The vascular endothelial growth factor receptor (VEGF) family is comprised of the ligands placental growth factor (PlGF), VEGF-A, VEGF-B, VEGF-C, and VEGF-D, and the receptors VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-A interaction with VEGFR-2 (fetal liver kinase 1 [Flk-1], kinase insert domain-containing receptor) has been demonstrated to be an essential modulator of tumor angiogenesis based on the results of an extensive range of human and preclinical investigations.1 VEGFR-1 (fms-like tyrosine kinase-1 [flt-1]) interacts with and is activated by the ligands PlGF, VEGF-A, and VEGF-B. Although VEGF-A is known to bind and activate both VEGFR-1 and VEGFR-2, VEGFR-1 is the only known receptor for the ligands PlGF and VEGF-B (Fig. 1).2, 3 VEGFR-1 differs from other members of the VEGF receptor family in that it is expressed as a soluble variant because of an alternative splicing event.
VEGFR-1 is expressed on the endothelial cells lining tumor blood vessels, myoepithelial (stromal) cells, and on human cancer cells of diverse tumor origin.4 Immunohistochemical studies have indicated frequent VEGFR-1 expression in multiple human malignancies, including breast, nonsmall cell lung, squamous cell head and neck, prostate, pancreas, ovarian, colon, bladder, hematologic, and other cancers.2, 5, 6 VEGFR-1 expression in resected breast cancer and early stage nonsmall cell lung cancer has been associated with shorter survival.7, 8
VEGFR-1 in Carcinogenesis and Cancer Pathogenesis
VEGFR-1 activation has been implicated in both investigational carcinogenesis and tumor pathogenesis. Constitutive activation of VEGFR-1 results in fibroblast transformation and morphogenesis.9 In colorectal cancer models, VEGFR-1 up-regulation and phosphorylation occur in conjunction with epithelial-mesenchymal transformation (EMT), associated with progression to an invasive phenotype, and VEGFR-1 signaling is essential for the survival of transformed carcinoma.10 VEGFR-1 signaling in a pancreatic cancer model has been shown to cause epithelial-mesenchymal transformation, characterized by loss of cellular polarity, pseudopod formation, and increased intercellular separation mediated by cytoplasmic translocation of E-cadherin and beta-catenin.11 VEGFR-1 signaling in colorectal cancer cells causes activation of mitogen-activated protein kinase and stress-activated protein kinase/c-jun NH2-terminal kinase, and mitogen- activated protein kinase and PI3-kinase/Akt signaling in breast cancer cells. VEGFR-1 signaling increases cell motility and invasiveness and reduces apoptotic indices in in vitro breast, colorectal, and pancreatic cancer models.12-15
VEGFR-1 and Cancer Angiogenesis
The role of VEGFR-1 in malignant angiogenesis has remained uncertain, in part because the receptor has substantially weaker tyrosine kinase activity than VEGFR-2, and exhibits a 10-fold higher binding affinity for VEGF-A.16, 17 It has been hypothesized that VEGFR-1 may act as a decoy receptor that sequesters circulating VEGF-A and thereby diminishes VEGFR-2 signaling; this view is based on the expression of the alternatively spliced soluble VEGFR-1 and on the viability of mice that express nonsignaling VEGFR-1 (deleted kinase domain).18, 19 However, VEGFR-1 signaling has been shown to increase endothelial activity and angiogenesis in both in vitro and animal models. Activation of VEGFR-1 by VEGF-A and PlGF enhances endothelial migration and survival; VEGFR-1 antagonist peptides reduce in vitro endothelial migration and capillary tube formation.20-22 VEGFR-1 signaling increases tumor angiogenesis in PlGF-expressing Lewis lung carcinoma murine xenografts.18 Inhibition of VEGFR-1 by inhibitory antibodies or peptides reduces in vivo angiogenesis of human epidermoid A431 and SW480 colorectal cancers.23, 24 Activation of VEGFR-1 by PlGF has been shown to cause VEGFR-1/VEGFR-2 heterodimerization and transphosphorylation in endothelial cells, resulting in enhanced VEGF-mediated VEGFR-2 signaling and angiogenesis.25
