Anticalins: Exploiting a non‐Ig scaffold with hypervariable loops for the engineering of binding proteins

Abstract

a b s t r a c tAntibodies, which can recognize a plethora of possible antigens, have been considered as a paradigm of protein engineering performed by nature itself. Lipocalins constitute a distinct family of proteins with functions in ligand binding and transport that occur in many organisms, including man. Like antibodies, lipocalins exhibit a structurally conserved framework -a b-barrel with an attached ahelix -which supports four structurally hypervariable loops forming a cup-shaped binding site. Thus, lipocalins offer an ideal platform for protein engineering to generate novel binding reagents. Using recombinant/synthetic DNA technology and methods of combinatorial library selection, 'Anticalins' with prescribed target specificities can be easily generated. Anticalins with picomolar affinities have been developed for three classes of ligands having relevance in basic research and/ or medical application: small molecules, peptides, and proteinaceous signalling molecules as well as cell surface receptors. Anticalins derived from human lipocalins have already reached the clinical trial stage. Due to their very small size and simple composition of a single polypeptide chain, which also facilitates the construction of bifunctional fusion proteins, Anticalins promise benefits as a next class of biopharmaceuticals.

Genetically modified proteins: functional improvement and chimeragenesis

“…The synthesis of hybrid proteins by domain fusion or the ligation of chemically or biologically synthesized peptides improves apparently a functional unit utility and is generally used for biological probing approach such as biosensors and bioassays, molecular imaging, targeted cancer or antiinflammatory therapy, regulated drug delivery. 41,42,43,44,78,79,80,81 Hybrid proteins may possess two or more functional activities realized by different modes of action indicating that the molecules of hybrid act as several distinct pharmacophores. Many pharmaceutically or industrially relevant enzymatic processes can also utilize hybrid enzymes with multiple domains.…”

“…Such molecules with a narrow specificity, which are able to bind selectively to carbohydrates, have also a key importance in the development of research related with mechanism of cancerogenesis or inflammation at the molecular level as well as for designing drugs targeted to a relevant molecule. 44,78,79,81 To develop a less complex chimeric fusion protein then the native human lectin MBL with similar ligand recognition and enhanced effector functions, the MBL carbohydrate recognition domain and L-ficolin (L-FCN) collagenous domain were fused and applied to reduce more significantly infection by wild type Ebola virus (Table 1). 79 A method for producing a highly carbohydrate-specific bifunctional hybrid holothurian lectin MBL-AJ, whose expression and purification were monitored by the alkaline phosphatase CmAP activity, has been developed for improving the enzyme-linked MBL-AJ-based assay (ELLA) for cervical cancer diagnosis (Table 1).…”

β-Lactoglobulin interactions with local anaesthetic drugs – Crystallographic and calorimetric studies

“…Lipocalins are often involved in the binding, storage and transport of low-molecular weight hydrophobic ligands [2]. The ability to bind small molecules has made lipocalins an interesting target for utilisation as a new class of drug transporters [3]. Several proteins from this family have been modified and engineered to bind molecular targets with high specificity and selectivity [4].…”

Plant Molecular Farming: Much More than Medicines

“…The ability to produce antibodies as recombinant proteins and create libraries for screening against target antigens has enabled the development of smaller derivatives such as single-chain variable fragment(s) (scFv) and nanobodies [also known as variable heavy-chain antibody fragments (VHHs)] that facilitate screening for improved specificity, affinity, and stability (58,59). Alternative binding scaffolds such as affibodies and anticalins can also be produced as recombinant proteins (60)(61)(62). Recombinant antibodies, fragments, and novel binders can also be produced as fusion proteins that extend their…”