Emerging Biomarkers for Monitoring Chimeric Antigen Receptor T-Cell Therapy

Abstract

BACKGROUND

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized treatment of hematologic malignancies and holds promise for solid tumors. While responses to CAR T-cell therapy have surpassed other available options for patients with refractory malignancies, not all patients respond the same way. The reason for this variability is not currently understood. Therefore, there is a strong need to identify characteristics of patients as well as cellular products that lead to an effective response to CAR T-cell therapy.

CONTENT

In this review, we discuss potential biomarkers that may predict clinical outcomes of CAR T-cell therapy. Based on correlative findings from clinical trials of both commercially available and early-phase products, we classify biomarkers into categories of pre- and post-infusion as well as patient and product-related markers. Among the biomarkers that have been explored, measures of disease burden both pre- and post-infusion, as well as CAR T-cell persistence post-infusion, are repeatedly identified as predictors of disease response. Higher proportions of early memory T cells at infusion appear to be favorable, and tracking T-cell subsets throughout treatment will likely be critical.

SUMMARY

There are a growing number of promising biomarkers of CAR T-cell efficacy described in the research setting, however, none of these have been validated for clinical use. Some potentially important predictors of response may be difficult to obtain routinely under the current CAR T-cell therapy workflow. A collaborative approach is needed to select biomarkers that can be validated in large cohorts and incorporated into clinical practice.

Introduction

Chimeric antigen receptor (CAR) T-cell therapy has transformed treatment options for relapsed hematologic malignancies, and efforts are underway to modify strategies for solid tumors and even some autoimmune diseases such as multiple sclerosis (1–3). As of June 2023, there are 4 FDA-approved CAR T-cell products for leukemia or lymphoma, and 2 for multiple myeloma (fda.gov). There are >100 active CAR T-cell therapy clinical trials listed in the United States, with many more trials running internationally (clinicaltrials.gov). Advances in manufacturing methods, such as the introduction of closed-system manufacturing, have led to a more automated and faster process with potential for large-scale or point-of-care production (4).

Despite the success of these therapies, outcomes remain inconsistent, with reported response rates around 60% among patients with acute lymphoblastic leukemia (ALL) (5, 6), 28%–65% among those with lymphoma (7–9), and 72%–97% for patients with multiple myeloma (10, 11). Furthermore, even with excellent initial responses in some products, relapse rates remain substantial, with median progression-free survival ranging from 3 to 24 months in these trials (5–11). CAR T-cell production and treatment remains a time- and resource-intensive process. Typically, manufacturing takes 7 to 10 days and costs up to US$500 000 per patient (12). Patients undergo conditioning chemotherapy before infusion and frequently have prolonged hospital admissions after infusion for monitoring and treatment of toxicities. During the time that CAR T cells are being produced, infused, and monitored, patients cannot pursue other therapy options. Given the rise of numerous novel cellular therapies and the commitment required not only from the institutions to produce CAR T cells but also from patients to undergo this lengthy and risky process, it is imperative to identify biomarkers that can predict short- and long-term disease response to CAR T-cell therapy.

Biomarkers are measurable characteristics that can be used as an indicator of normal biologic processes, pathogenic processes, or response to intervention (13). Potential biomarkers identified in small cohorts need to be validated and tested prospectively prior to being incorporated into practice. In 2020, the American Society of Hematology Taskforce for Immunotherapies published recommendations for development of biomarkers for immunotherapies such as CAR T cells (14). They emphasized the need for large-scale, centralized sample collection to identify and validate assays that will be informative biomarkers of CAR T efficacy.

The goal of this review is to summarize the findings of studies that have identified potential biomarkers predictive of response to CAR T-cell therapy. We will also discuss the feasibility and potential value of incorporating these methods into routine clinical CAR T-cell protocols.

CAR T-Cell Manufacturing

A CAR is composed of a targeting element that binds to tumor antigens (a single chain variable fragment, scFv), a spacer/hinge, a transmembrane domain, and an intracellular portion containing costimulatory domains such as CD28 or 4-1BB as well as CD3z, which drive signal activation and amplification of CAR T cells (Fig. 1) (15). Modifications to these basic components have led to various generations of CARs (16). The structure employed in currently approved CAR T products and in most of the studies discussed in this review is referred to as the second-generation CARs.

