exhausted CD39+ T-cells. Figure 1: Progression-free survival (PFS) stratified by leukapheresis CD8+CD39+ T-cells in the leukapheresis product. Blue - below cohort median percentage of CD8+T-cells that are CD39+ (low); Red - above median percentage of CD8+T-cells that are CD39+ (high). P-value by Log-Rank test. Overall survival (not shown) P-value 0.01. Figure 2: Proportion of CD8+CD39+ T-cells found in patients with low or high baseline metabolic tumor volume (MTV) based on a previously established cutoff (Dean et al. Blood Adv. 2020). MTV was measured on the pre-CAR T-cell PET/CT. P-value by T-test. MDJ, XY, and RMA are co-first authors with equal contribution.

Increased Proliferative Drive of Lymphocytes in Sickle Cell Disease

Introduction Patients with sickle cell disease (SCD) have higher levels of inflammation which plays a prominent role in disease pathogenesis. It is well known that individuals with SCD have a higher erythropoietic drive given the ~20 day life span of their red blood cells. We asked whether this increased proliferative drive also occurred in white blood cells (WBC). To characterize WBC turnover we focused on the longest-lived CD4+ and CD8+ T cells. Methods Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient from red cell waste products from consented individuals with sickle cell disease and from leukapheresis products of healthy donors. In ten healthy donors and ten donors with SCD, CD4 and CD8 T cells were purified using EasySep Cell Isolation, labeled with eFluor 670 proliferation dye, and cultured in RPMI with 30% FBS for seven days. Samples were collected daily and stained with naïve and memory T cell markers. CountBright beads were added to obtain absolute cell counts and eFluor 780 live/dead dye was used to capture daily snapshots of cell death. Flow cytometric analysis was performed to track absolute cell numbers, cell division and indirectly monitor cell death. B cells, NK, macrophages, and dendritic cells were similarly tracked. To further probe T cell turnover, T cell receptor (TCR) sequencing was performed on twelve separate aliquots from purified CD4+ T cells at two different timepoints in healthy donors and individuals with SCD. Turnover of clonal sequences was analyzed using the Morisita index. Results CD4+ and CD8+ lymphocytes from individuals with SCD experienced more death in culture by day 7 compared to healthy donor controls. In addition, T lymphocytes from donors with SCD were observed to undergo 15-fold more proliferation compared to healthy donors (p=0.0002, unpaired t-test). Immunophenotyping performed on the CD4 and CD8 T lymphocytes showed a propensity for memory cells to undergo proliferation. We also found individuals with SCD had higher turnover in TCR sequences in two timepoints compared to healthy donors (p=0.05, Chi-square). Notably, we also saw increased proliferation of B cells, NK cells, macrophages, and dendritic cells in individuals with SCD. Conclusion Our data suggest that T lymphocytes and other PBMCs undergo faster turnover in SCD than in healthy donors, suggesting a stronger hematopoietic drive. Our findings may have important implications for gene therapy since an increased proliferative drive may contribute to an increased risk of developing leukemia in SCD.

Predicting the CD34 content of mobilized peripheral blood leukapheresis products: single institution experience over 20 years

Background aimsSince the standardization of CD34 measurement by flow cytometry, predictors of leukapheresis CD34 yield have played a pivotal role in planning donor leukaphereses. We describe here a single institution's experience with a multivariate predictor that was used for 2,929 products without alteration for 20 years. MethodsThe ordinary least squares regression model variables included log peripheral CD34 count, collection duration (3- versus 4-hours), collection number, donor sex, and transplant type. ResultsDuring the study period we changed flow cytometers twice and leukapheresis instruments once. During the Cobe Spectra era the predictor explained 90% of the variability in CD34 collection yield for autologous transplants (r2 = 0.90), and 70% for allogeneic transplants with an overall sensitivity to predict a CD34 yield of ≥ 1 × 106/kg of 97.7%, and specificity of 81.4%. ConclusionsImplemented prospectively with real-time result reporting, the model allowed us to predict CD34 yield with both 3- and 4-hour collection scenarios. Given this guidance, 3-hour collections were selected by the clinical team 25% of the time, saving patient leukapheresis time and resources. When faced with a prediction of < 1 × 106 CD34/kg, the clinical team chose to defer collection 72% of the time. In instances where leukapheresis was performed despite a poor predicted outcome, 85% of patients collected on the Cobe Spectra, and 92% of patients collected on the Optia, failed to collect at least 1 × 106 CD34/kg. A revised model is tested retrospectively on Optia data, and suggestions for further improvements are discussed.

