Reliable repurposing of antibody interactome inside the cell

In biology proximity is paramount and eighty-five percent of the human proteome has at least one documented interacting monoclonal antibody. These molecules penetrate the cytoplasm poorly and are very often non-functional within the cell. Sequence analysis of 10 ⁶ antibody variable domains alongside the cytoplasmic human proteome shows charge and isoelectric point are characteristics ill adapted to intracellular monodispersity. Characterisation of forty-five single-chain variable fragment (scFv) intrabodies expressed in human cells confirmed charge to have the greatest impact on solubility. We created new interdomain linkers, optimised scFv domain orientation and found variable heavy domain framework sites to be generally positively charged, and promote insolubility, but be amenable to optimisation. This is applied in combination to reduce the search space and refine the products of AI-led inverse folding to create highly soluble, abundant and thermally stable intrabodies that maintain parent antibody epitope recognition. Over six hundred intrabody sequences are described targeting sixty cytoplasmic proteins with linear, conformational, post-translational modification or oligomeric state specificity. Interactions were validated for p53, α-synuclein, SOD1, polyQ, FUS/TLS, UCHL1 and GFP. This approach removes obstacles hindering intracellular repurposing of the vast sequenced antibody interactome with applications relevant to many human disease states.