Apheresis Material Research Articles

Using Real-World Remanufacturing and Recollection Data to Optimize Time-to-Treatment in Patients with out-of-Specification Ciltacabtagene Autoleucel

Ciltacabtagene autoleucel (cilta-cel) is a B-cell maturation antigen (BCMA)-directed genetically modified autologous chimeric antigen receptor-T cell (CAR-T) immunotherapy approved for the treatment of adults with relapsed/refractory multiple myeloma. Manufacturing of cilta-cel can take approximately 4 weeks from receipt of apheresis material until release for cilta-cel infusion. Though manufacturing is a well-controlled biological production process, delays in release can occur when the final product does not meet predetermined release specifications. Such products are referred to as being out-of-specification(s) (OOS). In the United States, OOS CAR-T products may be released for use via an exceptional release process as part of an expanded access clinical trial (NCT05346835). Additional time is needed as the process requires the prescriber's request for release, an internal safety review to assess benefit/risk, and US Food and Drug Administration (FDA) notification and/or approval. Further delays to treatment may occur if a prescriber requests to remanufacture and potentially recollect new apheresis material, attempting to obtain an in-specification product. In some instances, this increased time has resulted in clinically significant disease progression. We reviewed and characterized the experience from discussions with prescribers regarding OOS cilta-cel over the past 18 months. Options presented to the prescriber included: (1) remanufacture using the original apheresis product, (2) recollection of new apheresis material, (3) exceptional release of the OOS cilta-cel, and (4) cancellation of the order. The outcomes for cases of remanufacturing or recollection were analyzed. To date, remanufacturing from the initial apheresis material has been requested in 37% of OOS cases. Of these cases, 60% of remanufacturing attempts resulted in the same or worse outcomes (ie, additional OOS outcomes or worse performance on the original OOS outcomes). Recollection of new apheresis material was requested in 15% of OOS cases, either because of insufficient remaining material or concern about the impact of therapy received prior to collection. Upon recollection, 65% of these attempts resulted in the same or worse outcomes. The reason cilta-cel was determined to be OOS varied. Of remanufacturing attempts for OOS due to a low proportion of CAR+ cells, none were successful in obtaining an in-specification product. Cases where OOS was due to low cell viability were unsuccessfully remanufactured or recollected 71% and 75% of the time, respectively, whereas OOS due to low dose was unsuccessfully remanufactured 95% of the time. On occasion, the second attempt at remanufactured OOS cilta-cel due to low dose has been combined with the OOS cilta-cel generated in the first manufacture to achieve a combined dose that meets or comes close to the specified dose range. Remanufacturing or recollection success rates were increased in cases where the root cause of the OOS was due to something inadvertent that occurred during the manufacturing process. Our findings on remanufacturing and recollection outcomes may inform physician decisions and help optimize time-to-treatment in patients receiving cilta-cel. Additional studies (eg, NCT05347485) are underway to evaluate the safety and efficacy of OOS cilta-cel.

Role of Cell Therapy Product Characteristics in Patient Response to TAK-940 (CD19(T2)28z1xx Chimeric Antigen Receptor (CAR) T): Biomarker Analyses from a Phase I Study in Adult Patients with Relapsed or Refractory B-Cell Malignancies

TAK-940 (CD19(T2)28z1XX Chimeric Antigen Receptor (CAR) T) is an autologous CD19 CAR T cell therapy utilizing an optimized 1XX signaling domain substituting for the natural CD3ζ immunoreceptor tyrosine-based activation motifs. This 1XX design extends the functional persistence of CAR T cells with increased potency in mice (Feucht J et al. Nat Med 2019). TAK-940 is being evaluated in a Ph1 trial in patients with Relapsed or Refractory B-cell malignancies (NCT04464200) with demonstrated early signals of clinical efficacy (Park J et al. ASH 2022). The present study aims to provide a better understanding of the clinical performance of CD19(T2)28z1XX CAR T cells by in depth characterization of the cellular products and patient samples from this Ph1 trial. A total of 28 subjects were enrolled and treated in the dose escalation and expansion cohorts of the Ph1 trial. Responses were observed across all dose levels (25x10 6 to 200x10 6 CAR T cells), achieving ORR 82%, CR 71%. Immunophenotyping by flow cytometry was conducted on patient apheresis material, un-transduced cells, transduced cellular drug product and peripheral blood samples from infused subjects. Circulating cytokine profiling was performed on patient serum samples collected pre and post treatment with TAK-940. In the transduced cellular drug products, CAR+ and CAR- CD8+ central memory and transitional memory T cells were more abundant than naïve, stem cell memory or terminal effector populations. This elevation in central and transitional memory T cells seen in the drug product was not observed in the patient apheresis material, indicating that this effect is likely driven by the production process. CD57 and TIM3 expression in both CD4+ and CD8+ CAR+ T cells in the drug product showed a positive association with clinical response, while CTLA4 or LAG3 expression independently was negatively associated with response. The drug product also contained low numbers of dysregulated/exhausted T cells, with no observable difference between responders and non-responders. Interestingly, in the starting apheresis material, increased expression of TIM3 as well as decreased expression of either LAG3 or KLRG1 in CD8 T cells, independently associated with responses. Current analysis is based on associations with complete response. Additional analyses to be reported will include associations with ORR and durability of responses. These findings suggest potential utility of these T cell markers in predicting patient response and warrant evaluation in a larger population. Although CD57 expression has been associated with a senescent phenotype, we hypothesize that the observed increase in CD57 expression in CD4+ and CD8+ CAR+ T cells in the product may indicate higher cytotoxic potential of those cells. Increase in CD57+ CAR+ T cell subsets has been previously associated with durable CRs in large B cell lymphoma patients treated with axi-cel (Good Z et al. Nat Med 2022). Additional analyses to further characterize the CD57+ cell populations and establish their role in clinical responses to CAR T cell therapy with the 1XX CAR design are in progress. The increased expression of CD57 and the activation marker TIM3, and decreased expression of inhibitory receptors CTLA4 and LAG3 in the drug product from responders, together with the robust clinical responses observed with doses as low as 25x10 6 CAR T cells, suggest that the product consists of highly potent T cells, in accordance with the preclinical results obtained with the 1XX CAR. Low levels of circulating cytokines were observed following treatment with TAK-940, including IFNg, IL-6, IL-10, indicating a safe and tolerable CAR T product consistent with the observed clinical safety profile of TAK-940. Correlative analysis of T cell immunophenotypes observed in the product to clinical parameters including durability of responses, metabolic tumor volume/tumor burden, and induction of cytokines and chemokines is ongoing and will be reported. This will further contribute to our understanding of CD19 CAR T cells endowed with a 1XX signaling domain and potentially support the identification of product and/or apheresis biomarkers that may predict patient response.