Phase I Trial of Allogeneic Donor Gamma Delta T Cell Infusion Post-Hematopoietic Cell Transplantation, an Interim Safety Report

Background Patients with 2017 European LeukemiaNet (ELN) adverse risk acute myeloid leukemia (AML) are at 40% risk of relapse leading to poor survival after allogeneic hematopoietic cell transplantation (allo-HCT) (Hansen TCT 2021, Jimenez BMT 2021). Ex vivo donor-derived gamma delta T cells (GDT) expanded with artificial antigen presented calls (aAPC) are a novel therapy with potent MHC-independent antineoplastic cytotoxicity. An ongoing phase 1/1b study is evaluating the safety and potential efficacy of ex vivo expanded donor GDT after reduced-intensity conditioning (RIC) allo-HCT in patients with ELN adverse risk AML ( NCT05015426). We report on manufacturing, safety, and correlative biology of expanded donor GDT after allo-HCT in the first 5 treated patients (Table 1). Methods The primary endpoint is the maximum-tolerated dose (MTD) of allogeneic donor GDT as a single infusion at day 60 after RIC allo-HCT. Next generation sequencing was used for measurable residual disease (MRD) assessment pre-HCT and post-GDT infusion. Donor GDT collected from the same donor of the allo-HCT were enriched from non-mobilized leukapheresis product by stimulation with zoledronic acid and IL-2 for 7 days followed by alpha beta T cell depletion using the Miltenyi CliniMACS. Enriched GDT (&amp;gt;90% TCRVδ2) were then co-cultured with irradiated aAPCs in Wilson Wolf G-Rex 100MCS over 10 days (Boucher J. Immunother 2023). This single center, investigator-initiated phase 1 trial is testing 3 distinct dose cohorts of infused GDT (5 x 10 6 cells/kg, 2.5 x 10 7 cells/kg, and 1 x 10 8 cells/kg) with acceptable range of 25% margin. A Bayesian optimal interval design is used to guide dose escalation/de-escalation decisions. Results First 3 patients enrolled in dose level 1 (DL1) cohort received GDT dose of 6.25 x 10 6 cells/kg, while last 2 patients enrolled in DL2 cohort received 3.13 x 10 7 cells/kg dose. Dose-limiting toxicities (DLT) were evaluated for 42 days following GDT infusion and no DLT were observed. None of the treated patients experienced cytokine release syndrome (CRS), Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) or graft-versus-host disease (GVHD) during DLT period. Among patients treated on DL1, patient #01 had ASXL1 AML with MRD pos (VAF 43.9%) prior to RIC haploidentical (Haplo) HCT. He is in ongoing complete remission (CR) 9 months post-HCT and has had no GVHD. Patient #02 had MRD pos (VAF 61.7%) AML with TP53 mutation/complex karyotype prior to RIC matched sibling donor allo-HCT. He is in CR and has mild chronic GVHD at 7 months post-HCT. Patient #03 with pre-HCT MRD negRUNX1 AML is in ongoing CR 7 months post-RIC Haplo HCT with no GVHD. At day 28 and day 120 post-GDT, all 3 patients were MRD neg in bone marrow and had 100% bone marrow and peripheral blood leukocyte and granulocyte chimerism. In DL2 cohort, patient #04 with RUNX1 AML and patient #05 with t(9;22) AML both were MRD neg prior to RIC Haplo HCT and remain in CR with no GVHD at 5 and 4 months post-HCT, respectively. At day 28 post-GDT, both patients were MRD neg and had 100% chimerism. Patient #05 had a fall during DLT period in a setting of orthostatic hypotension requiring hospitalization for 24 hours for hydration. This was the only grade 3 adverse event observed in this trial. Serum cytokines analyzed included IL2, IL6, IL15, IFNγ, TNFα, Angiopoietin 1&amp;2, and GM-CSF (Figure 1). We compared results prior and up to 90 days post-GDT infusion and found no significant differences, which can explain no CRS or ICANS events yet observed on this trial. Immunophenotype (TCRVδ2, TCRVδ1, PD1, CTLA4, TIGIT and CD28) of GDT was analyzed in samples of donor apheresis, pre- and post-enrichment, infused GDT product and at days 28 and 90 post-infusion. GDT (TCRVδ2) persisted at day 28 and day 90 post-infusion and expressed low levels of cell surface inhibitory receptors. Conclusion This preliminary interim report demonstrates favorable safety and tolerability of donor GDT therapy with no CRS, ICANS or GVHD in first 5 patients treated in this trial. All these patients with adverse risk AML are in ongoing MRD neg CR following RIC allo-HCT, including 2 pre-HCT MRD pos cases with TP53 AML and AXSL1 AML. Early results of this trial are promising, which provides validation for continued testing of adoptive transfer of ex vivo expanded donor GDT. Updated results of this trial will be presented at the annual conference.

