Advanced RNA ISH Assay for Preclinical CAR-T Cell Target Safety and Biodistribution Analysis


himeric antigen receptor (CAR)-T cell therapy is highly effective in treating hematologic malignancies,

while major efforts are being made to achieve similar efficacy in solid tumors. The greater potency of CAR-T cells compared to antibody therapeutics demands a more stringent CAR-T target safety assessment to avoid adverse events resulting from “on-target/off-tumor” activity. Furthermore, it is critical to track and monitor the pharmacokinetics of CAR+ T cells within the context of intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. Current methods, however, are either not sensitive enough to detect low levels of expression or not capable of detecting CAR+ T cells in the tissue context.

In this study, we illustrate a way to detect activated CAR-T cells in the tissue context, as well as low expression of CAR target antigen, using the RNAscope®

5’ UTR

Signaling Domain

Tumor Antigen (CAR Target) Binding Domain

Figure 1. RNAscope probes were designed to target the 3’ UTR of the CAR vector.

RNAscope Probe 3’ UTR


The RNAscope assay revealed ROR1 was expressed in the xenograft tumor as well as at low levels in mouse liver (Fig. 2). The RNAscope Duplex assay with probes targeting the CAR 3’ UTR and GZMB mRNA allowed highly sensitive and specific detection of CAR T cells and their activation state in both tumor and normal tissues from anti-ROR1 CAR T cell treated mice. Activated anti-ROR1 CAR T cells were found in mouse liver with very few anti- ROR1 CAR T cells being found in the xenograft tumor.

in situ hybridization (ISH)

technology. The RNAscope ISH assay was used to assess CAR-T target expression specificity for one candidate CAR-T target, ROR1, in xenograft and host tissues using the RPMI-8226 xenograft mouse model. We also utilized the RNAscope Duplex assay to track CAR-T cell distribution and activation.


RNAscope probes were designed to target the 3’ UTR of the CAR vector (Fig. 1). Sections from formalin-fixed, paraffin embedded (FFPE) mouse multi-tissue arrays and xenograft tumors were used. Tissues were from untreated mice or mice treated with anti-ROR1 CAR-T cells. The RNAscope assays used to assess RNA expression patterns included the RNAscope 2.5 LS Red Assay and the RNAscope 2.5 LS Duplex Assay.

Tissues from anti-ROR1 CAR-T cell treated mice CAR 3’UTR + GZMB



Given the high sensitivity of CAR-T cells to very low densities of target antigen, it is critical to employ a highly stringent and sensitive assay for preclinical target safety assessment. The RNAscope assay can be incorporated into this assessment workflow to:

• Detect low levels of gene expression across all normal tissues in animal models and human samples

• Assess cell-specific target expression in normal tissues and expression heterogeneity in tumor tissues

• Visualize and quantify CAR-T cell infiltration and activation in tumor and off-tumor tissues

• Simultaneously detect CAR/TCR vector and cytokines, T cell markers, or other cell-type markers


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Figure 2. Activated anti-ROR1 CAR-T cells detected in liver and xenograft tumor, revealing on-target/off-tumor activity. The RNAscope 2.5 LS Duplex Assay was used to simultaneously detect the 3’ UTR from the CAR vector (red) and GZMB in liver and tumor xenograft from RPMI- 8226 mice treated with anti-ROR1 CAR-T cells.



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