Purification and characterization of the extracellular region of human receptor tyrosine kinase like orphan receptor 2 (ROR2).

Receptor tyrosine kinase like orphan receptor 2 (ROR2) is a co-receptor for some Wnt proteins including Wnt5a that activate the noncanonical Wnt/planar cell polarity (PCP) signaling pathway. Upregulation of ROR2 is associated with several cancer forms. The extracellular region of ROR2, which contains an immunoglobulin (Ig)-like domain, a Frizzled like cysteine-rich domain (CRD) and a Kringle domain, is a potential anticancer drug target. The structural and biochemical properties of the ROR2 extracellular region remain largely unexplored. Here we describe the mapping and purification, using a baculovirus - insect cell system, of a near-full-length ROR2 extracellular fragment (residues 53-402), which is well-behaved and suitable for future structural and biochemical analysis. We show that the extracellular region of ROR2 per se is monomeric in solution. Different monoclonal antibodies raised against the purified ROR2 protein can specifically recognize the protein and can either inhibit or activate the PCP activity in a cell-based assay, and are thus potentially useful for future mechanistic and therapeutic/diagnostic studies. The biological relevance of these antibodies further demonstrates that the purified recombinant ROR2 protein is properly folded and biochemically active.

Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues.

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 nonmutated 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 formalin-fixed, paraffin-embedded 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 show 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. Clin Cancer Res; 23(12); 3061-71. ©2016 AACR.