VEGFR-1 Specific Ligands PlGF and VEGF-B
The VEGFR-1 specific ligand PlGF is up-regulated in human cancer and disruption of PlGF signaling inhibits cancer growth. Increased PlGF expression has been observed in advanced (vs localized) colorectal cancer, and has been associated with shorter survival in patients with advanced (stage III-IV) colorectal cancer.26 PlGF expression is up-regulated in breast cancer; after resection with curative intent, patients whose tumors demonstrated higher PlGF expression had reduced disease-free survival.27 PlGF expression has also been observed in conjunction with adverse clinical or prognostic features in other tumor types, including human melanoma, gastric, and hepatocellular cancers.28-30 Inhibition of PlGF by means of a specific anti-PlGF antibody reduces tumor growth in B16 melanoma, Panc02 pancreatic, and CT26 colorectal cancer xenograft models.31
The VEGFR-1-specific ligand VEGF-B is among the least well-characterized members of the VEGF family, although some studies indicate that it is up-regulated in cancer. VEGF-B expression correlates with the presence of lymph node metastases and shorter disease-free and overall survival in a small number of breast cancer series.7, 32 VEGF-B expression has also been observed in conjunction with aggressive phenotypic features, including vascular invasion in hepatocellular cancer and increased microvessel density in oral squamous cancer.33, 34
VEGFR-1 and Tumor Inflammation
VEGFR-1 expression and signaling in inflammatory and other non-neoplastic cells may contribute to cancer pathogenesis (Fig. 2). Tumor inflammation and the presence of VEGFR-1 expressing tumor infiltrating macrophages correlates with a more aggressive clinical phenotype in human breast cancer and other malignancies, although the specific contribution of VEGFR-1 mediated signaling in this setting has not been elucidated.35, 36 VEGFR-1 activation in endothelial cells and macrophages has been shown to promote pulmonary metastases by means of matrix metalloproteinase-9 (MMP-9) up-regulation in Lewis lung carcinoma and B16 melanoma xenografts.37 Ligand-mediated activation of VEGFR-1 causes up-regulation of angiogenic factors FGF, SDF1, PDGF, and others by tumor-associated macrophages and stromal cells.38 Antibody-mediated PlGF inhibition decreases recruitment of macrophages in murine pancreatic and colorectal cancer models.31 Disruption of VEGFR-1 tyrosine kinase activity in Lewis lung carcinomas reduces the presence of tumor-associated macrophages.18
VEGFR-1 Expressing Hematopoietic Progenitor Cells and Cancer Metastasis
VEGFR-1 is expressed by circulating bone marrow-derived hematopoietic progenitor cells (HPCs). These cells have been shown to function as essential components of experimental tumor metastasis.39 These circulating progenitor cells, which also express CD34, CD11b, and other hematopoietic markers, interact with the tumor microenvironment before the development of metastases, and they elicit integrin alteration and growth factor up-regulation that enable tumor implantation and recruitment of VEGFR-2 expressing endothelial progenitors, essential for angiogenesis and growth.40 Antibody-mediated VEGFR-1 inhibition significantly reduces both the premetastatic localization of HPCs expressing VEGFR-1 and the development of metastasis in BB16 melanoma and Lewis lung carcinoma xenograft models.41 The localization of HPCs in advance of the arrival of circulating cancer cells has been termed the “premetastatic niche.” Interactions between tissue fibronectin and the integrin VLA-4 (α4β1; also expressed on VEGFR-1 positive HPCs) and degradation of basement membranes by HPC-derived MMP-9 are also essential for premetastatic HPC localization and metastasis formation.
