Predict Non-Targeted Clearance of Therapeutic Antibodies Using a Human FcRn Dependent in vitro Transcytosis Assay

Abstract

The advancement in the protein engineering of therapeutic antibodies has enhanced biologic treatments of antibody-based proteins. The industry is evolving from monoclonal immunoglobulin (IgG) format to IgG antibody-based proteins capable of binding multiple epitopes either on the same or distinct targets. By design, these uniquely structured and functional proteins have the capability to address medical needs that require a multi-targeted approach. There are many challenges these new multispecific IgG-based proteins encounter including understanding what effects the complexities of their structure and function will have not only on actual drug development, but also on their pharmacokinetics (PK) compared to their monoclonal counterparts. Non-targeted biological features that guide the disposition of these multispecific proteins are not fully understood. In vitro assays are one way to predict and evaluate PK parameters and are a great alternative to the animal studies typically used for these types of evaluations, especially in circumstances when the animals do not demonstrate human characteristics. Implementation of a cell-based assay that is designed in such a way to overcome limitations of an in vitro assay approach to PK prediction provides an alternative to expensive, low-throughput, non-translatable in vivo studies. Non-specific clearance of an antibody-based biotherapeutic can be influenced by several different properties, one of which is the interactions with neonatal Fc receptor (FcRn) which plays an important role in serum immunoglobulin (IgG) homeostasis. The Fc region of the antibody interacts with the Fc receptor and contributes to the recycling of the antibody back to the cell surface and releasing it into the extracellular fluid (Kamath, 2016). An internally produced Madin-Darby canine kidney (MDCK II) cell line overexpressing huFcRN was used to establish a transcytosis-based assay format used as a surrogate for the in vitro recycling process. This method proved capable of predicting non-specific clearance for monoclonal antibodies demonstrated by a correlation between in vitro and in vivo results (Chung et al., 2019). Will the same hold true for multispecific antibody-based biotherapeutics? Various marketed multispecific antibody therapeutics were tested in the huFcRN transcytosis assay. The goal of this work was to establish correlation between huFcRN cell transcytosis levels and in-house rat clearance data and published human clearance data for these marketed multispecific antibody therapeutics. The purpose of this work was to assess a huFcRn in vitro transcytosis assay as a potential method to quantitatively predict non-targeted clearance of multispecific antibody therapeutics in humans, ultimately supporting drug candidate selection and or optimization. Under the conditions tested and presented in this paper the assay is not suited to provide this type of analysis for multispecific antibodies. There is still work that can be done to optimize the current assay format to better align with the huFcRn recycling process.