The manufacturing process of autologous CAR T cells begins with the cell collection at the clinical site, followed by shipment of the collected cells to the manufacturing site, modification of cells to express the CAR construct, and return of the final product to the clinical site for infusion (17). The major steps involved at each step are summarized in Fig. 2.

The starting materials for CAR T-cell manufacturing are peripheral blood mononuclear cells, collected by leukapheresis. Obtaining optimal leukapheresis product depends on appropriate vascular access, achieving sufficient T-cell yield, eliminating contaminating cell types in the leukapheresis product, determining washout periods for medications, and managing adverse events at collection (18). The collected cells are then shipped to the manufacturing facility either fresh or cryopreserved or put directly into culture if CAR T-cell manufacturing occurs at the clinical site (19).

At the manufacturing site, T cells are isolated, purified, and activated using antibody-coated paramagnetic beads. While anti-CD3 antibodies alone or in combination with feeder cells and growth factors were used for T-cell activation for many years, beads coated with anti-CD3/anti-CD28 monoclonal antibodies or cell-based artificial antigen presenting cells have been found to be more effective and are now the common practice (20). The activation process is followed by introduction of the CAR into T cells through transduction with a lentiviral or retroviral vector containing the transgene that encodes the specific CAR (20). Once transduction is complete, excess vector and other residual agents are washed from the culture and the T cells that now express CAR are expanded in culture (21). Expansion continues until there are sufficient cells to meet the final product dose requirements (19). Expanded CAR T cells are then separated from beads, washed, and formulated in infusion media (19). The cells in the final solution are then cryopreserved prior to shipment back to the clinical center in a liquid nitrogen (LN2) dry shipper.

At each manufacturing step, samples are removed for quality control testing (20, 21). Quality control testing of apheresis product involves enumeration of T-cell content, measured by CD3⁺ T-cell yield (21). The final CAR T-cell product also undergoes product release testing including product identity, purity, viability, and transduction efficiency (21). Potency testing is not currently standardized, although some centers use transduction efficiency and expression of the CAR construct assessed by flow cytometry as surrogate markers. The relation between these surrogate markers and in vivo cytotoxicity is not clear, leading to the need for reliable biomarkers of efficacy.

Biomarkers for Efficacy of CAR T-Cell Therapy

Biomarkers of CAR T-cell efficacy may be obtained and measured at various points during the production or treatment process (Fig. 3). For this review, we will broadly classify them into pre-infusion biomarkers, those that are measured before the CAR T cells are infused into patients, and post-infusion biomarkers, those measured in the days, weeks, or months following infusion. We also classify biomarkers as patient related, which focus on a patient’s clinical or disease status, and product related, those that directly evaluate features of the CAR T cells themselves or their precursors in apheresis products.

Pre-infusion Biomarkers

Patient-related pre-infusion biomarkers

Before cells are even collected for production of CAR T-cell therapy, a patient’s baseline status provides important information that can predict response to therapy. The major pre-infusion biomarkers are summarized in Table 1 and discussed in detail in the following section (not all the biomarkers discussed are included in Table 1).

Baseline disease burden

In patients with lymphoma, clinical markers of advanced disease burden, such as disease stage or tumor volume, have clearly been predictive of poor CAR T-cell therapy clinical outcomes (22, 23). Therefore, there is increasing interest in developing more sensitive laboratory-based markers to identify patients with more aggressive diseases. Elevated levels of circulating tumor DNA in patients with lymphoma prior to CAR T-cell therapy have been shown to predict relapse (24). Similarly, having <5% blasts on bone marrow evaluation was predictive of long-term survival after CD19 CAR T-cell therapy in patients with ALL (25). Assessment of disease burden in another study of patients with B-ALL treated with CAR T-cell therapy revealed bone marrow blasts and leukocyte count at initial diagnosis to be predictors of complete response and minimal residual disease (MRD)-negative complete response, respectively (26). In pediatric patients receiving CD19 CAR T-cell therapy, high baseline disease burden (>5% blasts on bone marrow examination) was also associated with nonresponse (27).

