Manufacture Of Chimeric Antigen Receptor Research Articles

Impact of Bridging Chemotherapy on Clinical Outcomes of CD19-Specific CAR T Cell Therapy in Children/Young Adults with Relapsed/Refractory B Cell Acute Lymphoblastic Leukemia

Chimeric antigen receptor (CAR) T cells achieve response and durable remission in patients with relapsed/refractory (R/R) B cell malignancies. Following collection of patient T cells, chemotherapy ("bridging chemotherapy") is utilized during the manufacture of CAR T cells. However, the optimal bridging chemotherapy has yet to be defined. Our objective in this study was to report clinical outcomes following bridging chemotherapy in a cohort of pediatric/young adult patients with R/R B cell acute lymphoblastic leukemia (B-ALL) treated with CAR T cell therapy. This retrospective study included patients enrolled on clinical trial NCT01860937 or referred to Memorial Sloan Kettering Cancer Center for commercial CAR T cell therapy (tisagenlecleucel). Bridging chemotherapy (given after T cell collection and before CAR T cell infusion) was defined as high intensity if myelosuppression was expected for >7 days. Outcome comparison analyses were performed in high-intensity versus low-intensity bridging chemotherapy, 1 cycle versus ≥2 cycles of bridging chemotherapy, disease burden at the start of bridging chemotherapy, disease burden at the start of bridging chemotherapy with chemotherapy intensity, tumor debulking by bridging chemotherapy, and disease burden pre-lymphodepleting chemotherapy (LDC) for CAR T cell treatment. The outcomes of this analysis showed that the incidence of grade ≥3 infection was significantly higher (94% versus 56%; P=.019) and overall survival (OS) was significantly lower (hazard ratio, 3.73; 95% confidence interval, 1.39 to 9.97; P=.006) in patients who received ≥2 cycles versus 1 cycle of bridging chemotherapy. No difference in incidence was found for cytokine release syndrome (P > .99) or neurotoxicity/immune effector cell-associated neurotoxicity syndrome (P=.70). Disease burden at the start of bridging chemotherapy, disease burden prior to LDC, and tumor debulking by bridging chemotherapy also did not significantly affect outcomes after CAR T cell therapy in this cohort. In this study, patients receiving ≥2 cycles of bridging chemotherapy had higher rates of infection and lower OS but no difference in CAR-specific toxicity. Clinicians should carefully consider the use of additional cycles of chemotherapy during the bridging period as it delays treatment with CAR T cells and increases the risk of infectious complications. © 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3′ and 5′ arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 108 CAR19 T cells per one electroporation of 1 × 107 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products.

Formulation Considerations for Autologous T Cell Drug Products.

Genetically modified autologous T cells have become an established immunotherapy in the fight against cancer. The manufacture of chimeric antigen receptor (CAR) and αβ-T cell receptor (TCR) transduced T cells poses unique challenges, including the formulation, cryopreservation and fill–finish steps, which are the focus of this review. With an increasing number of marketing approvals for CAR-T cell therapies, comparison of their formulation design and presentation for administration can be made. These differences will be discussed alongside the emergence of automated formulation and fill-finish processes, the formulation design space, Monte Carlo simulation applied to risk analysis, primary container selection, freezing profiles and thaw and the use of dimethyl sulfoxide and alternative solvents/excipients as cryopreservation agents. The review will conclude with a discussion of the pharmaceutical solutions required to meet the simplification of manufacture and flexibility in dosage form for clinical treatment.

Scalable Manufacturing of CAR T cells for Cancer Immunotherapy.

As the demand for CAR T cell cancer therapy increases, several closed and automated production platforms are being deployed, and others are in development.This review provides a critical appraisal of these technologies that can be leveraged to scale and optimize the production of next generation CAR T cells.

