ADDENDUM: T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells (2016)

In this article (Cancer Immunol Res 2016;4:498–508), which appeared in the June 2016 issue of Cancer Immunology Research, we reported the design and optimization of bispecific, OR-gate chimeric antigen receptors (CARs) that can trigger robust T-cell activation in response to target cells that present either CD19 or CD20 (1), thus preventing malignant B cells from escaping T-cell therapy through loss of CD19 expression. In our original study, although OR-gate CAR T cells could significantly delay tumor expansion and prolonged survival, tumors were not eradicated. Tumor persistence was not due to the loss of both CD19 and CD20 antigens, nor due to the inability of the adoptively transferred T cells to survive or expand in vivo (1). We concluded that the failure to eradicate tumors was due to suboptimal tumor or T-cell dosing and/or the aggressiveness of the Raji tumor model.As the Raji tumor model had been used in other studies at dosages comparable with those employed in our own experiments (2), complete tumor clearance should be possible in this xenograft model using adoptively transferred CAR T cells. Here, we report follow-up studies showing that the viability levels of CAR T cells, both immediately after thawing and after 1–2 days of in vitro culture, are an important indicator of the CAR T cells' antitumor capability in vivo. Healthy T cells with OR-gate CARs could efficiently eradicate established lymphoma xenografts even if the lymphoma cells had spontaneously lost CD19 expression, whereas single-input CD19 CAR T cells succumbed to the selective expansion of CD19− mutants.The in vivo protocol employed in our study called for the tail-vein injection of luciferase-expressing Raji tumor cells, followed by the tail-vein injection of CAR T cells upon confirmation of tumor establishment, which was defined as clear tumor signal observed on two consecutive days via bioluminescence imaging (BLI). The CAR T cells used in these studies were previously frozen and thawed on the day of injection, and cells with >70% viability were considered suitable for adoptive transfer. To better characterize the CAR T cells (from donor A) used in our initial animal study reported in (1), we thawed additional stocks of the same donor's cells (donor A) and compared them with CAR T cells derived from other healthy donor blood samples (donors B and C) for cell viability, T-cell subtype distribution, and exhaustion marker expression. T-cell subtype distribution, exhaustion marker expression, and viability at the time of thawing were similar for all donors (Fig. 1). However, cell viability of CAR T cells from donors A and B drastically declined after 24 hours in culture, whereas donor C cells remained >70% viable, indicating that donor A cells may not have been of optimal quality (Fig. 1C). Although we had confirmed that donor A CAR T cells could survive and expand in vivo (1), they may not have proliferated well enough to completely eliminate the tumor xenograft.To investigate this hypothesis, we repeated the in vivo study using donor C cells, which showed superior viability in culture after thawing (Fig. 1C). As in the previous study, 5 × 105 Raji cells were delivered by tail-vein injection and allowed to engraft prior to treatment with 10 × 106 CAR T cells from donor C. Animals were injected with either purely wild-type (WT; CD19+/CD20+) Raji cells or a mixture of 75% WT and 25% CD19−-mutant Raji tumors. In contrast to the results obtained with donor A cells, we observed complete clearance of WT tumors treated with both single-input and OR-gate CAR T cells (Fig. 2A and B). Only OR-gate CAR T cells, however, could eradicate mixed (75% WT, 25% CD19−) Raji tumors (Fig. 2A and B). Animals engrafted with mixed Raji cells and treated with single-input CD19 CAR T cells eventually succumbed to tumor growths that consisted almost exclusively of the CD19−-mutant phenotype (Fig. 2C), confirming the selective expansion of antigen-negative clones when treated with single-input CAR T cells. Thus, unlike single-input CD19 CAR T cells, OR-gate CAR T cells limited the escape of CD19−-mutant tumors.Having confirmed the ability to eradicate both WT and CD19-knockout tumors using high-quality CAR T cells, we next investigated whether OR-gate CAR T cells would have superior resistance compared with single-input CD19 CAR T cells against the spontaneous loss of CD19 expression by tumor cells. Specifically, we investigated whether antigen loss is dependent on the timing of CAR T-cell treatment relative to the size of tumor burden, and whether the OR-gate CAR T cells could safeguard against the spontaneous emergence of CD19− mutants.Mice were injected with 5 × 105 WT Raji cells, which were allowed to grow either until first confirmation of engraftment (i.e., yielding clear tumor signal observed on 2 consecutive days via BLI, which occurred on day 6 in this set of studies), or until the tumor signal reached a 6-fold increase in radiance intensity compared with the first group (which occurred on day 9). Animals were then treated with 10 × 106 donor C T cells expressing either the single-input CD19 CAR or the OR-gate CAR. Consistent with previous results, animals treated at the earlier time point with either single-input or OR-gate CAR T cells were cleared of tumor engraftment within 6 days of T-cell injection (Fig. 3A). In contrast, all animals treated with CAR T cells on day 9 after tumor injection showed continued tumor progression for 3 days before tumor size began to decline (Fig. 3B). Animals treated with OR-gate CAR T cells completely cleared their tumors and showed no sign of relapse at the time of this writing (through day 109). In contrast, animals treated with single-input CD19 CAR T cells experienced only temporary tumor regression before relapsing, ultimately succumbing to multifocal cancer growth (Fig. 3B). Postmortem analysis revealed tumor engraftments in the liver and the chest cavity (Fig. 3C). The recovered Raji tumors, which were identified by their luciferase and EGFP expression (Fig. 3C), were CD20+ but had either no CD19 expression or significantly reduced CD19 expression (Fig. 3D), highlighting the risk of antigen escape with single-input CD19 CAR T cells and the OR-gate CAR T cells' ability to safeguard against this tumor defense mechanism.Patient relapse due to loss of CD19 antigen expression has been observed in multiple clinical trials, and antigen escape has been identified as a potential pitfall of CD19-directed therapies for B-cell malignancies (3, 4). The CD19/CD20 bispecific, OR-gate CAR demonstrates the ability to address this critical medical challenge and increase the efficacy of adoptive T-cell therapy for cancer.