Extended characterization of anti-CD19 CAR T cell products manufactured at the point of care using the CliniMACS Prodigy system: comparison of donor sources and process duration.

BACKGROUND AIMS: The CliniMACS Prodigy closed system is widely used for the manufacturing of chimeric antigen receptor T cells (CAR-T cells). Our study presents an extensive immunophenotypic and functional characterization and comparison of the properties of anti-CD19 CAR-T cell products obtained during long (11 days) and short (7 days) manufacturing cycles using the CliniMACS Prodigy system, as well as cell products manufactured from different donor sources of T lymphocytes: from patients, from patients who underwent HSCT, and from haploidentical donors. We also present the possibility of assessing the efficiency of transduction by an indirect method. METHODS: Seventy-six CD19 CAR-T cell products were manufactured using the CliniMACS Prodigy automated system. Immunophenotypic properties, markers of cell activation and exhaustion, antitumor, anti-CD19 specific activity in vitro of the manufactured cell products were evaluated. As an indirect method for assessing the efficiency of transduction, we used the method of functional assessment of cytokine secretion and expression of the CD107a marker after incubation of CAR-T cells with tumor targets. RESULTS: The CliniMACS Prodigy platform can produce a product of CD19 CAR-T cells with sufficient cell expansion (4.6 × 109 cells-median for long process [LP] and 1.6 × 109-for short process [SP]), transduction efficiency (43.5%-median for LP and 41.0%-for SP), represented mainly by T central memory cell population, with low expression of exhaustion markers, and with high specific antitumor activity in vitro. We did not find significant differences in the properties of the products obtained during the 7- and 11-day manufacturing cycles, which is in favor of reducing the duration of production to 7 days, which may accelerate CAR-T therapy. We have shown that donor sources for CAR-T manufacturing do not significantly affect the composition and functional properties of the cell product. CONCLUSIONS: This study demonstrates the possibility of using the CliniMACS Prodigy system with a shortened 7-day production cycle to produce sufficient amount of functional CAR-T cells. CAR transduction efficiency can be measured indirectly via functional assays.

Automatic generation of alloreactivity-reduced donor lymphocytes and hematopoietic stem cells from the same mobilized apheresis product.

INTRODUCTION: In vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαß/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. The current semi-automatic manufacturing process on the CliniMACS Plus, although robust, still requires a significant amount of manual labor to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαß/CD19 depletion module combined with the previously described CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy. METHODS: We evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split automatically by the Prodigy, one portion each depleted of CD45RA+ or of TCRαß+ and CD19+ cells. We investigated critical quality attributes for both products. Products were assessed for recovery of HSPCs and mature subsets, as well as depletion efficiency of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 2-6 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry. RESULTS: Ten sequential automatic processes were completed with minimal hands-on time beyond tubing set installation. Depletion efficiency of CD45RA+ resp. TCRαß+ and CD19+ cells was equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγδ+ and NK cells. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions remained efficient. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45+ and HSPCs was > 70% and in line with expectation. CONCLUSION: The closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαß/CD19-depleted products included HSPCs, TCRγδ+ and NK cells. Both products are predicted to be effectively depleted of GVH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.

Manufacturing chimeric antigen receptor T cells: issues and challenges.

Clinical trials of adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have delivered unprecedented responses in patients with relapsed refractory B-cell malignancy. These results have prompted Food and Drug Administration (FDA) approval of two CAR T-cell products in this high-risk patient population. The widening range of indications for CAR T-cell therapy and increasing patient numbers present a significant logistical challenge to manufacturers aiming for reproducible delivery systems for high-quality clinical CAR T-cell products. This review discusses current and novel CAR T-cell processing methodologies and the quality control systems needed to meet the increasing clinical demand for these exciting new therapies.

Depletion of αß+ T and B Cells Using the CliniMACS Prodigy: Results of 10 Graft-Processing Procedures from Haploidentical Donors.