Depletion of high-content CD14+ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing

With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin’s lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

Abstract LB105: In vivo CAR T therapy: targeted in vivo gene delivery of a CAR using a CD8-specific fusogen results in tumor eradication

Abstract CAR T cells are highly effective at inducing remissions in patients with refractory B cell malignancies. The ex vivo process used to manufacture these therapies limits patient access and exposes T cells to non-physiologic conditions in culture. Viral vectors are efficient at genetically modifying T cells and are used in the ex vivo manufacture of most CAR T cell products but lack target-cell specificity. Here we describe how viral envelope glycoproteins, termed fusogens, engineered to target the CD8 co-receptor can efficiently and specifically deliver a chimeric antigen receptor (CAR) to human T cells in vivo that support target cell killing and tumor eradication. Modification of viral envelope proteins to include antibody-based binding domains (CD8 binders) can produce fusogen-viral vector compositions that deliver genetic payloads to CD8 T cells with therapeutic effect (1, 2). Screening CD8-targeted binders identified fusogens with a range of on-target cell transduction efficiencies and off-target cell line specificities. CD8-targeted fusogens with the highest efficiency were comparable to VSVg-pseudotyped lentiviruses at transducing CD8 T cells, but unlike VSVg showed CD8 T cell specific transduction. Interestingly, CD8-targeted fusogens were superior to VSVg-pseudotyped lentivirus at transducing non-activated human T cells. In vivo fusogen-mediated delivery showed efficient and specific CD8 T cell transduction and expression of a GFP reporter gene. Second-generation CARs containing a CD19 binding domain and a 41BB costimulatory domain delivered by targeted fusogen demonstrated CD8 specific expression and functional activity against non-malignant B cells and CD19+ Nalm6 leukemia cells. To demonstrate targeted fusogen-mediated in vivo CAR delivery and activity, we established Nalm6 xenografts in mice into which unmodified activated human PBMCs were infused. Activated human PBMCs alone were unable to control tumor growth. A single intravenous delivery of a CD8 fusogen containing a second-generation CD19 CAR transgene across a range of functional titer doses resulted in the generation of CD8 CAR Ts that eradicated the CD19+ tumor xenografts. A high percentage of T cells demonstrated CAR expression after fusogen delivery with clear specificity for the CD8+ cells. Importantly, the fusogen was able to generate a functional CAR response regardless of prior activation status of the T cells. Targeted in vivo delivery of CAR into T cell subsets may represent a novel therapeutic approach for human B cell malignancies. The ability to specifically deliver a CAR gene to a T cell in vivo would be transformative in terms of patient access and is likely to generate a qualitatively superior therapeutic T cell.

Target and Manufacturing of Chimeric Antigen Receptor T cells for T-cell Malignancies--Review

T lymphoid malignancy is a group of highly heterogeneous hematological tumors. Disease recurrence and resistance to therapy are the common causes of failed treatment. Traditional therapy is radiotherapy and chemotherapy, although it has achieved great success. However, many patients still failed to survive following the treatment. With the introduction of monoclonal antibodies, immunotherapy and cellular therapy into clinical practice, the outcome of hematologic malignancies has been significantly improved. In particular, chimeric antigen receptor T cells (CAR-T) showed high efficacy in treating B-cell lymphoma and acute B lymphocytic leukemia and surpassed any previous therapeutic strategies. However, this treatment seldom succeeded in treating T cell malignancies. In this review, the history of CAR-T cells treating T cell malignancies, and the clinical trials, adverse events of previously reported were summarized briefly.

