Research in cell therapies has grown in the past few years with the FDA approval of Yescarta (axicabtagene ciloleucel) in 2017 for relapsed or refractory large B-cell lymphoma and Kymriah (tisagenlecleucel), also in 2017 for relapsed or refractory acute lymphoblastic leukemia, both of which are autologous Chimeric Antigen Receptor (CAR) T therapies. Currently research is underway for allogeneic ‘off-the-shelf’ CAR-T cells with the cell source being the major difference between the two therapies. While autologous CAR-T therapy utilizes patients’ cells, allogeneic CAR-T therapy takes advantage of the selection of healthy donors from which the cells can be isolated and, after modification, infused to patients from various genetic backgrounds. The main advantage is the availability of already modified cells that do not require the patient to wait for their own cells to be manufactured. Additionally, it allows manufacturers to obtain cells from carefully selected donors.1 The products generated from either of these processes results in a live, constantly changing product that requires sophisticated analytical flow cytometry techniques. Hence, flow cytometry has become an essential component of the CAR-T therapy field and is used for the release assays that are required by the FDA, for exploratory assays to understand the product before and after infusion as well as to track it over time. The analytical strategies used to characterize the autologous and allogeneic CAR-T therapies will be different because of the source of the cells, the development process and the questions that need to be answered. In addition, the attributes may differ based on the disease indication, the target and the Mechanism of Action (MoA) of the product. However, the recommendations for generating flow cytometry-based assays will be the same. Therefore, for the purpose of this review, I will focus only on the autologous platform.
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Release Versus Characterization Assays
The release assays required by FDA for which flow cytometry is essential are; identity of the drug product, its potency, the purity of the drug product and its viability.2,3 These assays may not be more than 5-8 parameter flow cytometry panels because they are designed specifically to answer the questions necessary by FDA for the approval and release of the product and may also be used during process development to understand certain aspects of the process. For example, the identity of the drug product may be determined by a simple flow cytometry panel including markers such as CD45, CD3 (for T cells), CD4 and CD8 along with the Anti-Idiotype to recognize the construct to understand the transduction efficiency of the process. CD4 to CD8 ratios, although not necessary for the release of the product, can serve as an important characteristic of the product.4 Another simple panel may include CD45, CD3 (to identify the T cell product) while making sure that the final product does not contain any B cells, NK cells, Dendritic cells or monocytes impurities. This same panel can be used during the process to understand the efficiency of positive or negative selection of the cells of interest. Cytotoxicity flow cytometry-based assays can also be developed to determine the potency of the product and can be used to compare the drug product to the drug substance because formulation can also affect the phenotype of the cells. While the release assays are necessary, they only give us limited knowledge, a more thorough examination of the product requires high parameter flow cytometry that looks at 20+ parameters on the cells at the same time providing information that helps in determining the critical quality attributes (CQA). The phenotype of the T cells informs whether the T cell product has only effector population or it consists of a combination of effector and memory functions.5 Exhaustion markers are a bit difficult to evaluate but important nonetheless due to the fact that an exhausted product will not be able to persist or perform its function for too long. Once developed, the assays can be used to answer various questions during the process development phase which is the best time to understand the process and the resulting product.
Development of Characterization Assays by Flow Cytometry
Designing and developing the flow cytometry assays is challenging and takes time, about four to six months, but there are some steps that can be a guidance for those attempting to develop one. While the parameters that can be utilized in a cytometer have increased, they are still limited by either the number of channels in a traditional flow cytometer or the number of prototype dyes available for a given marker, and therefore cannot encompass all the information desired for a particular cell product. The custom conjugation of dyes to a marker takes about a minimum of six weeks to develop and is not only costly, but can also affect the antibody performance. Therefore, it is necessary to define the purpose of the assay and identify the source material for which the assay will be designed for. For example, if the purpose is to characterize the CAR-T cells in depth, then the assay should have the markers that are relevant to the T-cell biology and the CAR construct that is driving the MoA of the CAR-T product. Due to the fact that each CAR-T product is unique, the expression pattern of markers is not always known. Hence, once identified, it is always good to test the expression and co-expression of these markers on the product using PE conjugated clones. Various clones can also be tested to determine specificity of the antibody for the marker of interest. During the design of the flow cytometry panel, a gating strategy is equally useful in determining which cells can be excluded or gated in to get to the desired parameter. Some of the basic panel design knowledge still applies to high parameter panel designs, such as pairing bright fluorochrome with low antigen density markers while pairing dim fluorochromes with high antigen density markers. However, this is complicated by the spread caused by the inherent nature of the fluorochromes and their spill over that can be visualized after compensation has been applied. Therefore, it is important to have knowledge of the instrument and the fluorochrome spreading matrix to avoid using fluorochromes that have a high spread for markers that are co-expressed on the same cell, for example CD3 and CD4 or CD8 to identify T cells. Additionally, this strategy should also be applied to markers that come early in the gating hierarchy versus those that come later to prevent the spread from carrying down the hierarchy. The panel design can also be affected by the ways the data is expected to be reported. The data in flow cytometry is either reported as percentage of a population or the median fluorescence intensity of that population. It is important to decide on these parameters in advance of panel development.