A Novel Model Combining Circulating Absolute Monocyte Counts and a Four-Gene Monocyte Signature in Leukapheresis Identifies Lymphoma Patients at Very High Risk of Progression after Tisagenlecleucel or Axicabtagene Ciloleucel Therapy

Introduction CD19-directed chimeric antigen receptor (CAR) T can induce durable remissions in a fraction of relapsed/refractory large B-cell lymphomas (R/R LBCL), but 60% of patients (pts) still relapse. Biological mechanisms explaining lack of responses are emerging but they are largely unsuccessful in predicting disease response at the patient level. Aim To address the potential role of lymphocyte features before CAR T manufacturing, we performed transcriptomic and functional evaluations of leukapheresis products (LK) in 95 R/R LBCL pts enrolled in a prospective observational study, to identify correlates of response and survival to tisagenlecleucel (Tisa-cel) and axicabtagene ciloleucel (Axi-cel). Methods RNA of sorted CD3+ cells from LK was digitally quantified using the nCounter CAR T Characterization Panel (NanoString). Flow cytometry (FCM) was used for CAR T enumeration and monocyte analysis. Disease response at day 90 was assessed by computed tomography (CT) and positron emission tomography-CT (PET/CT) according to Lugano criteria. Statistical analyses were performed by GraphPad Prism v.9.00 and R v.4.1.2. Results We identified a 4-gene transcriptional signature (including MS4A4A, CD86, CD163 and SIGLEC5) able to divide LBCL pts into two groups, [namely Expressors (EXP) and poor-Expressors (poor-EXP)], characterized by significantly different survival probabilities. EXP have shorter PFS (P&amp;lt;0.0001: median PFS for EXP was 61 days and not reached for poor-EXP) (Fig. 1) and are less likely to respond (P&amp;lt;0.01). As the identified genes are highly expressed in monocytes but not in T cells, we sought to define whether monocytes contained in LK have a role in determining post CAR T outcome. In line with the fact that EXP had higher frequencies of monocytes in LK by FCM, we confirmed elevated absolute monocyte counts (AMC) both in LK (P&amp;lt;0.01) and in peripheral blood (PB) (P&amp;lt;0.05) of EXP. We then checked if monocyte levels could influence response and survival. When compared to responders, non-responders (NR) had higher LK AMC (P&amp;lt;0.01), higher percentages of monocytes (P&amp;lt;0.01) but also decreased percentages of lymphocytes (P&amp;lt;0.001), a parameter that was also associated with reduced PFS [HR: 0.97 (CI 0.96 - 0.99) P=0.01]. Consistent with the fact that circulating monocytes significantly correlated with those in LK (r= 0.6621, P&amp;lt;0.0001), NR also had significantly higher AMC (P&amp;lt;0.001) and AMC levels in PB negatively correlated with PFS [HR: 1.8 (CI 1.2 - 2.9) P=0.01]. Additionally, pts with serum C-reactive protein (CRP) and ferritin levels higher than the upper normal limit, displayed significantly higher AMC (P&amp;lt;0.001 for CRP; P&amp;lt;0.05 for ferritin). Accordingly, univariate analysis indicated that high ferritin, CRP and LDH levels at the time of LK were associated with the lack of response, but only high serum ferritin levels correlated with shorter PFS [HR: 2.86 (CI 1.25-6.55) P=0.013]. We next investigated if the combination of the monocyte signature and high ferritin levels and/or high circulating monocytes could better identify a group of pts at high risk of progression, compared to the expression of the 4 genes alone. Remarkably, albeit high ferritin levels and monocyte counts are overall associated with significantly reduced survival, the shortest PFS was achieved in EXP with monocyte counts above the median (P=0.0234: median PFS for EXP with high monocytes was 32.5 days and for EXP with low monocytes 209 days) (Fig. 2), thus supporting the hypothesis that CAR T treatment outcome is influenced by the presence of elevated levels of monocytes expressing the 4 genes. Concatenated monocyte FCM data from 30 pts analyzed with FlowSOM, followed by a consensus clustering algorithm combined with t-SNE (t-stochastic neighbor embedding), indicated that EXP had higher frequencies of CD14hi monocytes co-expressing MS4A4A, CD86, CD163 and SIGLEC5 at the protein level than poor-EXP (P&amp;lt;0.05), and accordingly, the presence of this population was also associated with lack of response (P&amp;lt;0.05). Conclusions Our data suggest that the poor outcome of some LBCL pts receiving CAR T is due to the presence of high monocytes levels expressing a four-gene signature in an inflammatory microenvironment. Additionally, the pre-manufacturing combined analysis of the gene signature and of PB AMC, could be used to plan trials evaluating CAR T cells versus other newly approved therapies such as bispecific antibodies.