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

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

Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study

Lisocabtagene maraleucel (liso-cel) is an autologous, CD19-directed, chimeric antigen receptor (CAR) T-cell product. We aimed to assess the activity and safety of liso-cel in patients with relapsed or refractory large B-cell lymphomas. We did a seamless design study at 14 cancer centres in the USA. We enrolled adult patients (aged ≥18 years) with relapsed or refractory large B-cell lymphomas. Eligible histological subgroups included diffuse large B-cell lymphoma, high-grade B-cell lymphoma with rearrangements of MYC and either BCL2, BCL6, or both (double-hit or triple-hit lymphoma), diffuse large B-cell lymphoma transformed from any indolent lymphoma, primary mediastinal B-cell lymphoma, and follicular lymphoma grade 3B. Patients were assigned to one of three target dose levels of liso-cel as they were sequentially tested in the trial (50 × 106 CAR+ T cells [one or two doses], 100 × 106 CAR+ T cells, and 150 × 106 CAR+ T cells), which were administered as a sequential infusion of two components (CD8+ and CD4+ CAR+ T cells) at equal target doses. Primary endpoints were adverse events, dose-limiting toxicities, and the objective response rate (assessed per Lugano criteria); endpoints were assessed by an independent review committee in the efficacy-evaluable set (comprising all patients who had confirmed PET-positive disease and received at least one dose of liso-cel). This trial is registered with ClinicalTrials.gov, NCT02631044. Between Jan 11, 2016, and July 5, 2019, 344 patients underwent leukapheresis for manufacture of CAR+ T cells (liso-cel), of whom 269 patients received at least one dose of liso-cel. Patients had received a median of three (range 1-8) previous lines of systemic treatment, with 260 (97%) patients having had at least two lines. 112 (42%) patients were aged 65 years or older, 181 (67%) had chemotherapy-refractory disease, and seven (3%) had secondary CNS involvement. Median follow-up for overall survival for all 344 patients who had leukapheresis was 18·8 months (95% CI 15·0-19·3). Overall safety and activity of liso-cel did not differ by dose level. The recommended target dose was 100 × 106 CAR+ T cells (50 × 106 CD8+ and 50 × 106 CD4+ CAR+ T cells). Of 256 patients included in the efficacy-evaluable set, an objective response was achieved by 186 (73%, 95% CI 66·8-78·0) patients and a complete response by 136 (53%, 46·8-59·4). The most common grade 3 or worse adverse events were neutropenia in 161 (60%) patients, anaemia in 101 (37%), and thrombocytopenia in 72 (27%). Cytokine release syndrome and neurological events occurred in 113 (42%) and 80 (30%) patients, respectively; grade 3 or worse cytokine release syndrome and neurological events occurred in six (2%) and 27 (10%) patients, respectively. Nine (6%) patients had a dose-limiting toxicity, including one patient who died from diffuse alveolar damage following a dose of 50 × 106 CAR+ T cells. Use of liso-cel resulted in a high objective response rate, with a low incidence of grade 3 or worse cytokine release syndrome and neurological events in patients with relapsed or refractory large B-cell lymphomas, including those with diverse histological subtypes and high-risk features. Liso-cel is under further evaluation at first relapse in large B-cell lymphomas and as a treatment for other relapsed or refractory B-cell malignancies. Juno Therapeutics, a Bristol-Myers Squibb Company.

CD19 CAR T Cells for the Treatment of Pediatric Pre-B Cell Acute Lymphoblastic Leukemia.

The development of cluster of differentiation (CD)-19-targeted chimeric antigen receptor (CAR) T cells for the treatment of pre-B-cell acute lymphoblastic leukemia (B-ALL) is an exciting new advancement in the field of pediatric oncology. Tisagenlecleucel and axicabtagene ciloleucel are the first US FDA-approved CD19-targeted CAR T cells. While various different CD19 CAR T cells are in development, tisagenlecleucel is the only CAR T cell approved for pediatric patients. The multicenter phase II trial that led to the approval of tisagenlecleucel demonstrated excellent responses in individuals with highly refractory disease. Other high-risk groups of patients with B-ALL who experience poor outcomes with standard therapy may also benefit from treatment with tisagenlecleucel. After receiving CAR T cells, patients must be closely monitored for unique toxicities, including cytokine release syndrome, neurotoxicity, and B-cell aplasia. The management of patients with relapsed or refractory disease after administration of CD19 CAR T cells can be challenging, and treatment options vary according to the characteristics of the disease present at relapse. In the many patients who experience a complete response, CAR T cells can lead to a durable remission. This review describes the current design and manufacturing of CAR T cells. Data in the selection and management of pediatric patients are highlighted, as are areas where further studies are needed.

Shortened ex vivo manufacturing time of EGFRvIII-specific chimeric antigen receptor (CAR) T cells reduces immune exhaustion and enhances antiglioma therapeutic function.