Antibodies used to identify various populations or expressions are considered critical reagents in flow cytometry and must be titrated to optimal concentrations using a stain index. In high parameter flow, one marker can drastically affect the resolution of another, especially when expressed on the same cell and proper titration can resolve this issue. Another critical reagent in a CAR-T product panel is the anti-idiotype. The generation of an anti-idiotype takes a long time, therefore scientists have used protein-L as a surrogate. Protein-L binds to the kappa light chain of the CAR.6 However, due to high background binding seen in many processes as well as the fact that it interacts with other antibodies within the panel,7 it is ideal to have more specific detection methods such as the FMC63 clone for the detection of CD19-specific CAR-T cells.8 Appropriate controls in an experiment are necessary to differentiate the positive signal from the negative one, they are equally important to highlight any problems with the design of the panel. Isotype versus Florescence Minus One (FMO) control has been a debate for a long time and if it has not been resolved in a particular lab then it is recommended to test FMO controls against the isotype controls. In general, FMOs are preferred because they not only encompass the auto fluorescent background of the cells, but also give the fluorescence spill over coming from other channels into the channel of interest, therefore highlighting the problems within a panel. Isotypes, if and only if, have the same protein to fluorochrome conjugation ratio as the antibody itself can then be used. Each antibody should be titrated and a stain index should be determined to identify the best amount of antibody to stain the cells with. When acquiring the stained samples on the cytometer, it is important to adjust Photo Multiplier Tubes (PMTs) by maximizing the resolution between the positive and the negative and keeping the signal within a linear range of the detector. This practice allows for optimal panel setup on the instrument. As a rule of thumb, the total number of events acquired in a sample can depend on enough events to confidently identify the rarest population.
Development of any flow cytometry assay is usually performed on healthy donors leaving a gap of knowledge on how the panel will perform on patient cells. After development, the assay should be tested on at least three different patients, if not more, to account for patient-to-patient variability and to gain confidence in the assay. During testing phase and especially while testing patient samples, it is important to have a reference control. Depending on the purpose of the assay, the reference control can either be various lots of CAR-T cells or starting material. The same lot of cells that were used to develop the assay can also be a good reference control granted that all the markers within the panel were expressed on the cells. One of the main challenges in using a reference control is its availability for the course of the assay development and testing. However, since it is difficult to have a continued supply of assay appropriate reference control, we have tested lyophilized cells as a possible surrogate for reference control and have not been successful in that attempt so far.
Validation of Flow Cytometry Assays
Variability in flow cytometry data can come from reagents, the way the sample was prepared, instrument set-up, the operator as well as analysis. While a great deal of effort is spent in ensuring that the reagent lots are the same for the duration of the study, the sample preparation as well as staining was followed using a Standard Operating Procedure (SOP), the instrument was maintained in the same manner as it was during the development of the assay, and the analysis is standardized, the assay still needs to be validated. A thorough validation of the flow cytometry-based assays for regulatory agencies is a must and needs evaluation of parameters starting from instrument set-up, the analytical method itself and the analysis. The instrument set-up and maintenance needs to be performed on a daily basis, while the analytical method should be carefully planned out to include specificity, precision, sensitivity, robustness and stability. The data should be stored in a secured place and the gating strategy for the analysis plan needs to be determined.7,9,10 However, this thorough validation is impossible to implement for high parameter flow cytometry assays. The validation for 20+ parameters can be performed for a-fit-for-purpose and can include intra-assay and inter-assay variation along with inter-scientist variability, especially if more than one person maybe performing the same assay on patient samples.
Flow cytometry-based assays, when designed properly, can give a wealth of information about the cell product. The investment in developing these assays is critical at an early stage so that correlations can be made between processes and products manufactured. Well informed process changes can affect the manufactured product’s MoA, its infiltration into the tumor micro-environment and response of the patients to the drug.
References
- Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov. 2020;19(3):185-199.
- Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs). January 2020.
- Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products. January 2011.
- Turtle CJ, Hanafi LA, Berger C, et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest. 2016;126(6):2123-2138.
- Garfall AL, Dancy EK, Cohen AD, et al. T-cell phenotypes associated with effective CAR T-cell therapy in postinduction vs relapsed multiple myeloma. Blood Adv. 2019;3(19):2812- 2815.
- Zheng Z, Chinnasamy N, Morgan RA. Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry. J Transl Med. 2012;10:29.
- Sarikonda G, Mathieu M, Natalia M, et al. Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses. Cytometry B Clin Cytom. 2020.
- Jena B, Maiti S, Huls H, et al. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials. PLoS One. 2013;8(3):e57838.
- der Strate BV, Longdin R, Geerlings M, et al. Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum. Bioanalysis. 2017;9(16):1253-1264.
- Selliah N, Eck S, Green C, et al. Flow Cytometry Method Validation Protocols. Curr Protoc Cytom. 2019;87(1):e53.