Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin.

Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse.

Selection of catalytically active biotin ligase and trypsin mutants by phage display.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.

The use of biosensor technology for the engineering of antibodies and enzymes.

Recently developed scientific instrumentation featuring surface plasmon resonance detection allows the detection of biomolecular interactions in real time and without chemical modification of the binding partners. These biosensors are proving invaluable tools in protein engineering, particularly in research aimed at the isolation and improvement of protein binders and catalysts from macromolecular repertoires containing billions of individual members. This article reviews the use of biosensor technology for the isolation and characterization of engineered antibodies and enzymes.

A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage.

We have aimed at developing a general methodology for the isolation of enzymatic activities from large repertoires of protein displayed on the surface of a filamentous phage. When selecting for protein binders by phage display, phage particles with suitable specificities are physically isolated by affinity capture and amplified by bacterial infection. Selection for catalysis mediated by enzymes displayed on filamentous phage is more difficult, as reaction products (which represent the biochemical memory of the reaction catalysed by the phage particle) diffuse away after the reaction is complete. We reasoned that if we were able to anchor the reaction products on the phage surface, the catalytically active phages could then be physically isolated using specific anti-product affinity reagents. We achieve the conditional anchoring of reaction substrates and products on phage by displaying enzyme-calmodulin chimeric proteins on filamentous phage as gene III fusions. Such phage particles can be targeted in a stable fashion (koff<10(-4) s(-1)) by chemical derivatives of a calmodulin-binding peptide. The peptide-phage complexes are stable in purification procedures such as capture with magnetic beads and polyethylene glycol precipitation, and can be conditionally dissociated by addition of calcium chelators. Glutathione-S-transferase and an endopeptidase were used in model selection experiments to demonstrate that it is possible to isolate catalytic activities from calmodulin-tagged enzymes displayed on filamentous phage, with enrichment factors >50 per round of selection.