Integrin αIIbβ3 specific synthetic human monoclonal antibodies and HCDR3 peptides that potently inhibit platelet aggregation

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Abstract
SPECFIC AIMS The goal of this study was the in vitro evolution of integrin αΙΙbβ3 (platelet glycoprotein IIb/IIIa, GPIIb/IIIa) -specific synthetic human monoclonal antibodies as well as peptides whose sequences are derived from these antibodies. In addition to their high selectivity and affinity for integrin αΙΙbβ3, these antibodies and peptides were anticipated to inhibit platelet aggregation with relevance for antithrombotic therapy and prophylaxis. PRINCIPAL FINDINGS 1. Generation and selection of a synthetic human antibody library with an RAD motif grafted into HCDR3 The interaction of fibrinogen with integrin αΙΙbβ3 is an essential step in platelet aggregation (Fig. 1 ⤻ ). The RGD motif is found in variety of adhesive proteins interacting with integrins αvβ3, αvβ5, αIIbβ3, and α5β1. In previous studies, we showed that adhesive protein-mimicking synthetic human mAbs containing an RGD motif in HCDR3 flanked by six randomized residues could be selected from antibody libraries by phage display. Later, we found that the RGD motif can be diversified to (K/R)XD, where X stands for Val, Ala, Asn, Arg, Thr, Gln, Asp, Ser, or Trp. Although many of the selected antibodies showed a preference to a certain RGD motif binding integrin, none were specific for integrin αIIbβ3. To generate synthetic human antibodies that distinguish integrin αIIbβ3 from other RGD binding integrins, especially integrin αvβ3, the present study was based on a conceptually new library design. First, we used an RAD motif rather than an RGD motif as central recognition sequence. Second, a CX9C disulfide bridge surrounding the central recognition sequence in both previous studies was removed. Third, the randomized HCDR3 sequence VGXXXRADXXXYAMDV was grafted onto a naive human VH library amplified from human bone marrow cDNA. Thus, a second level of library complexity was introduced by diversifying VH. Selection of the new library against integrin αIIbβ3 yielded a new motif-displaying disulfide bridge of the type CX5C that was found in 90% of the selected antibody sequences with the consensus sequence V(V/W)CRAD(K/R)RC. The selected antibodies revealed high specificity toward integrin αΙΙbβ3 but not to other RGD binding integrins αvβ3, αvβ5, and α5β1. All antibodies were reactive with native integrin αIIbβ3 expressed on the surface of human platelets but did not bind to HUVEC cells, which mainly express integrin αvβ3. Figure 1. Schematic diagram illustrating the inhibition of platelet aggregation by integrin αIIbβ3-specific synthetic human monoclonal antibodies and HCDR3 peptides. The interaction of fibrinogen with integrin αΙΙbβ3, mediated in part by an RGD tripeptide motif, is an essential step in platelet aggregation. We used phage display to select monoclonal antibodies specific to integrin αIIbβ3 from a synthetic human antibody library based on the randomized HCDR3 sequence VGXXXRADXXXYAMDV. The selected antibodies revealed a strong consensus in HCDR3 (V(V/W)CRAD(K/R)RC) and high specificity toward integrin αΙΙbβ3 but not to other RGD binding integrins like αvβ3, αvβ5, and α5β1. The antibodies as well as three synthetic peptides (VWCRADRRC, VWCRADKRC, and VVCRADRRC), whose sequences were derived from HCDR3 sequences of the selected antibodies, strongly inhibited platelet aggregation ex vivo by interfering with the interaction of fibrinogen with integrin αΙΙbβ3. Download figure Download PowerPoint 2. The selected antibodies inhibit platelet aggregation by blocking the interaction between integrin αIIbβ3 and fibrinogen In a competitive ELISA assay based on immobilized integrin αIIbβ3, biotinylated fibrinogen, and avidin-HRP, the selected antibodies strongly inhibited the interaction between integrin αIIbβ3 and fibrinogen. Fab RAD87 blocked the interaction between integrin αΙΙbβ3 and fibrinogen in a dose-dependent manner with an IC50 of 8.0 × 10−8 M. Based on competitive ELISA, the Kd value of the monovalent Fab RAD87/integrin αΙΙbβ3 interaction was 3.3 × 10−9 M. An ex vivo platelet aggregation assay, in which platelet aggregation was induced by adding ADP to a final concentration of 20 μM to conditioned plasma prepared from human peripheral blood and monitored by a platelet aggregometer, revealed that Fab RAD87 potently inhibited platelet aggregation with an EC50 of 60 nM or 3 μg/mL (Fig. 2 ⤻ ). Figure 2. Fab RAD87 