Tumor-specific characteristics have also been evaluated. In a trial of CD19-targeted CAR T-cell therapy for lymphoma, patients whose diffuse large B-cell lymphoma (DLBCL) was transformed from follicular lymphoma had better survival (28). Patients who have a p53 mutation in their lymphoma, or those with higher levels of TP53-mutated circulating tumor DNA, also have poor response to CAR T-cell therapy (29, 30). The presence of CD58 on lymphoma cells is also important for effective adhesion and interaction with CD2 on CAR T cells, and loss of CD58 on pre-infusion tumor samples is associated with poor response (31). Among patients with leukemia, the impact of high-risk cytogenetics on CAR T-cell therapy outcomes has been evaluated, but no difference was observed between disease response or overall survival between patients with and without high-risk mutations (32). Although KMT2A was not associated with higher risk of relapse, patients with KMT2A rearrangement were more likely to have a lineage switch when they did relapse after CAR T-cell therapy (33).

Inflammation and overall clinical status

In addition to direct measures of disease burden, baseline laboratory values suggestive of inflammation and illness severity are also predictive of CAR T-cell outcomes. In patients with DLBCL, elevated lactate dehydrogenase (LDH) and C-reactive protein (CRP) levels are both independently predictive of CAR T-cell treatment failure, with LDH remaining significant in multivariate analyses (22). Normal LDH levels have been predictive of both complete response and progression-free survival in patients with lymphoma (34). Similarly, in a cohort of patients with B-cell ALL, lower LDH as well as higher platelet counts prior to infusion were associated with improved survival after CD19 CAR T-cell therapy (35). Higher absolute lymphocyte count, an important metric for feasibility of CAR T-cell production, has also shown some correlation with response rate and survival in patients with lymphoma and multiple myeloma (36, 37). In another study of patients who received CD19 CAR T-cell therapy for B-cell malignancies, immune status prior to infusion was a predictor of favorable response, defined by elevated levels of IL-12, DC-Lamps, FAS ligand, and TRAIL but low myeloid-derived suppressor cells, low IL-6, IL-8, NAP3, sPDL1, and sPDL2 (38). The gut microbiome can also serve as an informative marker of immune status, and there are growing data showing that microbiome profiling can predict long-term response to CAR T cells (39).

Response to lymphodepletion may also be an important predictor. Patients with non-Hodgkin lymphoma (NHL) who had elevated levels of IL-7 and MCP-1 after lymphodepletion had longer progression-free survival after CD19 CAR T-cell therapy (40). Another study found that higher levels of IL-15 after lymphodepletion were predictive of lymphoma remission after CAR T cells (41). Total fludarabine exposure has also been shown to predict positive response to CD19 CAR T cells in pediatric patients (42).

Product-related pre-infusion biomarkers

Detailed characterization of CAR T-cell products has been an important aspect of correlative studies for CAR T-cell clinical trials. A range of different cell markers, falling broadly into categories of T-cell phenotype and exhaustion or activation markers, have been found to be associated with response to CAR T-cell therapy. As shown in Fig. 3, product-related pre-infusion biomarkers can be obtained either from the leukapheresis product premanufacturing or the infusion product postmanufacturing. It is important to note that for commercially manufactured products, most of the final infused product biomarkers discussed next would have to be obtained at the manufacturing site, which has implications for their broad applicability.

CD4/8 ratio

In early CAR T-cell trials, it was observed that the proportion of CD8 cells correlated with greater toxicity (1). In later trials, CD4 and CD8 selection of apheresis products has led to greater expansion of CD22 CAR T cells as well as increased inflammatory response in patients with ALL (43). A predefined CD4:8 ratio of 1:1 in CD19 CAR T cells also led to increased potency in an adult cohort of B-ALL patients (44). Thus, although the ideal proportion of CD4:8 cells is yet to be defined and likely varies for different patient populations, these subsets are an essential part of characterizing CAR T-cell products.

Early memory T cells

Several studies have identified an important subset of early memory T cells, which are associated with improved expansion and persistence of CAR T cells in vivo. Fraietta et al. observed that cells identified by the flow signature CD27⁺CD45RO⁻CD8⁺, were associated with a sustained response among patients with chronic lymphocytic leukemia (CLL) who received CD19 CAR T cells (45). A similar population of CAR T cells was found to be associated with improved outcomes in patients with DLBCL, also treated with CD19 CAR T cells (46). Early memory T cells were also associated with response in NHL and multiple myeloma patients, with higher proportions of effector memory T cells identified in nonresponders (47). Lamure et al. observed a slightly different pattern in a cohort of adult patients with DLBCL, with higher proportions of effector memory to naïve T cells found in the infusion products of patients who responded to CAR T cells (48). In a trial of CAR T cells targeted against CAIX to treat renal cell carcinoma, higher proportions of naïve CD8 cells were similarly associated with greater CAR T-cell expansion in vivo (49). Interestingly, CAR T cells produced from healthy donors have a greater proportion of naïve T cells compared with those of lymphoma patients (50).

