Biochemical/Biophysical Approaches in the Development of Biotherapeutics

CANZIANI GA

Augusta, MI, USA

Otro; Annual MPI Research Educational Summit: Advances in Therapeutic Discovery and Drug Development. Novel Therapeutic Biologics: Innovative Molecules and mechanisms.; 2009

MPI-CRO

Antibodies are unique bioactive macromolecules that have shown incredible potential in pharmaceutical applications in the past century. Besides the precise recognition of antigens, IgGs mediate cellular cytotoxicity (ADCC), via macrophages, as well as complement-dependent cytotoxicity (CDC). Due to their integral role in mammalian immune regulation, antibodies are particularly effective in eliminating diseased cells such as tumor, viral-infected, and other pathogenic cells. Antibodies have been the biotherapeutics of choice for the past two decades. The structural and thermodynamic determinants for antigen-antibody binding ultimately define a therapeutic antibody lead. The determinants of specific antigen recognition may be explored by mapping a neutralizing antibody’s binding epitope. An epitope is defined by at least one key contact residue in the target protein, but includes near-all contact residues and their relative energetic contribution (or backbone contributions). The contribution of the antibody and the antigen to binding may be determined by various parameters such as binding affinity (KD), binding kinetics, ΔCp (specific heat change), change in solvent accessible surface area (△SASA), change in binding free energy (△△Gmt-wt, kcal/mol). Mutational analysis has been used to define the epitopes of therapeutic and diagnosis/tracer antibodies and how individual residues of the antigen impact the parameters mentioned above. The ultimate goal is to reveal which residues are involved in specific binding due to their charge, pI, hydrophobic, polar or apolar contribution and thus provide a rational for the design of high affinity antibodies and peptide antagonists for therapeutic use in vivo. The technologies available in the biophysics toolbox for this purpose, and described briefly in this presentation, include isothermal titration calorimetry (ITC), SPR detection of mass change (Biacore), and kinetic exclusion assay Kinexa. Experimentally, the task of measuring the kinetic and thermodynamic properties of biomolecular interactions is limited by the required isolation and purification of active reagents.