CAR-T Cell Manufacturing Research Articles

Identification of the conditions and minimum requirements necessary for the release of autologous fresh CAR T-cell products under hospital exemption: a position paper from the WP-bioproduction of the UNITC consortium.

The accessibility of CAR-T cells in centralized production models faces significant challenges, primarily stemming from logistical complexities and prohibitive costs. However, European Regulation EC No. 1394/2007 introduced a pivotal provision known as the hospital exemption. This exemption enables academic institutions to produce ATMPs, including autologous CAR-T cells, under GMP conditions tailored to specific needs. In response to this regulatory framework, our work group has launched a guideline initiative. This endeavor aims to bolster academic CAR T-cell manufacturing by implementing strategic workshops that serve a dual purpose: standardizing production processes and ensuring both efficacy and safety standards are met. This inaugural focused on delineating criteria for the release of fresh CAR-T cell batches. Key emphases included thorough product characterization, stringent safety and potency evaluations. These criteria are essential for compliance with regulatory mandates and aligning with industry best practices. Notably, the release of initial CAR T-cell batches will be facilitated through provisional certification, with final certification contingent upon the acquisition of comprehensive analytical control results. This procedural framework adheres to methodologies endorsed by the SFGM-TC and the EBMT. Such adherence underscores a commitment to harmonizing practices across academic manufacturing facilities, thus fortifying the accessibility, efficacy, and safety of point-of-care units.

Effects of an initial anti-CD19 CAR T-cell therapy on subsequent anti-CD22 CAR T-cell manufacturing and clinical outcomes in patients with r/r LBCL.

Patients with large B-cell lymphoma (LBCL) progressing after anti-CD19 CAR T-cell (CAR19) therapy have poor outcomes. Subsequent CAR T-cell therapy shows promise, but the impact of residual CAR19 and early relapse remains unclear. We evaluated 37 CAR19-refractory LBCL patients who received anti-CD22 CAR T-cell (CAR22) in a phase 1b trial (NCT04088890). Residual CAR19 was unquantifiable in 17 of 33 evaluable patients post-CAR22 infusion. Single-cell RNA sequencing revealed minimal CAR19/CAR22 co-transduction. Peak CAR19 transgene levels did not affect CAR22 efficacy or toxicities. CAR22 products from patients undergoing leukapheresis > 6 months post-CAR19 had higher CD4+ naïve T and CD4+/CD8+ T- central memory (TCM) cells, with lower CD4+ T-effector memory cells. High and low percentages of CAR22 TCM and TEM, respectively, were correlated with CAR22 transduction efficiency and achieving complete response. Residual CAR19 and leukapheresis timing did not significantly affect outcomes, while CAR22 product composition was significantly correlated with treatment response.

Current Non-Viral-Based Strategies to Manufacture CAR-T Cells

The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods. While viral vectors offer high transfection efficiencies, concerns regarding potential malignant transformation coupled with costly and time-consuming vector manufacturing are constant drivers in the search for cheaper, easier-to-use, safer, and more efficient alternatives. In this review, we examine different non-viral gene transfer methods as alternatives for CAR-T cell production, their advantages and disadvantages, and examples of their applications. Transposon-based gene transfer methods lead to stable but non-targeted gene integration, are easy to handle, and achieve high gene transfer rates. Programmable endonucleases allow targeted integration, reducing the potential risk of integration-mediated malignant transformation of CAR-T cells. Non-integrating CAR-encoding vectors avoid this risk completely and achieve only transient CAR expression. With these promising alternative techniques for gene transfer, all avenues are open to fully exploiting the potential of next-generation CAR-T cell therapy and applying it in a wide range of applications.

Charting the course for CAR T-cell manufacturing.

Charting the course for CAR T-cell manufacturing.

CAR Assembly Line: Taking CAR T-Cell Manufacturing to the Next Level

CAR Assembly Line: Taking CAR T-Cell Manufacturing to the Next Level

Combining the induced pluripotent stem cell (iPSC) technology with chimeric antigen receptor (CAR)-based immunotherapy: recent advances, challenges, and future prospects.