Outcomes of Patients with Relapsed/Refractory Mantle Cell Lymphoma (R/R MCL) Treated with Brexucabtagene Autoleucel (Brexu-cel) in ZUMA-2 and ZUMA-18, an Expanded Access Study

Introduction: Brexu-cel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved in the United States for the treatment of adults with R/R MCL and in the European Union for adults with R/R MCL who received ≥2 prior therapies, including a Bruton tyrosine kinase inhibitor (BTKi). After a median of 35.6-months follow-up in the pivotal Phase 2, multicenter ZUMA-2 study, brexu-cel demonstrated an objective response rate (ORR) of 91%, a complete response (CR) rate of 68%, and a median overall survival (OS) of 46.6 months in all 68 treated patients with R/R MCL and a median OS not reached in patients with a CR (Wang et al. J Clin Oncol. 2022). ZUMA-18 is a multicenter, open-label, expanded access study of brexu-cel for the treatment of patients with R/R MCL. Here, we report OS outcomes after 4 years in ZUMA-2 and the primary analysis of ZUMA-18. Methods: In ZUMA-2, adults (≥18 years) with R/R MCL with ≤5 prior regimens including a BTKi underwent leukapheresis and conditioning chemotherapy followed by one infusion of brexu-cel (2×10 6 anti-CD19 CAR T cells/kg). ZUMA-18 consisted of 2 cohorts. The primary objectives were to provide access to brexu-cel for patients with R/R MCL until it was commercially available (Cohort 1) and patients with R/R MCL whose manufactured product did not meet commercial release specifications (Cohort 2). In Cohort 1, adults (≥18 years) with R/R MCL with ≥1 prior regimen underwent leukapheresis and conditioning chemotherapy followed by a single infusion of brexu-cel at a target dose of 2×10 6 cells/kg (or fixed dose of 2x10 8 anti-CD19 CAR T cells for patients who are ≥100 kg). In Cohort 2, patients received Cohort 1 treatment without leukapheresis (initial leukapheresis product used). Key endpoints were safety, ORR, and OS. Results: As of July 23, 2022, median follow-up in all 68 treated patients in ZUMA-2 was 47.5 months (range, 37.9-68.3) and median OS was 46.4 months (range, 1.2-63.0) with 30 patients (44%) still alive. Median (range) OS for patients with CR (n=46), partial response (PR; n=16), and no response (n=6) were 58.7 (4.8-61.9), 16.3 (1.2-63.0), and 8.5 months (2.3-22.9), respectively. As of February 3, 2023, 27 patients were enrolled in ZUMA-18 and 23 (Cohort 1, n=21; Cohort 2, n=2) received brexu-cel with a median follow-up of 33.5 months (range, 24.5-35.3). Median age was 69.0 years (range, 43-79); 78% were male; and median number of prior regimens was 4 (range, 1-10). The investigator-assessed ORR was 87% (95% CI, 66.4-97.2); 13 patients (57%), including both patients in Cohort 2, had a CR (95% CI, 34.5-76.8), 7 patients (30%) had a PR (95% CI, 13.2-52.9), and 2 patients (9%) had progressive disease (PD) as their best response to brexu-cel (95% CI, 1.1-28.0; 1 patient had not been assessed at data cutoff). The median OS was not reached (95% CI, 10.4-not estimable) at data cutoff with a 58% OS rate at 24 months (Figure 1). At data cutoff, 14 patients (61%) were still alive and 9 patients (39%) had died; 5 due to adverse events (AEs), 2 due to PD, and 2 due to other causes. All 23 patients in ZUMA-18 experienced at least 1 Grade ≥3 AE and 18 patients (78%) experienced at least 1 Grade ≥3 treatment-related AE, of which, neutropenia (43%), anemia (30%), thrombocytopenia (30%), encephalopathy (22%), and leukopenia (22%) were most common. Grade ≥3 cytokine release syndrome and neurological events occurred in 1 patient (4%) and 8 patients (35%), respectively. Five Grade 5 AEs occurred, 1 that was deemed related to brexu-cel therapy (multiple organ dysfunction syndrome on Day 20) and 4 that were deemed unrelated to brexu-cel therapy (sepsis [2, Days 123 and 219], aspiration [1, on Day 49], and encephalopathy [1, on Day 68]). The combined median OS for ZUMA-2 and ZUMA-18 patients (N=91) was 46.4 months (range, 0.7-63.0) with a 62% OS rate at 24 months (Figure 2). Conclusions: With 4 years of median follow-up in ZUMA-2, patients continued to benefit from brexu-cel with a median OS of almost 5 years in patients with CR. Consistent with ZUMA-2 findings, brexu-cel demonstrated a high level of efficacy in patients with R/R MCL in ZUMA-18, with an ORR of 87% and median OS not yet reached at 33.5 months of follow-up. Additionally, no new safety signals were detected in ZUMA-18. Of note, given the small sample size (n=2), no definitive conclusions can be drawn from Cohort 2 outcomes alone. Together, these results support the continued use of brexu-cel in the R/R MCL setting.

Detailed immunophenotyping of the hematopoietic graft from patients with multiple sclerosis undergoing autologous hematopoietic stem cell transplant

