Identification of a novel anti‐ROR1 nanobody through phage display and its biochemical characterization

In this study, we aimed to develop nanobodies targeting receptor tyrosine kinase‐like orphan receptor 1 (ROR1) for cancer diagnosis and therapy. We immunized alpacas with ROR1, extracted RNA from their blood, and converted it to complementary DNA (cDNA) to amplify the VHH (variable domain of heavy‐chain antibodies) sequence. This sequence was used to construct a phage library with a capacity of 8 ×10 8 . Screening identified a high‐affinity nanobody, HCAbs1, which binds effectively to ROR1. ELISA and surface plasmon resonance analyses revealed HCAbs1's binding affinities to ROR1 at 4.42 and 12.9 nM, respectively. Functional tests showed HCAbs1 could reduce extracellular signal‐regulated kinase (ERK) phosphorylation levels induced by Wnt5a in ROR1‐transfected cells. Our findings highlight the potential of HCAbs1 nanobodies in diagnosing and treating cancers through targeting ROR1.