Screening and functional characterization of nanobodies targeting the transferrin receptor

The transferrin receptor (TfR1) mediates the cellular uptake of iron and other molecules, playing a vital role in hematology and tumor growth. Nanobodies (NBs) targeting TfR1 offer promising therapeutic potential due to their small size, high specificity and stability. However, rapid identification of effective nanobodies remains challenging.In this study, the truncated extracellular fragment of human TfR1 was expressed in a prokaryotic system and purified. Immunized camelids provided a source for nanobody libraries, which were screened using phage display and high-throughput strategies to identify candidates with specific TfR1 binding.NB 2D7 with nanomolar-level dissociation constants (KD) were successfully identified.The analysis of Cell Counting Kit-8(CCK8) experiments indicates that the combined treatment of NB2D7 with FeCl₃ can reduce the survival rate of LoVo cells.This research establishes an efficient platform for anti-TfR1 nanobody screening and highlights the therapeutic potential of these nanobodies in cancer treatment and iron metabolism disorders.