BACKGROUND: Depletion of TCRαß+ T cells and B cells with the CliniMACS Plus® has been used for haploidentical hematopoietic stem cell transplantation for a decade. The depletion procedure is time and labour demanding and with variable reported efficiencies. Recently, an automated procedure was launched for the CliniMACS Prodigy® (Miltenyi Biotec) but reported data are scarce. Here, we report the results of the first ten TCRαß+ and B cell depletion procedures for clinical use performed at our centre. MATERIALS AND METHODS: All transplants were from a parent to a child. Collection of peripheral blood stem cells was performed after filgrastim mobilisation by use of the Spectra Optia® (TerumoBCT) set on the MNC program. Because of insufficient hematopoietic stem cell mobilisation, 1 donor received additional plerixafor. RESULTS: We performed ten uncomplicated processes with the CliniMACS Prodigy. We found the results of the depletion procedures satisfactory with a median log reduction of TCRαß+ cells of -4.21 (range -3.98 to -4.74), resulting in a median number of TCRαß+ cells in the depleted product of 28.6 × 103/kg recipient weight (range 14.9-69.7 × 103/kg). The median CD34 recovery was 83% (range 70-100). To achieve a sufficient number of CD34+ cells, we performed an additional CD34+ enrichment procedure using the CliniMACS Plus for 3 patients. The B cell depletion was slightly less efficient with a median log reduction of -3.72 (range -2.83 to -4.20). CONCLUSION: Overall, we found the TCRαß and CD19 depletion procedure on the CliniMACS Prodigy easy to handle and reliable, providing reproducible good results.

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center.

BACKGROUND AIMS: Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. METHODS: The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days. RESULTS: Enrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3+ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5-10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82-100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. DISCUSSION: The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.

Generation of alloreactivity-reduced donor lymphocyte products retaining memory function by fully automatic depletion of CD45RA-positive cells.

BACKGROUND AIMS: For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA+) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products. METHODS: Unstimulated apheresis products from random volunteer donors were depleted of CD45RA+ cells on CliniMACS Prodigy, using Good Manufacturing Practice (GMP)-compliant reagents and methods throughout. Using phenotypic and functional in vitro assays, we assessed the cellular constitution of CD45RA-depleted products, including T-cell subset analyses, immunological memory function and allo-reactivity. RESULTS: Selections were technically uneventful and proceeded automatically with minimal hands-on time beyond tubing set installation. Products were near-qualitatively CD45RA+ depleted, that is, largely devoid of CD45RA+ T cells but also of almost all B and natural killer cells. Naive and effector as well as γ/δ T cells were greatly reduced. The CD4:CD8 ratio was fivefold increased. Mixed lymphocyte reaction assays of the product against third-party leukocytes revealed reduced allo-reactivity compared to starting material. Anti-pathogen responses were retained. DISCUSSION: The novel, closed, fully GMP-compatible process on Prodigy generates highly CD45RA-depleted cellular products predicted to be clinically meaningfully depleted of GvH reactivity.

BKV-specific T cells in the treatment of severe refractory haemorrhagic cystitis after HLA-haploidentical haematopoietic cell transplantation.

BACKGROUND: Haemorrhagic cystitis caused by BK virus (BKV) is a known complication of allogeneic haematopoietic cell transplantation (HCT) and is relatively common following HLA-haploidentical transplantation. Adoptive immunotransfer of virus-specific T cells from the donor is a promising therapeutic approach, although production of these cells is challenging, particularly when dealing with low-frequency T cells such as BKV-specific T cells. CASE REPORT: Here, we present a patient who, following haploidentical HCT, developed severe BKV haemorrhagic cystitis, resistant to standard therapy. He responded well to adoptive transfer of donor cells enriched in BKV-specific T cells using the new second-generation CliniMACS Prodigy and the Cytokine Capture System from Miltenyi Biotec. Treatment led to full resolution of both the symptoms and viraemia without unwanted complications. CONCLUSION: Our observations suggest that use of products enriched with BKV-specific T cells generated using this system is safe and efficient in HLA-haploidentical HCT where BKV cystitis can be a serious complication.

Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy.

BACKGROUND AIMS: Ex vivo expansion of natural killer (NK) cells is a strategy to produce large numbers of these effector cells for immunotherapy. However, the transfer of bench-top expansion protocols to clinically applicable methods is challenging for NK cell-based therapy because of regulatory aspects and scale-up issues. Therefore, we developed an automated, large-scale NK cell expansion process. METHODS: Enriched NK cells were expanded with interleukin-2 and irradiated clinical-grade Epstein-Barr virus-transformed lymphoblastoid feeder cells with the use of an automated system in comparison to manual expansion, and the cells were investigated for their functionality, phenotype and gene expression. RESULTS: Automated expansion resulted in a mean 850-fold expansion of NK cells by day 14, yielding 1.3 (± 0.9) × 10(9) activated NK cells. Automatically and manually produced NK cells were comparable in target cell lysis, degranulation and production of interferon-γ and tumor necrosis factor-α and had similar high levels of antibody-dependent cellular cytotoxicity against rituximab-treated leukemic cells. NK cells after automated or manual expansion showed similar gene expression and marker profiles. However, expanded NK cells differed significantly from primary NK cells including upregulation of the functional relevant molecules TRAIL and FasL and NK cell-activating receptors NKp30, NKG2D and DNAM-1. Neither automatically nor manually expanded NK cells showed reduced telomere length indicative of a conserved proliferative potential. CONCLUSIONS: We established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.

Automated and closed clinical-grade manufacturing protocol produces potent NK cells against neuroblastoma cells and AML blasts.

Natural killer (NK) cells are a promising allogeneic immunotherapy option due to their natural ability to kill tumor cells, and due to their apparent safety. This study describes the development of a GMP-compliant manufacturing protocol for the local production of functionally potent NK cells tailored for high-risk acute myeloid leukemia (AML) and neuroblastoma (NBL) patients. Moreover, the quality control strategy and considerations for product batch specifications in early clinical development are described. The protocol is based on the CliniMACS Prodigy platform and Natural Killer Cell Transduction (NKCT) (Miltenyi Biotec). NK cells are isolated from leukapheresis through CD3 depletion and CD56 enrichment, followed by a 12-hour activation with IL-2 and IL-15 cytokines. Three CliniMACS Prodigy processes demonstrated the feasibility and consistency of the modified NKCT process. A three-step process without expansion, however, compromised the NK cell yield. T cells were depleted effectively, indicating excellent safety of the product. Characterization of the NK cells before and after cytokine activation revealed a notable increase in the expression of activation markers, particularly CD69, consistent with enhanced functionality. Intriguingly, the NK cells exhibited increased killing efficacy against patient-derived CD33 + AML blasts and NBL cells in vitro, suggesting a potential therapeutic benefit in AML and NBL.

Effective virus-specific T-cell therapy for high-risk SARS-CoV-2 infections in hematopoietic stem cell transplant recipients: initial case studies and literature review.

