title: Antibodies as drug carriers III: design of oligonucleotides with enhanced binding affinity for immunoglobulin G.
abstract: OBJECTIVE
  To understand the structural requirements in designing epitope-bearing oligonucleotides with high antibody-binding affinity.
  
  
  METHODS
  Binding affinity (KA) and stoichiometry (n) of dinitrophenyl (DNP)-derivatized model 27-mer oligonucleotides (ODNs), GGG(AAA)7GGG, to monoclonal anti-trinitrophenyl (TNP) antibodies were determined using isothermal titration calorimetry (ITC). Structural variations were made in the ODNs to assess the effects of antigenic valence, epitope density, inter-epitope linker length, and linker flexibility. Binding isotherms were fitted with a single binding-site model to obtain K(A) and n, from which changes in Gibbs free energy (deltaG(0)), entropy (deltaS(0)), and enthalpy (deltaH(0)) were derived.
  
  
  RESULTS
  As expected, ligands displaying increased epitope density showed increases in K(A): for example, K(A) for (DNP)2-Cys is 3.3-fold greater than that for DNP-Lys. Introduction of multiple DNP groups via long and flexible linkers to one end of the 27-mer ODN resulted in a bivalent behavior with n value of 1. A bivalent ligand, derivatized at both ends with a long and flexible linker, failed to form an immune complex when hybridized to its antisense strand, presumably due to intercalation of the DNP moiety to the double strand. ODNs derivatized with flexible linkers exhibited a higher K(A) than those with a rigid linker. Ligands with flexible inter-epitope linkers measuring distances of 110, 60, and 40 angstroms yielded 13-, 30-, and 13-fold increases in K(A), respectively. The combination of these factors; namely, bivalence, flexible inter-epitope linkers, and optimal inter-epitope distance, resulted in an overall 66-fold increase in K(A). Thermodynamic analysis of binding indicates that the formation of high-affinity ODN-IgG complexes was a spontaneous and exothermic event, characterized by large negative deltaS degrees, deltaH degrees, and deltaG degrees values.
  
  
  CONCLUSIONS
  All four strategies tested during this investigation, namely bivalence, epitope density, inter-epitope linker flexibility, and optimal inter-epitope distance, proved to be useful in improving the binding affinity of DNP-labeled ODNs to anti-TNP IgG. The final ODN design incorporating these strategies will be used in testing the systemic pharmacokinetic advantage gained from complexing such ODNs to IgG.
authors.affiliation: Division of Drug Delivery and Disposition, School of Pharmacy, The University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Enzo
authors.initials: E
authors.lastname: Palma
authors.forename: David G
authors.initials: DG
authors.lastname: Klapper
authors.forename: M J
authors.initials: MJ
authors.lastname: Cho
mesh_terms: D001666:Binding Sites, Antibody; D004337:Drug Carriers / Q000138:chemical synthesis / Q000378:metabolism*; D015195:Drug Design; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D004337:Drug Carriers; D007074:Immunoglobulin G; D009841:Oligonucleotides