Background aimsAutologous hematopoietic stem cell transplantation (AHSCT) is a highly effective therapy for relapsing multiple sclerosis. Re-infused stem cells provide “rescue” from the pancytopenia induced by immuno-chemotherapy. To date, no study has analyzed the non-stem cell content of the leukapheresis product (graft) in regards to its influence on disease remission in AHSCT for multiple sclerosis (MS). MethodsDetailed immunophenotyping of the stem cell graft was performed in a cohort of highly active patients with MS (n = 22) followed for a median of 6 years’ post-AHSCT. ResultsEffector memory populations thought to house pathogenic clones including Th17 cells and central nervous system homing T cells were detected in the graft at similar proportions to pre-AHSCT. There was no association between absolute counts of these populations in the graft and treatment response. Only in responder patients was there evidence of a significant decrease in these putative pro-inflammatory populations by 3 months’ post-transplant. Although there was no statistical difference in the number of T regulatory cells (Tregs) in the graft between responders and relapsing patients, the absolute count of Tregs in the graft correlated with circulating Tregs in the first 6 months post-AHSCT in responders alone. ConclusionsOur results collectively suggest that the early establishment of immune tolerance post-AHSCT appears to relate to a decrease in putative pathogenic cell populations following reinfusion, and that Treg load in the leukapheresis product is less relevant to treatment response than the early expansion of graft-derived Tregs. It therefore remains unclear whether employment of CD34 selection to manipulate the graft may offer additional benefit in remission rates post-AHSCT for MS. Cellular therapy targeted toward early Treg expansion may provide recourse for long-term remission rates in MS.

The Effect of Iron Overload on the Mobilization of Peripheral Blood Hematopoietic Stem Cells in Pediatric Patients with Thalassemia Major

Introduction: The purpose of this study was to examine the effect of iron overload on the mobilization of peripheral blood stem cells (PBSCs) in pediatric patients with β-thalassemia major (TM). Methods: We retrospectively reviewed the records of 226 patients with TM from whom PBSCs were collected. Iron overload was based on serum ferritin level, and liver and cardiac iron overload was measured by magnetic resonance imaging (MRI) T2*. Results: The mean age of the TM patients was 7.35 ± 3.41 years. Of the patients, only 171 received MRI. Of the 171 patients, 35 had normal liver iron levels, 39 mild liver iron overload, 90 intermediate liver iron overload, and 7 severe liver iron overload. The intermediate + severe group was associated with significantly higher age and BMI and lower leukapheresis product white blood cell count and CD34⁺ cell levels (all, p < 0.05). Conclusion: Leukapheresis indices were similar between patients with different degrees of iron overload according to the ferritin level and cardiac iron overload, in which the later might be due to the small number of patients with cardiac overload. In patients with TM, the intermediate and severe liver iron overload was associated with poorer mobilization of PBSCs.

Metabolic Disruption Induced by mTOR Signaling Pathway Inhibition in Regulatory T-Cell Expansion for Clinical Application.

Regulatory T cell (Treg) therapy is considered an alternative approach to induce tolerance in transplantation. If successful, this therapy may have implications on immunosuppression minimization/withdrawal to reduce drug-induced toxicity in patients. The aim of this study was to assess the efficacy of the mTORC1/C2 inhibitor, AZD8055, in the manufacturing of clinically competent Treg cells and compare the effects with those induced by rapamycin (RAPA), another mTOR inhibitor commonly used in Treg expansion protocols. Primary human Treg cells were isolated from leukapheresis product. Cell viability, expansion rates, suppressive function, autophagy, mitochondrial unfolded protein response (mitoUPR), and cell metabolic profile were assessed. We observed a stronger inhibition of the mTORC2 signaling pathway and downstream events triggered by Interleukin 2 (IL2)-receptor in AZD8055-treated cells compared with those treated with RAPA. AZD8055 induced progressive metabolic changes in mitochondrial respiration and glycolytic pathways that disrupted the long-term expansion and suppressive function of Tregs. Unlike RAPA, AZD8055 treatment impaired autophagy and enhanced the mitoUPR cell stress response pathway. A distinct pattern of mTOR inhibition by AZD, compared with RAPA, induced mitochondrial stress response and dysfunction, impaired autophagy, and disrupted cellular bioenergetics, resulting in the loss of proliferative potential and suppressive function of Treg cells.

Improved enrichment of circulating tumor cells from diagnostic leukapheresis product.