The COVID-19 pandemic has exacerbated mortality rates among immunocompromised patients, accentuating the need for novel, targeted therapies. Transplant recipients, with their inherent immune vulnerabilities, represent a subgroup at significantly heightened risk. Current conventional therapies often demonstrate limited effectiveness in these patients, calling for innovative treatment approaches. In immunocompromised transplant recipients, several viral infections have been successfully treated by adoptive transfer of virus-specific T-cells (VST). This paper details the successful application of SARS-CoV-2-specific memory T-cell therapy, produced by an interferon-γ cytokine capture system (CliniMACS® Prodigy device), in three stem cell transplant recipients diagnosed with COVID-19 (case 1: alpha variant, cases 2 and 3: delta variants). These patients exhibited persistent SARS-CoV-2 PCR positivity accompanied by bilateral pulmonary infiltrates and demonstrated only partial response to standard treatments. Remarkably, all three patients recovered and achieved viral clearance within 3 to 9 weeks post-VST treatment. Laboratory follow-up investigations identified an increase in SARS-CoV-2-specific T-cells in two of the cases. A robust anti-SARS-CoV-2 S (S1/S2) IgG serological response was also recorded, albeit with varying titers. The induction of memory T-cells within the CD4 + compartment was confirmed, and previously elevated interleukin-6 (IL-6) and IL-8 levels normalized post-VST therapy. The treatment was well tolerated with no observed adverse effects. While the need for specialized equipment and costs associated with VST therapy present potential challenges, the limited treatment options currently available for COVID-19 within the allogeneic stem cell transplant population, combined with the risk posed by emerging SARS-CoV-2 mutations, underscore the potential of VST therapy in future clinical practice. This therapeutic approach may be particularly beneficial for elderly patients with multiple comorbidities and weakened immune systems.

Propagation of SARS-CoV-2 in a Closed Cell Culture Device: Potential GMP Compatible Production Platform for Live-Attenuated Vaccine Candidates under BSL-3 Conditions?

Live-attenuated SARS-CoV-2 vaccines present themselves as a promising approach for the induction of broad mucosal immunity. However, for initial safety assessment in clinical trials, virus production requires conditions meeting Good Manufacturing Practice (GMP) standards while maintaining biosafety level 3 (BSL-3) requirements. Since facilities providing the necessary complex ventilation systems to meet both requirements are rare, we here describe a possibility to reproducibly propagate SARS-CoV-2 in the automated, closed cell culture device CliniMACS Prodigy® in a common BSL-3 laboratory. In this proof-of-concept study, we observed an approximately 300-fold amplification of SARS-CoV-2 under serum-free conditions with high lot-to-lot consistency in the infectious titers obtained. With the possibility to increase production capacity to up to 3000 doses per run, this study outlines a potential fast-track approach for the production of live-attenuated vaccine candidates based on highly pathogenic viruses under GMP-like conditions that may contribute to pandemic preparedness.

CAR-T cells targeting IL-1RAP produced in a closed semiautomatic system are ready for the first phase I clinical investigation in humans.

PURPOSE OF THE STUDY: The use of chimeric antigen receptor (CAR)-T cells has demonstrated excellent results in B-lymphoid malignancies. The Advanced Therapy Medicinal Products (ATMP) status and good manufacturing practice (GMP) of CAR-T cells require particular conditions of production performed in a pharmaceutical establishment. Our team developed a new medical drug candidate for acute myeloid leukemia (AML), a CAR targeting interleukin-1 receptor accessory protein (IL-1RAP) expressed by leukemia stem cells, which will need to be evaluated in a phase I-IIa clinical trial. During the preclinical development phase, we produced IL-1RAP CAR-T cells in a semi-automated closed system (CliniMACSࣨ Prodigy) using research grade lentiviral particles. PATIENTS AND THE METHODS: The purpose of this work was to validate our production process and to characterize our preclinical GMP-like medicinal product. IL-1RAP CAR-T cells were produced from healthy donors in 9 days, either in an semi-automated closed system (with GMP-like compliant conditions) or according to another research protocols, which was used as a reference. RESULTS: Based on phenotypic, functional and metabolic analyses, we were able to show that the final product is ready for clinical use. Finally, in a xenograft AML murine model, we demonstrated that the IL-1RAP CAR-T cells generated in a GMP-like environment could eliminate tumor cells and increase overall survival. CONCLUSION: We demonstrated that our IL-1RAP CAR-T cell preclinical GMP-like production process meets standard regulatory requirements in terms of CAR-T cell number, subpopulation phenotype and cytotoxic functionality. Our CAR-T cell production process was validated and can be used to produce medicinal IL-1RAP CAR-T cells for the first phase I clinical trial.

