Phage Display Technology for Human Monoclonal Antibodies.

During the last 20 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection and screening procedures to identify lead candidates. One of the most successful methods is based on the use of filamentous phages. Functional Antibodies (Abs) fragments can be displayed on the surface of phages by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridoma technology can be isolated by a series of cycles of selection on the antigen of interest. In this system, antibody genes can be recovered simultaneously with selection and can be easily further engineered, for example by increasing their affinity to levels unobtainable in the immune system, or by modulating their specificity and their effector functions (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction and selection of binder with desired specificity.

An albumin-derived peptide scaffold capable of binding and catalysis.

We have identified a 101-amino-acid polypeptide derived from the sequence of the IIA binding site of human albumin. The polypeptide contains residues that make contact with IIA ligands in the parent protein, and eight cysteine residues to form disulfide bridges, that stabilize the polypeptide structure. Seventy-four amino acids are located in six α-helical regions, while the remaining thirty-seven amino acids form six connecting coil/loop regions. A soluble GST fusion protein was expressed in E. coli in yields as high as 4 mg/l. This protein retains the IIA fragment's capacity to bind typical ligands such as warfarin and efavirenz and other albumin's functional properties such as aldolase activity and the ability to direct the stereochemical outcome of a diketone reduction. This newly cloned polypeptide thus represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency.