Abstract
Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scFV), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scFV binding, stability, quaternary structure, and other biophysical properties. Comprehensive studies of these attributes in a single scaffold have not been reported, making design and optimization of Ab fragments challenging. Here, we constructed libraries of 3E8, an Ab specific to tumor-associated glycoprotein 72 (TAG-72), a mucinous glycoprotein overexpressed in 80% of adenocarcinomas. We cloned, expressed, and characterized scFVs, diabodies, and higher-order multimer constructs with varying linker compositions, linker lengths, and domain orientations. These constructs dramatically differed in their oligomeric states and stabilities, not only because of linker and orientation but also related to the purification method. For example, protein L-purified constructs tended to have broader distributions and higher oligomeric states than has been reported previously. From this library, we selected an optimal construct, 3E8.G4S, for biodistribution and pharmacokinetic studies and in vivo xenograft mouse PET imaging. These studies revealed significant tumor targeting of 3E8.G4S with a tumor-to-background ratio of 29:1. These analyses validated 3E8.G4S as a fast, accurate, and specific tumor-imaging agent.
Highlights
S these small batch purifications showed less than 10% deviation of dimer to higher oligomeric state ratio (Fig. S3)
The data from the IMAC-purified constructs demonstrate the versatility of scFvs with respect to linker length, amino acid composition, and domain orientation
Antibody fragments have great potential for clinical use as enhanced cancer imaging tools, but the widespread use of engineered fragments is hindered by their decreased binding affinity and stability and by the resulting difficulty of large-scale production
Summary
Design of tagless3E8 libraryAfter completion of the antibody linker library, a construct was selected for further in vivo characterization. The dimeric nature of this construct created a complex greater than 50 kDa and was predicted to have a longer serum half-life than its monomeric counterparts. The resistance gene from pHLIC was changed from ampicillin to kanamycin to create the new expression vector pHLICK. The His[6] tag was removed from the sequence, and an alternate method of purification by protein L was adapted. This new expression format and purification process increased our protein yields substantially. The protein eluted from a single protein L column was equal, if not greater, in purity than our previously described two column Ni–NTA purification method (Fig. S2)
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