Probing Interactions of Therapeutic Antibodies with Serum via Second Virial Coefficient Measurements

Antibody-based therapeutics are the fastest growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. While the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, in ways that are incompletely understood, thereby altering therapeutic effect and disposition. While excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B2) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy (FCS) is able to measure diffusive B2 values directly in undiluted serum. Apparent second virial coefficient (B2,app) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the common approach of isolating specific components and completing independent cross-term virial coefficient measurements is an incomplete representation of nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general, and advance the development of therapeutic antibodies and other protein-based therapeutics. STATEMENT OF SIGNIFICANCEWe present FCS as an orthogonal method to traditional methods for characterizing weak, nonspecific homo- and hetero-interactions through determination of self- and cross-term second virial coefficients, respectively. We also characterize weak interactions between therapeutic antibodies and serum components through determination of an apparent second virial coefficient (B2,app) directly in undiluted serum. Our results suggest that global nonideality effects are antibody-dependent, and that attractive and repulsive interactions with co-solutes are occurring simultaneously. This approach could advance our understanding of the impact of nonideality to the biophysical and pharmacological properties of therapeutic antibodies and other engineered proteins in relevant biological environments, and could accelerate the development and optimization of protein-based therapeutics.