Limited diversity of human scFv fragments isolated by panning a synthetic phage-display scFv library with cultured human melanoma cells.

To broaden the specificity of the Abs recognizing human melanoma-associated Ags (MAAs), we have isolated human single-chain fragment of the V region (scFv) fragments by panning the synthetic phage Ab library (#1) with the human melanoma cell lines S5 and SK-MEL-28. All of the isolated scFv fragments reacted with the mouse mAb defined high molecular weight melanoma-associated Ag (HMW-MAA). scFv #70 immunoprecipitates the two characteristic subunits of HMW-MAA, while scFv #28 only immunoprecipitates its large subunit. These results challenge the current view regarding the structure of HMW-MAA and indicate that it consists of two independent subunits. The human scFv fragments share some similarities with the mouse anti-HMW-MAA mAb. Like mAb 149.53 and 225.28, scFv #28 reacts with rat B49 neural cells that express a homologue of HMW-MAA. scFv #70 reacts with a determinant that is spatially close to the one identified by mAbs 149.53, VT68.2, and VT86. Besides suggesting similarities in the recognition of human melanoma cells by the mouse and human Ab repertoire, these results indicate that the Abs isolated from synthetic Ab libraries resemble those that are found in natural Ab repertoires. The restricted diversity of the scFv fragments that were isolated by panning synthetic Ab libraries with different melanoma cell lines suggests that certain Ags, like HMW-MAA, are immunodominant in vitro. This phenomenon, which parallels the in vivo immunodominance of certain Ags, implies that the antigenic profile of the cells used for panning determines the specificity of the preponderant population of isolated Abs.

High resolution mapping of the B cell epitopes of staphylokinase in humans using negative selection of a phage-displayed antigen library.

Staphylokinase (Sak), a 16-kDa protein secreted by Staphylococcus aureus, induces fibrin-specific thrombolysis in patients with thrombotic disorders. However, Sak also elicits high titers of neutralizing Abs that persist for several months and preclude its repeated use in humans. To identify the antigenic determinants of Sak recognized by humans, a phage-displayed library of Sak variants was selected for mutants that escape binding to an affinity matrix derivatized with patient-specific polyclonal anti-Sak Abs. Fifty-six escape Sak variants were identified after three selection cycles using human polyclonal anti-Sak IgGs obtained from four different patients. DNA sequencing revealed 213 amino acid substitutions, of which 73% were found at 25 positions clustered in eight discontinuous Sak antigenic segments. Although each antigenic segment was recognized to a variable extent by each patient antiserum, the main epitopes of Sak in all patients were roughly targeted to two large discontinuous areas covering 35% of the solvent-accessible surface of Sak. The antigenic area I comprises three segments centered on residues 66, 73, and 135, while the antigenic area II consists of four segments centered on positions 20, 95, 102, and 121. These results suggest that a secondary immune response against Sak can occur in patients, and confirm an initial site-directed mutagenesis study wherein amino acid Lys74 was shown to play a prominent antigenic role. Comprehensive mapping of the most relevant sites of Sak that are antigenic for humans will guide efforts to modulate the immunogenicity of this therapeutically important molecule.