Abstract
Background aims:
The successful development of CD19-targeted chimeric antigen receptor (CAR) T-cell therapies has led to an exponential increase in the number of patients recieving treatment and the advancement of novel CAR T products. Therefore, there is a strong need to develop streamlined platforms that allow rapid, cost-effective, and accurate measurement of the key characteristics of CAR T cells during manufacturing (i.e., cell number, cell size, viability, and basic phenotype).
Methods:
In this study, we compared the novel benchtop cell analyzer Moxi GO II (ORFLO Technologies), which enables simultaneous evaluation of all the aforementioned parameters, with current gold standards in the field: the Multisizer Coulter Counter (cell counter) and the BD LSRFortessa (flow cytometer).
Results:
Our results demonstrated that the Moxi GO II can accurately measure cell number and cell size (i.e., cell volume) while simultaneously assessing simple two-color flow cytometry parameters, such as CAR T-cell viability and CD4 or CAR expression.
Conclusions:
These measurements are comparable with those of gold standard instruments, demonstrating that the Moxi GO II is a promising platform for quickly monitoring CAR T-cell growth and phenotype in research-grade and clinical samples.
Keywords: CAR T-cell manufacturing, cellular Immunotherapies, flow cytometry
Introduction
Chimeric antigen receptor (CAR) T-cell immunotherapy has revolutionized the field of medicine, as evidenced by the Food and Drug Administration approval of six products for B-cell leukemias, B-cell lymphomas and multiple myeloma [1–10]. This therapy is based on the genetic engineering of patients’ T cells with the expression of a CAR capable of redirecting them specifically against antigen-positive cancer cells. As the field of adaptive immunotherapy continues to evolve, thousands of patients are being treated, and novel CAR T products are being developed, necessitating continuous advancements in monitoring and characterizing CAR T cells during the manufacturing process. Moreover, several studies have demonstrated that response to CAR T-cell therapy depends heavily on the quality of manufacturing and the subsequent fitness of the final infusion product, emphasizing that the robust production of a patient’s CAR T cells is crucial for their positive clinical response [11–13].
CAR T-cell manufacturing involves a series of steps, beginning with isolating patients’ white blood cells through leukapheresis. From the leukapheresis, T lymphocytes are purified, activated via anti-CD3 (+/− anti-CD28) stimulation, and transduced with the CAR transgene using a lentivirus or gamma retrovirus. CAR T cells are then expanded over several days and cryopreserved for future use. During CAR T-cell manufacturing, it is essential to have accurate measurements of several metrics that are critical to assessing the success of manufacturing and releasing the product for clinical use. Among them, cell concentration, cell size, cell viability, percent-age of CD3+ cells from leukapheresis, proportion of of CD4+ and CD8+ cells, and CAR expression (i.e., transduction efficiency) are essential not only from a purely scientific perspective but also as a regulatory requirement [14]. Cell concentration is needed to monitor cell growth and determine the need for additional medium [15]. Knowledge of cell size is also critical, as it mirrors the activation status of the CAR T cells. T-cell size dramatically increases upon activation, and progressively returns closer to a resting state at cryopreservation, depending on the duration of manufacturing [16]. In addition, viability measurement is critical throughout the process to monitor the T cells’ health and growth during the different manufacturing steps. Finally, the percentage of CD3+ cells in the leukapheresis and the CAR transduction efficiency in the final product are traditionally monitored by flow cytometry and are critical factors for quality control [17]. In short, ensuring accurate and reproducible determination of these four criteria is crucial for the generation of a robust CAR T-cell infusion product [14,15,17].