inhibits platelet aggregation ex vivo. The platelet aggregation assays were derived from a whole blood lumi-aggregometer. For each assay, 15 μL of Fab RAD87 (A) or Fab abciximab (ReoPro; B) solution was mixed with 435 μL of platelet-rich plasma to reach a final concentration between 20 and 100 nM. Then ADP was added to a final concentration of 20 μM and the aggregation was monitored for 10 min. Plotted mean values and standard deviations of 3 independent experiments are shown in panel C. Download figure Download PowerPoint 3. Disulfide constrained nonapeptides derived from the selected HCDR3 sequences of the integrin αΙΙββ3-specific antibodies retained their inhibitory characteristics Four nonapeptides whose sequences were derived from the selected HCDR3 sequences of the integrin αIIbβ3 binding antibodies were chemically synthesized. This panel included three cyclic peptides, VWCRADKRC, VWCRADRRC, and VVCRADRRC, and linear peptide THSRADRRE. Based on competitive ELISA, all cyclic peptides were found to inhibit the interaction between integrin αΙΙbβ3 and fibrinogen at micromolar concentrations. By contrast, the linear peptide and three control peptides—two with an inversed RAD motif (VVCDARRRC and THSDARRRE) and one with an unrelated sequence—were not inhibitory. In the ex vivo platelet aggregation assay, all three cyclic peptides but neither linear nor control peptides completely inhibited platelet aggregation at a concentration of 90 μM (Fig. 3 ⤻ ). The most potent inhibitor was cyclic peptide VWCRADRRC, which inhibited platelet aggregation at a concentration as low as 9 μM (Fig. 3)⤻ . Figure 3. Synthetic peptides derived from selected HCDR3 sequences inhibit platelet aggregation ex vivo. Platelet aggregation assays were performed as described in Fig. 2⤻ in the presence of peptides. Note that all three cyclic peptides with the RAD motif potently inhibit platelet aggregation (A–C) whereas a linear peptide with the RAD motif and two control peptides with an inversed RAD motif have no effect over background (D). Abciximab Fab (ReoPro) was used as positive control in all platelet aggregation assays. Download figure Download PowerPoint CONCLUSIONS AND SIGNIFICANCE Three integrin αΙΙbβ3 inhibitors—abciximab, eptifibatide, and tirofiban—have clinical approval in the United States. Abciximab, the first one to be approved and the most widely used agent, is a chimeric Fab with mouse variable and human constant domains. Due to its chimeric composition, thrombocytopenia, often severe, occurs in 4% of patients treated with abciximab. Eptifibatide and tirofiban are small molecule drugs that specifically bind to the RGD ligand interaction site of integrin αΙΙbβ3. Typical of small molecule drugs, eptifibatide and tirofiban are of lower affinity and shorter circulatory half-life than abciximab. While their potential application as antithrombotic drugs overlaps with abciximab, the RAD antibodies we describe here have several distinctive features. First, they are human antibodies, which are less likely to induce an immune response in the patients. Second, the RAD antibodies directly block the RGD binding site of integrin αΙΙbβ3. Abciximab binds to an epitope adjacent to the ligand binding region and inhibits fibrinogen binding by steric hindrance. Third, the RAD antibodies selectively bind to integrin αΙΙbβ3 whereas abciximab cross-reacts with αvβ3 and αMβ2. The display of peptide libraries within the antibody immunoglobulin variable domain provides an intriguing link between antibody, peptide, and peptidomimetic drug discovery. The placement of bioactive and/or binding peptides within an antibody scaffold or their directed evolution within the scaffold provides for the rapid development of immunological agents that can be used as biological tools or therapeutics. The display of peptides within an antibody addresses detection problems as well as problems associated with proteolysis of peptides and their rapid clearance, since antibodies and antibody fragments exhibit relatively predictable pharmacokinetic behavior. Where desirable—for example, in a cancer setting—the Fc region of the antibody can bestow cell-killing properties onto the peptide sequence as a result of immune effector coupling. We believe this approach can be applied to a wide range of peptides with binding activity to rapidly generate useful immunological reagents. 1To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0586fje
Funding Information
  • Skaggs Institute for Chemical Biology, Scripps Research Institute