A new story for an old challenge: Would flow cytometry beat molecular biology in monitoring chimeric antigen receptor T cell pharmacokinetics?

A recently published paper in Cytometry A by Jiali Cheng et al. [1] compared the performances of flow cytometry and PCR in detection of rare chimeric antigen receptor (CAR) T cells. This reminds us of the heavy debates that appeared several years ago on choosing the best method to monitor the efficacy of leukemia treatments by counting residual tumor cells circulating in blood, named minimal residual disease (MRD).

The new challenge addresses the monitoring of CAR T cell therapy. CAR T cells consist of autologous T cells which were genetically modified to kill tumor cells. In brief, the capacity to recognize tumor associated antigen (TAA) is genetically transferred as a T cell receptor substitute to as many T cells as possible. Then, these genetically modified T cells are eventually trained (i.e., using polyclonal activator and interleukines) and selected to get the most efficient cytotoxic capacity before re‐infused back to the patient [2, 3].

The CAR T cell receptor is composed of an antigen binding site, made of a heavy and light chain variable fragment from a monoclonal antibody, linked together as a single‐chain variable fragment (scFv). The scFv part is attached to the membrane receptor by a spaced region derived from IgG4 or CD8 molecules [4]. The intracellular part of the receptor can be the CD3zeta that has the ability to transduce the signal to the nucleus. Additional costimulatory domains can be associated from various costimulatory molecules, such as 4‐1BB (CD137), CD28, OX40 (CD134), or induction T cell stimulator (ICOS or CD278) [4, 5].

After collection from the patient by leukapheresis, the peripheral blood mononuclear cells (PBMC) or magnetically sorted T cells are genetically modified using retrovirus or lentivirus. T cells are then preferentially expanded ex vivo and activated to kill using stimulants like anti‐CD3 antibodies and interleukins like IL2, IL‐7, and IL‐15 [3, 4].

After several days, transfected T cells are reinfused back to the patient who has been conditioned with lymphocyte depletion regimens like Fludarabine, R‐CHOP (Rituximab, Cyclophosphamide, Adriamycin, and Prednisone) or chemotherapy, to essentially destroy the T and B cells [4]. Thus, in the first few days, recipient circulating lymphocytes are mainly CAR T cells with possibly some residual NK cells that could have escaped from lymphodepletion (Figure 1).

FIGURE 1.

Schematic representation of CAR‐T cell fate in peripheral blood accessible for flow cytometry monitoring. In brief, polyclonal lymphocytes are given ex vivo one artificial recognition receptor, are stimulated for expansion and maturation, favoring T cells, and are re‐infused into the patients to migrate to tissues for tumor killing or to lymph nodes for long term memory and treatment sustainability. CAR‐T kinetics have several steps: (1) prior to gene modification normal lymphocytes are present (except in case of depletion of B cells). (2) Prepared CAR‐T ready to be reinfused are in majority CD4+ T and fewer CD8+ T cells, that in great part, express the CAR receptor (). (3) Shortly after re‐infusion, CAR‐T are the majority in peripheral blood, with different effector/homing capacities as well as immunoregulatory co‐receptors. (4) Part are driven to lymph nodes () while (5) others are going to tissues and tumor (). (6) Few weeks later, normal lymphocyte populations are reconstituted while CAR‐T are exhausted, declining and become rare events to detect. Flow cytometry makes possible to monitor CAR‐T number but also homing (, ) and functional capacity like cytotoxicity, at any stage of the treatment [Color figure can be viewed at wileyonlinelibrary.com]

Most CAR T cells are strongly armed to kill tumor cells, and they act rapidly and very efficiently. However, this can lead also to side effects like tumor lysis syndrome (TLS), cytokine release syndrome (CRS) and long‐lasting neurotoxicity syndrome (ICANS) [6]. These effects depend on CAR T efficacy, but also on the quantity of CAR T cells that are reinfused and on the tumor burden.

The most used CAR‐T are targeting B cell homeopathies. Since CAR T cells have shown to have great efficacy in clinical use, their applications have expanded since the initial approval of 2017 toward solid tumors or even sever auto‐immune diseases. Consequently, this raises the question of developing the appropriate evaluation tools to monitor their efficacy.

CAR T cells have an initial expansion phase upon reinfusion and this is improved using low‐affinity CD19 CAR receptor [7]. The proportion of CAR T cells decreases exponentially due to their exhaustion combined with the progressive reconstitution of natural T cell population [8, 9]. A few weeks after reinfusion the number of circulating CAR T cells becomes low but can persist for several months ensuring a prolonged protection again tumor relapse. This is where the detection of residual CAR T cells in peripheral blood raises the same questions as MRD: which technique to choose for the detection, the very precise and rapid flow cytometry or the highly sensitive PCR? These questions were addressed by Jiali Cheng et al. [1] PCR methods detect the presence of transgenic DNA in circulating cells. PCR can detect as low as five copies per μg of DNA (limit of detection, LOD). On the other hand, flow cytometry characterized by its exceptional precision, has reached a very high sensitivity with a LOD below 0.05% of lymphocytes analyzed.