Several commercial instruments can be used to evaluate these characteristics during CAR T-cell manufacturing. One such standard instrument is the Multisizer Coulter Counter (Beckman Coulter, Brea, CA, USA), or MS, which is used to evaluate cell concentration and cell size [18]. This instrument takes advantage of the Coulter principle, an electrical approach to determining the size of particles by measuring changes in electrical impedance caused by particles passing through an orifice in which constant current is being applied. The amplitude of the voltage change can be directly related to the size of the particle, thus enabling the measurement of cell size [19]. In addition, monitoring the frequency of these voltage pulses provides a means of counting the number of particles. Separate metering of the fluid is then applied to convert the pulse event counts into particle concentrations. However, the limitation of the MS is that it cannot measure the viability or phenotype of CAR T cells. For these measurements, flow cytometry (e.g., the BD LSRFortessa [LSR]; BD Biosciences, San Diego, CA, USA) is typically used separately to measure viability, CAR expression, and T-cell markers [20]. Recently, the Moxi GO II (ORFLO Technologies, Ketchum, ID, USA) (MGII) was developed to simultaneously assess cell concentration, cell size, cell viability, and two-color flow cytometry [21]. The MGII incorporates both the Coulter principle and features of conventional flow cytometry. Despite the MGII’s compact benchtop design, the use of polyfunctional tools raises concerns about the accuracy and reliability of each measurement taken. This methods article compares the ability of the MGII to monitor the critical measurements of CAR T cells during manufacturing with the reference standards of the MS and LSR.
Methods
Lentiviral vector production and titration
The detailed protocol for lentiviral vector production and titration is described in supplementary Materials.
CAR T-cell manufacturing
The detailed protocol for CAR T-cell manufacturing is available in the supplementary Materials [22].
Instruments used in the study
The technical characteristics of MGII, MS, LSR, and general flow cytometry protocols are described in supplementary Materials and Supplementary Figure 1.
Protocol to measure cell number, size, viability, and two-color phenotype using the MGII platform
For the MGII, cell count and volumetric size were determined by using the system’s “Cell Counts (Size +Viability)” assay with Type S+ cassettes (cat. no. MXC032; ORFLO Technologies) (Supplementary Figure 1). Cell samples were initially diluted into a working range for both the MS and MGII instruments. The MGII samples were then further processed from that initial dilution using the MoxiCyte Viability reagent (cat. no. MXA055; ORFLO Technologies), adhering to the manufacturer’s standard 10× dilution protocol of 15 μL of cell sample mixed into 135 μL of MoxiCyte Reagent (2.25 μg/mL propidium iodide [PI]). Samples were run on the system using the standard protocol of selecting the assay and loading 60 μL of cells into the micro-fluidics chip when prompted. Measurements were extracted using the on-system software. For CAR T-cell phenotype analysis, samples were diluted according to MGII protocol, then the 561-nm/LP band-pass filter was used for the second fluorescence channel and the Open Flow Cytometry system assay was selected to record the data. The X-axis was set to size (diameter), and the Y-axis to PMT2 (long pass, PE channel). All tests were performed using the Type S+ (MXC032, ORFLO) cassettes.
Protocol to measure cell number and size with the Beckman MS
The cell counts and volumetric size were gathered using Beckman Coulter MS. The cells were first collected and then transferred into individual cuvette ST vials containing ISOTON II diluent. Samples were loaded into the MS one at a time, and cell concentration was based on volumetric sampling (500 μL) under the following conditions: 100-μm aperture, 1600-μA current and a gain setting of two. Before running samples, the MS was unblocked, and “Start” was initiated to run the machine. The MS data were collected using Beckman Coulter particle characterization software using the “Statistics” function. Histogram gating was applied to all samples to have consistent, unbiased gating across samples.
CD4 expression evaluation
Serial dilution starting from a pure CD8+ T-cell sample was used to reach concentrations ranging from 100% down to 10% of CD4+ T cells in 10 different samples. Those samples were then run on the MGII or LSR using Anti-CD4 (RPA-T4: FITC, cat. no. 300505; BioLegend, San Diego, CA, USA). Results were compared using pairwise comparison.
