In the development of novel drug conjugates alternatives to monoclonal antibodies (mAbs) have started to emerge. While antibodies have proven to be useful tools in a broad range of applications, including the development of antibody-drug conjugates, they also possess some inherent limitations. For example, as a result of their complex structure and extensive glycosylation, antibodies are fragile molecules with low yields in bacterial expression systems, having a high temperature sensitivity, and high production costs.
Furthermore, the correct folding relies on the formation of intramolecular disulphide bonds and as a consequence they frequently do not function in the reducing environment inside eukaryotic cells. Finally, it is often very challenging or impossible to raise antibodies that are able to distinguish between different isoforms or mutants of proteins, an often wished for tool in clinical diagnostics.
Hence, scientists have been looking for alternatives to antibodies. Among these alternatives, Engineered Scaffold Proteins or (ESPs) are, buy some scientists, considered to be the next-generation non-immunoglobulin-based therapeutics. These are derived from small, robust non-immunoglobulin proteins, which are used as “scaffolds” for supporting a surface with the ability to specifically interact with the desired target antigens with high affinity, such as receptors overexpressed on cancer cells. 
Affibody molecules are small affinity-engineered scaffold proteins (6-7 kDa) which can be engineered to bind to a number of desired targets are one of the most studied ESPs. They are engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies, and are therefore a member of the family of antibody mimetics.
The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated in a variety of studies.
Biodistribution studies in mice demonstrated elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody-drug conjugates have shown a moderate- to high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs.
Scientists from Uppsala University in Uppsala, Sweden, aimed to reduce the hepatic uptake of AffiDCs, while, at the same time optimizing their in vivo targeting properties. In their study, they investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (ZHER2:2891)2-ABD-MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake.
As part of their study, two new AffiDCs including either a triglutamate-spacer-, (ZHER2:2891)2-ABD-E3-MC-DM1, or a hexaglutamate-spacer-, (ZHER2:2891)2-ABD-E6-MC-DM1 next to the site of MC-DM1 conjugation were designed.
The scientists radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (ZHER2:2891)2-ABD-MC-DM1 drug conjugate without a glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity.
A comparative biodistribution study of the three radiolabeled AffiDCs demonstrated that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake.
The observed results confirmed, according to the Swedish researchers, the relation between DM1 hydrophobicity and liver accumulation. And based on their findings, the scientists believe that the drug development approach they described may be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.
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 Ding H, Altai M, Rinne SS, Vorobyeva A, Tolmachev V, Gräslund T, Orlova A. Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody-DM1 Drug Conjugates. Cancers (Basel). 2019 Aug 14;11(8). pii: E1168. doi: 10.3390/cancers11081168. [Pubmed][Article]