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J. Biol. Chem., Vol. 269, Issue 52, 32788-32795, Dec, 1994
JW Smith, D Hu, A Satterthwait, S Pinz-Sweeney and CF Barbas 3rd
An antibody engineering strategy was employed to build high affinity
ligands and antagonists of integrins alpha v beta 3 and alpha IIb beta 3.
Previously, we inserted the integrin recognition motif, RGD, into the
antigen binding site of a human antibody and selected the optimal flanking
sequences from a phage-display library (Barbas, C. F., Languino, L. R., and
Smith, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10003-10007). The
resulting antibody, Fab-9, blocked the function of integrin alpha v beta 3
but also bound to the ligand binding site of platelet integrin alpha IIb
beta 3. In this report, the antibody engineering effort has been extended
by 1) redesigning Fab-9 to achieve specificity for platelet integrin alpha
IIb beta 3, 2) building non-RGD- containing antibodies that bind the ligand
binding site of both beta 3- integrins, and 3) testing the hypothesis that
peptides derived from complementarity determining regions (CDR) can be used
to emulate the activity of the parent synthetic antibody. These goals were
accomplished by subjecting the original antibody, Fab-9, to a "motif
optimization" (MTF). A phage library was constructed in which the residues
flanking the RGD motif in Fab-9 were maintained, but the RGDX sequence was
randomized. This library was panned on purified alpha IIb beta 3 to
identify high affinity binders. Four function-blocking antibodies lacking
RGD, but with specificity for alpha IIb beta 3, were characterized. The
antibody with the highest preference for alpha IIb beta 3, MTF-10, had an
adhesion sequence of KGDN. This sequence is similar in primary structure to
the active sequence within the disintegrin barbourin, which also
antagonizes alpha IIb beta 3 (Scarborough, R. M., Rose, J. W., Hsu, M. A.,
Phillips, D. R., Fried, V. A., Campbell, A. M., Nannizzi, L., and Charo, I.
F. (1991) J. Biol. Chem. 266, 9359-9362). MTF-10 had a 70-fold higher
affinity for alpha IIb beta 3 than alpha v beta 3. Through our selection
strategy, we also identified several antibodies that lack RGD but still
blocked ligand binding to both integrins with high affinity. Therefore, the
RGD sequence is not necessary for a high affinity interaction with the
ligand binding site of beta 3-integrins. Further investigation showed that
the activity of inhibitory antibodies could be emulated by synthetic
peptides derived from the protein sequences of the antibody's HCDR3.
CDR-derived peptides blocked ligand binding to integrins and maintained
essentially the same specificity as the parent antibody.
Building synthetic antibodies as adhesive ligands for integrins
Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037.
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