Towardin vitro-to-in vivotranslation of monoclonal antibody pharmacokinetics: Application of a neonatal Fc receptor-mediated transcytosis assay to understand the interplaying clearance mechanisms

Abstract

Monoclonal antibodies (mAbs) are a rapidly growing drug class for which great efforts have been made to optimize certain molecular features to achieve the desired pharmacokinetic (PK) properties. One approach is to engineer the interactions of the mAb with the neonatal Fc receptor (FcRn) by introducing specific amino acid sequence mutations, and to assess their effect on the PK profile with in vivo studies. Indeed, FcRn protects mAbs from intracellular degradation, thereby prolongs antibody circulation time in plasma and modulates its systemic clearance. To allow more efficient and focused mAb optimization, in vitro input that helps to identify and quantitatively predict the contribution of different processes driving non-target mediated mAb clearance in vivo and supporting translational PK modeling activities is essential. With this aim, we evaluated the applicability and in vivo-relevance of an in vitro cellular FcRn-mediated transcytosis assay to explain the PK behavior of 25 mAbs in rat or monkey. The assay was able to capture species-specific differences in IgG-FcRn interactions and overall correctly ranked Fc mutants according to their in vivo clearance. However, it could not explain the PK behavior of all tested IgGs, indicating that mAb disposition in vivo is a complex interplay of additional processes besides the FcRn interaction. Overall, the transcytosis assay was considered suitable to rank mAb candidates for their FcRn-mediated clearance component before extensive in vivo testing, and represents a first step toward a multi-factorial in vivo clearance prediction approach based on in vitro data.

Anin vitroFcRn- dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans

“…A number of cell-based assays employing MDCK cells transfected with human FcRn have been developed to support development of therapeutic proteins with prolonged half-lives in humans. [31][32][33][34][35] While some of these assays showed notable correlations between assay output and clearance of engineered molecules with enhanced FcRn binding, none were able to predict clearance of conventional mAbs. In these assays, test molecules were typically loaded into the inner chamber of transwells with acidic buffer (pH <6.0) and the transcytosed molecules were harvested in the outer chamber with basic buffer (pH >7.4).…”

“…Transcytosis assays using Madin-Darby canine kidney (MDCK) cells stably expressing human FcRn have been developed to support development of engineered antibodies or antibody domains with enhanced FcRn binding and engineered FcRn-binding peptide fusion proteins. [31][32][33] The transcytosis readouts from these assays appeared to correlate with test molecules' in vivo clearance. Similar assays have been used to characterize FcRn binding of therapeutic antibodies and Fc-fusion proteins, including wild-type (WT) and engineered Fc variants with varying FcRn binding affinities, as well as oxidized and aggregated antibody samples.…”

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

“…[2,3] Various smaller formats, including single chain variable fragments (scFv), antigen binding fragments (Fab), minibodies etc. [4] as well as antibody structures from alternative sources like camelid antibodies or immunoglobulin new antigen receptors [5] were investigated for their potential use as therapeutics.…”

Radiolabeled IgG antibodies: Impact of various labels on neonatal Fc receptor binding

“…Conjugations of 4‐SCN‐Bz‐TCMC were performed in HEPES (4‐(2‐hydroxyethyl)‐1‐piperazineethansulfonic acid, Gibco, Paisley, UK), at pH 7.5 by a protein concentration of 20 to 30 mg/mL. The conjugations with [ 3 H]NSP and IgGs were prepared according to previously published procedures …”

“…The conjugations with [ 3 H] NSP and IgGs were prepared according to previously published procedures. 24…”