Fundamental information that enhances our understanding of biospecific interactions can be obtained using a new analytical system based on biosensor technology. The functional characteristics of biospecific interaction, such as kinetics, affinity and binding position, are examined by label-free analysis of proteins in free solution binding to an immobilized ligand at a hydrophilic sensor surface.

Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V‐genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V‐genes were shuffled at random and cloned for display as single‐chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V‐genes, with high specificities of binding to human self‐antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)‐10(6) M‐1s‐1, k(off) = 10(−2)s‐1 and Ka = 10(7) M‐1). The kinetics of association are typical of known Ab‐protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.

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Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen. This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response. Here we have improved the affinity of one such "primary" antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V-genes (chain shuffling) obtained from unimmunized donors. For a human phage antibody for the hapten 2-phenyloxazol-5-one (phOx) (Kd = 3.2 x 10(-7) M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity. By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15-fold improved affinity. The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd = 1.1 x 10(-9) M) was comparable to that of mouse hybridomas from the tertiary immune response. Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of 125I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with 125I-EGF.