Advancing CAR-T Therapy in Acute Lymphoblastic Leukemia: Multi-Omic Analyses of CD19-Directed CAR-T Cells Enabled By an Ex Vivo Co-Culture Platform

Background: Acute Lymphoblastic Leukemia (ALL) has witnessed remarkable progress in the treatment of patients with Chimeric Antigen Receptor T-cell (CAR-T) therapies, leading to high response rates and durable remissions. However, challenges persist for patients who do not respond to CAR-T treatment, experience relapse, or encounter adverse events such as cytokine release syndrome. Overcoming these challenges necessitates a deeper understanding of mechanisms and the development of advanced technologies and assays. This study focuses on harnessing the potential of an innovative microfluidic platform for high-throughput cell-based assays, enabling multi-omics and functional analysis using a minimal amount of primary patient cells. By utilizing this platform, we aim to gain insight into the dynamics of primary patient material by evaluating responses to aid in selection of promising CAR-T enhancements or combination therapies designed to overcome mechanisms of CAR-T failure or relapse to improve clinical outcomes. Methods: For the cytotoxicity assay, we utilized autologous or allogeneic pairs of CD19-directed CAR-T cells and T-cell negative fractions from ALL patients' blood apheresis samples, containing CD19-expressing cancer cells. A negative control was established using untransduced T-cells. To ensure high cell viability and purity, the cells underwent careful processing and were labeled with long-lived dyes for tracking. Multiple cell suspensions with precise Effector to Target to Negative Fraction ratios were prepared. Only a few thousand cells from each coculture suspension were seeded in microfluidic culture chambers, which required no fluidic control system. Experiments were conducted for up to 96 hours with media replacement every 24 hours. At each endpoint, media was collected for cytokine quantification, and cells were stained for live/dead counting as well as quantification of intracellular or surface marker expression by imaging. After image acquisition, cells were collected for RNA extraction and gene expression analysis. Results: Results demonstrated that the developed co-culture platform allows precise control over culture conditions, ensuring culture of accurate ratios of highly complex cell populations. The viability of primary cells in the device guaranteed functional relevance of the cytotoxicity assays for up to a 96-hour timepoint. Strong CAR-T-mediated cell killing was observed, with a dose-dependent effect depending on the co-culture's relative CAR-T content. Less than 50% cell survival was observed in cocultures with Effector to Target ratio 1:1, as compared to the untransduced T-cell control. Furthermore, strong CAR-T activation was observed with over 60% of CART expressing CD25 and CD69 surface markers, as measured by imaging. A multi-color staining panel allowed for precise monitoring of changes over time within the assay of immune cell populations, including cancer cells, monocytes/macrophages, and NK cells, among others. Cytokine analysis revealed an increased secretion of IL-2, TNFa, IFNg, and Granzyme B in coculture with CAR-T cells compared to untransduced T-cells, with a dose-dependent effect based on the CAR-T cell ratio. Finally, RNA isolated from remaining cells at the end of the assay was of high quality with yields sufficient to enable transcriptome analysis for each treatment condition. Discussion: This study contributes to the development of innovative multiplexed high throughput strategies for in vitro functionality assessments in cell therapy, including combination studies and deeper mechanistic understandings. The platform's ability to generate fully integrated, multi-omics readings from complex allogeneic and autologous cocultures of CAR-T and patient cells offers a new frontier in early discovery and translational cancer research. This opens new possibilities for drug discovery, drug screening, and disease modeling, ultimately advancing our understanding of complex biological processes and accelerating therapeutic development. Future directions include exploring mechanisms of resistance and sensitivity, including donor-specific effector cell functionality, combination studies, impacts of immunosuppressive cell types, patient heterogeneity of target antigen expression or other co-stimulatory molecules, and evaluation of product potency or fitness of incoming apheresis material.

Determinants of Response to Anti CD-19 CAR-T Cells for Diffuse Large B-Cell Lymphoma in Pre-Treatment Peripheral Blood Mononuclear Cells Using Single-Cell RNA-Seq

Background: Chimeric antigen receptor T (CAR-T) cell therapy has become the standard of care in patients with relapsed/refractory diffuse large cell lymphoma (R/R DLBCL), providing durable remission in 40% of patients. Lack of response is likely to be attributed to inter- and intra-patient variability, including PBMC heterogeneity and apheresis material composition, CAR-T infusion product, in-vivo expansion and activity of CAR-T cells and the development of tumor-intrinsic mechanisms. Current methods for evaluating CAR-T cell activation and potency have limited predictive value for clinical outcomes. To address this challenge, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) obtained from patients with R/R LBCL patients at the time of apheresis for the manufacturing of anti-CD19 CAR-T cell therapy. Methods: Between 10/2020 and 03/2022, 31 patients who underwent lymphopheresis were enrolled. Samples were obtained - 1) At day of lymphopheresis from peripheral blood; 2) On the day of infusion of CAR-T from the product; and 3) On day +7 after infusion from peripheral blood. All patients had day +30 PET-CT. In addition, PBMCs samples from healthy donors were used as control to analyze baseline characteristics compared to the DLBCL patients. PBMCs from patients of healthy donors were purified from frozen blood samples by density gradient separation, stained using human CD45 antibody and loaded onto a 10x Chromium system. libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit. The data was processed and analyzed using the Seurat R package. The study was approved by the local Ethic committee. Results: Patient characteristics: 31 RR DLBCL patients (42% females; median age 72, range 47-81 years), all treated with CAR-T cells therapy as third line of therapy, participated in the study. All patients underwent lymphopheresis, followed by CAR-T production. Disease status at lymphodepletion was complete remission (CR, n=4, 13%), partial remission (n=6, 19%), and progressive disease (n=21, 68%) and patients received either tisagenlecleucel (n=20, 65%) or axicabtagene ciloleucel (n=11, 35%) . PET scan performed on day 30 post treatment revealed that 68% and 32% obtained CR, and PD, respectively. scRNA analysis revealed differences in PBMC composition between healthy donors and LBCL patients (Figure 1A), with lower levels of naïve and memory T cells and elevated levels of activated monocytes in LBCL patients, suggesting a shift towards an inflamed and pre-dysfunctional immune state. Categorization of LBCL patients into responders (PR/CR on day +30 PET-CT) and non-responders (PD on day +30 PET-CT) revealed differences in immune cell subsets between responders and non-responders (Figure 2A), with enriched inflammation-related pathways in non-responders, In contrast, responders exhibited a distinct healthy-like immune profiles, particularly high levels of naive T cells, similar to that obtained in their age matched healthy controls. Of note, non-responders showed alterations in immune cell subsets, including increased levels of T regulatory cells and effector NK cells. Furthermore, non-responders demonstrated a prominent inflammatory immune profile, characterized by gene pathways related to inflammation and activation, such as type I and III IFN-G, and TNFb response (Figure 1B). Notably, an IFN-G score was significantly higher in non-responder's monocytes subpopulations, compared to responders. Conclusions: These findings indicate that the success of CAR-T therapy could be determined by the molecular characteristics and subpopulation distribution of each patient's PBMC. The diverse immune profiles of DLBCL patients might impact the effectiveness of their immune cells in generating successful CAR-T products or providing a conducive environment for CAR T cells to combat cancer. Furthermore, these findings emphasize the potential of scRNA-seq in evaluating PBMC from CAR-T patients prior to treatment, allowing the potential calculation of an “immune potency score” associated with the probability of a positive outcome from CAR-T treatment of DLBCL patients. This score could potentially be used by oncologists in decision-making processes to determine the appropriate candidates for CAR-T therapy.