The T-cell phenotype patterns identified by cell-surface markers have also been supported by transcriptomic data. Early memory T-cell signatures were identified in infusion products from CLL and DLBCL patients who responded to CD19 CAR T cells (45, 47, 51). Disease response in patients with lymphoma who were treated with CD19 CAR T cells was associated with CAR T-cell gene expression signatures characteristic of early memory T cells (51). Early memory signatures were also found more frequently among CAR T cells with 4-1-BB costimulatory domains, compared with CD28zeta CAR T cells, consistent with the finding that 4-1BB CAR T cells tend to have greater potential for in vivo expansion (52). Furthermore, patients who had longer CAR T-cell persistence were found to not only have more early memory CAR T cells in the infusion product, but also to have effector cells that expressed genes typically associated with early memory cells (30).

Other T-cell subsets

More recently, there has been increasing attention to the T-regulatory (Treg) cell population in CAR T-cell products. A lower proportion of Tregs among CAR T cells, identified by the flow markers CD4⁺CD25⁺CD127^low, was associated with greater chances of achieving remission (53). This study identified that the level of CAR Tregs, measured pre-infusion as well as 1-week post-infusion, was significantly higher among patients who did not achieve remission. It is not clear whether these are all Tregs that are transduced with the CAR construct or whether the cells change phenotype during the process of CAR T-cell manufacturing. Similarly, increased populations of CAR Tregs found in infusion products were associated with nonresponse among patients with lymphoma being treated with CD19-targeted CAR T-cell therapy (54). A small study of 12 ALL patients who had been treated with CD19 CAR T-cell therapy also showed that a lack of T2 helper cells in the infusion product was associated with greater chance of relapse (55).

T-cell exhaustion

Another characteristic of CAR T cells that is important in predicting outcome is T-cell exhaustion, a term that encompasses a terminally differentiated state affecting ability of cells to secrete cytokines, kill target cells, and proliferate (56). T-cell exhaustion is characterized by expression of inhibitory markers such as PD-1, PD-L1, TIM3, and LAG3 (56). A higher proportion of exhausted cells, identified as CD3⁺CD28⁻CD27⁻ by flow cytometry, was predictive of poor response to CAR T-cell therapy in patients with DLBCL (57). In another analysis of T-cell exhaustion, Beider et al. demonstrated a high percentage of CD57⁺CD39⁺CD28⁻ cytotoxic CD8⁺ T cells in final CAR T-cell products of patients with primary CAR T-cell therapy failure (58). TIM3, in combination with several other exhaustion markers, including PD-1 and LAG-3, was also found more commonly in CAR T cells derived from lymphoma patients compared with healthy controls (50). Novel assays may help identify exhaustion markers earlier than existing methods. For example, DNA methylation profiling has been shown to identify effector cell genes and exhaustion genes earlier than changes in cell-surface markers (59). Similarly, epigenetic profiling can potentially predict which cells are more prone to exhaustion (60).

T-cell functionality

In addition to cell-surface markers, an important method of evaluating CAR T cells is through the measurement of cytotoxic potential. While in vitro targeting and killing of malignant cells can be observed and is tested in product development, the more efficient way of gathering similar information during CAR T-cell manufacture is with cytokine production assays. Rather than levels of individual cytokines, it is the overall cytokine production, termed “polyfunctionality,” which has been associated with disease response and higher grades of cytokine release syndrome (CRS) (61).