Development, Preclinical, and Preliminary Clinical Experience With IMC-18F1 (Human Anti-VEGFR-1 Antibody)
Because of the significant association of VEGFR-1 with carcinogenesis and tumor pathogenesis, the high-affinity, human IgG1, anti-VEGFR-1 antibody IMC-18F1 has been developed as an anti-cancer therapeutic agent. IMC-18F1 was developed using anti-VEGFR1-producing hybridomas generated from human IgG transgenic mice, with specific selection and additional polymerase chain reaction amplified subcloning resulting in generation of full-length IgG1 with potent and specific blockade of human VEGFR-1. Recombinant IMC-18F1 binds to VEGFR-1 with high affinity (Kd = 5.0 × 10−11M) and inhibits ligand binding with an IC50 (concentration that inhibits 50% of ligand binding or cell proliferation) of 0.2 to 1.2 nM. IMC-18F1 does not bind to or recognize VEGFR-2 or murine VEGFR-1. Because IMC-18F1 interacts exclusively with the human VEGFR-1, the rat anti-murine anti-VEGFR-1, MF1 was developed to better study potential anti-endothelial and anti-stromal mechanisms in human tumor murine xenograft models. In these xenografts, IMC-18F1 activity is confined to direct inhibition of VEGFR-1 on tumor cells, and MF1 inhibits VEGFR-1 activity on host (murine) elements such as stroma and blood vessels. The use of both antibodies in combination enables an assessment of activity that may better approximate activity in human cancer.
IMC-18F1 monotherapy has been associated with growth inhibition in several murine human breast cancer xenograft models including MDA-MB-435, DU4475, and MDA-MD-231 tumors. In several of these xenograft models, the addition of MF1 (specific to the murine VEGFR-1) conferred additional growth inhibition, suggesting that VEGFR-1 inhibition impaired both direct tumor growth (via the IMC-18F1 component) and angiogenesis (or effect on stromal or other host-derived tumor supporting mechanisms, by means of the antimouse MF1 component). In MDA-MD-231 xenografts, the IMC-18F1/MF1 combination augmented the growth inhibition associated with the cytotoxic agents 5-fluorouracil, doxorubicin, and cyclophosphamide in several xenograft investigations. The combination of IMC-18F1 and MF1 also conferred enhanced growth inhibition when combined with IMC-DC101 (antimurine VEGFR2) in MDA-MD-231 xenografts. In the MDA-MD-231 xenograft models, antitumor activity of IMC-18F1 was observed when plasma concentrations were maintained in the range of 88 to 200 μg/mL (or above), with average interdose plasma concentrations in the range of 200 to 319 μg/mL.3
Phase I investigation of IMC-18F1 has indicated a favorable adverse event profile across a range of schedules and intravenous doses. Krishnamurthi and colleagues have administered IMC-18F1 at weekly doses of 2, 3, 6, 12, and 15 mg/kg every 2 weeks, and 20 mg/kg every 3 weeks. Preliminary pharmacokinetic analyses indicate that doses of 12 mg/kg/week generate serum trough levels comparable to (or greater than) those associated with anticancer activity in preclinical models. IMC-18F1 related adverse events and serious adverse events have occurred infrequently; a maximum tolerated dose has not been identified.42
VEGFR-1 inhibition via IMC-18F1 may impede cancer growth via several mechanisms, including direct tumor inhibition, inhibition of angiogenesis, and more indirect mechanisms involving inhibition of VEGFR-1 signaling on circulating hematopoietic precursor cells, tumor-infiltrating macrophages, and stromal cells, which have been associated with cancer pathogenesis in diverse investigational models. Preclinical investigation of IMC-18F1 (and the antimouse counterpart MF-1) has resulted in inhibition of cancer growth in several in vivo models; more robust growth inhibition has occurred with combination IMC-18F1/MF-1 therapy, suggesting that both antitumor and antiangiogenic mechanisms are operant. When combined with cytotoxic chemotherapy, IMC-18F1 (or IMC-18F1/MF1) was associated with enhanced growth inhibition in some xenograft models. Preliminary clinical studies to date have indicated a favorable safety profile associated with IMC-18F1 monotherapy.