The median number of circulating tumor cells (CTCs) detected in 7.5 mL of peripheral blood by CellSearch (PB-CS) in patients with metastatic prostate cancer is in the order of 1-10, which means many samples have insufficient tumor cells for comprehensive characterization. A significant increase is obtained through diagnostic leukapheresis (DLA), however, only 2%-3% of the DLA product can be processed per CellSearch test, limiting the gain. We processed aliquots from 30 DLA products of metastatic prostate cancer patients consisting of 0.2 × 109 leukocytes using CellSearch (DLA-CS) as well as the newly introduced reduced enrichment reagent protocol (RER), which uses 10-fold less enrichment reagents than DLA-CS. The number of tumor cells and the total number of captured cells were determined using the CellTracks Analyzer. Additionally, for six DLA samples, a 1.0 × 109 leukocyte aliquot was processed (RER+), using twofold less enrichment reagents than DLA-CS. A median 2.7-fold reduction in leukocyte co-enrichment was found between DLA-CS and RER methods without any loss in tumor cell recovery (Wilcoxon Signed Ranks Test, p = 0.953). Using 1.0 × 109 leukocyte aliquots a fourfold increase in tumor cells was found compared to DLA-CS and a 19-fold increase compared to PB-CS was obtained. The here-introduced RER protocol results in a higher final sample purity without any loss in tumor cell recovery while using 10-fold less CellSearch capture reagent. With this improved method, 26% of the leukapheresis sample can now be processed using reagents from a single CellSearch test, enabling the obtainment of a sufficient number of CTCs for comprehensive characterization in most metastatic prostate cancer patients.

Variable recovery of cryopreserved hematopoietic stem cells and leukocyte subpopulations in leukapheresis products

IntroductionDue to the expansion of cell therapy using not only haematopoietic stem cells (HSC) but also other leukocyte subpopulations, the loss of these cells in cryopreserved apheresis products needs to be evaluated. Various factors that could negatively affect post-thaw recovery, such as leukapheresis product characteristics, storage time and cryopreservation protocols have been identified. MethodsThe post-thaw recovery of HSCs, lymphocytes, NK cells and monocytes, as well as the factors that could adversely affect it were analysed in autologous and allogeneic leukapheresis products. ResultsThe lowest post-thaw recovery was observed in autologous and allogeneic CD34+ cells, with the median of 73.7% and 68.1%, respectively. In leukocyte subpopulation, the lowest post-thaw recovery was observed for CD14+ cells, both autologous and allogeneic. The highest post-thaw recovery was observed for CD3+/CD8+ cells in autologous, and for CD19+ cells in allogeneic samples. The statistically significant difference was observed between autologous and allogeneic PBSC products for CD3+ cell recovery (P = 0.031) and CD3+/CD8+ cell recovery (P = 0.009). The evaluation of factors that could adversely affect the post-thaw recovery in autologous samples showed weak negative correlations between platelet concentration and CD3+ recovery, as well as between storage time and CD3+CD8+ recovery. In allogeneic samples, a strong negative correlation was observed only between the percentage of granulocytes and CD3+, CD3+/CD8+ and CD3+/CD4+ cell recoveries. ConclusionSince various post-thaw recoveries of leukocyte subpopulations were observed, the cell therapy manufacturing centers should evaluate how their cryopreservation method and other factors affect the recovery of cell population of interest in their settings.

Easy 250 EasySep&amp;[trade] Magnet: A Novel Magnetic Platform For Large-Volume CD34+ Hematopoietic Stem and Progenitor Cell Isolation from Leukapheresis Products