Hematological Parameters: Comparative Evaluation of Performance between Dxh 500 and XN-10 Equipments

Introduction The development of hematopoietic stem cell transplantation (HSCT) was an extremely important milestone for treating certain malignant and non-malignant hematological diseases. The HSCT process contemplates collecting hematopoietic progenitor cells (HPC) from the donor, conditioning chemotherapy, infusion of HPC, and engraftment. Mobilized peripheral blood is the most commonly used HPC source for HSCT due to its ease of obtaining through an apheresis procedure and high progenitor cell count yields compared to bone marrow source, which requires a bone marrow aspiration procedure, usually performed in a surgical center under general anesthesia, and requires the collection of higher volumes to reach the amount of HPC desired. The total nucleated cell count (TNC) corresponds to the sum of all nucleated cells in the sample and is used as a reference for the target dose for both sources of HPC. The TNC provides essential information on the quality of the sample obtained, since an infusion of insufficient number of cells affects the ability to reconstitute the recipient's hematopoietic system. An insufficient TNC can result in poor engraftment, leading to a slow recovery of blood cell production and increasing the risk of complications such as infections and bleeding. Determination of the number of nucleated cells in bone marrow and peripheral blood samples can be performed automatically by hematological analyzers such as the Beckman Coulter DxH 500 and Sysmex XN-10. This study aims to compare the performance of those equipments in determining hematological parameters in bone marrow and apheresis material. Materials and methods We compared the results of 40 HPC samples (20 bone marrow samples and 20 peripheral blood samples collected by apheresis) analyzed in the Sysmex XN-10 and Beckman Coulter DxH 500 equipments. All these specimens were analyzed without dilution and diluted 1:10. The evaluated parameters were: TNC, hematocrit, platelets, lymphocytes, monocytes, and granulocytes. Results The analysis of the results showed a positive Pearson correlation coefficient (above 0.9) between XN-10 and DxH 500 equipment for the evaluation of TNC in diluted samples obtained by apheresis (figure 1) and by bone marrow (figure 2). In pure samples obtained by apheresis the DxH 500 equipment showed non-linearity for TNC but revealed a positive correlation with the XN-10 when evaluating pure bone marrow samples (figure 2). On the other hand, XN-10 equipment showed linearity when correlating TCN between pure and diluted samples obtained by apheresis (figure 1) and bone marrow aspiration (figure 2). Hematocrit analysis in samples obtained by apheresis showed 90% and 100% of agreement for diluted samples between the equipment and for diluted versus pure samples, respectively, when using the cutoff value of 12%. Hematocrit validation using the Beckman Coulter equipment was impaired due to the sample's viscosity and limited linearity. Also, there was a satisfactory hematocrit correlation for pure bone marrow samples and comparing diluted versus pure bone marrow samples with both equipment. The results also showed a good correlation when analyzing the platelet count in pure and diluted samples (bone marrow and apheresis) with XN-10 or DxH 500, as well as comparing the diluted samples versus the pure samples. The use of differential count of nucleated cells (lymphocytes, monocytes and granulocytes) realized by the two evaluated devices was not approved for bone marrow or apheresis samples. Conclusion The determination of TNC, platelet and hematocrit counts using the Sysmex XN-10 equipment were satisfactory for both pure and diluted samples obtained by apheresis or bone marrow. For pure samples obtained by apheresis analyzed by DxH 500, we could observe absence of linearity for TNC and hematocrit. This situation could be explained by the high number of cells in this sample and the consequent high viscosity.

CAR+ T-Cell Lymphoma Post Ciltacabtagene Autoleucel Therapy for Relapsed Refractory Multiple Myeloma

