Wishing on a CAR: Understanding the Scope of Intrinsic T-cell Deficits in Patients with Cancer

Two different formulations of chimeric antigen receptor (CAR) T cells targeting the B-cell antigen CD19 have received FDA approvals in the last 2 years. Tisagenlecleucel (Kymriah) has approvals for both pediatric/young adult relapsed/refractory acute lymphoblastic leukemia (ALL) as well as adult relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Axicabtagene ciloleucel (Yescarta) has a single approval for relapsed/refractory DLBCL. Remarkably, despite insertion of the same transgene to patients' T cells during the manufacturing of tisagenlecleucel, adult patients with DLBCL and pediatric patients with ALL have dramatically different durable complete response rates after at least 3 months of follow-up (29% vs. 82%, respectively; refs. 1, 2). Disease-specific response rates in DLBCL between tisagenlecleucel and axicabtagene ciloleucel are comparable among adult patients (3).

Impaired therapeutic efficacy has been attributed to antigen escape, a tumor characteristic, as well as patient characteristics such as T-cell deficits. Tumor antigen escape as a means of evading CAR-T therapy has been well characterized in patients with ALL treated with anti-CD19 CAR T cells. Leukemic blasts in up to 25% of pediatric patients may lose (or decrease) CD19 expression, rendering them resistant to CD19-targeted CAR-T–mediated killing (4).

The presence of early-lineage T cells (naïve and early memory) prior to genetic engineering has been found to correlate with better expansion and to be enriched in patients with ALL compared with patients with non-Hodgkin lymphoma (NHL; ref. 5). A similar population of naïve T cells (CD27⁺CD45RO⁻CD8⁺) in the starting lymphocyte material of adult patients with chronic lymphocytic leukemia (CLL) correlated with a favorable subsequent clinical response to tisagenlecleucel (6). Importantly, naïve T-cell numbers are known to decline with age, which could partially explain the decreased response rates in adults compared with children with regard to CAR-T therapy (7). Furthermore, intensive cytotoxic chemotherapy disproportionately depletes this key subset of naïve lymphocytes, which may ultimately become an important consideration for patients with cancer considering adoptive T-cell therapy (8).

In this issue of Cancer Discovery, Das and colleagues perform an extensive analysis of T-cell fitness and phenotyping in a large cohort of pediatric patients with cancer over time as they received progressive cycles of chemotherapy (9). The authors undertook the herculean effort of prospectively consenting and enrolling 195 newly diagnosed pediatric patients with cancer to the study, which involved serial blood draws at baseline and then after every cycle of chemotherapy (see Fig. 1). Each sample was then analyzed at the single-cell level by flow cytometry for key T-cell subsets based on the expression of surface markers, and cultured individually in a functional expansion assay that measures T-cell growth in response to stimulation with beads coated with anti-CD3 and anti-CD28 antibodies. Importantly, this is essentially the same culture process that is used in the manufacturing of CAR T cells. A previously defined threshold of in vitro T-cell expansion that correlated with successful manufacturing of CAR T cells was used to split patients into three groups: those whose T-cell expansion would be a “pass,” “indeterminant,” or “fail.”

Das and colleagues found that patients with standard risk (SR) ALL had the highest initial “pass” rate at diagnosis (69%), which trended down with subsequent cycles of chemotherapy to less than 25% by cycle seven. Patients with lymphoma had lower pass rates at diagnosis (20%–30%), which also worsened over time. Patients with Ewing sarcoma had the worst pass rate (15%) at diagnosis before falling even further. Importantly, expansion potential could not be inferred from the readily available clinical metric of absolute lymphocyte count.

Prior work from the authors had shown that coculture with IL7 and IL15 enriched early-lineage T cells and rescued T-cell expansion capacity from the chemotherapy-induced depletion among patients with NHL (5). Upon addition of these two cytokines to the bead expansion assay, the authors saw a universal increase in the percentage of stem central memory T cells in the final postexpansion samples (primarily correlating with a loss of terminal effector T cells). Reassuringly, no samples that initially “passed” without the addition of the cytokines “failed” when the cytokines were added. On immunophenotyping analysis, patients with SR ALL had the most naïve cells at diagnosis and this population never fell below 25% of the total T-cell population, whereas most other malignancies saw declines in this subset. Importantly, surface phenotyping was not sufficient to explain the variance in the functional expansion assay, suggesting some latent functional defects.

This work has important implications. With the increasing interest in developing and applying the CAR-T cell therapies to solid tumors, it is critical to consider the fitness of the T cells being used for the manufacture of CAR-T products, especially given the mounting evidence of a correlation with clinical outcomes. Studies like this may also provide insight into CAR-T therapy for adult malignancies like CLL, which are known to harbor inherent T-cell deficits. The authors highlight the important and unexpected finding of very different naïve T-cell populations between solid tumors and leukemias, remarking that, “With the available evidence, it is not clear whether children who have these intrinsic T-cell differences are more at risk for solid tumors, or whether the tumors themselves are altering the T-cell compartment” (9).

One consideration for solid tumor and adult CAR-T applications is that many of the pediatric CAR-T patients have previously undergone allogeneic bone marrow transplantation as part of their treatment and thus had CAR-T products manufactured with a largely chemotherapy-naïve hematopoietic system. Ongoing efforts are under way to improve T-cell fitness via several strategies. One such option includes use of a PI3K inhibitor during manufacturing, which has been shown to preserve a less differentiated state (10). Alternatively, it may be possible to simultaneously give patients a drug that augments CAR-T cell function while receiving therapy. The Bruton tyrosine kinase inhibitor ibrutinib has been shown to enhance CAR-T engraftment, tumor clearance, proliferation, and survival as well as clinical response in patients with CLL (11). The use of allogeneic CAR-T cells manufactured from the blood of healthy donors may be another strategy to circumvent the “defective” T cells in patients with cancer. Some allogeneic CAR-T cells are currently in clinical trials and have shown efficacy, but rejection in both the host and graft directions remains a concern. One final strategy involves simply shifting CAR-T cell therapy (or at least cell collection) to the first line, prior to receiving cytotoxic chemotherapy, to preserve lymphocyte integrity. This paradigm is commonly employed in patients with multiple myeloma in which stem cells are collected and stored for a future autologous transplant prior to the receipt of (or early in the course of) immunomodulatory imides, which are known to impair stem-cell function. To this end, a clinical trial utilizing tisagenlecleucel in the up-front setting is planned (but not yet recruited) for pediatric and young adult patients with very high-risk B-cell acute lymphoblastic leukemia (NCT03792633). This trial will allow the collection of T cells untouched by chemotherapy and hopefully obviate the need for successive rounds of chemotherapy and bone marrow transplantation in this high-risk population. The use of up-front T-cell therapy holds the promise of sparing these pediatric patients the long-term sequalae from intensive chemotherapy and bone marrow transplantation.

In summary, this study by Das and colleagues provides the largest and most comprehensive analysis to date of T-cell phenotype and functional fitness over time in the course of multiple malignancies in pediatric patients. This data will help inform the design and testing of CAR-T cells in both pediatric and adult populations across all malignancies.

M.V. Maus has received commercial research grants from Agentus, Crispr Therapeutics, Kite Pharma, and TCR2; has ownership interest (including stock, patents, etc.) in Agentus; and is a consultant/advisory board for Adaptimmune, Agentus, Cellectis, Crispr Therapeutics, Kite Pharma, Novartis, Takeda, TCR2, Windmill, and Bluebird Bio. No potential conflicts of interest were disclosed by the other author.