Low viability assay
To reduce the viability of CAR T samples, we exposed them to 56°C for 1 hour. We then mixed dead cells with viable CAR T in six different samples to reach a final viability of 0% up to 50%. Cells were sub-sequently stained with PI, and viability was evaluated using MGII or LSR. One donor with two biological replicates was used. Interrogation of viability on the Yellow/Green channel was measured as previously stated. Results were compared using pairwise comparison.
CAR19 expression in research-grade samples
From lentivirus titration, we performed serial dilutions as previously stated and incubated for 72 hours with T cells. Samples were stained using CAR19 anti-idiotype antibody and incubated under no light for 20 minutes. After the staining, samples were washed with 2% fluorescence-activated cell sorting twice and resuspended. Samples for the LSR were run directly from the resuspended medium. Samples for the MGII were washed two more times to prevent oversaturation on the instrument and resuspended on fluorescence-activated cell sorting. PE fluorescence was interrogated with both flow cytometry instruments.
Clinical specimens
We collected leukapheresis and infusion products from consented patients treated with commercial CART19 products at the University of Pennsylvania (institutional review board no. 831474). Aliquots of infusion products were obtained by rinsing the patient infusion bag using a cold phosphorus-buffered solution (phosphate-buffered saline 1×). The washed cells were then resuspended in freezing media (90% fetal bovine serum and 10% dimethyl sulfoxide) and cryopreserved. Before the experiments, samples were thawed in R10 media and rested overnight. Cell counts were performed according to the described protocols. Leukapheresis samples were stained for CD3 (OKT-3; fluorescein isothiocyanate [FITC]; cat. no. 317305; BioLegend) and 7-aminoactinomycin D (cat. no. 420403; BioLegend) and run on MGII and LSR. Infusion products were stained for CAR19 anti-idiotype antibody (phycoerythrin [PE], provided by Novartis, Basel Switzerland). Results were compared using pairwise comparison.
Statistical analysis
Fluorescence plots of viability (PI fluorescence or 7-aminoactinomycin D versus cell size), FITC-CD4/CD3 expression (FITC fluorescence versus cell size) and CAR19 expression (PE fluorescence versus cell size) were generated using FlowJo, version 10 (FlowJo, Ashland, OR, USA). GraphPad Prism, version 9.5 (Dotmatics; Boston, MA, USA) and Igor Pro (WaveMetrics, Bend, OR, USA) were used to analyze the data and generate all the related plots. For matched comparisons, pairwise t-tests were used. For all tests, the alpha error was set to 0.05. The latest MGII system firmware v2.12 was used in this study. BioRender.com was used to draw out schematics.
Results
Measurement of CAR T-cell concentration and size during manufacturing
Initial CD4+ and CD8+ T-cell numbers, Cell size and CAR T cell expansion are critical parameters to monitor during CAR T manufacturing. Therefore, we first investigated the reliability of the MGII in measuring these parameters during CAR T manufacturing (the schematic of manufacturing is shown in Figure 1a).
Figure 1.
Comparison of size and population doubling measurement using MGII versus MS. (a) CAR T-cell expansion timeline and measurement time course. (b) CD4+ T cells and CD8 + T cells count before and after combining them in the 1:1 mix before starting the CAR T expansion (n = 2). (c) Comparing population doubling with MGII versus MS during CAR T-cell manufacturing for two different donors in a repeated experiment (P = 0.66 and P = 0.81, respectively). (d) Comparing cell size measurement with MGII and MS during CAR T-cell manufacturing for two different donors in repeated experiments (P = 0.48 and P = 0.50, respectively). Groups 1—3 are repeated experiments with 4–1BB—based CAR19 construct mentioned in the supplementary Methods section. ns = P > 0.05.
