Abstract

Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery.Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro.Results: T8scFv exhibited nanomolar affinity for TfR1 (KD= 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, KD= 18.5 ± 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro.Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems.