Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues

Abstract

Purpose This study examines cell-surface ROR1 expression in human tumors and normal tissues. ROR1 is considered a promising target for cancer therapy due to putative tumor-specific expression and multiple groups are developing antibodies and/or chimeric antigen receptor-modified T cells to target ROR1. On-target, off-tumor toxicity is a challenge for most non-mutated tumor antigens, however prior studies suggest that ROR1 is absent on most normal tissues. Experimental Design Our studies show that published antibodies lack sensitivity to detect endogenous levels of cell-surface ROR1 by immunohistochemistry (IHC) in FFPE tissues. We developed a ROR1-specific monoclonal antibody (mAb) targeting the carboxy-terminus of ROR1, and evaluated its specificity and sensitivity in IHC. Results The 6D4 mAb is a sensitive and specific reagent to detect cell-surface ROR1 by IHC. The data shows that ROR1 is homogenously expressed on a subset of ovarian cancer, triple negative breast cancer and lung adenocarcinomas. Contrary to previous findings, we found ROR1 is expressed on several normal tissues including parathyroid, pancreatic islets and regions of the esophagus, stomach and duodenum. The 6D4 mAb recognizes rhesus ROR1, and ROR1 expression was similar in human and macaque tissues suggesting that the macaque is a suitable model to evaluate safety of ROR1 targeted therapies. Conclusions ROR1 is a promising immunotherapeutic target in many epithelial tumors, however high cell-surface ROR1 expression in multiple normal tissues raises concerns for on-target off-tumor toxicities. Clinical translation of ROR1 targeted therapies warrants careful monitoring of toxicities to normal organs, and may require strategies to ensure patient safety.

Interaction between ROR1 and MuSK activation complex in myogenic cells

“…The highest expression, by median value, was observed in preadipocytes, heart, and myocytes. We noted less than 1 TPM expression for ROR1 in liver (no expression), as has also been previously observed [22].…”

“…4C. We observe significant expression in muscle and fat related cell types, as well as in previously observed tissues of pancreas and lung [22,31]. The highest expression, by median value, was observed in preadipocytes, heart, and myocytes.…”

Therapeutic T cell engineering

“…In contrast to CD19, CD22 or BCMA, which are expressed in normal but non-vital or temporarily dispensable tissues, other commonly cited targets such as ROR1, mesothelin or prostate-specific membrane antigen (PSMA), are detected in a subset of normal tissues 47,93,94 . The low expression of these antigens in normal tissues may avert significant toxicity, but the toxicities encountered in clinical trials targeting carbonic anhydrase IX or carcinoembryonic antigen with T cells suggest caution for clinical efforts targeting such molecules 95,96 .…”

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility

“…Note that the IHC detection of endogenously expressed ROR1 on the cell surface of fixed human cells and tissues by mAbs to the ECD of ROR1 is known to be difficult. 58 Fittingly, a high concentration of rabbit mAbs (20 μg/mL) was required for staining the MDA-MB-231 cells. All light microscopy images were taken at a magnification of 40× (total magnification of 400×).…”