After experiencing many ups and downs, chimeric antigen receptor (CAR)-T cell therapy has reached a milestone as an anti-cancer method, as evidenced by the increasing number of clinical trials and approved products. Nonetheless, there is a real need to optimize CAR-T cell therapy and overcome its existing limitations. The importance of cellular starting material for generating CAR-T cells is undeniable, as the current personalized manufacturing approach is the main roadblock to providing a fast, affordable, and standard treatment for patients. Thus, developing an off-the-shelf CAR-T product is a leading focus in adoptive cell therapy. Several biotech companies worldwide are focused on developing an off-the-shelf CAR-T product from allogeneic sources. Induced pluripotent stem cells (iPSCs) have unique characteristics, making them highly attractive among various allogeneic sources. IPSCs can be modified with CAR, undergo other intended gene manipulations, and then be differentiated into functional hematopoietic lineages with anti-cancer activity. Moreover, iPSCs provide an unlimited cell source, simplifying the setting of a standard treatment protocol by generating a homogenous population of resulting cells and reducing batch-to-batch inconsistency. In this review, we delve into the manufacturing of iPSC-derived CAR-T (iCAR-T) cells and discuss the path and challenges of their clinical translation. We also introduce some iPSC-derived cellular alternatives to conventional iCAR-αβ-T cells, including iCAR-T cells with a limited TCR diversity, iCAR-NK, iCAR-macrophages, and iCAR-neutrophils and discuss their relative advantages and disadvantages as well as their differentiation from iPSCs in compliance with cGMP. Finally, we reviewed iPSC-derived CAR-engineered cells being evaluated in clinical trials.

Innovative Bone Marrow CAR-T Cell Manufacturing for AML: Enhancing Viability and Tumor Migration

Innovative Bone Marrow CAR-T Cell Manufacturing for AML: Enhancing Viability and Tumor Migration

Automated Rapid CAR-T Cell Manufacturing Process, Starting from Whole Blood, on a Novel Closed Platform

Automated Rapid CAR-T Cell Manufacturing Process, Starting from Whole Blood, on a Novel Closed Platform

Rapid manufacture of low-seed CAR-T cells in a GMP-grade hollow-fiber bioreactor platform

Both quality of product and rapidity of manufacture are critical parameters if ex vivo manufacturing of autologous chimeric antigen receptor T cell (CAR-T) therapies is to reach its full potential. The Quantum Flex Cell Expansion System from Terumo Blood and Cell Technologies (Terumo BCT), a hollow-fiber bioreactor platform, is one of several cell expansion systems available to cell and gene therapy manufacturers to generate such cells in a GMP-compliant manner. In this study, the dynamic range of the Quantum Flex platform to expand CD19 CAR-T cells from variable quantities of starting material was investigated. Reflecting the industry's utilization of contract development manufacturing organizations (CDMOs) for accelerating clinical timelines, Terumo Blood and Cell Technologies performed a technology transfer of application protocols for study execution. Four different amounts of starting material (1, 3, 6 and 15 million cells) were expanded on Quantum Flex, using a unique donor's cells for each run. In this study, CAR-T cells were created using commercially obtained T cells and an anti-CD19 CAR-T lentiviral construct. The resultant heterogenous cell populations were expanded for 7 days in the functionally closed bioreactor platform. Expansion kinetics for all 4 starting material amounts were remarkedly similar, resulting in a 150- to 200-fold increase in cell numbers. This allowed for a study maximum of 2.6 billion cells from loading 15 million cells. Viability remained high throughout the expansion process with >93% for all 4 donors at harvest. To complete the manufacturing cycle, the automated and functionally closed Finia Fill and Finish System (Terumo Blood and Cell Technologies, Lakewood, CO) was used to formulate the cells for cryopreservation. Postprocedure analysis for potency and cytotoxicity demonstrated the production of efficacious cells. With this range of starting numbers, the platform is relevant to adult, pediatric and compassionate CAR-T expansion dosing. Today, several platforms are available to achieve sufficient cell yields for therapeutic applications of CAR-T, and awareness of the capabilities, pros and cons of each platform is critical to drive progress.

Efficient Manufacturing of CAR-T Cells from Whole Blood: A Scalable Approach to Reduce Costs and Enhance Accessibility in Cancer Therapy

BackgroundChimeric antigen receptor T (CAR-T) cells have significantly advanced the treatment of cancers such as leukemia and lymphoma. Traditionally, T cells are collected from patients through leukapheresis, an expensive and potentially invasive process that requires specialized equipment and trained personnel. Although whole blood collections are much more technically straightforward, whole blood starting material has not been widely utilized for clinical CAR-T cell manufacturing, in part due to lack of manufacturing processes designed for use in a good manufacturing practice (GMP) environment. Collecting cellular starting material from whole blood without leukapheresis could reduce manufacturing complexity and cost, thereby improving accessibility to CAR-T cell therapy. MethodsWhole blood samples were collected from eight healthy donors and one pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient. These samples were processed using the Sepax C-Pro (Cytiva) instrument to isolate mononuclear cells (MNCs) via density gradient separation. CAR-T cells were then manufactured from the isolated MNCs using a GMP-compliant 7-day protocol, whereby T cells were activated with anti-CD3 and IL-2, transduced with GMP lentiviral vector encoding a CD19/CD22 bispecific CAR, and expanded in gas permeable cell culture bags. The resulting CAR-T cells were then evaluated for their phenotypic and functional properties using flow cytometry, cytokine release, and cytotoxicity assays. ResultsFrom an average 77.7 mL of whole blood from healthy donors (range = 29 - 96 mL), we isolated an average of 42.2 × 106 CD3⁺ T cells (range 7.3 - 63.0) post processing. CAR-T cell cultures were initiated from thaw using 1 - 10 × 106 starting CD3+ T cells, yielding a median T cell number of 105 × 106 cells on day 7 (range 61 - 188 × 106). We observed 66 ± 11% mean transduction efficiency and produced a mean of 77.4 × 106 transduced CAR-T cells (range 30.8 - 143.5 × 106). Similar results were obtained when using a blood sample (28mL) obtained from a patient with relapsed B-ALL who had received recent chemotherapy. ConclusionsTherapeutically relevant doses of CD19/CD22 CAR-T cells can be successfully manufactured from whole blood. On average, 80 mL of whole blood yields enough CAR-T cells to create a single dose for a pediatric patient (50 kg) at a dosage of 1 × 106 CAR-T cells/kg. For larger patients, scaling up is straightforward by collecting a larger blood volume. This method also demonstrates a cost-effective approach to T cell activation and expansion which, alongside a more straightforward collection of whole blood, makes it more widely accessible especially for middle- and low-income countries. By reducing costs and labor, this strategy has the potential to significantly expand global access to CAR-T cell therapy.

Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand

Anti-CD19 chimeric antigen receptor (CAR)-T cell therapy has evolved as a standard of care for various forms of relapsed/refractory B cell malignancies in major developed countries. However, access to industry-driven CAR-T cell therapy is limited in developing countries, partly due to the centralized manufacturing system. Here, we demonstrated the feasibility of the point-of-care (POC) manufacturing of anti-CD19 CAR-T cells from heavily pretreated patients and healthy graft donors at an academic medical center in Thailand using a closed semi-automated production platform, CliniMACS Prodigy, and established in-process quality control and release testing to ensure their identity, purity, sterility, safety, and potency. Nine out of the nine products manufactured were used in a pilot study (ISRCTN17901467). However, we did observe that starting Tcells with CD4/CD8 ratios of less than one-third had a high chance of manufacturing failure, which could be minimized by serum supplementation. Further analysis of Tcell phenotypes in the infused versus circulating CAR-T cells revealed the differentiation from early memory subtypes toward effector cells invivo. The POC manufacturing and quality control settings herein could be applied to other CAR-T cell products and may benefit other academics, especially those in developing countries, making CAR-T cells more accessible.

Local radiation enhances systemic CAR T-cell efficacy by augmenting antigen crosspresentation and T-cell infiltration

AbstractChimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CART-19) represents a significant advance in the treatment of patients with relapsed or refractory CD19+ B-cell lymphomas. However, a significant portion of patients either relapse or fail to respond. Moreover, many patients have symptomatic disease, requiring bridging radiation therapy (RT) during the period of CAR T-cell manufacturing. To investigate the impact of 1 to 2 fractions of low-dose RT on CART-19 treatment response, we developed a mouse model using A20 lymphoma cells for CART-19 therapy. We found that low-dose fractionated RT had a positive effect on generating abscopal systemic antitumor responses beyond the irradiated site. The combination of RT with CART-19 therapy resulted in additive effects on tumor growth in irradiated masses. Notably, a significant additional increase in antitumor effect was observed in nonirradiated tumors. Mechanistically, our results validate activation of the cyclic guanosine adenosine synthetase/stimulator of interferon genes pathway, tumor-associated antigen crosspriming, and elicitation of epitope spreading. Collectively, our findings suggest that RT may serve as an optimal priming and bridging modality for CAR T-cell therapy, overcoming treatment resistance and improving clinical outcomes in patients with CD19+ hematologic malignancies.

Good manufacturing practice-grade generation of CD19 and CD123-specific CAR-T cells using piggyBac transposon and allogeneic feeder cells in patients diagnosed with B-cell non-Hodgkin lymphoma and acute myeloid leukemia.

The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.

Maximizing process control and efficiency in CAR-T cell manufacturing

Maximizing process control and efficiency in CAR-T cell manufacturing

Nanomaterials Boost CAR-T Therapy for Solid Tumors.

T cell engineering, particularly via chimeric antigen receptor (CAR) modifications for enhancing tumor specificity, has shown efficacy in treating hematologic malignancies. The extension of CAR-T cell therapy to solid tumors, however, is impeded by several challenges: The absence of tumor-specific antigens, antigen heterogeneity, a complex immunosuppressive tumor microenvironment, and physical barriers to cell infiltration. Additionally, limitations in CAR-T cell manufacturing capacity and the high costs associated with these therapies restrict their widespread application. The integration of nanomaterials into CAR-T cell production and application offers a promising avenue to mitigate these challenges. Utilizing nanomaterials in the production of CAR-T cells can decrease product variability and lower production expenses, positively impacting the targeting and persistence of CAR-T cells in treatment and minimizing adverse effects. This review comprehensively evaluates the use of various nanomaterials in the production of CAR-T cells, genetic modification, and in vivo delivery. It discusses their underlying mechanisms and potential for clinical application, with a focus on improving specificity and safety in CAR-T cell therapy.

