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Integrin _IIbß₃-specific synthetic human monoclonal antibodies and HCDR3 peptides that potently inhibit platelet aggregation¹

JUNHO CHUNG^*,,, CHRISTOPH RADER^*, MIKHAIL POPKOV^*, YOUNG-MI HUR, HYUN-KYUNG KIM, YOUNG-JOON LEE and CARLOS F. BARBAS, III^*

^* The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA;
Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul National University College of Medicine, Jongro Gu, Seoul, 110-799 Korea, and
National Cancer Center 809 Madu-dong, Ilsan-gu, Goyang, Gyeonggi, 411-764, Korea

² Correspondence: The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. E-mail: carlos{at}scripps.edu

SPECFIC AIMS

The goal of this study was the in vitro evolution of integrin _IIbß₃ (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 _IIbß₃, 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 _IIbß₃ is an essential step in platelet aggregation (Fig. 1 ). The RGD motif is found in variety of adhesive proteins interacting with integrins _vß₃, _vß₅, _IIbß₃, and ₅ß₁. 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 _IIbß₃. To generate synthetic human antibodies that distinguish integrin _IIbß₃ from other RGD binding integrins, especially integrin _vß₃, 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 V_H. Selection of the new library against integrin _IIbß₃ 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 _IIbß₃ but not to other RGD binding integrins _vß₃, _vß₅, and ₅ß₁. All antibodies were reactive with native integrin _IIbß₃ expressed on the surface of human platelets but did not bind to HUVEC cells, which mainly express integrin _vß₃.

View larger version (72K): [in this window] [in a new window]   Figure 1. Schematic diagram illustrating the inhibition of platelet aggregation by integrin IIbß3-specific synthetic human monoclonal antibodies and HCDR3 peptides. The interaction of fibrinogen with integrin IIbß3, 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 IIbß3 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 IIbß3 but not to other RGD binding integrins like vß3, vß5, and 5ß1. 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 IIbß3.

2. The selected antibodies inhibit platelet aggregation by blocking the interaction between integrin _IIbß₃ and fibrinogen
In a competitive ELISA assay based on immobilized integrin _IIbß₃, biotinylated fibrinogen, and avidin-HRP, the selected antibodies strongly inhibited the interaction between integrin _IIbß₃ and fibrinogen. Fab RAD87 blocked the interaction between integrin _IIbß₃ and fibrinogen in a dose-dependent manner with an IC₅₀ of 8.0 x 10^-8 M. Based on competitive ELISA, the K_d value of the monovalent Fab RAD87/integrin _IIbß₃ interaction was 3.3 x 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 ).

View larger version (31K): [in this window] [in a new window]   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.

3. Disulfide constrained nonapeptides derived from the selected HCDR3 sequences of the integrin _IIßß₃-specific antibodies retained their inhibitory characteristics
Four nonapeptides whose sequences were derived from the selected HCDR3 sequences of the integrin _IIbß₃ 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 _IIbß₃ 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) .

View larger version (23K): [in this window] [in a new window]   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.

CONCLUSIONS AND SIGNIFICANCE

Three integrin _IIbß₃ 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 _IIbß₃. 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 _IIbß₃. 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 _IIbß₃ whereas abciximab cross-reacts with _vß₃ and _Mß₂.

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.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0586fje

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