Development of an automated manufacturing process for large-scale production of autologous T cell therapies.

Engineered T cell therapies have shown significant clinical success. However, current manufacturing capabilities present a challenge in bringing these therapies to patients. Furthermore, the cost of development and manufacturing is still extremely high due to complexity of the manufacturing process. Increased automation can improve quality and reproducibility while reducing costs through minimizing hands-on operator time, allowing parallel manufacture of multiple products, and reducing the complexity of technology transfer. In this article, we describe the results of a strategic alliance between GSK and Miltenyi Biotec to develop a closed, automated manufacturing process using the CliniMACS Prodigy for autologous T cell therapy products that can deliver a high number of cells suitable for treating solid tumor indications and compatible with cryopreserved apheresis and drug product. We demonstrate the ability of the T cell Transduction - Large Scale process to deliver a significantly higher cell number than the existing process, achieving 1.5 × 1010 cells after 12 days of expansion, without affecting other product attributes. We demonstrate successful technology transfer of this robust process into three manufacturing facilities.

Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy.

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation-expansion process and its validation on clinical-scale. METHODS: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. RESULTS: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. CONCLUSIONS: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

Automated generation of gene-edited CAR T cells at clinical scale.

The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/ß-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/ß-free CAR19 T cells at clinical scale.

Optimal large-scale CD34+ enrichment from a leukapheresis collection using the clinimacs prodigy platform.

Optimization of Hematology Patient's treatment: It is possible to obtain a 100% CD34+ recovery after CD34+ selection using the CliniMACS Prodigy.

Point-Of-Care CAR T-Cell Production (ARI-0001) Using a Closed Semi-automatic Bioreactor: Experience From an Academic Phase I Clinical Trial.

Development of semi-automated devices that can reduce the hands-on time and standardize the production of clinical-grade CAR T-cells, such as CliniMACS Prodigy from Miltenyi, is key to facilitate the development of CAR T-cell therapies, especially in academic institutions. However, the feasibility of manufacturing CAR T-cell products from heavily pre-treated patients with this system has not been demonstrated yet. Here we report and characterize the production of 28 CAR T-cell products in the context of a phase I clinical trial for CD19+ B-cell malignancies (NCT03144583). The system includes CD4-CD8 cell selection, lentiviral transduction and T-cell expansion using IL-7/IL-15. Twenty-seven out of 28 CAR T-cell products manufactured met the full list of specifications and were considered valid products. Ex vivo cell expansion lasted an average of 8.5 days and had a mean transduction rate of 30.6 ± 13.44%. All products obtained presented cytotoxic activity against CD19+ cells and were proficient in the secretion of pro-inflammatory cytokines. Expansion kinetics was slower in patient's cells compared to healthy donor's cells. However, product potency was comparable. CAR T-cell subset phenotype was highly variable among patients and largely determined by the initial product. TCM and TEM were the predominant T-cell phenotypes obtained. 38.7% of CAR T-cells obtained presented a TN or TCM phenotype, in average, which are the subsets capable of establishing a long-lasting T-cell memory in patients. An in-depth analysis to identify individual factors contributing to the optimal T-cell phenotype revealed that ex vivo cell expansion leads to reduced numbers of TN, TSCM, and TEFF cells, while TCM cells increase, both due to cell expansion and CAR-expression. Overall, our results show for the first time that clinical-grade production of CAR T-cells for heavily pre-treated patients using CliniMACS Prodigy system is feasible, and that the obtained products meet the current quality standards of the field. Reduced ex vivo expansion may yield CAR T-cell products with increased persistence in vivo.

Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing.

Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1®, to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering.

In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 109 leukocytes (including 1.1-2.3 × 109 NK cells) collected by small-scale lymphapheresis (n = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log -3.6 after CD3 depletion and log -3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult® chamber of Prodigy resulted in approximately 4.2-8.5-fold NK cell expansion rates by adding of NK MACS® basal medium containing NK MACS® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector-target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.