title: Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site.
abstract: The conserved nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS (K(d) = 1-8 μM) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1-2 J/cm(2) yielded the most efficient photocrosslinking and resulted in 1-2 conjugations per antibody, while preserving the antigen binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Alves
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Champion
authors.forename: Jared F
authors.initials: JF
authors.lastname: Stefanick
authors.forename: Michael W
authors.initials: MW
authors.lastname: Handlogten
authors.forename: Demetri T
authors.initials: DT
authors.lastname: Moustakas
authors.forename: Yunhua
authors.initials: Y
authors.lastname: Shi
authors.forename: Bryan F
authors.initials: BF
authors.lastname: Shaw
authors.forename: Rudolph M
authors.initials: RM
authors.lastname: Navari
authors.forename: Tanyel
authors.initials: T
authors.lastname: Kiziltepe
authors.forename: Basar
authors.initials: B
authors.lastname: Bilgicer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000378:metabolism; D000941:Antigens / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002021:Buffers; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D011485:Protein Binding / Q000528:radiation effects; D011961:Receptors, Fc / Q000378:metabolism; D000069283:Rituximab; D013816:Thermodynamics; D014466:Ultraviolet Rays
chemical_list: D000906:Antibodies; D058846:Antibodies, Monoclonal, Murine-Derived; D000941:Antigens; D002021:Buffers; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D007211:Indoles; D008024:Ligands; C569656:N-end cysteine peptide tumor-homing peptide; D009711:Nucleotides; D009842:Oligopeptides; D011961:Receptors, Fc; C014612:indolebutyric acid; D000069283:Rituximab; D001710:Biotin; D017239:Paclitaxel