Introduction:Rareevents of T-cell lymphoma (TCL) derived from CAR-T cells (2 cases) have been reported in patients receiving nonviral piggyBac transposon-based CAR-T therapy (Micklethwaite et al, Blood, 2021). Ciltacabtagene autoleucel (cilta-cel) is an anti-BCMA CAR-T therapy produced via conventional lentiviral transduction. In the randomized, phase 3 CARTITUDE-4 study (NCT04181827), cilta-cel significantly improved PFS (HR=0.26) vs standard of care in lenalidomide-refractory patients with multiple myeloma and 1-3 prior lines of therapy. We present the clinicogenomic characterization of a CARTITUDE-4 patient who developed a CAR+ TCL post cilta-cel. Methods: Diagnostic and staging workup included biopsy analyses and FDG-PET scan. Presence of CAR+ cells in lymph node biopsy (LNB) was assessed by quantitative polymerase chain reaction (qPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). Whole genome sequencing (WGS), transcriptome sequencing, whole exome sequencing (WES), T-cell receptor (TCR) sequencing, and genome-wide CAR integration analyses were conducted. Results:A 51 y/o male patient received cilta-cel; CAR+ T cells in blood peaked 14 d post-infusion (77 cells/μL) and decreased to 3 cells/μL at d 92 post-infusion, when he achieved stringent complete response (sCR) and MRD negativity at 10 -5. At 5 mo post-infusion, a relatively rapidly growing erythematous nasofacial plaque developed. TCL was diagnosed based on facial lesion biopsy showing an infiltrate of atypical T cells positive for CD2 and CD3 but negative for CD4, CD8, CD7, CD56, ALK, EBER-ISH, TdT, CD30, and cytotoxic T cell markers. FDG-PET showed bilateral FDG-avid cervical lymphadenopathy, with similar T cell infiltrate in submandibular LNB. qPCR and ISH/IHC revealed 90-100% of LNB cells to be CAR+. At d 162 post-infusion (after TCL diagnosis but before TCL-directed chemotherapy) CAR+ T cells in blood had re-expanded, independent of BCMA antigen, to 378 cells/μL. CAR integration analysis of LNB revealed a dominant insertion into the 3′UTR of PBX2 (91.1% of total reads from all integration sites), suggesting tumor monoclonality. TCR sequencing (1.8×10 -6 sensitivity) of LNB showed a monoclonal sequence in 91% of all T cells. Analysis of the drug product revealed the same unique TCR sequence from the monoclone at low frequency (~2×10 -6), suggesting the presence of this clone in apheresis material. WGS showed low overall mutational burden in the LNB (1.26 mutations/megabase) with 37 coding and 3286 non-coding variants, including 2 predicted loss-of-function TET2 mutations; a PTPRB truncation; and a focal duplication involving the 5′UTR to intron 16 of NFKB2. No dominant mutational signatures or gross copy number changes were observed. WES of the LNB indicated that a TET2 mutation (p.H1416R), which was not due to CAR insertion, was heterozygous and likely clonal. Targeted sequencing (0.5% sensitivity) of stored CD34+ cells and bone marrow aspirate collected from the patient 2 y earlier showed no abnormalities. Germline samples revealed the presence of a heterozygous JAK3 variant (p.V722I) that has been described as an activating variant, implicated in TCL, and detected in germline samples from patients with antigen-induced TCL (Blombery et al, Haematologica, 2016). The patient received CHOEP-21 (cyclophosphamide-doxorubicin-vincristine-prednisone-etoposide) and achieved metabolic CR but relapsed soon after treatment was stopped. Subsequent treatment with gemcitabine-dexamethasone-cisplatin-alemtuzumab was followed by consolidation with fludarabine plus melphalan and matched allogeneic stem cell transplant. He relapsed with cutaneous disease within 3 mo. Conclusions: To our knowledge, this is the first case of CAR+ TCL occurring after infusion of a CAR-T therapy produced via lentiviral transduction (cilta-cel). This rare malignancy was potentially driven by genetic mutations (e.g., TET2, NFKB2, PTPRB and/or JAK3), some of which may have existed in the form of a clone with malignant potential before cilta-cel manufacturing (e.g., TET2 p.H1416R and JAK3 p.V722I variants). A potential contributory role of the CAR insertion in the 3′ untranslated region of PBX2 to TCL development remains unclear and cannot be excluded at this time. Further investigation is needed to elucidate the differential contributions of these genomic factors to the etiology of this TCL case.

Manufacturing of a Subsequent Autologous CAR-T Product after Prior CAR-T Is Safe and Feasible

Introduction: Patients with large B-cell lymphoma frequently fail to achieve a durable remission following CD19-directed chimeric antigen receptor T-cell therapy (CAR-T). Unfortunately, the outcomes for patients who progress after CAR-T are dismal with the median overall survival of approximately 6 months. Trials are now demonstrating the possibility of a subsequent autologous CAR-T for treating these patients. We demonstrated a CD22-directed CAR-T (CAR22) achieved a 68% and 53% overall (ORR) and complete response (CR) rate in 38 subjects who progressed after a CD19 CAR-T (CAR19) (NCT04088890). Of the 20 patients who achieved CR, 17 never relapsed after a median follow up of 22.8 months. The safety and feasibility of manufacturing a second (or more) CAR-T from patients has largely not been addressed and some concerns have been raised regarding the theoretical risk of malignant transformation due to the repeatedly introduction of new genomic material through serial CAR-T manufacturing. Here we address the impact of residual CAR19 on CAR22 production and patient outcomes. Method: We evaluated the patients who progressed after CAR19 and underwent manufacturing for CAR22. Single-cell RNA-sequencing (scRNAseq; 10X) was used to quantify the frequency of axi-cel transcripts in CAR22 products. Patients' peripheral blood mononuclear cells (PBMCs) were collected on day 7, 14, 21, and 28 after CAR22 infusion. The FMC63 and CD22.BB.z sequence (with albumin as a control) were quantified using quantitative polymerase chain reaction (qPCR) to determine the presence of CAR19 and CAR22 within the PBMCs. The assay's limit of detection and quantification (LOQ) were calculated as 1.68 and 5.1 copies per 50ng of DNA reaction with 95% confidence, respectively. Results: Among 40 patients underwent manufacturing for CAR22, 2 failed manufacturing due to insufficient T cells in the apheresis material. One of the two patients received prior alemtuzumab in combination with a CAR19 product, likely contributing to the failure. Among the 38 who were successfully manufactured, 37 patients had received prior CAR19, including axi-cel (n = 28), tisa-cel (n = 3), liso-cel (n = 3), autologous CAR19 in clinical trial (n = 1), bispecific CD19/CD22 CAR-T (n = 1), and bispecific CD19/CD20 CAR-T (n = 1). The median progression-free survival (PFS) from the previous CAR19 infusion was 3.3 months (range from 1.0 to 40.4 months), with a median age of 64.4 years old (ranging from 24.9 to 83.9 years old) at the time of relapse. The median time from CAR19 infusion to leukapheresis for CAR22 manufacturing was 6.8 months (ranging from 1.2 to 42.9 months). Thirteen CAR22 products were available for scRNAseq; all from patients who had received axi-cel previously. The median frequency of CAR19 positive cells was approximately 68 (ranging from 0 to 2146) per million cells per CAR22 product. Residual CAR19 cell proportions were not statistically significantly different between patients who underwent CAR22 manufacturing within or after 6 months of CAR19 infusion. Thirty-one patients had available PBMCs post-CAR22 infusion for analysis. Twelve patients (39%) exhibited an increase in CAR19 qPCR transgene levels above the LOQ during CAR22 treatment. Most of these patients (n = 8) underwent leukapheresis for CAR22 manufacturing within 6 months after CAR19 infusion. The median CAR19 qPCR transgene levels increased by 1.2 log (ranging from 0.2 to 2.1 log; equivalent to 1.4 to 133.7 copies per 50ng of DNA), while CAR22 transgene levels increased by a median of 3.7 log (ranging from 0.7 to 3.9 log; equivalent to 4.7 to 9027.1 copies per 50ng DNA). In the comparison between patients who underwent leukapheresis within or beyond 6 months after CAR19 infusion, there were no statistically significant differences in the maximal CAR19 and CAR22 qPCR transgene levels. The ORR, cytokine release syndrome severity, immune effector cell-associated neurotoxicity syndrome severity and PFS after CAR22 were not statistically significantly different between patients with detectable CAR19 qPCR or not. Conclusion: We found that CAR19 was detectable in the majority of CAR22 products, but the levels of CAR19 transcripts were rare. While CAR19 cells were detected post-CAR22 infusion, the levels were frequently just at the LOQ and did not appear to contribute to toxicity or clinical outcomes. Overall, we find that serial manufacturing of autologous CAR-T products is safe and feasible.

