To understand this intriguing finding, the molecular properties of VEGF and the binding sites of its antagonists must be considered. VEGF
165 consists of a receptor-binding domain (AS8–109) and of the heparin-binding domain (AS111–165). The receptor-binding domain binds to VEGFR-2 and induces signal transduction and, consequentially, migration and proliferation. The primary purpose of the heparin-binding domain is to enhance VEGF-induced signaling by binding to the cell surface or the neuropilin-1 coreceptor.
9 10 11 The binding sites of bevacizumab and ranibizumab are in the receptor-binding region (AS82–91).
12 Pegaptanib, in contrast to bevacizumab and ranibizumab, has been developed to bind to VEGF
165. VEGF
165 and VEGF
121 differ in the heparin-binding domain (AS111–165); this is the only region in which pegaptanib can bind to VEGF
165 without inhibiting other isoforms.
13 Consequently, it does not affect the receptor-binding domain (AS8–109) because the domains are identical in VEGF
165 and VEGF
121. Bevacizumab and ranibizumab, on the other hand, bind to all isoforms.
12 14 15 Bevacizumab and ranibizumab bind only weakly to rodent VEGF, as in rodent VEGF, the glycine at AS88 is exchanged with a serine.
10 They do bind to porcine VEGF, which does not carry this mutation. The company that produces the ELISA (R&D Systems) could not provide the exact epitope to which their VEGF antibody binds (personal communication, November 16, 2007), but the antibody does bind to VEGF
165 and VEGF
121. Therefore, it must be in the receptor-binding domain. It is reasonable that the binding of bevacizumab or ranibizumab to VEGF sterically inhibits the recognition of VEGF by the ELISA antibody; hence, neutralization is seen. Pegaptanib binds VEGF at a different epitope that does not impede the binding of ELISA. (The heparin-binding domain of porcine and human VEGF is identical.) Although one could argue that the difference between pegaptanib and the other VEGF antagonists is related to the experimental design, this does in fact offer an explanation as to why pegaptanib is not as effective in vivo as ranibizumab. The heparin-binding site assists the association of VEGF
165 with the cell membrane, ameliorating receptor activation, and binds to NP-1, enhancing the effect of VEGF
165,
13 16 and it has some importance for the mitogenic potency of VEGF,
11 which explains why pegaptanib shows clinical effects. Binding to the heparin-binding site, however, as indicated in this article, does not efficiently prevent the binding of VEGF
165 to its receptor site. Hence, it does not inhibit VEGF-induced angiogenesis. Pegaptanib inhibits the amplification of VEGFR signaling, whereas bevacizumab and ranibizumab inhibit VEGFR signaling itself
(Fig. 6) .