Danlin Yang, University of New Hampshire, Durham


A central dogma in immunology is that the in vivo functionality of an antibody is mediated by two independent events: antigen binding by the variable (V) region, followed by effector activation by the constant (C) region. It is widely accepted that the antibody effector functions are achieved by associative interactions, through crosslinking of multiple IgGs with both target antigen and effector molecules of the immune system. However, this view has been recently challenged, by reports suggesting allostery exists between the V and C regions, triggered by C-region-subclass configurational differences. The possibility of allosteric signals propagating through the IgG domains complicates our understanding of the antibody structure-function relationship, and challenges the current subclass selection process in therapeutic antibody design. The goal of this dissertation was to clarify the existence of allosteric (intramolecular) and associative (intermolecular) cooperativity in IgG antibodies by examining the extent of V-C domain cooperativity and the C-region-subclass mediated effects in solution.

The research was conducted using a panel of 12 monoclonal antibodies comprising 3 unique V regions each grouped into 4 human IgG subclasses, constructed and produced by NS0 stable cell lines. This IgG matrix was characterized by various biophysical techniques in storage and physiological solution conditions to address two central questions related to intramolecular and intermolecular cooperativity. The first question concerned the origin of allostery and the underlying mechanism for intramolecular signaling through the IgG domains. Binding studies by surface plasmon resonance combined with thermal denaturation studies by differential scanning calorimetry delivered fundamental results that contradicted the hypothesis of C-region-mediated configurational allostery. Neither technique detected V-C signal transmission in either direction or change in respective V and C interactions. The equivalence in unfolding enthalpy between the sum of cleaved fragments and the full length IgG further confirmed the lack of intramolecular interactions. Instead, changes in V-region antigen binding were found to be caused by impure reagent and not by C-region subclass switch. In addition to the data being consistent with V-C independence, the intramolecular stability of the IgG molecules was found to be influenced by both V- and C- regions as a result of their intrinsic, sequence-dependent properties.

The second question centered on IgG nonspecific intermolecular interactions in high concentration solutions. Using analytical ultracentrifugation with fluorescence detection coupled with SEDANAL global analysis, a matrix of hydrodynamic nonideality coefficient (ks) was determined from tracer IgGs sedimenting in solutions of identical, similar, and different background IgG molecules. Comparison of self- and cross- nonideality ks terms revealed the relative interactions between various IgG:IgG pairwise interactions. Assessments of ks at elevated salt concentration and temperature indicated that the detected weak interactions were enthalpic in origin and were pairwise specific (V- and C- region dependent). Notably, native IgGs from human serum exhibited the strongest attractive interaction, perhaps due to charge diversity and sequence heterogeneity. It was also found that these native IgGs exerted associative effect on all other monoclonal antibodies. These findings, therefore, provided evidence for weak intermolecular interactions by IgG molecules and demonstrated proof-of-concept that ks can be used as an indicator for weak protein-protein interactions.

Finally, on the basis of negative findings for allosteric signaling between IgG V and C regions and positive findings for associative interactions in native human IgGs, the combined studies from this research are in accord with the immunological dogma of V-C independence. Furthermore, our results are consistent with a model in which intermolecular interactions are a source of cooperative free energy for effector and complement activation. The weak attractive interactions in human IgGs suggest that the immune system can use the promiscuity of this cooperative energy to take advantage of the polydiversity in the immune response to ensure efficient immunological functions. It is recognized that antigens can perform an associative role by bringing IgGs into close proximity with each other to facilitate Fc crosslinking with immune cells and C1q. But, in scenarios where the epitopes are distributed far apart on the antigen surface, bound IgGs may not be spaced close enough for the necessary crosslinking to occur. However, because it seems that all human IgGs partake in weak attractive interactions, it is not necessary for a particular epitope to be spaced properly to allow intermolecular interactions. The generality of the IgG::IgG cooperative free energy allows for a wider array of epitope spacing to initiate effector functions and complement activation.