Label-free epitope binning assays of monoclonal antibodies enable the identification of antigen heterogeneity.

Label-free biosensors are often used in the discovery of therapeutic antibodies to characterize the epitope binding regions of a panel of monoclonal antibodies that target a specific antigen, thus facilitating their organization into epitope groups or "bins". When tested in a pairwise combinatorial manner, two antibodies that compete with one another for binding to a specific antigen may be grouped into the same epitope bin - that is, they recognize similar or overlapping epitopes - whereas two antibodies that bind simultaneously to the antigen are placed into different epitope bins. However, depending on the assay format used, results from such experiments can sometimes contradict one another. Here, we provide two examples that illustrate how antigen heterogeneity, either inherent in an antigen sample, or induced by the assay conditions, can confound the interpretation of epitope binning results and, in some cases, lead to erroneous conclusions. We highlight why assays that employ solution antigen are often more reliable than those that employ immobilized antigen and, by corroborating our binning results with assays that utilize native antigen, we determine which subpopulations of our heterogeneous antigen samples are biologically relevant and thus improve the correlation between epitope bins and functional activity. Furthermore, we provide recommendations for performing definitive binning assays and a diagnostic assay procedure that can be followed when antigen heterogeneity is suspected.

Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D) values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent K(D) and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.

The application of target information and preclinical pharmacokinetic/pharmacodynamic modeling in predicting clinical doses of a Dickkopf-1 antibody for osteoporosis.

PF-04840082 is a humanized prototype anti-Dickkopf-1 (Dkk-1) immunoglobulin isotype G(2) (IgG(2)) antibody for the treatment of osteoporosis. In vitro, PF-04840082 binds to human, monkey, rat, and mouse Dkk-1 with high affinity. After administration of PF-04840082 to rat and monkey, free Dkk-1 concentrations decreased rapidly and returned to baseline in a dose-dependent manner. In rat and monkey, PF-04840082 exhibited nonlinear pharmacokinetics (PK) and a target-mediated drug disposition (TMDD) model was used to characterize PF-04840082 versus Dkk-1 concentration response relationship. PK/pharmacodynamic (PK/PD) modeling enabled estimation of antibody non-target-mediated elimination, Dkk-1 turnover, complex formation, and complex elimination. The TMDD model was translated to human to predict efficacious dose and minimum anticipated biological effect level (MABEL) by incorporating information on typical IgG(2) human PK, antibody-target association/dissociation rates, Dkk-1 expression, and turnover rates. The PK/PD approach to MABEL was compared with the standard "no adverse effect level" (NOAEL) approach to calculating clinical starting doses and a pharmacological equilibrium method. The NOAEL method gave estimates of dose that were too high to ensure safety of clinical trials. The pharmacological equilibrium approach calculated receptor occupancy (RO) based on equilibrium dissociation constant alone and did not take into account rate of turnover of the target or antibody-target complex kinetics and, as a result, it likely produced a substantial overprediction of RO at a given dose. It was concluded that the calculation of MABEL according to the TMDD model was the most appropriate means for ensuring safety and efficacy in clinical studies.