Specific Antibody Fragment Ligand Traps Blocking FGF1 Activity.

Fibroblast growth factor 1 (FGF1) and its receptors (FGFRs) regulate crucial biological processes such as cell proliferation and differentiation. Aberrant activation of FGFRs by their ligands can promote tumor growth and angiogenesis in many tumor types, including lung or breast cancer. The development of FGF1-targeting molecules with potential implications for the therapy of FGF1-driven tumors is recently being considered a promising approach in the treatment of cancer. In this study we have used phage display selection to find scFv antibody fragments selectively binding FGF1 and preventing it from binding to its receptor. Three identified scFv clones were expressed and characterized with regard to their binding to FGF1 and ability to interfere with FGF1-induced signaling cascades activation. In the next step the scFvs were cloned to scFv-Fc format, as dimeric Fc fusions prove beneficial in prospective therapeutic application. As expected, scFvs-Fc exhibited significantly increased affinity towards FGF1. We observed strong antiproliferative activity of the scFvs and scFvs-Fc in the in vitro cell models. Presented antibody fragments serve as novel FGF1 inhibitors and can be further utilized as powerful tools to use in the studies on the selective cancer therapy.

Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells.

The family of fibroblast growth factor receptors (FGFRs) plays an important role in cell growth, survival, differentiation and angiogenesis. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) are critical for ligand binding and specificity towards fibroblast growth factor and heparan sulfate. Fibroblast growth factor receptors are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we have cloned, expressed in CHO cells and purified Fc-fused extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2b-Fc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGFR4b-Fc), which could be used as molecular targets for the selection of specific antibodies. The fusion proteins were analyzed using gel electrophoresis, Western blotting and mass spectrometry. To facilitate their full characterization, the fusion proteins were deglycosylated using PNGase F enzyme. With an optimized transient transfection protocol and purification procedure we were able to express the proteins at a high level and purify them to homogeneity.

FGF2-Derived PeptibodyF2-MMAE Conjugate for Targeted Delivery of Cytotoxic Drugs into Cancer Cells Overexpressing FGFR1.

Fibroblast growth factor receptors (FGFRs) are emerging targets for directed cancer therapy. Presented here is a new FGFR1-targeting conjugate, the peptibodyF2, which employs peptibody, a fusion of peptide and the Fc fragment of human IgG as a selective targeting agent and drug carrier. Short peptide based on FGF2 sequence was used to construct a FGFR1-targeting peptibody. We have shown that this peptide ensures specific delivery of peptibodyF2 into FGFR1-expressing cells. In order to use peptibodyF2 as a delivery vehicle for cytotoxic drugs, we have conjugated it with MMAE, a drug widely used in antibody-drug conjugates for targeted therapy. Resulting conjugate shows high and specific cytotoxicity towards FGFR1-positive cells, i.e., squamous cell lung carcinoma NCI-H520, while remaining non-toxic for FGFR1-negative cells. Such peptibody-drug conjugate can serve as a basis for development of therapy for tumors with overexpressed or malfunctioning FGFRs.

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

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 smoking-associated 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. ©2017 AACR.