Antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, repeated thromboembolic phenomena, and thrombocytopenia. The syndrome is believed to be caused by antiphospholipid beta-2-glycoprotein-I (␤2GPI)-dependent Abs or anti-␤2GPI Abs by themselves. Using a hexapeptide phage display library, we identified three hexapeptides that react specifically with the anti-␤2GPI mAbs ILA-1, ILA-3, and H-3, which cause endothelial cell activation and induce experimental APS. To enhance the binding of the peptides to the corresponding mAbs, the peptides were lengthened to correspond with the site of the ␤2GPI epitope being recognized by these mAbs. As a result, the following three peptides were prepared: A, NTLKTPRVGGC, which binds to ILA-1 mAb; B, KDKATFGCHDGC, which binds to ILA-3 mAb; and C, CATLRVYKGG, which binds to H-3 mAb. Peptides A, B, and C specifically inhibit both in vitro and in vivo the biological functions of the corresponding anti-␤2GPI mAbs. Exposure of endothelial cells to anti-␤2GPI mAbs and their corresponding peptides led to the inhibition of endothelial cell activation, as shown by decreased expression of adhesion molecules (E-selectin, ICAM-1, VCAM-1) and monocyte adhesion. In vivo infusion of each of the anti-␤2GPI mAbs into BALB͞c mice, followed by administration of the corresponding specific peptides, prevented the peptide-treated mice from developing experimental APS. The use of synthetic peptides that focus on neutralization of pathogenic anti-␤2GPI Abs represents a possible new therapeutic approach to APS.

Basic fibroblast growth factor (bFGF) is known to bind to its cell-surface receptors with high aitmity and in a heparin-dependent manner. In an attempt to predict the receptor recognition site on bFGF we screened phageepitope libraries with monoclonal antibodies DG2 and DE6, which inhibit bFGF binding to its receptor. On the affinityisolated phages, we identified several peptide sequences as the putative antibody-binding epitopes on bFGF. Two monoclonal antibodies (mAbs) raised against human recombinant bFGF were found to efficiently block binding of bFGF to its cell-surface receptors (17). These two mAbs, designated DG2 and DE6, inhibited bFGF-stimulated proliferation of cultured human glioma cells and retarded rat C6 glioma growth in nude mice (18). A recent study showed that the administration of neutralizing mAbs to bFGF caused significant alterations in tumor growth in vivo and that these changes were specific for tumor type and bFGF characteristics (8). The inhibitory effects of the antibodies on bFGF binding and biological activities suggest that the bFGF epitopes recognized by the antibodies may coincide with sequences recognized by the receptor. To identify these sequences we used these antibodies to screen a phageepitope library (19).The library consists of phages bearing random hexapeptides fused to the amino terminus of the coat protein PIII. Screening of the library is accomplished by the use of a monospecific binding protein, such as an antibody, to affinity-purified phages that display a high-avidity binding peptide. The amino acid sequences ofthe hexapeptides displayed on the phage are then determined by sequencing the corresponding coding region in the phage DNA (19). Here we describe the characterization of a series of hexapeptides synthesized according to the epitope sequences identified in the phage-epitope library by screening with mAbs DG2 and DE6. These peptides show a significant ability to inhibit high-affinity receptor binding and biological activity of bFGF. MATERIALS AND METHODSMaterials. Human recombinant bFGF was from Takeda (Osaka). The mAbs DG2 and DE6 have been described (17). Rabbit antibodies against human placenta alkaline phosphatase (AP) or against phage M13 were generated by immunization using a standard procedure (20). Heparin was from Hepar (Franklin, OH). The hexapeptide phage-epitope library was from G. Smith (University of Missouri, Columbia) (19).

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