Apheresis products

A few studies analyzed apheresis material as well as infusion products and have found similar patterns. The central memory T-cell subset that Fraietta et al. identified in CAR T cells (CD45RO⁻, CD27⁺CD28⁺) was also present in apheresis product from patients with CLL and predictive of response to CD19 CAR T-cell therapy (45). In the DLBCL cohort examined by Lamure et al., T-cell phenotypes change significantly between the apheresis and infusion product, with increased naïve and central memory cells, and decreased effector memory cells present after CAR T-cell manufacturing. However, it was a higher proportion of effector cells at baseline, as well as in the infusion product, that correlated with CAR T-cell response (48). One prior study has focused on apheresis material from pediatric patients: Finney et al. found no association with T-cell phenotypes, but observed that greater expression of an exhaustion marker, LAG-3, as well as low TNF-alpha production were predictive of nonresponse (62). Further research in this area is needed, as identification of biomarkers in apheresis products allows for the opportunity to intervene by cell selection or other manufacturing adjustments to optimize CAR T-cell potency.

In sum, clinical experience to date has demonstrated the strongest correlation between disease response and proportions of early memory T cells, exhausted T cells, and Treg populations among CAR T-cell products. Identifying these T cells subsets will be important in predicting disease response and could guide potential interventions.

Post-infusion Biomarkers

Post-infusion biomarkers are those that can be measured within days, weeks, or months after the infusion of CAR T-cell therapy. The patient-related markers evaluate the biological effects of CAR T-cell therapy on the disease or the patients’ overall clinical status, and the product-related markers are measures of CAR T cells once they are infused into the patients. The major post-infusion biomarkers are summarized in Table 2, and these and some additional biomarkers are discussed in detailed in the section that follows.

Patient-related post-infusion biomarkers

After infusion of CAR T cells, both their direct effect on target cells and subsequent activation of immune response can be predictive of response. Some of these effects may lead to therapy-related side-effects or adverse events and may require further management. Here, we will focus on the effects that have been shown to predict positive disease response.

B-cell aplasia

One of the expected effects of B-cell targeting CAR T-cell therapy is suppression of B-cell counts along with remission of the disease, a process often referred to as B-cell aplasia (63). It has been shown that CD19-targeted CAR T cells proliferate immediately after infusion, leading to the elimination of detectable normal B-cells as well as leukemic cells in >80% of pediatric patients with ALL (5). Moreover, the level of B-cell aplasia has been shown to positively correlate with therapeutic response to CAR T cells (5). Conversely, loss of B-cell aplasia implies loss of functional CAR T cells.

In a Phase 1 trial of 45 children and young adults with relapsed or refractory B-ALL who received CD19-targeted CAR T cells, a longer duration of B-cell aplasia correlated significantly with the durability of remission (64). When the 40 patients who achieved MRD-negative remission in this study were analyzed, Gardner et al. found that loss of B-cell aplasia increased risk of relapse. On long-term follow-up of this cohort, with a median follow-up period of 26.4 months, the duration of B-cell aplasia positively correlated with leukemia free survival (62). Patients who lost B-cell aplasia within the first 63 days after infusion were at a significantly higher risk of relapse. These results indicate that following the level and duration of B-cell aplasia could be an important biomarker to predict the long-term outcome of B-cell targeted CAR T-cell therapy.

While the persistence of B-cell aplasia is clearly an important predictor of disease response, some studies have shown that it is not necessary to maintaining remission in patients with NHL. Patients with ongoing responses up to 24 months after CAR T-cell therapy do show evidence of B-cell recovery (65, 66).

Systemic inflammatory response

CRS is a life-threatening systemic inflammatory syndrome that involves an increase in circulating cytokines, triggered by infusion of therapies such as CAR T cells (67). While it is a serious side-effect of CAR T-cell therapy, some degree of CRS has also been correlated with positive outcomes (68). The retrospective analysis of multiple clinical trials has shown that moderate (grade 2) CRS following CAR T-cell infusion significantly correlates with therapeutic response (69, 70). In a study that included 102 CAR T-cell treated patients with relapsed or refractory lymphoma, a multivariable regression showed that moderate CRS was associated with higher complete response at 1 year and longer progression-free survival as well as overall survival with median follow-up of 16.5 months (69). Similarly, a retrospective study of 34 NHL patients treated with CAR T cells showed that higher grade CRS correlated with better outcomes (70). In both studies, the median time of onset for CRS was within 2–3 days after infusion. Overall, these studies show the presence of moderate grade CRS following CAR T-cell infusion could be an important predictor of outcome.