As shown in Figure 1b, MGII and MS gave comparable results in measuring CD4+ and CD8+ T cell used to generate the 1:1 CD4+:CD8+ mix to begin CAR T manufacturing (n = 2). After the debeading at day six, repeated measurements of cell growth and size were performed to assess the expansion of the CAR T cells and to determine the correct size to freeze the final product (~300 fL). We first measured cell growth in terms of population doubling, using the MGII and MS, during the expansion for two distinct healthy donors (ND1 and ND2) and three groups of CART (4–1BB-based CAR19 construct). As shown in Figure 1c, there was no difference between the two instruments (ns, P = 0.66 for ND1 and ns, P = 0.81 for ND2, with P > 0.05 for every matched comparison).
We then measured median cell size, using the MGII and MS in the same independent experiments and found no difference, based on the instrument used, both in the global evaluation and in all the matched comparisons (ns, P = 0.48 ND1, ns, P = 0.50 ND2) as shown in Figure 1d.
Finally, we sought to analyze cell size and growth before debeading, i.e., in the early phases of activation. As expected, cell size increased early after T-cell activation, but overall cell numbers remained stable or decreased early on (Supplementary Figure 2). Also, in this setting, the two instruments were comparable (P > 0.05 for matched comparisons for both population doubling and cell size), although a trend toward higher median cell size was present using MGII, probably due to difficulties in discriminating single cells and doublets in this more heterogeneous population.
Measurement of CAR T-cell viability during manufacturing
Another critical parameter for CAR T culture is cell viability. Therefore, we decided to compare the assessment of this parameter using the MGII or LSR on the same samples. Supplementary Figure 3 shows a qualitative comparison of the output for the MGII system (left) and for the LSR (right). There was no statistical difference in the paired analysis of quantitative repeated evaluation of CAR T-cell viability, with the MGII (blue) and LSR (red), (ns, P = 0.81) as highlighted in Figure 2a. We also studied viability over time at eight different time points post—T-cell activation (CART manufacturing), measured with the MGII (blue) or LSR (red) (ND2) (Figure 2b). In both data sets, viability increased with time, reaching values approaching 100% at the end of manufacturing. Moreover, there was no difference between the measurements with the two instruments (P = 0.87). Highlighted by the shadow area is the 95% confidence interval, which overlaps for every time point. Finally, we demonstrated a close relationship between the MGII and LSR platforms in repeated experiments, as shown by the linear regression (r2 = 0.97 and P < 0.0001) (Figure 2c). Of note, during the expansion, the viability of the cells was constantly above 60 percent; for that reason, we decided to investigate the abilities of the two instruments to detect and quantify less viable populations. We diluted viable CAR T with dead T cells to reach scalable values from 50% to 0% (Figure 2d). Even in this stress test, the two measurements were comparable at pairwise statistics (P > 0.05) with a clear linear relation between the two values (r2 = 0.91, P = 0.001), highlighting the reliability of MGII in evaluating this critical parameter (Figure 2e).
Figure 2.
Viability measurements during manufacturing using MGII and LSR. (a) Paired measurement of viability by MGII and LSR from D6 (debeading day) to the CART rest down (cell size <300 fl, day 13—23) (P = 0.81). (b) Viability over time during expansion by MGII or LSR. The transparency area represents the confidence interval of 95%. Results from one representative donor using eight time points. (c) Linear correlation of the MGII and LSR viability results at multiple time points (r2 = 0.97 and P < 0.0001). (d) Viability comparison for serial dilution from 0% up to 50% (ns). (e) Linear correlation of the MGI and LSR for viability between 0 and 50% (r2 = 0.91 and P = 0.001). ns = P > 0.05.