• The first question this paper is raising is: do we really need to detect CAR T cells in peripheral blood after infusion? The answer depends on the underlying rationale of monitoring CAR T cells expansion and/or exhaustion: how this data will be used for treatment efficacy? The final aim of CAR T cell therapy is the elimination of tumor cells; hence it might be more appropriate to measure this parameter.

• The second question is: can we detect efficiently CAR T cells by flow cytometry? This question has been already addressed and several tools have been developed to efficiently enumerate CAR T cells by flow cytometry. These include a generic immunoglobulin binding protein, such as Protein L, that recognizes kappa and lambda light chains, or a recombinant TAA target aimed to be recognized and stably bound by the transgenic CAR T cell receptor. Finally, a very specific antibody recognizing the binding site (anti‐idiotype) is commonly used. Most of CAR T cell applications are targeting the same CD19 molecule; common B cell derived malignancies such as B cell acute leukemia or non‐Hodgkins lymphoma (NHL), specifically diffuse large B cell lymphoma (DLBCL) [4]. This needs to be adapted to monitor new CAR programs using other cell types like NK cells [9, 10] and other tumor types like solid tumors, for which, TAAs are highly variable [11, 12].

Jiali Cheng et al. [1] answered positively this question, which has also been confirmed in previous studies [13, 14]. CAR T cell were efficiently detected by flow cytometry, especially using an anti‐idiotype antibody, with a LOD at 0.03678% of lymphocytes (mean + 3SD in healthy whole blood) with reasonable coefficient of variance below 11%.

• The next question constitutes the main objective of the study: Would flow cytometry compete with droplet digital PCR (dd‐RT PCR) efficiency in detecting rare CAR T cells?

The authors respond positively in this question, as expected in a Cytometry journal. They showed very comparable sensitivities and a reasonable correlation coefficient (r = 0.68 in clinical samples) between the two methods. This shows that flow cytometry is well adapted in monitoring CAR‐T in early phase as well as in rare residual cells in the late stage of the treatment.

However, this work also, raises some questions:

• do we really measure the same parameters, comparing a number of DNA copies, measured by dd‐RT PCR, and a cell count among peripheral blood CD45 bright leukocytes, measured by flow cytometry? Does one CAR DNA copy represent one transfected T cell?

• a technical question is also warranted: the authors state that CAR T cell detection was performed on whole blood unless leukocytes counts were very low; in the latter case samples were lysed and washed prior to staining. Can this pre‐analytical process affect the relative cell count in these two types of preparation?

• Furthermore, flow cytometry analysis in the study uses a ratio of relative number of CAR T cells (numerator) out of total lymphocytes (denominator). It has been shown that CAR T follow a kinetics of initial expansion following by a decrease in numbers [2], whereas the opposite is usually true for normal T cells; the initial numbers are low, due to the lymphodepletion, and subsequently increase due to T cell reconstitution. It will be informative if the authors provide the original absolute T cell counts in their study subjects.

Finally, flow cytometry has an advantage in the short response delay and quantitative values but most of all; it can provide qualitative information on cells. These can help understand the efficacy and functional parameters of immune cells [15]. Transfused cells are mainly T cells, of CD4 as well as CD8 types, but it might worth considering other cytotoxic cells like NK or other ILC that may have been genetically modified and have survived the expansion stage. CD4+ T cells are usually the most frequent in PBMC and apparently the most frequent among transfused CAR [3]. However, it is well known that CD8+ T cell are more efficient than CD4+ T cells in cytotoxicity. So, should not we consider separately CD4+ and CD8+ CAR‐T cells? Furthermore, effector CD8 T cells are known to be more efficient than naïve cells [2]. Flow cytometry has the advantage of looking at specific T cell subsets of the memory phenotype, either central memory or effector memory. Considering the maturation stage of T cells is important in the clinical outcome and relapse of the tumor. Shouldn't we consider separately central versus effector memory CAR‐T cells as suggested by preliminary works [16, 17]?

The advantage of multiparametric flow analysis capacity gives precise results, which allow to report on the heterogeneity of immune cell subsets their effector status and homing capabilities. On this point, we can anticipate that homing to lymph node (such as CD62L or CCR7) used to identify “central memory” may not have the same significance in terms of anti‐tumor efficacy whether the tumor cells reside in the lymph node as it is usual for B cell malignancies or in bone marrow for myeloblastic leukemia or myeloma or in other tissues for solid tumor. Furthermore, expression of co‐stimulatory molecules and check points receptors would certainly be of high interest in in vivo pharmacokinetics, especially since CAR‐T can be associated with check point inhibitor to complete the treatment efficacy [18, 19].

In conclusion, these results indicate that flow cytometry has a great place in monitoring not only CAR T cell number in a rapid and cost‐effective way and possibly even more by also considering CAR cell phenotype, homing and functionality and/or fitness.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/cyto.a.24695.

Lambert C, Ntrivalas E, Sack U, The flow cytometry working group of the international Federation of Clinical Chemistry (IFCC). A new story for an old challenge: Would flow cytometry beat molecular biology in monitoring chimeric antigen receptor T cell pharmacokinetics? Cytometry. 2023;103(1):8–11. 10.1002/cyto.a.24695