While CRS is mainly a clinical diagnosis, there are also measurable laboratory values that could be used as indicators of CRS. Among cytokines released during CRS, the levels of IL-6, IL-10, IFN-γ, and MCP-1 have been shown to be strong predictors (71–73). Levels of ferritin and CRP have also been associated with increased CRS grade (74). Therefore, while they may not be direct or stand-alone predictors, the measurement of cytokine levels, ferritin, and CRP after CAR T-cell infusion could aid in predicting efficacy. The release of cytokines in response to CAR T-cell therapy has also been correlated with clinical response, regardless of presence of clinical CRS. In a study of 41 patients with advanced, heavily pretreated, and high-risk CLL, investigators observed higher peaks of serum IL-6 and IL-15 within the first 28 days after infusion among patients that responded to treatment (45).

Minimal residual disease (MRD)

In line with its importance as a predictor of leukemia response after chemotherapy, MRD has also become important in the setting of CAR T-cell therapy. In a study of 53 patients with relapsed/refractory B-ALL treated with CAR T-cell therapy, investigators found that patients with MRD-negative complete remission 3 weeks after CAR T-cell infusion (evaluated by absence of leukemia clone of immunoglobulin heavy chain by high-throughput sequencing) had improved overall and event-free survival (35). Additionally, bone marrow assessment for MRD status after CAR T-cell infusion could also be a predictor of outcome for patients. In a retrospective study of 60 multiple myeloma patients who received CAR T-cell therapy, bone marrow MRD-negative status at month 1 correlated with deep response and prolonged progression-free survival (75). These studies suggest that MRD status 3–4 weeks after CAR T-cell infusion could be a good biomarker for long-term disease response. Circulating tumor DNA has also emerged in more recent studies as a sensitive predictor of disease response and relapse after CAR T-cell therapy (24, 76).

Antigen loss

Another biomarker closely related to MRD status is antigen loss or recurrence of antigen negative cancer after CAR T-cell therapy, which predicts poor outcome. In the pediatric B-ALL trial described by Gardner et al., 7 of the 18 patients with relapse had an associated loss of cell-surface expression of CD19 at 1 month after CAR T-cell infusion (64). One of these relapses even included a lineage switch to acute myeloid leukemia. These observations have led to the development of CD22-targeted CAR T cells either as dual targeting with CD19 or as a stand-alone therapy to treat those with resistance to CD19 CAR T-cell therapy (77, 78). Therefore, along with MRD status, measuring the level of antigen expression within 1 month of infusion could help in predicting response to CAR T-cell therapy.

Product-related post-infusion biomarkers

Some characteristics of the CAR T cells themselves can also be measured after infusion into patients to serve as predictors of outcome. These characteristics, such as expansion and persistence, as well as the presence of other T-cell subtypes within circulation, may provide insight into the robustness of CAR T cells.

CAR T-cell expansion

CAR T-cell expansion, measured through flow cytometry, has been shown to be an important predictor of outcome. In a landmark multicenter, Phase 2 trial of CD19 CAR T cells for 111 patients with lymphoma, higher peak CAR T-cell expansion, defined as number of CAR T cells per unit of blood volume, was associated with both objective and durable response (23). In the same study, cumulative CAR T-cell levels over the first 28 days, measured in blood by area under the curve, were also associated with objective and durable responses (23). Similarly, among patients with CLL who received at least 1 dose of CD19-directed CAR T cells, those who responded to the CAR T-cell therapy exhibited dramatic in vivo expansion of CAR T cells in the first 2 weeks after infusion (45). Therefore, measuring the CAR T-cell levels in blood and monitoring expansion in the first 14–28 days could be another biomarker to determine response to therapy.

CAR T-cell persistence

In addition to short-term expansion, CAR T-cell persistence within circulation, measured through flow cytometry, is one of the most important predictors of efficacy. Persistence of high levels of CAR T cells in peripheral blood to at least day 28 after infusion correlates with objective response (44, 79). In the multicenter Phase 2 trial described above involving CD19 CAR T-cell treatment of patients with lymphomas, CAR T cells persisted up to 180 days after infusion in most patients, and all those with ongoing complete remission at 24 months had persistently detectable CAR T cells in the blood (79). In another study that involved 22 patients with advanced-stage lymphoma, the peak level of CAR T cells in the blood were significantly higher in patients who achieved complete or partial responses (41). Although the level of CAR T cells in blood is one of the important post-infusion biomarkers for efficacy, further studies are needed to determine the length of persistence needed for optimal therapeutic response.

