High-Affinity Internalizing Human scFv-Fc Antibody for Targeting FGFR1-Overexpressing Lung Cancer

Abstract

Targeted delivery of anticancer drugs using antibodies specific for tumor-associated antigens represents one of the most important approaches in current immuno-oncology research. Fibroblast growth factor receptor 1 (FGFR1) has been demonstrated to be a high-frequency targetable oncogene specific for smokingassociated lung cancers, present in over 20% of lung squamous cell carcinoma cases. This report describes the generation of a potent, fully human antibody fragment in scFv-Fc format efficiently targeting FGFR1. Antibody phage display was used to select high-affinity scFv antibody fragments against the extracellular domain of FGFR1(IIIc). Enzyme immunoassay (ELISA) and surface plasmon resonance (SPR) analysis were used for antibody screening and characterization. The best binder (named D2) was cloned to diabody and Fc fusion formats. All D2 antibodies demonstrated high affinity for FGFR1 with dissociation constants of 18 nmol/L (scFvD2), 0.82 nmol/L (scFvD2 diabody), and 0.59 nmol/L (scFvD2-Fc). scFvD2 was found to be exquisitely selective for FGFR1 versus other FGFR family members and bound FGFR1 even in the presence of its natural ligand FGF2, as shown by competitive analysis. Confocal microscopy revealed that scFvD2-Fc was specifically and rapidly internalized by a panel of cell lines overexpressing FGFR1. Finally, it was demonstrated that scFvD2-Fc mediated specific delivery of a cytotoxic payload into lung cancer cells harboring oncogenic FGFR1 gene amplifications.Implications: This study reports a highly specific internalizing antibody fragment that can serve as a therapeutic targeting agent for efficient delivery of cytotoxic drugs into FGFR1-positive lung cancer cells. Mol Cancer Res; 15(8); 1040-50. Ó2017 AACR.

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

“…We have recently reported a phage display‐based selection of high‐affinity antibody fragments that selectively recognize epitopes within the D1 domain of the FGFR1 [25]. Furthermore, we have demonstrated that the bivalency and the high affinity promote internalization of FGFR1/engineered antibody complexes [5,6,49].…”

“…Additionally, in our study we used previously reported bivalent anti‐FGFR1 engineered antibody, B‐Fc, composed of single scFv fused with the Fc (Fig. 1A) [25]. Both proteins were efficiently overproduced in CHO cells and purified using affinity chromatography (Fig.…”

“…T‐Fc was constructed based on the B‐Fc by the in frame fusion of second anti‐FGFR1 scFv coding sequence [25]. The scFv proteins in the Fc format (scFv‐Fc) were expressed in CHO‐S cells.…”

“…Up to date, several ADCs have been approved and are commercially available for treatment of various cancers [18]. Importantly, a number of selective cytotoxic conjugates including ADCs against cancers overproducing FGFRs were generated,however, their in vivo therapeutic potential awaits further evaluation [20–27]. A critical step in the anti‐cancer therapy with ADCs is selective and efficient delivery of the cytotoxic drug to the cell interior [28].…”

Targeted Protein Liposomes-Mediated AChE Gene Therapy for Effective Liver Cancer Treatment

“…Transferrin (Tf) is a well-known plasma protein responsible for transporting iron into cells via the transferrin receptor (TfR) that binds to the cell membrane [20][21][22]. TfR is overexpressed in many human tumors, and it is possible to improve the uptake e ciency of liver cancer cells by transferrin [23][24][25][26]. Transferrin receptors are present on the membrane of liver parenchyma, mannose receptors are distributed on non-parenchymal membranes, and low-density lipoprotein is regulated [27][28][29].…”