Non-viral manufacturing of CAR T cells via the Sleeping Beauty transposon is cost effective and reduces the risk of insertional mutagenesis from viral transduction. However, the current gold standard methodology requires ex vivo numerical expansion of these cells on artificial antigen-presenting cells (AaPCs) for 4weeks to generate CAR T cells of presumedsufficient quantity and function for clinical applications. We engineered EGFRvIII-specific CAR T cells and monitored phenotypic changes throughout their ex vivo manufacturing. To reduce the culture time required to generate the CAR T-cell population, we selected for T cells in peripheral blood mononuclear cells prior to CAR modification (to eliminate the competing NK cell population). While we found increased expression of exhaustion markers (such as PD-1, PD-L1, TIM-3, and LAG-3) after 2weeks in culture, whose levels continued to rise over time, we were able to generate a CAR+ T-cell population with comparable CAR expression and cell numbers in 2weeks, thereby reducing manufacturing time by 50%, with lower expression of immune exhaustion markers. The CAR T cells manufactured at 2weeks showed superior therapeutic efficacy in mice bearing established orthotopic EGFRvIII+ U87 gliomas. These findings demonstrate a novel, rapid method to generate CAR T cells by non-viral modification that results in CAR T cells superior in phenotype and function and further emphasizes that careful monitoring of CAR T-cell phenotype prior to infusion is critical for generating an optimal CAR T-cell product with full antitumor potential.

Naïve T-cell Deficits at Diagnosis and after Chemotherapy Impair Cell Therapy Potential in Pediatric Cancers.

Translational data on chimeric antigen receptor (CAR) T-cell trials indicate that the presence of naïve T cells in the premanufacture product is important to clinical response and persistence. In anticipation of developing CAR trials for other tumors, we investigated the T-cell distribution from children with solid tumors and lymphomas at diagnosis and after every cycle of chemotherapy. We found that patients with T cells enriched for naïve and stem central memory cells expanded well in vitro, but the majority of tumor types showed chemotherapy-related depletion of early lineage cells with a corresponding decline in successful ex vivo stimulation response. Unexpectedly, many pediatric patients with solid tumors had low numbers of naïve T cells prior to any therapy. These data indicate the ex vivo manufacture of CAR T cells may need to be customized based on the nature of T cells available in each disease type. SIGNIFICANCE: Cumulative chemotherapy cycles deplete naïve T cells in many pediatric cancer regimens, reducing expansion potential associated with successful adoptive cellular therapies. Naïve T-cell deficits can be seen at diagnosis as well, implying immune deficits that exist prior to chemotherapy, which may also affect the development of immune-based therapies.See related commentary by Leick and Maus, p. 466.This article is highlighted in the In This Issue feature, p. 453.

Factors affecting lymphocyte collection efficiency for the manufacture of chimeric antigen receptor T cells in adults with B-cell malignancies.

The clinical and procedural parameters that affect the optimal collection of lymphocytes for the production of chimeric antigen receptor (CAR) T cells remain undefined but are increasingly important, as commercial products are now available. We evaluated determinants of low lymphocyte collection efficiency (CE) and the rate of successful CAR T-cell manufacture in middle-aged and older adults with advanced B-cell malignancies. Mononuclear cell collections using two apheresis platforms (COBE Spectra and Spectra Optia, Terumo BCT) from patients participating in a CD19-directed CAR T-cell therapy trial were reviewed. Patient- and disease-specific factors, peripheral blood counts, apheresis parameters, and product cell counts were analyzed to determine effects on lymphocyte CE. Ninety-two apheresis events from patients with acute lymphocytic leukemia (ALL) (n = 28), chronic lymphocytic leukemia (n = 18), and non-Hodgkin lymphoma (n = 46) were available for analysis. Forty-one collections (45%) had a lymphocyte CE of <40%. On multivariable analysis, age (every 10-year increase, odds ratio [OR] = 1.51; p = 0.034), disease type (chronic lymphocytic leukemia vs. ALL, OR = 0.24; p = 0.052; non-Hodgkin lymphoma vs. ALL, OR = 0.20; p = 0.009) and precollection platelets (every 10 × 103 /μL increase, OR = 1.07; p = 0.005) were appreciably associated with a lymphocyte CE of <40%. No major apheresis complications occurred. Lymphocyte collection at our center was well tolerated and 100% successful in manufacturing CD19-directed CAR T cells from adult patients with B-cell malignancies despite low CE in some patients. A diagnosis of ALL, advancing age, and higher preapheresis platelet counts were observed to be associated with low CE.