Regulatory T (Treg) cells

As discussed in the pre-infusion biomarker section, elevated levels of CAR Treg cells, both pre- and post-infusion, are associated with progressive disease (53, 54). In a study of 32 patients with large B-cell lymphoma treated with CD19-CAR, CD4⁺Helios⁺ CAR T cells identified on day 7 after infusion were associated with progressive disease (80). These cells were identified using single-cell proteomic profiling of circulating CAR T cells and deep profiling demonstrated they manifest hallmark features of Treg cells. Therefore, measuring the level of Tregs 1 week after CAR T-cell infusion cells could be another efficacy biomarker.

Conclusion

Identification of biomarkers in the setting of CAR T-cell therapy is a rapidly growing effort as we continue to learn more about product characteristics as well as immune response to CAR T-cell therapy. Most of the biomarkers described here have been identified in a research setting, few have been tested prospectively, and none have been validated for clinical use. It is important for researchers, clinicians, and clinical laboratorians to come together in this field to select biomarkers that can be validated in large cohorts and incorporated into clinical practice.

Among biomarkers that have been reported in correlative studies from clinical trials, good candidates for clinical development will be those that (a) have consistent correlation to short and long-term disease response, (b) can be measured with clear, clinically relevant cutoff values, and (c) can be measured reliably with standard clinical laboratory equipment. The advantages and limitations of the various types of biomarkers discussed in this review are summarized in Table 3.

From the biomarkers described in this review, measures of disease burden and response have been most consistently related to clinical outcomes. Sensitive measures of disease burden pre-infusion will be important in determining timing of CAR T-cell therapy as well as the value of any bridging chemotherapy. Similarly, close monitoring of disease levels as well as CAR T-cell persistence post-infusion will have implications for subsequent therapies such as consolidative bone marrow transplant. Emerging data also indicate that higher proportions of early memory T cells are favorable at infusion, and tracking T-cell subsets throughout treatment will likely be critical.

Pre-infusion evaluation of apheresis material and CAR T cells have been the focus of much research and merit consideration as they can help stratify patients or personalize manufacturing. However, it is important to consider the setting in which CAR T cells are administered. Under current workflows, many patients are receiving CAR T cells at a clinical center separate from the pharmaceutical company where cells are produced. This means that any evaluation of the final infusion products would occur at the site of production rather than the clinical center where patients are being treated. Samples from the leukapheresis product could potentially be retained for biomarker testing at the clinical center, but the most feasible biomarkers would be those that use peripheral blood of patient samples.

In summary, developing reliable predictors of response to CAR T-cell therapy can affect clinical practice in multiple ways. Identifying patients who are likely to respond to CAR T can help direct resources to those most likely to benefit and can guide decisions about alternative therapies for others. Prediction of CAR T-cell expansion and persistence can also inform cell dosing as well as dosing of preconditioning chemotherapy. Identification of biomarkers in leukapheresis products prior to CAR T-cell manufacture can inform optimization of the manufacturing process. Finally, prediction of clinical response can guide the need for adjunct therapies and consolidative bone marrow transplant following CAR T-cell. A collaborative approach to gathering patient samples and analyzing correlative data from ongoing clinical trials is needed to identify meaningful biomarkers and improve patient outcomes.

Nonstandard Abbreviations

CAR, chimeric antigen receptor; ALL, acute lymphoblastic leukemia; MRD, minimal residual disease; DLBCL, diffuse large B-cell lymphoma; LDH, lactate dehydrogenase; CRP, C-reactive protein; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin lymphoma; Treg, T-regulatory; CRS, cytokine release syndrome.

Author Contributions

The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list.

Theodros Mamo, Alexandra Dreyzin, David Stroncek, and David H. McKenna (none declared).

Authors’ Disclosures or Potential Conflicts of Interest

Upon manuscript submission, all authors completed the author disclosure form.

Research Funding

None declared.

Disclosures

D.H. McKenna has worked with and received support from the following companies on cell therapy development and product manufacturing: Fate Therapeutics, Intima Bioscience, Gamida Cell, Immusoft, and Diabetes Free. T. Mamo has received advisory board consulting fees from BioLineRx USA on new mobilizing agent for apheresis cell collection.

References

Author notes