Abstract We have developed a novel magnetic platform for large-volume CD34+ hematopoietic stem and progenitor cell (HPSC) isolation from both mobilized and non-mobilized leukapheresis products. As the frequency of CD34+ cells is low, and cell quality and viability can be variable between samples, it takes great effort to isolate these cells with consistently high purities and yields. Working with large volume leukapheresis products can be challenging as the sample often needs to be split and processed in parallel, which can be both time-consuming and labor-intensive. The Easy 250 EasySep™ Magnet simplifies cell isolation procedures when processing leukapheresis samples. It is based on column-free immunomagnetic EasySep™ cell isolation technology and can process sample volumes up to 225 mL. We have developed a negative selection protocol for the enrichment of CD34+ HPSCs (mean purity ± SD of 74.6 ± 10.5%; n = 8) from non-mobilized leukapheresis products. For mobilized leukapheresis products, an EasySep™ positive selection protocol has been optimized for the isolation of highly purified CD34+ HPSCs (mean purity ± SD of 92.0 ± 1.7%; n = 7). Notably, these protocols can be completed in as little as 25 minutes, making the Easy 250 EasySep™ Magnet the fastest and simplest method for enriching highly purified HPSCs from large-volume leukapheresis products—critical for addressing the growing clinical demands for CD34+ cell-based therapies.

Abstract LB187: Case report: APN401, a novel cancer therapy using Cbl-b silenced autologous PBMCs, induced stable disease in two patients with advanced solid tumors

Abstract Immune checkpoint control is one of several mechanisms that negatively influence the immune system and contribute to the development and progression of cancer. To enhance anti-tumor immune responses in patients with advanced solid tumors, we employ a novel autologous cancer cell therapy, APN401, which allows transient and highly specific silencing of Casitas B-lineage lymphoma-b (Cbl-b) in patients’ peripheral mononuclear cells (PBMCs). Cbl-b is an E3 ubiquitin protein ligase that plays a central role in both innate and adaptive immune responses. Here we present two patients with advanced, metastatic solid tumors: one patient diagnosed with appendix carcinoma, and one patient diagnosed with head and neck squamous cell carcinoma (HNSCC). Both showed stable disease (SD) during treatment with APN401. In an open-label, multicenter Phase Ib trial, two increasing dose levels of APN401 were evaluated in patients with advanced solid tumors. Using invIOs’s closed cell-processing platform - Enhancement Platform for immune Cells (EPiC) - patients’ PBMCs were purified from leukapheresis products and subsequently transfected with a small interfering ribonucleic acid (siRNA) to specifically block Cbl-b expression. The entire manufacturing process required less than six hours, and the drug product could be reinfused on the same day. The trial evaluated safety and clinical outcomes, as well as the activity and potency of the drug product. Additionally, various biomarkers were analyzed in patients’ peripheral blood, which was collected prior to each treatment cycle. Two patients, one with appendix carcinoma and one with HNSCC, showed stable disease after repeated APN401 treatments at the lowest and/or intermediate dose levels. Subsequent drug product analyses revealed increased IL-2 levels and elevated CD8/CD4 ratios, suggesting potential cytotoxic efficacy. In stimulation assays with HLA class I-restricted viral or tumor antigens, increased IFN-γ levels were detected and served as surrogate markers for improved immunity and tumor reactivity. 10X genomics and TCR clonotyping revealed clonal expansion of T cell subsets throughout the course of the treatment. Taken together, these initial results indicate that APN401, a novel personalized cancer cell therapy based on targeted silencing of Cbl-b in PBMCs, may be a safe and effective immunotherapy for solid tumors. This cell therapy will be further evaluated as a monotherapy along with potential combinations in clinical trials in various solid tumors. Ethics approval: The clinical trial was approved by the Medical University of Vienna institution’s independent Ethics Committee, approval number 1778/2020. Citation Format: Mario Kuttke, Alexander Dohnal, Andreas Tanzmann, Beate Pribitzer, Felix Batrina, Stefan Bunka, Sophia Spagl, Manuela Branka, Sarah Bischof, Kathrin Thell, Maria Urban, Hannes Muehleisen, Bernhard Peball, Bianca Gapp, Angela Halfmann, Markus Raderer, Gerald Prager, Thorsten Fuereder, Romana Gugenberger, Nina Worel. Case report: APN401, a novel cancer therapy using Cbl-b silenced autologous PBMCs, induced stable disease in two patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB187.