Advantages of high cell concentration prior to cryopreservation of initial leukapheresis in CAR-T cell therapy.

Chimeric antigen receptor (CAR) T-cell therapy is increasingly used in patients affected by B-cell lymphoma and acute lymphoblastic leukemia. For logistical reasons, initial apheresis products may be cryopreserved for shipment to manufacturing centers. Due to the characteristics of these patients, cells are often collected in large volumes, meaning more bags must be cryopreserved. This requires increased storage, time and monetary costs. In this context, we aimed to evaluate a high cell concentration cryopreservation protocol by centrifugation to standardize the initial CAR-T manufacturing procedure. Sixty-eight processes of leukapheresis of 57 patients affected by refractory/relapsed B cell lymphoma and 9 patients affected by acute lymphoblastic leukemia who were eligible for anti-CD19 CAR-T cell treatment performed between June 2019 and October 2022 were analyzed. Whole blood count, percentage and number of T cells were assessed on the apheresis final product. The apheresis product, which was alternatively stored overnight at 4°C, was centrifuged, adjusting the volume to approximately 40 mL. The product was immediately cryopreserved to achieve a final cell concentration of 50-200×106 cells/ml for cryopreservation. Leukapheresis volume was reduced by almost fivefold (median: 185 to 40 mL), resulting in a higher product concentration in one bag. In addition, the number of non-target cells (monocytes, platelets and erythrocytes) was also reduced during the development of CAR-T cell therapy, thereby maintaining T lymphocyte levels and providing a purer starting material. The advantages of the protocol include reducing economic costs, saving storage space, simplifying the manufacturing process, and facilitating shipping logistics. In conclusion, we present a validated, simple, and cost-effective cell enrichment processing protocol that provides high-quality cryopreserved products as starting material for the CAR-T cell manufacturing process.

Revolutionary Selection, Activation, and Expansion with Microbubbles for CAR-T Cell Manufacturing

Abstract Despite several chimeric antigen receptor-modified T cells (CAR-T) products being approved and commercialized, CAR-T cell therapy has manufacturing challenges. Conventional CAR-T therapies are manufactured by collecting apheresis material from a patient’s blood, then modifying the isolated T cells from the apheresis material to express a cancer cell-specific CAR, eventually infusing the CAR-T cells into a patient. Challenges include early exhaustion of the therapeutic cells, manual/laborious processes, and complex workflows. Akadeum’s innovative microbubble technology streamlines human T cell selection, activation, and expansion into a single step. Here, we demonstrate over 90% capture efficiency while reducing the selection process to under one hour. Because of the microbubbles’ natural buoyancy, T cell activation occurs at the top of the culture. As the T cells divide and the bound receptors are recycled, T cells fall to the bottom of the media, avoiding CD3/CD28 overstimulation. PD-1 expression is reduced throughout expansion compared to traditional activation workflows. T cell activation was not compromised, as evidenced by the upregulation of CD69, CD25, and HLA-DR+. Additionally, microbubble technology can increase the percentage of desired effector T cell populations. As CAR-T therapy expands, innovations like Akadeum's microbubble technology play a crucial role in overcoming manufacturing challenges and ensuring the development of high-quality, effective therapies.

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics

BackgroundChimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity.MethodsThis retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products.ResultsA pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response.ConclusionspH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

In vivo manufacture and manipulation of CAR-T cells for better druggability.

The current CAR-T cell therapy products have been hampered in their druggability due to the personalized preparation required, unclear pharmacokinetic characteristics, and unpredictable adverse reactions. Enabling standardized manufacturing and having clear efficacy and pharmacokinetic characteristics are prerequisites for ensuring the effective practicality of CAR-T cell therapy drugs. This review provides a broad overview of the different approaches for controlling behaviors of CAR-T cells in vivo. The utilization of genetically modified vectors enables in vivo production of CAR-T cells, thereby abbreviating or skipping the lengthy in vitro expansion process. By equipping CAR-T cells with intricately designed control elements, using molecule switches or small-molecule inhibitors, the control of CAR-T cell activity can be achieved. Moreover, the on-off control of CAR-T cell activity would yield potential gains in phenotypic remodeling. These methods provide beneficial references for the future development of safe, controllable, convenient, and suitable for standardized production of CAR-T cell therapy products.

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

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