Manufacture of Chimeric Antigen Receptor T Cells from Mobilized Cyropreserved Peripheral Blood Stem Cell Units Depends on Monocyte Depletion

Cytotoxic chemotherapy and radiation can render lymphocyte repertoires qualitatively and quantitatively defective. Thus, heavily treated patients are often poor candidates for the manufacture of autologous chimeric antigen receptor (CAR)-T cell products. In the United States and Europe, children with high-risk neuroblastoma undergo apheresis early in the course of treatment to collect peripheral blood stem cells (PBSCs) for cryopreservation in preparation for high-dose chemotherapy followed by autologous stem cell rescue. Here, we investigate whether these cryopreserved chemotherapy and granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs can serve as starting material for CAR-T cell manufacturing. We evaluated T cell precursor subsets in cryopreserved PBSC units from 8 patients with neuroblastoma using fluorescent activated cell sorting-based analysis. Every cryopreserved unit collected early in treatment contained both CD4 and CD8 precursors with significant numbers of naïve and central memory precursors. Significant numbers of Ki67+/PD1+ T cells were detected, presumably the result of chemotherapy-induced lymphopenia and subsequent homeostatic proliferation. Cryopreserved PBSC units containing 56 to 112 × 106 T cells were amenable to immunomagnetic selection, CD3 × 28 bead activation, lentiviral transduction, and cytokine-driven expansion, provided that CD14 monocytes were depleted before the initiation of cultures. Second- and third-generation CD171 CAR+ CD4 and CD8 effector cells derived from cryopreserved units displayed antineuroblastoma lytic potency and cytokine secretion comparable to those derived from a healthy donor and mediated in vivo antitumor regression in NSG mice. We conclude that cryopreserved PBSCs procured via standard methods during early treatment can serve as an alternative starting source for CAR-T cell manufacturing, extending the options for heavily treated patients.

CAR T Cells in Trials: Recent Achievements and Challenges that Remain in the Production of Modified T Cells for Clinical Applications.

The adoptive transfer of chimeric antigen receptor (CAR)-modified T cells is attracting growing interest for the treatment of malignant diseases. Early trials with anti-CD19 CAR T cells have achieved spectacular remissions in B-cell leukemia and lymphoma, so far refractory, very recently resulting in the Food and Drug Administration approval of CD19 CAR T cells for therapy. With further applications and increasing numbers of patients, the reproducible manufacture of high-quality clinical-grade CAR T cells is becoming an ever greater challenge. New processing techniques, quality-control mechanisms, and logistic developments are required to meet both medical needs and regulatory restrictions. This paper summarizes the state-of-the-art in manufacturing CAR T cells and the current challenges that need to be overcome to implement this type of cell therapy in the treatment of a variety of malignant diseases and in a greater number of patients.

Automated Manufacturing of Potent CD20-Directed Chimeric Antigen Receptor T Cells for Clinical Use.

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

Clinical Development and Manufacture of Chimeric Antigen Receptor T cells and the Role of Leukapheresis

Adoptive transfer of chimeric antigen receptor (CAR) T cells is a powerful targeted immunotherapeutic technique. CAR T cells are manufactured by harvesting mononuclear cells, typically via leukapheresis from a patient’s blood, then activating, modifying the T cells to express a transgene encoding a tumour-specific CAR, and infusing the CAR T cells into the patient. Gene transfer is achieved through the use of retroviral or lentiviral vectors, although non-viral delivery systems are being investigated. This article discusses the challenges associated with each stage of this process. Despite the need for a consistent end product, there is inherent variability in cellular material obtained from critically ill patients who have been exposed to cytotoxic therapy. It is important to carefully select target antigens to maximise effect and minimise toxicity. Various types of CAR T cell toxicity have been documented: this includes “on target, on tumour”, “on target, off tumour” and “off target” toxicity. A growing body of clinical evidence supports the efficacy and safety of CAR T cell therapy; CAR T cells targeting CD19 in B cell leukemias are the best-studied therapy to date. However, providing personalised therapy on a large scale remains challenging; a future aim is to produce a universal “off the shelf” CAR T cell.

