Comment on “Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma”

The authors of this study¹ should be commended on the important and complex undertaking of identifying multiple antigens for chimeric antigen receptor (CAR) T-cell therapy, which is one of several key limitations to the field. There are 2 levels of antigenic heterogeneity at play that require consideration—intrapatient variability, in which the existence of antigen-negative tumor cells may result in eventual recurrence, and interpatient variability, in which different expression patterns across all patients make identification of a single or “universal” therapeutic for treatment difficult, if not impossible. The authors state that by targeting 3 antigens: human epidermal growth factor receptor 2 (HER2), interleukin-13 receptor alpha 2 (IL13Rα2), and ephrin-A2 (EphA2) using a trivalent UCAR, they can “overcome intrapatient and interpatient variability” and that “trivalent T cells were able to approach killing in 100% of tumor cells in nearly all patients modeled.” As investigators working in the field of immuno-oncology, we very much wish this to be the case; however, these conclusions are not supported by the data presented in the manuscript.

First, the investigators claim that these 3 antigens are present in most patients and have sufficient expression in each patient to cover all tumor cells. However, their own data specifically demonstrate that at least 20% of patients have at least 10% of glioblastoma (GBM) cells that are negative for all 3 antigens (Figure 1). Furthermore, these data are incongruous with those presented in articles from The Cancer Genome Atlas wherein 56% to 68% of GBM patients fail to express even one of these antigens.^2,3 This is a significant percentage of cells in a significant proportion of patients. The small sample size and the discrepancy with larger published datasets demand further inquiry before stating that targeting these 3 antigens offers a universal treatment that overcomes interpatient heterogeneity.

Second, although the authors include in their study cutting edge modeling of autologous CAR T cells and patients’ GBMs, these experiments fall short of addressing interpatient heterogeneity, as 4 patients with very similar antigen expression profiles were chosen for in vitro studies. Moreover, the 2 patient xenografts, UPN001 and UPN005, selected for the autologous in vivo xenograft model show an almost identical frequency of expression of the 3 antigens (Figure 1, Supplementary Table S1). Thus, the claim of harnessing the power of autologous patient xenografts for modeling interpatient heterogeneity is overstated.

These investigators also attempt to claim that there is a more robust immunological engagement using immune synapse distance of the UCAR construct relative to monovalent HER2 CAR T cells. However, the immune synapse distance between HER2 CAR T cells and nontransduced cells does not appear to be different (Figure 3). This calls into question whether the HER2 CAR T-cell population is representative of monovalent CAR T-cell function, which was used as the comparator to the UCARs. Inclusion of the IL13Rα2 CAR or EphA2 CAR T cells for this comparison may have been more appropriate.

Finally, the authors state in the abstract that the UCAR T cells “exhibited improved cytotoxicity and cytokine release over the best monospecific and bispecific T cells per patient tumor profile.” However, the UCAR T cells have comparable cytotoxicity to monovalent IL13Rα2 and EphA2 CAR T-cell populations (Figure 2). The cytotoxicity “superiority” of the UCAR constructs over biCAR T cells was only demonstrated in a very small sample of autologous tumor patient gliomas (n = 2/4), which is insufficiently statistically powered to support this conclusion.

In summary, although this study does attempt to address an important limitation in the field of targeting glioblastoma antigen heterogeneity using CAR T cells and uses a cutting edge murine model of GBM, the authors undercut the value of the model by choosing patients’ xenografts with nearly identical antigenic frequency in vitro and in vivo, and as such, do not really address interpatient variability. In addition, the presentation of the UCAR as “universal” for all GBM patients is misleading when considering the antigen expression profiling of 15 GBM patients, which is incongruent with data from TCGA. Thus, these fundamental flaws in the presented study do not support the interpretation that “trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma” and have the potential to misrepresent the progress in the field.