89 zr‐labeled domain ii‐specific scfv‐fc immunopet probe for imaging epidermal growth factor receptor in vivo

Abstract

Epidermal growth factor receptor I (EGFR) is overexpressed in many cancers. The extracellular domain of EGFR has four binding epitopes (domains I‐ IV). All clinically approved anti‐EGFR antibodies bind to domain III. Imaging agents that bind to domains other than domain III of EGFR are needed for accurate quantification of EGFR, patient selection for anti‐EGFR therapeutics and monitoring of response to therapies. We recently developed a domain II‐specific antibody fragment 8709. In this study, we have evaluated the in vitro and in vivo properties of89Zr‐ 8709‐scFv‐Fc (105 kDa). We conjugated 8709‐scFv‐Fc with the deferoxamine (DFO) chelator and radiolabeled the DFO‐8970‐scFv with89Zr. We evaluated the binding of89Zr‐DFO‐8709‐scFv‐Fc in EGFR positive and negative cell lines DLD‐1, MDA‐MB‐231 and MDA‐MB‐435, respectively, and in mouse xenograft models. Simultaneously, we have compared the binding of89Zr‐8709‐scFv‐Fc with 111 In‐nimotuzumab, a domain III anti‐EGFR antibody. DFO‐8709‐scFv‐Fc displayed similar cell binding specificity as 8709‐scFv‐Fc. Saturation cell binding assay and immunoreactive fraction showed that radiolabeling did not alter the binding of 8709‐scFv‐Fc. Biodistribution and microPET showed good uptake of89Zr‐8709‐scFv‐Fc in xenografts after 120 h post injection (p.i). and was domain‐specific to EGFR domain II.89Zr‐8709‐scFv‐Fc did not compete for binding in vitro and in vivo with a known domain III binder nimotuzumab. The results show that89Zr‐8709‐scFv‐Fc is specific to domain II of EGFR making it favorable for quantification of EGFR in vivo, hence, patient selection and monitoring of response to treatment with anti‐EGFR antibodies.