Abstract 2296: Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple in vivo tumor models

Abstract Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells for treatment requires ex vivo culture to facilitate CAR gene transfer and to achieve a therapeutic dose of the modified cells. Recent data suggests that specific T cell subtypes can provide enhanced anti-tumor efficacy, spurring efforts to optimize the production of therapeutic T cells via the cumbersome physical isolation of central memory T cells or culture in cytokines such as IL-7 and IL-15. Here we explored the potential for a simple culture modification to improve the therapeutic potential of CAR T cells without adding manufacturing complexity. To this end, we produced CAR T cells specific to B cell maturation antigen (BCMA) using standard IL-2 culture conditions supplemented with a PI3K inhibitor, or with IL-7 and IL-15 in place of IL-2. The in vivo activity of CAR T cells was studied in mouse models of human Burkitt's lymphoma (Daudi) and multiple myeloma (RPMI-8226), both of which express BCMA. In the Daudi model, NSG mice were injected intravenously with 2 × 106 tumor cells and allowed to accumulate a large tumor burden to model late stage disease observed in relapsed and refractory lymphoma. In this advanced disease model, anti-BCMA CAR T cells (4 × 106/mouse) cultured either in IL-2 or IL-7 and IL-15 had little or no effect on tumor growth (p = 0.22 and 0.23, respectively) and all mice succumbed to tumors within two weeks of treatment. In contrast, all animals treated with the same number of anti-BCMA CAR T cells cultured with PI3K inhibition survived and had complete long-term tumor regression (p = 0.003). The same anti-BCMA CAR T cells were studied in a model of multiple myeloma. NSG mice were injected subcutaneously with 107 RPMI-8226 cells and 22 days later received a single administration of anti-BCMA CAR T cells (4 × 105/mouse) cultured under various conditions. In this model, tumor regression occurred regardless of in vitro culture conditions. To model tumor relapse and evaluate CAR T cell durability, surviving animals were re-challenged with RPMI-8226 cells on the opposite flank two weeks after initial tumor clearance. In contrast to other conditions, all animals treated with anti-BCMA CAR T cells cultured with PI3K inhibition were protected against subsequent tumor challenge (p = 0.005). This improved therapeutic activity of anti-BCMA CAR T cells cultured with PI3K inhibition was associated with an increased frequency of CD62L+ CD8+ T cells in the drug product (p &amp;lt; 0.001) suggesting enrichment of this distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate an improved anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Citation Format: Shannon Grande, Molly R. Perkins, Amanda Hamel, Holly M. Horton, Fay Eng, Claire J. Rhodes, Tracy E. Garrett, Sara M. Miller, John W. Evans, Howard J. Latimer, Christopher Horvath, Michael Kuczewski, Kevin Friedman, Richard A. Morgan. Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple in vivo tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2296.

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.

Translational Implications for Off-the-shelf Immune Cells Expressing Chimeric Antigen Receptors

Chimeric antigen receptor (CAR) endows specificity to T-cells independent of human leukocyte antigen (HLA). This enables one immunoreceptor to directly target the same surface antigen on different subsets of tumor cells from multiple HLA-disparate recipients. Most approaches manufacture individualized CAR(+)T-cells from the recipient or HLA-compatible donor, which are revealing promising clinical results. This is the impetus to broaden the number of patients eligible to benefit from adoptive immunotherapy such as to infuse third-party donor derived CAR(+)T-cells. This will overcome issues associated with (i) time to manufacture T-cells, (ii) cost to generate one product for one patient, (iii) inability to generate a product from lymphopenic patients or patient's immune cells fail to complete the manufacturing process, and (iv) heterogeneity of T-cell products produced for or from individual recipients. Establishing a biobank of allogeneic genetically modified immune cells from healthy third-party donors, which are cryopreserved and validated in advance of administration, will facilitate the centralizing manufacturing and widespread distribution of CAR(+)T-cells to multiple points-of-care in a timely manner. To achieve this, it is necessary to engineer an effective strategy to avoid deleterious allogeneic immune responses leading to toxicity and rejection. We review the strategies to establish "off-the-shelf" donor-derived biobanks for human application of CAR(+)T-cells as a drug.