Thermodynamic and Binding Effects of Antibody Affinity Maturation

S. Sen , F. Wang , S. Sun , T. J. Magliery , P. G. Schultz 4 1 WIL Research Laboratories, LLC, 2 Scripps Research Institute, 3 Ohio State University, 4 The Scripps Research Institute Purpose Affinity maturation involves production of matured antibodies that has increased specific affinity towards an antigen duri the course of an immune response, brought about by B cells. Matured antibodies differ from its germline precursor not on by active site mutations (functional) but also by many additional peripheral non-functional mutations. The role of these no functional, somatic hypermutations are not known. In this study we analyzed the effects of individual somatic mutations a combinations thereof on both antibody binding affinity and thermal stability for different antibody fragments, thereby providing generalized results. Methods We have used site directed mutagenesis and an extrinsic fluorophore based method (Differential Scanning Fluorimetry or DSF) to understand the stability effects of individual and grouped mutations along with their effects on hapten binding us surface plasmon resonance (SPR). Differtial scanning calorimetry or DSC has been used to validate the thermodynamic stability obtained by DSF. We have also expressed the antibodies in insect cells to quantitate the yields for different constructs. Finally, X-ray crystallography was used to gain structual insights of different antibody constructs. Results Individual somatic mutations that enhance binding affinity to hapten decrease the stability of the germline antibody; combining these binding mutations produced a mutant with high affinity for hapten but exceptionally low stability (Figure Adding back each of the remaining somatic mutations restored thermal stability. The expression levels of different antibo constructs correlated with their stability, thereby indicating the exact role of why additional stabilizing mutations are requ during affinity maturation. Crystal structural revealed additonal hydrogen bonding and Van-der-Waals interactions in stabilized antibodies brought about by the non-functional mutations. Conclusion This study provides a generalized mechanism of how and why additonal non-functional mutations occur during the proce of affinity maturation of antibodies. Germline antibodies are stable but lack specificity. Active (functional) mutations enhances affintity in matured antibodies towards specific antigens but at the cost of its overall stability (Figure 2). These additonal mutations help to restore stability without compromising on the function (specificity) of matured antibodies. ng ly nnd