Short FcRn-Binding Peptides Enable Salvage and Transcytosis of scFv Antibody Fragments

Abstract

Therapeutic antibodies have become one of the most widely used classes of biotherapeutics due to their unique antigen specificity and their ability to be engineered against diverse disease targets. There is significant interest in utilizing truncated antibody fragments as therapeutics, as their small size affords favorable properties such as increased tumor penetration as well as the ability to utilize lowercost prokaryotic production methods. Their small size and simple architecture, however, also lead to rapid blood clearance, limiting the efficacy of these potentially powerful therapeutics. A common approach to circumvent these limitations is to enable engagement with the half-life extending neonatal Fc receptor (FcRn). This is usually achieved via fusion with a large Fc domain, which negates the benefits of the antibody fragment's small size. In this work, we show that modifying antibody fragments with short FcRn-binding peptide domains that mimic native IgG engagement with FcRn enables binding and FcRn-mediated recycling and transmembrane transcytosis in cell-based assays. Further, we show that rational, single amino acid mutations to the peptide sequence have a significant impact on the receptor-mediated function and investigate the underlying structural basis for this effect using computational modeling. Finally, we report the identification of a short peptide from human serum albumin that enables FcRnmediated function when grafted onto a single-chain variable fragment (scFv) scaffold, establishing an approach for the rational selection of short-peptide domains from full-length proteins that could enable the transfer of non-native functions to small recombinant proteins without significantly impacting their size or structure.

A molecular toolkit for heterologous protein secretion across Bacteroides species

“…The sequences of Nluc and 7D12 were cloned from plasmids pNBU2_erm-TetR-P1T_DP-GH023-NanoLuc (Addgene #117728) and pTrcHIS-wt7D12 (Addgene #125268). The scFv-HER2 was constructed from trastuzumab as previously described 68 . All the promoters, RBS, secretion carriers, and protein cargoes were cloned into pBT vector at the sequence between ampR and ermG , and 3xFLAG as well as a rrnb T1 terminator of E. coli was further introduced at the end of the protein cargo coding region by overhang primer PCR.…”

“…The HER2 extracellular domain (ECD) was purified as previously described 68 . For sdAb-TcdA and toxin A fragment (TcdAf) purification, an overnight culture of E. coli BL21(DE3) harboring the 2Bc-T-TcdAf plasmid was grown overnight at 37 °C with shaking, then diluted 50-fold in 50 mL terrific broth (yeast extract 24g, tryptone 20g, glycerol 4mL, 100 mL KPO 4 buffer [0.17 M KH 2 PO 4 , 0.72 M K 2 HPO 4 ] per liter of medium) with 50 µg/ml kanamycin.…”

“…1E) to secrete six additional proteins with therapeutic and/or diagnostic functions. Three of the six proteins are disease-targeting antibody fragments, including an sdAb targeting tumor necrosis factor alpha (sdAb-TNFα) 64 , an antigen associated with chronic conditions such as inflammatory bowel disease (IBD) 65 ; another sdAb targeting epidermal growth factor receptor (sdAb-EGFR) 66 , commonly overexpressed in colon cancer 67 ; and a single-chain variable fragment targeting human epidermal growth factor receptor-2 (scFv-HER2) 68 , mainly known for its role in breast cancer, but also implicated in colon cancer 69 . This set of proteins allows us to evaluate secretion efficiency across diverse antibody fragment formats while still focusing on targets relevant to gastrointestinal delivery by engineered living therapeutics.…”