The production, characterisation and enhanced pharmacokinetics of scFv–albumin fusions expressed in Saccharomyces cerevisiae

Monovalent Fc receptor blockade by an anti–Fcγ receptor/albumin fusion protein ameliorates murine ITP with abrogated toxicity

“…The inhibitor constants for 3G8 and 3G8 scFv-MSA are ;1 nM and 40 nM, respectively ( Figure 1B), demonstrating lowered binding efficiency of 3G8 scFv-MSA compared with its parent antibody 3G8 ( Figure 1B), likely as a result of reduced multivalency and protein domain rearrangement. [24][25][26] This may be important when the construct is developed for human ITP. To investigate the in vivo efficacy and adverse event profile of monovalent targeting, we next generated 2.4G2 scFv-MSA fusion protein, the murine counterpart of 3G8 scFv-HSA that targets murine FcgRIII/IIB.…”

Single-chain Variable Fragment Albumin Fusions Bind the Neonatal Fc Receptor (FcRn) in a Species-dependent Manner

“…Albumin Fusion Formats-To address how genetic fusion of proteins to HSA affects binding of HSA to FcRn, several HSA fusion variants were constructed using a synthetic HSA gene that is codon-optimized for expression in S. cerevisiae (25). A model peptide HSA fusion was constructed by adding the sequence corresponding to the FLAG tag (DYKDDDDK (2 kDa)) to the N-or C-terminal end, whereas an scFv fragment (12 kDa) with specificity for FITC was fused to either the N-terminal or the C-terminal end or both.…”

“…Plasmids containing expression cassettes for the production of scFv genetically fused to HSA, at either the N terminus or the C terminus or both, and a FLAG (FG) sequence (DYKDDDDK) genetically fused to HSA were used as described (25). Expression plasmids were used to transform Saccharomyces cerevisiae D638 cir 0 (pmt1 mutant derived of DYB7) using methods described (25,26). An expression cassette containing the FLAG tag genetically fused to the N terminus of HSA was prepared using PCR and in vivo cloning (i.e.…”

“…pDB4643 was digested with NsiI/ PvuI, and the DNA was purified using a Qiagen PCR purification kit as per the manufacturer's instructions before being used, along with Acc65I/BamHI-digested pDB3936, to co-transform S. cerevisiae D638 cir 0 as described (15,26). Construction and production of N-and C-terminal fusions of an scFv fragment with specificity to fluorescein isothiocyanate (FITC) have previously been described (25). All fusions of scFv to the C terminus of HSA have a linker, (GGS) 4 GG, referred to as GS in Fig 1. Construction of Mouse, Rat, and Rhesus Monkey AlbuminExpression cassettes for mouse, rat, and rhesus macaque albumin (MSA, RSA, and MySA) (accession numbers: BC024643, BC085359, and NP_001182578.1, respectively) were prepared by GeneArt GmbH.…”

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

“…The albumin variants were produced in Saccharomyces cerevisiae and monomeric fractions were purified using the AlbuPure TM matrix (ProMetic BioSciences) followed by chromatography as described (10). Construction and production of WT HSA, K500A, and K573P with N-or C-terminal fusions of a scFv fragment with specificity for fluorescein isothiocyanate were done in accordance with previously described procedures (22,26). WT HSA and K573P with N-terminal fusions of a c-Myc tag (EQKLISEEDL) without a linker sequence were constructed essentially as previously described (10), followed by purification using AlbuPure TM , diethylaminoethyl weak anion exchange Sepharose Fast Flow (GE Healthcare), and Sephacryl S200 high resolution gel filtration (GE Healthcare) as to reduce the level of a ϩ2058-Da miscleaved leader to below 5% (w/v).…”