Various important biological pathways are modulated by TGFb isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFb neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFb3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFb1 and TGFb2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFb1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFb2 to 2.83 Å . The structures provide further insight into the details of TGFb recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoformspecific antibodies using a fresolimumab scaffold.

Most antibodies display very low brain exposure due to the blood-brain barrier (BBB) preventing their entry into brain parenchyma. Transferrin receptor (TfR) has been used previously to ferry antibodies to the brain by using different formats of bispecific constructs. Tetravalent bispecific tandem immunoglobulin Gs (IgGs) (TBTIs) containing two paratopes for both TfR and protofibrillar forms of amyloid-beta (Aβ) peptide were constructed and shown to display higher brain penetration than the parent anti-Aβ antibody. Additional structure-based mutations on the TfR paratopes further increased brain exposure, with maximal enhancement up to 13-fold in wild-type mice and an additional 4–5-fold in transgenic (Tg) mice harboring amyloid plaques, the main target of our amyloid antibody. Parenchymal target engagement of extracellular amyloid plaques was demonstrated using in vivo and ex vivo fluorescence imaging as well as histological methods. The best candidates were selected for a chronic study in an amyloid precursor protein (APP) Tg mouse model showing efficacy at reducing brain amyloid load at a lower dose than the corresponding monospecific antibody. TBTIs represent a promising format for enhancing IgG brain penetration using a symmetrical construct and keeping bivalency of the payload antibody.

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