Two-color phenotyping of T cells in leukapheresis and infusion products in research and clinical-grade products
One of the advantages of the MGII platform is the possibility not only to measure cell size, cell numbers and viability but also to perform basic two-color flow cytometry. To assess this capability, we compared MGII with LSR in defining specific cell subsets in clinical and research-grade samples. CAR T production requires an adequate leukapheresis collection. The Food and Drug Administration—approved CAR T therapies require that leukapheresis have adequate mononucleate cell numbers and CD3+ T cells [23]. For this reason, we investigated the reliability of MGII in measuring these parameters in patient leukapheretic samples. As shown in Figure 3a total CD3+ T cell percentage, viability, and mononucleate cell counts (Supplementary Figure 4 illustrates the gating strategies used for the analysis of CD3+ T cells and viability in MGII and LSR) are globally comparable using the MGII or the gold standard MS and LSR (for phenotyping and cell counts, respectively, P > 0.05 for paired analysis). However, minor differences can be detected in the single comparisons, suggesting some variability. Once leukapheresis is collected, the CD4+:CD8+ T-cell ratio can affect CAR T manufacturing and subsequent clinical efficacy [24]. Therefore, we compared the ability of MGII to identify CD4+ T-cell populations in samples of known concentrations of CD4+ T cells (black), with the gold standard LSR (gating strategies for MGII and LSR are shown in Supplementary Figure 5). The overall pairwise comparison showed no significant differences (ns, P = 0.42) between the MGII and LSR values (Figure 3b). The scatter plot comparison (Figure 3c) demonstrates a high correlation in the data (r2 = 0.997) as well. The CAR T percentage (percent of T cells that express the CAR) in the final cellular product is a crucial factor for CAR T cytotoxicity in in vitro and in vivo models. Therefore, we investigated whether the MGII could accurately quantify CAR expression in research and clinical grade CART samples (gating strategies for CAR19+ detection is shown in Figure 3d). We first measured CAR T percentage during repeated titration experiments using PE anti-CAR19 immunolabeling. The two instruments showed no statistical difference for the measured CAR+ populations (Figure 3e, ns, P = 0.30). The scatter plot also demonstrates the linear relationship between the two measured sets (Figure 3f, r2 = 0.90). We finally, as a proof-of-concept, evaluated the CAR19 expression in CART19 products used from patients’ treatment (n = 3), showing again comparable results, even if LSR was more sensitive for low-level of CAR+ cells (P > 0.05, Supplementary Figure 6).
Figure 3.
Simple T-cell phenotyping and cell counts in research-grade and clinical-grade leukapheresis and CART19 infusion products. (a) Pairwise comparison of CD3+ T-cell percentage, viability and total counts using MGII versus LSR and MS, for phenotyping and cell counts, respectively (P > 0.05 for all the comparisons). (b) Pairwise comparison of CD4+ T-cell percentage at different dilutions MGII and LSR with one representative donor (P = 0.42). (c) Linear correlation of CD4+ T-cell measurement with MGII and LSR (r2 = 0.997). (d) Dot plot of CART19 samples using MGII or LSR stained with anti-CAR19 antibody. (e) Pairwise comparison of CAR19 percent expression in MGII and LSR. Five healthy donors with six serial dilutions (P = 0.30). (f) Linear regression of MGII and LSR. The dotted line represents the confidence interval of 95% (r2 = 0.90). ns = P > 0.05.
Discussion and Conclusions
The application of CAR T cells is rapidly expanding to several clinical settings, from solid cancers to autoimmune diseases. An increasing number of laboratories and organizations are working to improve the efficacy of this treatment. Instruments that can reduce the hands-on work without reducing the accuracy of the measurements could represent the next breakthrough in laboratory operations. This article compares, for the first time, the measurement of cellular size, cell growth, viability, and T-cell marker expression using the polyfunctional MGII with the reference standards MS and LSR for cell number/size and phenotype, respectively.
Manufacturing engineered immune cells requires repeated measures of cell growth, size, CAR expression, and cell phenotypes to establish the optimal expansion settings and the ideal time to harvest the product. Both the MGII and MS measurements of cell numbers and size are based on the Coulter principle [19]. In our study, we showed that quantitative measurements of cell size and number, using MGII and MS, are comparable. These findings suggest that the MGII could be a valid alternative to the gold standard MS during routine CAR T expansion in a research-grade setting.
