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1.
MAbs ; 10(8): 1157-1167, 2018.
Article in English | MEDLINE | ID: mdl-30183492

ABSTRACT

Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells.


Subject(s)
Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Carcinoma, Pancreatic Ductal/immunology , Frizzled Receptors/immunology , Pancreatic Neoplasms/immunology , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/therapeutic use , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cells, Cultured , Frizzled Receptors/antagonists & inhibitors , Frizzled Receptors/metabolism , HEK293 Cells , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Protein Binding , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/immunology , Protein Isoforms/metabolism , Sequence Homology, Amino Acid
2.
MAbs ; 8(6): 1167-76, 2016.
Article in English | MEDLINE | ID: mdl-27185291

ABSTRACT

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.


Subject(s)
Antibodies/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/immunology , Immunoglobulin Fab Fragments/chemistry , Antibodies/genetics , Antibody Affinity , Epitopes/chemistry , Humans , Immunoglobulin Fab Fragments/genetics , Magnetic Resonance Spectroscopy , Peptide Library , Phosphorylation , Protein Domains , Protein Engineering , Protein Folding , Surface Plasmon Resonance
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