Paired Analysis from Patients with Myeloma Elucidates Favorable CAR-T Manufacture and Phenotype, Starting from Mobilized Pre-Transplant Apheresis Vs. Non-Mobilized Apheresis after Four Prior Lines

Introduction B-Cell Maturation Antigen (BCMA) targeting chimeric antigen receptor T-cells (CAR-T) have demonstrated unprecedented efficacy in patients with relapsed and refractory multiple myeloma, approved after 4 or more prior lines of therapy (PLOT). Given the known T-cell dysfunction that develops over the disease trajectory, autologous T-cells harvested after many PLOT may not be as biologically active as those collected earlier. Estimates suggest 93% of stored stem cell products collected from MM patients are never used (Liang et al, 2021). We sought to demonstrate the feasibility of using stored mobilized apheresis products to generate anti-BCMA CAR-T cells. Given the heterogeneity that can exist between MM patients, we evaluated the differences in starting T-cell phenotype, manufacturing characteristics, and CAR-T phenotype and function side by side using paired samples from the same patient, obtained prior to transplant or prior to commercial CAR-T. Methods We identified a cohort of paired samples from: 1) Mobilized unfractionated apheresis obtained prior to ASCT (preASCT); and 2) Apheresis for CAR-T manufacture after ≥4 PLOT (preCAR). The flow cytometry panel used for phenotyping included antibodies against: CD3, CD4, CD8, CCR7, CD45RO, CD39, TIGIT, PD1, LAG3 and TCF7. Second generation anti-aBCMA.4-1BB.CD3z (aBCMA.BBz) CAR-T cells were manufactured from CD3+ T cells negatively selected from either sample using a Pan T cell isolation kit (Miltenyi). Post manufacture flow cytometry was used to characterize immunophenotype and transduction efficiency. Doubling time was calculated using trypan blue exclusion on D+2 to D+5 after transduction. A BCMA-expressing myeloma cell line, MM.1S, was used to stimulate and evaluate cytotoxicity. Results In 10 patients with paired preASCT and preCAR samples, the median time from ASCT to CAR-T infusion was 4.2 years (range 2.5-12.5 years), all patients had received three drug induction regimens, and 50% had >1 line of combination therapy prior to ASCT. PreASCT samples ranged in collection from Nov 2008 - April 2019. All patients were triple class exposed prior to collection for CAR-T and treated with median 6 PLOT (range 4-13). Comparison of the T-cell composition in both apheresis samples, revealed a higher CD4:CD8 ratio in preASCT samples (p=0.006), and a higher proportion of stem central memory/SCM (CCR7+CD45RO-, p<0.05, Figure 1) in both CD4 and CD8 compartments. Additionally, in preASCT samples, patients with only 1 line of induction, exhibited a trend toward a higher proportion of SCM T-cells relative to more heavily treated patients. PreASCT CD4 (p=0.002) and CD8 (p=0.01) T-cells expressed more TCF7 compared with preCAR, however T-cells with an effector phenotype were proportionally higher in preCAR samples across both CD4 and CD8 compartments (CCR7-CD45RO-, p<0.05, Figure 1). PreCAR T-cells express higher levels of checkpoints (CP) PD1 and TIGIT, with dual positive CP more prevalent in CD4+ T-cells. CAR-T cells manufactured from preASCT samples expanded better (p=0.048) and also had increased CD4:CD8 ratios (p=0.011) as compared to preCAR, without significant differences in transduction efficiency, CAR density or viability. After 24h stimulation with BCMA+ MM.1S, CD8+ CAR-T cell phenotype was different between preASCT and preCAR products, with more SCM in preASCT (p=0.004) and more effector memory/EM (CCR7-CD45RO+) in preCAR (p=0.008); however, the killing capacity after 24h was similar for both CAR-T products at different E:T ratios. The CAR-T cells generated from T-cells preCAR (later in the disease course) expressed higher TIGIT, LAG3 and CD39 after stimulation, and more dual (p=0.027) or triple (p=0.027) positive CP than those from preASCT T-cells ( Figure 2). Conclusion This study suggests CAR-T cells manufactured from banked mobilized unfractionated stem cell product expand better during manufacture, yield an equally cytotoxic, less differentiated, and less exhausted product than those manufactured from later timepoints (preCAR samples). Importantly, we demonstrate two major findings: (1) that a possibly more efficacious anti-BCMA CAR-T product can be manufactured from cryopreserved, mobilized apheresis material regardless of storage time and (2) cryopreserved preASCT samples may expand autologous CAR-T cell therapy access to heavily treated patients with poor T cell quality. CLF & JAM contributed equally

Idecabtagene vicleucel for relapsed and refractory multiple myeloma: post hoc 18-month follow-up of a phase 1 trial

Idecabtagene vicleucel (ide-cel) is a B-cell-maturation antigen (BCMA)-directed chimeric antigen receptor T cell therapy. We performed a post hoc analysis of a single-arm phase 1 multicenter study in relapsed/refractory multiple myeloma (CRB-401) (n = 62; median follow-up, 18.1 months). The primary endpoint was safety outcomes, and secondary endpoints included overall response rate (ORR), complete response (CR) and very good partial response (VGPR). The study met its primary endpoint with low rates of grade 3/grade 4 cytokine release syndrome (6.5%) and neurotoxicity (1.6%). ORR was 75.8%; 64.5% achieved VGPR or better and 38.7% achieved CR or stringent CR. Among exploratory endpoints, median duration of response, progression-free survival (PFS) and overall survival were 10.3, 8.8 and 34.2 months, respectively, and ide-cel expansion in blood and bone marrow correlated with clinical efficacy and postinfusion reduction of soluble BCMA. Patients with PFS ≥ 18 months had more naive and less exhausted T cells in apheresis material and improved functional T cell phenotype in the drug product compared with those with less durable responses. These results confirm ide-cel safety, tolerability and efficacy and describe T cell qualities that correlate with durable response. Clinicaltrials.gov identifier : NCT02658929.

First in Human Study of an on/Off Switchable CAR-T Cell Platform Targeting CD19 for B Cell Malignancies (CLBR001 + SWI019)

First in Human Study of an on/Off Switchable CAR-T Cell Platform Targeting CD19 for B Cell Malignancies (CLBR001 + SWI019)

Effects of Prior Alkylating Therapies on Preinfusion Patient Characteristics and Starting Material for CAR T Cell Product Manufacturing in Late-Line Multiple Myeloma

Effects of Prior Alkylating Therapies on Preinfusion Patient Characteristics and Starting Material for CAR T Cell Product Manufacturing in Late-Line Multiple Myeloma

Tumor Inflammation and Myeloid Derived Suppressor Cells Reduce the Efficacy of CD19 CAR T Cell Therapy in Lymphoma

Introduction: Approximately 60% of Large B cell Lymphoma (LBCL) patients that receive CD19 CAR T cell therapy with axicabtagene ciloleucel (axi-cel) experience lymphoma progression (Locke et al. Lancet Oncol. 2019) and the likelihood of response to subsequent therapy is low (Spiegel, Dahiya et al. ASCO 2019). Target loss of CD19 is observed in less than a third of patients experiencing relapse. Alternative mechanisms of resistance to axi-cel are poorly understood. Lymphoma patients with elevated serum markers of systemic inflammation, such as ferritin and IL-6, have worse outcomes following axi-cel (Locke, Neelapu et al. Mol.Ther.2017; Faramand et al. ASH 2018). We hypothesized that suppressive monocytic myeloid derived suppressor cells (M-MDSCs), which are associated with worse chemotherapy outcomes in LBCL (Azzaoui et al. Blood 2016), and tumor driven inflammation may be present and responsible for decreased efficacy of axi-cel in LBCL. Methods: LBCL patients undergoing axi-cel treatment were enrolled onto prospective sample collection protocols. Patients were stratified for analysis into ongoing responders (complete response or partial response) or relapsed (progressive disease) after a minimum of 3 months follow-up (range 3 - 15 months). M-MDSCs, defined as a Lin-, CD11b+, CD33+, CD15-, CD14+, HLA-DRlow population, were sorted from leftover apheresis material after collection for axi-cel manufacture. M-MDSC ability to suppress proliferation of autologous T cells stimulated with CD3/CD28 coated beads was measured by 3H thymidine incorporation. Circulating peripheral blood M-MDSCs, quantified by % of live cells by flow cytometry, were measured at the time of apheresis and serially after axi-cel infusion until day 30. In vitro mouse experiments utilized a CD19-CD28 CAR and cytokine-induced bone marrow MDSCs (Thevenot et al. Immunity 2014). Cytokines were measured by ELISA and cytotoxicity against CD19 bearing cell lines used xCELLigence real-time cell analysis, as we have done previously (Li et al. JCI Insight 2018).Tumor biopsies were taken within 1 month prior to infusion of axi-cel. Limited gene expression profiling of tumor microenvironment (TME) genes used the Nanostring IO360 panel (770 genes). Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Results: First, we demonstrated that M-MDSCs sorted from patient apheresis material suppressed the proliferation of autologous T cells (n=6). We next enumerated M-MDSCs in the peripheral blood (n = 32). M-MDSC numbers initially decreased after lymphodepleting chemotherapy but recovered to baseline levels by day +10. The level of M-MDSCs following CAR T cell therapy strongly correlated with pre-CAR T baseline levels (R = 0.871, p <0.0001), suggesting that the number of M-MDSCs present during CAR T cell expansion is dependent on factors already present before therapy began. M-MDSC levels were significantly higher in patients who subsequently relapsed, both at baseline (p= 0.01) and after axi-cel (p=0.04), as compared to patients with durable response. Mouse MDSCs were able to suppress CAR T cell IFN-gamma excretion (p<0.0001) and cytotoxicity (p<0.0001) in vitro. To evaluate the role of the TME we interrogated limited set gene expression profiling on patient (n=27) pre-axi-cel tumor biopsies. By cell type scoring, the macrophage gene score was significantly higher in patients who relapsed after CAR T therapy (p <0.001). By differential gene expression and gene set enrichment, patients who relapsed had a significantly higher expression (p <0.01) of multiple genes indicative of chronic interferon (IFN) signaling including higher levels of OAS2, OAS3, IFI6 and IFIT1, as well as the IFN-stimulated macrophage gene SIGLEC-1/CD169. Conclusions: Systemic inflammatory myeloid cytokines, circulating M-MDSCs in the blood and chronic IFN in the TME all associate with LBCL relapse after axi-cel CAR T cell therapy. Our observations support that CAR T cells can be suppressed by baseline patient and tumor-related factors and strategies to overcome these factors should be targeted to improve patient outcomes. MDJ and HZ contributed equally. Disclosures Jain: Kite/Gilead: Consultancy. Bachmeier:Kite/Gilead: Speakers Bureau. Chavez:Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; Spectrum/Astrotech: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria. Mullinax:Iovance: Research Funding. Davila:Celgene: Research Funding; GlaxoSmithKline: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; Atara: Research Funding; Bellicum: Consultancy; Adaptive: Consultancy; Anixa: Consultancy. Locke:Kite: Other: Scientific Advisor; Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy.

