Use of multicellular human liver models to investigate clearance of therapeutic antibodies.

Bispecific antibodies (BsAbs) can bind two distinct epitopes simultaneously unlike monovalent mAbs. These novel mAb scaffolds are of great interest to the Biopharmaceutical industry with the potential to provide improved efficacy and safety over monospecific or dual therapies. However, the ability to bind bispecifically may predispose them to form immune complexes, with the potential for accelerated clearance.
It has been proposed that liver sinusoidal endothelial cells (LSECs) could be the main cellular route by which BsAbs are cleared (13) although there is some debate in the literature as to the main routes of elimination and cell types involved (e.g. (14).
The ability to access and utilise primary human cells and tissue would provide a significant advantage in the study of antibody clearance mechanisms. Of particular interest are the whole liver perfusion technology as there is evidence that hepatic cells undergo phenotypic changes upon continued tissue culture thus are not fully representative of in-vivo pharmacokinetics (15 & GSK internal data).
We propose using the models described in this proposal to study binding, internalisation and subsequent clearance of multiple antibody formats in order to gain a better understanding of the molecular mechanisms involved. Of particular interest are elucidation of cell-type and cell-surface receptors responsible for internalisation, identification of antibody-format dependant pathways, understanding of antibody - receptor binding and evaluation of possible engineering strategies designed to mitigate the effects of such clearance pathways.

Specific aims of this project:
Identification of tissue localisation of binding and cell-types involved as well as critical cell surface receptors responsible for binding and internalisation.
Understand the impact of differing antibody formats on internalisation and identify any format specific pathways involved.
Elucidate key structural motifs involved in enhanced clearance and the effect of suitable modifications on binding and internalisation rates

Suitable methodology includes IHC & immunofluorescence, Flow cytometry, genetic ablation of receptors (siRNA / CRISPR), protein engineering, Affinity determination.