Isolation of a Highly Specific Ligand for the as/3t Integrin from a Phage Display Library

Our previous studies showed that the txsB~ integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to OL5/31 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by a5/31. Selection for high affinity peptides for o~5/31 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of c~5/31-mediated cell attachment to fibronectin. This peptide is nearly specific for the usB~ integrin, because much higher concentrations were needed to inhibit the Uv#~ integrin, and there was no effect on ot,/33and ot,/35-mediated cell attachment to vitronectin. The peptide also did not bind to the otmfl3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in t~5/31 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-contalning peptide. These results reveal a novel binding specificity in the usBl integrin. F mRONECTiN is the only known protein ligand for the cts~ integrin (Pytela et al., 1985). This binding is mediated by the RGD sequence in the 10th type III repeat of fibronectin (Pierschbacher and Ruoslahti, 1984). Studies with mutated forms of fibronectin have suggested that regions in the 8th and 9th type III repeats are also needed for the full adhesive activity of fibronectin (Obara et al., 1988; Aota et al., 1991; Nagai et al., 1991). These synergistically acting regions may contribute to the specific recognition of fibronectin by OtsB~. The ot5/3~ integrin is important in promoting the assembly of fibronectin matrix and initiating cell attachment to fibronectin (Akiyama et al., 1989; Zhang et al., 1993). Moreover, o~5/~1 also appears to play crucial roles in cell migration (Bauer et al., 1992, 1993; Zhang et al., 1993) as well as tumor invasion and metastasis (Gehlsen et al., 1988; Humphries et al., 1986; Saiki et al., 1989; Seftor et al., 1993). Probing of Otsfl~ function with RGD-containing peptides has suffered from the drawback that these peptides also bind to several other integrins such as the av~3 and a,B5 vitronectin receptors and the Ot~rd~3 fibrinogen receptor of platelets (Ruoslahti and Pierschbacher, 1987). Peptide libraries (Smith and Scott, 1993) offer a way of Address all correspondence to Erkki Ruoslahti, Cancer Research Center, La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037. searching for peptides with improved binding affinities and selectivities for integrins. In previous experiments, we searched for peptides binding to aSBl from a hexapeptide library expressed on the surface of filamentous phage (Koivunen et al., 1993). Among the several phage sequences that bound to asB~ was the cyclic peptide GA*CRGDC*LGA. This peptide, while not specific for asBl, has a 10-fold higher affinity for otsBt than the standard linear RGD peptides such as GRGDSP. The hexapeptide library, even though capable of presenting peptides containing disulfide bonds, seemed to have only a limited repertoire of such peptides. We, therefore, constructed a heptapeptide library in which a random heptapeptide insert is flanked by a cysteine residue on each side. A disulfide bond formed by the cysteines cyclized the random sequences, and as a result the library expresses conformationally constrained peptides capable of high-affinity interactions with receptors. Potent ligands for the o~r~3 integrin have been isolated from such a library based on a cyclic hexapeptide (O'Neil et al., 1992). We describe here novel O¢sB~binding sequences selected from the cyclic heptapeptide library. Materials and Methods