Viability and T-cell subsets are other critical parameters during CART expansion. Standard flow cytometers, like the LSR, measure the emission of several different fluorophores at the same time, allowing accurate evaluation of different markers. This deeper characterization level comes with greater costs in terms of analysis time, training time, and instruments-related expenses. Conversely, the compact and ready-to-use MGII is not only equipped with a quantitative Coulter principle—based volumetric sizing, but it also incorporates a blue (488 nm) laser and photomultiplier tube detection of fluorescence emission to allow for simultaneous electrical (Coulter principle) and fluorescence (flow cytometry) measurements of up two parameters. We showed that MGII performance is overall comparable with the LSR; however, some differences are present in point-to-point comparisons. This is not surprising, given the much greater resolution and sensitivity of the LSR compared with the MGII. Our results confirm the need for LSR or a similar flow cytometer when complex and precise phenotyping is required. Despite this limitation, we proved that the MGII is useful for rapid check of CAR percentages and critical T cell markers in both research and clinical-grade samples.
In conclusion, we showed that the MGII is a useful instrument for the rapid and streamlined measurement of cell counts, size, viability and simple phenotyping. As expected, the MGII performance in terms of flow cytometry is reduced compared with bigger and more sophisticated flow cytometers. Given its benchtop nature and the relative ease of use, its results are useful to assess CART manufacturing. Further studies are needed to fully assess whether such an instrument can be used for release criteria or potency assays in the clinical setting.
Supplementary Material
Acknowledgments
The authors acknowledge the Human Immunology Core at UPenn for the selection of CD4+ and CD8+ normal donor T cells. They also thank the patients and families.
Funding
This work was supported by the Laffey McHugh Foundation (to MR, no grant number) and the Berman and Maguire Funds for Lymphoma Research at Penn (to MR, no grant number).
Footnotes
Supplementary materials
Supplementary material associated with this article can be found in the online version at doi: 10.1016/j.jcyt.2024.01.007.
Declaration of Competing Interest
MR holds patents related to CAR T cells that are licensed to Novartis, Tmunity (Kite) and viTToria biotherapeutics. MR has served as a consultant for nanoString, BMS, GSK, GLG, GuidePoint, Sana, Bayer and AbClon. MR receives research funding from AbClon, Beckman Coulter, Oxford Nano Imaging, Curiox and viTToria biotherapeutics. MR is the scientific founder of viTToria biotherapeutics. The Ruella laboratory was provided a Moxi GO II device and supplies to perform this study. GD is an employee of ORFLO Technologies. All other authors declare no competing interests.
References
- [1].Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, Braunschweig I, Oluwole OO, Siddiqi T, Lin Y, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377(26):2531–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [2].Locke FL, Neelapu SS, Bartlett NL, Siddiqi T, Chavez JC, Hosing CM, Ghobadi A, Budde LE, Bot A, Rossi JM, et al. Phase 1 results of ZUMA-1: a multicenter study of KTE-C19 anti-CD19 CAR T cell therapy in refractory aggressive lymphoma. Mol Ther 2017;25(1):285–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [3].Schuster SJ, Bishop MR, Tam CS, Waller EK, Borchmann P, McGuirk JP, Jäger U, Jaglowski S, Andreadis C, Westin JR, et al. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma. N Engl J Med 2019;380(1):45–56. [DOI] [PubMed] [Google Scholar]
- [4].Munshi NC, Hege K, San-Miguel J. Idecabtagene vicleucel in relapsed myeloma. Reply. N Engl J Med 2021;384(24):2357–8. [DOI] [PubMed] [Google Scholar]