Influence of CD27- CD28- T-Cells on the Therapeutic Outcome in Adult Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma after CART-Infusion

Background: Therapies involving adoptive transfer of chimeric antigen receptor-modified T-cells (CARTs) targeting CD19-expressing B-cells have shown remarkable efficacy in patients with relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL). We hypothesized, that a higher fraction of more differentiated, senescent and exhausted T-cells affects negatively ex vivo T-cell expansion in the manufacturing phase of CARTs and the in vivo function of CARTs after infusion. Of note, naïve CD4+ T-cells were shown to uniformly express the co-stimulatory receptors CD27 and CD28, while repeated cycles of activation led to their progressive loss (van Leewen et al, J. Immunol, 2004), accompanied by telomere erosion and replicative senescence (van Baarle et al., Immunol. Lett. 2005; Effros RB et al., Dev Comp Immunol 1997). CD28 expression on T-cells within the tumor environment was shown to be a decisive factor for the efficacy of anti-PD-1 therapy (Kamphorst RO et al, Science 2017). We have therefore analyzed the CD27 and CD28 expression status of CD3+ T-cells from the peripheral blood and apheresis products of adult r/r DLBCL patients at the day of leukapheresis. Methods: Peripheral blood and apheresis samples of 22 consecutive r/r DLBCL patients scheduled for CART therapy were analyzed by flow cytometry to assess their CD27 and CD28 expression status on CD3+ T-cells. Samples were stained with fluorochrome conjugated antibodies (anti-CD3 PerCP, anti-CD27 FITC, CD28 PE, anti-CD4 FITC, BioLegend) and analyzed using a FACScalibur flow cytometer supported by CELLQUEST software (Becton Dickinson, BD, Palo Alto, CA). Results: To rule out an apheresis-related bias within cell populations we analyzed peripheral blood and apheresis samples for each patient. No differences in the distribution of CD27-, CD28-, CD27-/CD28- or CD27+/CD28+ T-cells between peripheral blood and apheresis product were detected. Mean CD3+ cell count in blood samples before apheresis was 624±399/µl (range, 75-1853cells/µL) with only about 25% of the patients presenting with CD3+ cell counts within the normal range (690-3320/µL) and 70% of the patients showed an inverse CD4/CD8 ratio (<1.0). We observed a considerable heterogeneity within the T-memory cell compartment. In all samples high percentages of CD27- (39.7±18.1%), CD28- (40.2±19.0%) and CD27-/CD28- (30.7±19.8%) T-cells were seen when compared to healthy controls (CD27-: 15.2±9.9%; CD28-: 18.4±8.9%; CD27-/CD28-: 9.9±8.7%; p<0.05). Patients receiving CARTs were embedded within three different CART-trials (NCT02445248, NCT03630159, NCT03484702). A significantly lower frequency of CD27-/CD28-T-cells (20.8±18.8 vs 42.4±13.7%; p=0.045 Mann Whitney U) was found in responders (n=8) compared to non-responders (n=5; Figure 1a). We also noticed a higher frequency of CD27+/CD28+ T-cells (57.5±22.7% vs 44.3±11.7%; Figure 1b) and a trend towards a normal (>1.0) CD4/CD8 ratio (Figure 1c) in responding patients. Furthermore, we did not observe significant differences in CD27-and CD28- expression in samples derived from patients who died prior to receiving CART therapy (n=6) when compared to patients responding or progressing after CART therapy. Three patients have not been infused yet. Conclusion: We demonstrate in this small patient cohort that individuals with a lower percentage of more differentiated, senescent or exhausted T-cells are more likely to respond to CART therapy. Our observation underscores the importance of T-cells with normal replicative capacity in the apheresis material for consecutive CART production to achive therapeutic success. Further analysis is needed to determine the effect of cytotoxic pretreatment on the fraction of immunosenescent/exhausted T-cells. However, to confirm our findings additional investigations including the T-cell status of manufactured cells are warranted. Disclosures Worel: Sanofi Genzyme, Malinckrodt Therakos: Speakers Bureau; Sanofi Genzyme, Malinckrodt Therakos: Research Funding; Jazz, Sanofi, Celgene, Novartis, Malinckrodt Therakos: Honoraria. Jaeger:Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria. Hopfinger:Celgene, Gilead, GlaxoSmithKline, Janssen, Novartis, Roche, Takeda,: Honoraria; Gilead: Research Funding.

Application of a Standardized Flow Cytometry Panel for Defining and Monitoring the Immunophenotype of CAR-T Cells

Chimeric antigen receptor T-cells (CAR-T) have emerged as an extremely promising therapy for hematological malignancies. The immunophenotype of apheresis material and the CAR-T cell product is known to be predictive of the likelihood of response to treatment of certain malignancies. Central memory and stem cell-like memory T cell phenotypes are associated with a more sustained proliferative response and long-term CAR-T persistence (Fraietta et al, Nature Medicine, 2018). There is an unmet need for standardized methods and reagents to reliably profile the memory phenotype of CAR-Ts to better evaluate product quality, and support improvements in CAR-T manufacturing. The BD Biosciences dried memory T-cell panel contains a pre-validated mixture of 7 antibodies for the identification of naïve, stem cell memory, central memory and effector memory CD4+ and CD8+ T cell subsets. The pre-mixed dried antibody tube offers consistency in staining profiles over time and reduces the risk of operator errors. Additional drop-in antibodies can complement the panel and enable more in-depth evaluation of the T cell phenotype. Here we demonstrate the use of this panel with drop-in markers to monitor changes in expression of PD-1, TIM-3, LAG-3, HLA-DR, CD45RO, and CXCR3 on T cells transduced to express our novel anti-CD37 CAR. Cells were stained at day 0 prior to transduction, day 7, and following resting and re-stimulation, and acquired on a 12 color BD FACS Lyric. The use of a standardized memory T-cell panel will allow us to more accurately evaluate how T-cell phenotype impacts on the efficacy and longevity of response in patients receiving CAR-T therapies. Disclosures Maus: INFO PENDING: Other: INFO PENDING. Bornheimer:BD Biosciences: Employment. Hanley:BD Biosciences: Employment. Frigault:Novartis: Patents & Royalties: Royalty; Arcellx, Celgene, Foundation Medicine, Kite/Gilead, Nkarta, Novartis, and Xenetic: Consultancy.

