The vascular endothelial growth factors (VEGFs) are a major family of angiogenic proteins involved in endothelial cell activation, proliferation, and survival, particularly during retinal proliferative diseases and tumorigenesis. VEGFs belong to the VEGF-PDGF (platelet-derived growth factor) super-gene family, and are small glycoprotein dimers that bind receptors expressed on vascular and lymphatic endothelial cells. There are currently seven known ligands in the VEGF family: VEGF-A (VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E (viral-derived), and placental growth factor (PlGF)-1 and -2. These VEGF ligands mediate their effects by binding to one or more of the three known VEGF receptors (VEGF-Rs), each of which possess receptor tyrosine kinase activity. VEGF-R1 (Flt-1) is predominantly expressed on endothelial cells and monocytes, binds VEGF and VEGF-B, and appears to mediate endothelial and monocyte migration. VEGF-R2 (i.e., human KDR or murine Flk-1) is mainly expressed on endothelial cells, is selective for VEGF (and particular fragments of VEGF-C and VEGF-D), and mediates VEGF-induced endothelial cell proliferation, survival, and migration, as well as vascular permeability. VEGF-R3 (Flt-4) is mainly expressed on lymphatic endothelial cells and binds VEGF-C and VEGF-D to promote lymphangiogenesis. VEGF-R1, -R2, and -R3 are each expressed on some tumor cells. Binding of VEGF to the VEGF receptors triggers receptor dimerization, leading to subsequent receptor activation and signal transduction. VEGF binding to VEGF-R2 initiates a signal transduction pathway that is dominant in promoting angiogenesis. This pathway involves receptor activation with subsequent induction of intracellular signaling. Receptor activation in this case entails three basic events: (i) VEGF binding to VEGF-R2, (ii) receptor dimerization, and (iii) receptor autophosphorylation (and hence activation) of the receptor tyrosine kinase. Intracellular messengers such as phospholipase C and phosphatidylinositol-3-kinase bind directly to the autophosphorylated form of VEGF-R2 and become phosphorylated by the receptor tyrosine kinase, which subsequently triggers an intracellular cascade of signaling events leading to nuclear signals that ultimately promote cell proliferation, migration, and survival (anti-apoptosis), and increase vascular permeability.
Aberrant angiogenesis is associated with a variety of disease states, including cancer (Holash 2002). VEGF signaling has been verified to play a role in both normal vascular development and the pathologic angiogenesis associated with various diseases (Erikkson 1999; Ferrara 1999; Yancopoulos 2000). VEGF promotes vascular endothelial cell growth and increases vascular permeability (Ferrara 2004).
Previous studies have revealed elevated VEGF expression levels in a majority of tumor types (Berkman 1993; Brown 1993; Brown 1995; Dvorak 1995; Mattern 1996). Studies have also revealed increased VEGF levels in subjects with ocular angiogenic diseases such as wet AMD. Wet AMD accounts for only around 10% of total AMD cases, but causes approximately 90% of blindness arising from AMD. Wet AMD is characterized by choroidal neovascularization (CNV), the development of abnormal blood vessels beneath the retinal pigment epithelium layer of the retina. VEGF-A is believed to play a major role in the formation of these vessels, which leak beneath the macula and cause retinal distortion and vision deterioration.
Therapeutic anti-VEGF antibodies are currently available. For example, the humanized IgG1 monoclonal antibody Bevacizumab (a.k.a., Avastin®, sold by Genentech, San Francisco, Calif.; also referred to herein as BM-1) binds human VEGF with an affinity (KD) of approximately 500 pM. While Bevacizumab has been used to treat a variety of cancers, there is a need in the art for antibodies with greater in vivo efficacy. Such antibodies present significant technical challenges and are highly elusive because merely increasing the binding affinity of a VEGF antibody does not necessarily increase its in vivo efficacy (Liang 2006).