Affilins are genetically engineered proteins with the ability to selectively bind antigens. They are structurally derived from one of two proteins, gamma-B crystallin or ubiquitin, both occurring in humans. Affilins are constructed by modification of near-surface amino acids of these proteins and isolated by display techniques such as phage display. They resemble antibodies in their affinity to antigens but not in structure, which makes them a type of antibody mimetic. Affilins are being developed as potential new biopharmaceutical drugs.
Two proteins, gamma-B crystallin and ubiquitin, have been described as scaffolds for affilins. Certain amino acids in these proteins can be substituted by others without losing structural integrity, a process creating regions capable of binding different antigens, depending on which amino acids are exchanged. In both types, the binding region is located in a beta sheet structure, whereas the binding regions of antibodies, called complementarity determining regions, are flexible loops.
Based on gamma crystallin
Gamma crystallin is a family of proteins found in the eye lens of vertebrates, including humans. It consists of two identical domains with mainly beta sheet structure and a total molecular mass of about 20 kDa. The eight near-surface amino acids 2, 4, 6, 15, 17, 19, 36, and 38 are suitable for modification.
Based on ubiquitin
Ubiquitin, as the name suggests, is a highly conserved protein occurring ubiquitously in eucaryotes. It consists of 76 amino acids in three and a half alpha helix windings and five strands constituting a beta sheet. The eight surface-near exchangeable amino acids 2, 4, 6, 62, 63, 64, 65, and 66 are located at the beginning of the first N-terminal beta strand (2, 4, 6), at the nearby beginning of the C-terminal strand and the loop leading up to it (63–66). The resulting affilins are about 10 kDa in mass.
The molecular mass of crystallin and ubiquitin based affilins is only one eighth or one sixteenth of an IgG antibody, respectively. This leads to an improved tissue permeability, heat stability up to 90 °C (195 °F), and stability towards acids and bases. The latter enables affilins to pass through the intestine, but like most proteins they are not absorbed into the bloodstream. Renal clearance, another consequence of their small size, is the reason for their short plasma half-life, generally a disadvantage for potential drugs.
A molecular library of affilins is generated by random mutagenesis. Substituting some or all of the eight amino acids at the potential binding site with one of the nineteen proteinogenic amino acids excluding cysteine gives 198 ≈ 17,000,000,000 possible combinations. Cysteine is excluded because of its liability to form disulfide bonds. In a dimeric affilin, up to 14 amino acids are exchanged, resulting in 8 × 1017 combinations, but not all of these are realised in a given library.
The next step is the selection of affilins that bind the desired target protein. To this end display techniques such as phage display or ribosome display are used. The fitting species are isolated and characterised physically, chemically and pharmacologically. Subsequent dimerisation or multimerisation can increase plasma half-life and, due to avidity, affinity to the target protein. Radionuclides or cytotoxins can be conjugated to affilins, making them potential tumour therapeutics and diagnostics. Conjugation of cytokines has also been tested in vitro.
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- Scil Proteins, the developer