Outcomes in Aggressive B-Cell Non-Hodgkin Lymphomas with Anti-CD19 CAR T-Cell (CTL019) Products Not Meeting Commercial Release Specifications

Outcomes in Aggressive B-Cell Non-Hodgkin Lymphomas with Anti-CD19 CAR T-Cell (CTL019) Products Not Meeting Commercial Release Specifications

Characterization and Isolation of Very Small Embryonic-like (VSEL) Stem Cells Obtained from Various Human Hematopoietic Cell Sources.

Stem cell transplantation is one of the available treatments for leukemia, lymphoma, hereditary blood diseases and bone marrow failure. Bone marrow (BM), peripheral blood progenitor cells (PBPC), and cord blood (CB) are the predominant sources of stem cells. Recently a new type of stem cell with a pluripotent potential has been identified. These cells were named "very small embryonic like stem cells (VSELs)". It is claimed that VSEL stem cells can be found in adult BM, peripheral blood (PB), CB and other body tissues. This study is designed to characterize and isolate VSEL stem cells from different human hematopoietic sources; CB, PB and apheresis material (PBPC). VSEL stem cells were isolated from MNC and erythrocyte layers for all materials by using centrifugation and ficoll gradient method. We determined embryonic markers by flow cytometry, immunofluorescence and western blotting methods. Results from western blotting and immunofluorescence show high level of NANOG and OCT4 protein expression in PB, apheresis material and CB. Immunofluorescence images showed cytoplasmic and nuclear presence of these proteins. Flow cytometry results exhibited a higher expression of VSELs markers on debris area than CD45- population and higher expression on CB than PB. As a result, these findings have shown that it is necessary to investigate the function of pluripotent stem cell markers in differentiated adult cells. We further conclude that erythrocyte lysis method had the highest cell recovery amount among erythrocyte lysis and ficoll gradient methods. Consequently, this study gives us new information and viewpoints about expression of pluripotent stem cell (PSC) markers in adult tissues.

Fingerprints of CD8+ T cells on human pre-plasma and memory B cells.

Differentiation of B cells is a stringently controlled multi-step process, which is still incompletely understood. Here we identify and characterize a rare population of human B cells, which surprisingly carry CD8AB on their surface. Existence of such cells was demonstrated both in tonsils and in human apheresis material. Gene expression profiling and real time PCR detected however no CD8A or CD8B message in these cells. Instead, we found that surface CD8 was hijacked from activated CD8+ T cells by a transfer process that required direct cell-to-cell contact. A focused transcriptome analysis at single cell level allowed the dissection of the CD8 positive B cell population. We found that the affected cells are characteristically of the CD27+CD200- phenotype, and consist of two discrete late-stage subpopulations that carry signatures of activated memory B like cells, and early plasmablasts. Thus, there is only a restricted time window in the differentiation process during which B cells can intimately interact with CD8+ T cells. The findings point to a novel link between the T and B arms of the adaptive immune system, and suggest that CD8+ T cells have the capability to directly shape the global antibody repertoire.

Sustained Disease Control for Adult Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma: An Updated Analysis of Juliet, a Global Pivotal Phase 2 Trial of Tisagenlecleucel

Sustained Disease Control for Adult Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma: An Updated Analysis of Juliet, a Global Pivotal Phase 2 Trial of Tisagenlecleucel

Abstract 5644: Generation of clinical grade autologous TLR 7,8-polarized fast dendritic cell vaccines for active immunotherapy of patients with AML

Abstract Although progress has been made in the treatment of AML in recent years, relapse rates following chemotherapy are still high resulting in only low long-term patient survival rates. Treatment options are often limited following relapse, emphasizing the continued need for new and more effective treatments options.We have developed an active immunotherapy using TLR7/8-polarized fast dendritic cells (DCs), which is currently under evaluation for treatment of patients with AML in morphological remission in a non-randomized phase I/II clinical trial (NCT02405338). The trial subjects receive repeated immunotherapy with autologous vaccine cells, transfected with RNA encoding two leukemia-associated antigens WT1 and PRAME. Patients are vaccinated for two years or until disease progression. Here we present data on the feasibility of clinical grade production of our TLR7/8-polarized fast DC vaccine for long-term vaccination of chemotherapeutically pretreated AML patients. A total of 20 patients with a median age of 59 years (range 24 - 73 years) were recruited to this ongoing trial. For vaccine production, autologous apheresis material was collected from each patient and fast DC generation was performed following isolation of DC precursor cells using a TLR7/8-agonist containing maturation cocktail. Final vaccine cells were cryopreserved in multiple aliquots prepared to deliver 5x106 - 10x106 cells per vaccine dose. Vaccine cells could be generated from all 20 AML patients. A second apheresis was performed during the trial for a second production in order to generate sufficient vaccine doses for the intended treatment period for 4 of 20 patients. The majority of production runs (17 out of 24) yielded 20 or more vaccine doses (8 batches: 20-29 doses, 6 batches: 30-39 doses, 2 batches: 40-49 doses and more than 60 doses from one batch). For seven patients less than 20 vaccine doses were cryopreserved from one production run. To evaluate vaccine cell purity, presence of autologous non-DC cells was determined and revealed a purity of more than 70% for 22 vaccine batches (17 batches 80% - 99%, 5 batches 71% - 77%). Only two productions yielded lower purities (range 64% - 69%). The average post-thawing vaccine cell viability was 83% (range 68%-95%) with the majority of cells showing a viability of >70%. Additionally, DCs generated from all patients showed a mature surface phenotype as demonstrated by high expression of typical DC surface markers such as CD80, CD86, CD83, CD40 and HLA-DR. Furthermore, high expression of the lymphocyte homing receptor CCR7 could be detected for all cell preparations. Taken together, these results clearly demonstrate the feasibility and robustness of our protocol for production of mature clinical grade TLR7/8-polarized fast DCs in high numbers from heavily pretreated post-remission AML patients allowing for long-term vaccination of trial subjects. Citation Format: Iris Bigalke, Lisbeth Johanne Skoge, Kirsti Hønnåshagen, Christiane Geiger, Gunnar Kvalheim, Dolores J. Schendel. Generation of clinical grade autologous TLR 7,8-polarized fast dendritic cell vaccines for active immunotherapy of patients with AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5644.

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.