- [5].Schuster SJ, Svoboda J, Chong EA, Nasta SD, Mato AR, Anak O, Brogdon JL, Pruteanu-Malinici I, Bhoj V, Landsburg D, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377(26):2545–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [6].Berdeja JG, Madduri D, Usmani SZ, Jakubowiak A, Agha M, Cohen AD, Stewart AK, Hari P, Htut M, Lesokhin A, et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study. Lancet 2021;398(10297):314–24. [DOI] [PubMed] [Google Scholar]
- [7].Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, Bader P, Verneris MR, Stefanski HE, Myers GD, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378(5):439–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [8].Park JH, Riviere I, Gonen M, Wang X, Senechal B, Curran KJ, Sauter C, Wang Y, Santomasso B, Mead E, et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med 2018;378(5):449–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [9].Jacobson CA, Chavez JC, Sehgal AR, William BM, Munoz J, Salles G, Munshi PN, Casulo C, Maloney DG, de Vos S, et al. Axicabtagene ciloleucel in relapsed or refractory indolent non-Hodgkin lymphoma (ZUMA-5): a single-arm, multi-centre, phase 2 trial. Lancet Oncol 2022;23(1):91–103. [DOI] [PubMed] [Google Scholar]
- [10].Wang M, Munoz J, Goy A, Locke FL, Jacobson CA, Hill BT, Timmerman JM, Holmes H, Jaglowski S, Flinn IW, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382(14):1331–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [11].Watanabe N, Mo F, McKenna MK. Impact of manufacturing procedures on CAR T cell functionality. Front Immunol 2022;13:876339. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [12].Blache U, Popp G, Dunkel A, Koehl U, Fricke S. Potential solutions for manufacture of CAR T cells in cancer immunotherapy. Nat Commun 2022;13(1):5225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [13].Ruella M, Korell F, Porazzi P, Maus MV. Mechanisms of resistance to chimeric antigen receptor-T cells in haematological malignancies. Nat Rev Drug Discov 2023;22(12):976–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].Wang X, Riviere I. Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol Ther Oncolytics 2016;3:16015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [15].Milone MC, Xu J, Chen SJ, Collins MA, Zhou J, Powell DJ, Jr, Melenhorst JJ. Engineering enhanced CAR T-cells for improved cancer therapy. Nat Cancer 2021;2 (8):780–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [16].Poulton TA, Gallagher A, Potts RC, Beck JS. Changes in activation markers and cell membrane receptors on human peripheral blood T lymphocytes during cell cycle progression after PHA stimulation. Immunology 1988;64(3):419–25. [PMC free article] [PubMed] [Google Scholar]
- [17].Vormittag P, Gunn R, Ghorashian S, Veraitch FS. A guide to manufacturing CAR T cell therapies. Curr Opin Biotechnol 2018;53:164–81. [DOI] [PubMed] [Google Scholar]
- [18].Beckman Coulter. Multisizer Coulter Counter. <https://www.beckman.com/cell-counters-and-analyzer/multisizer> [accessed 01.07.23].
- [19].Graham MD. The Coulter principle: a history. Cytometry A 2022;101(1):8–11. [DOI] [PubMed] [Google Scholar]
- [20].BD Biosciences. LSRFortessa. <https://www.bdbiosciences.com/content/dam/bdb/marketingdocuments/bd_lsr_fortessax20_techspecs.pdf> [accessed 01.07.23].
- [21].Orflo. Moxi GO II. <https://www.orflo.com/moxi-go-ii/> [accessed 01.07.23].
- [22].Lee YG, Guruprasad P, Ghilardi G, Pajarillo R, Sauter CT, Patel R, Ballard HJ, Hong SJ, Chun I, Yang N, et al. Modulation of BCL-2 in both T cells and tumor cells to enhance chimeric antigen receptor T-cell immunotherapy against cancer. Cancer Discov 2022;12(10):2372–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [23].Qayed M, McGuirk JP, Myers GD, Parameswaran V, Waller EK, Holman P, Rodrigues M, Clough LF, Willert J. Leukapheresis guidance and best practices for optimal chimeric antigen receptor T-cell manufacturing. Cytotherapy 2022;24 (9):869–78. [DOI] [PubMed] [Google Scholar]
- [24].Turtle CJ, Hanafi LA, Berger C, Gooley TA, Cherian S, Hudecek M, Sommer-meyer D, Melville K, Pender B, Budiarto TM, et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest 2016;126(6):2123–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.