A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.

292:E964-E976, 2007. First published 28 November 2006; Am J Physiol Endocrinol Metab Peter Bohlen, Rudolph L. Leibel and Paul Kussie Carrick, Huiyuan Zheng, Hans-Rudolf Berthoud, Owen P. McGuinness, Juqun Shen, Haijun D. Sun, Maria Malabunga, James R. Tonra, Roberto DiRenzo, Francine E. rodents and monkeys cause potent but reversible hypophagia and weight loss in Monoclonal antibody antagonists of hypothalamic FGFR1

We generated three fully human monoclonal antibody antagonists against fibroblast growth factor receptor-1 (FGFR1) that potently block FGF signaling. We found that antibodies targeting the c-splice form of the receptor (FGFR1c) were anorexigenic when administered intraperitoneally three times weekly to mice, resulting in rapid, dose-dependent weight loss that plateaued (for doses>4 mg/kg) at 35-40% in 2 wk. Animals appeared healthy during treatment and regained their normal body weights and growth trajectories upon clearance of the antibodies from the bloodstream. Measurements of food consumption and energy expenditure indicated that the rapid weight loss was induced primarily by decreased energy intake and not by increased energy expenditure or cachexia and was accompanied by a greater reduction in fat than lean body mass. Hypophagia was not caused through malaise or illness, as indicated by absence of conditioned taste aversion, pica behavior, and decreased need-induced salt intake in rats. In support of a hypothalamic site of action, we found that, after intraperitoneal injections, anti-FGFR1c (IMC-A1), but not a control antibody, accumulated in the median eminence and adjacent mediobasal hypothalamus and that FGFR1c is enriched in the hypothalamus of mice. Furthermore, a single intracerebroventricular administration of 3 microg of IMC-A1 via the 3rd ventricle to mice caused an approximately 36% reduction in food intake and an approximately 6% weight loss within the ensuing 24 h. Our data suggest that FGF signaling through FGFR1c may play a physiological role in hypothalamic feeding circuit and that blocking it leads to hypophagia and weight loss.

Fibroblast growth factors (FGFs) are important regulators of hematopoiesis and have been implicated in the tumorigenesis of solid tumors. Recent evidence suggests that FGF signaling through FGF receptors (FGFRs) may play a role in the proliferation of subsets of acute myeloid leukemias (AMLs). However, the precise mechanism and specific FGF receptors that support leukemic cell growth are not known. We show that FGF-2, through activation of FGFR1β signaling, promotes survival, proliferation and migration of AML cells. Stimulation of FGFR1β results in phosphoinositide 3-kinase (PI3-K)/Akt activation and inhibits chemotherapy-induced apoptosis of leukemic cells. Neutralizing FGFR1-specific antibody abrogates the physiologic and chemoprotective effects of FGF-2/FGFR1β signaling and inhibits tumor growth in mice xenotransplanted with human AML. These data suggest that activation of FGF-2/FGFR1β supports progression and chemoresistance in subsets of AML. Therefore, FGFR1 targeting may be of therapeutic benefit in subsets of AML.

Vascular endothelial growth factor receptor 1 (VEGFR‐1) is present on endothelial cells and subsets of human tumor cells, raising the hypothesis that angiogenic factors may promote tumor growth both by inducing angiogenesis and directly signaling through activation of VEGFR‐1 on tumor cells. Here, we report that VEGFR‐1 is expressed on a panel of 16 human breast tumor cell lines, and the vasculature and the tumor cell compartment of a subset of breast carcinoma lesions, and that selective signaling through VEGFR‐1 on breast cancer cells supports tumor growth through downstream activation of the p44/42 mitogen‐activated protein kinase (MAPK) or Akt pathways. Ligand‐stimulated proliferation of breast tumor cells was inhibited by specific blockade with an anti‐VEGFR‐1 neutralizing monoclonal antibody. Treatment with anti‐VEGFR‐1 mAb significantly suppressed the growth of DU4475, MCF‐7, BT‐474 and MDA‐MB‐231 breast xenografts in athymic mice. Histological examination of anti‐VEGFR‐1 mAb treated tumor xenografts showed a significant reduction of activation of the p44/42 MAPK or Akt pathways in tumor cells resulting in an increase in tumor cell apoptosis. Importantly, cotreatment with mAbs targeting human VEGFR‐1 on tumor cells and murine VEGFR‐1 on vasculature led to more potent growth inhibition of breast tumor xenografts. The results suggest that VEGF receptors may not only modulate angiogenesis, but also directly influence the growth of VEGF receptor expressing tumors. © 2006 Wiley‐Liss, Inc.

Inhibition of vascular endothelial growth factor (VEGF) signaling, a key regulator of tumor angiogenesis, through blockade of VEGF receptor (VEGFR)-2 by the monoclonal antibody DC101 inhibits angiogenesis, tumor growth, and invasion. In a surface xenotransplant assay on nude mice using a high-grade malignant squamous cell carcinoma cell line (A-5RT3), we show that DC101 causes vessel regression and normalization as well as stromal maturation resulting in a reversion to a noninvasive tumor phenotype. Vessel regression is followed by down-regulation of expression of both VEGFR-2 and VEGFR-1 on endothelial cells and increased association of alpha-smooth muscle actin-positive cells with small vessels indicating their normalization, which was further supported by a regular ultrastructure. The phenotypic regression of an invasive carcinoma to a well-demarcated dysplastic squamous epithelium is accentuated by the establishment of a clearly structured epithelial basement membrane and the accumulation of collagen bundles in the stabilized connective tissue. This normalization of the tumor-stroma border coincided with down-regulated expression of the stromal matrix metalloproteinases 9 and 13, which supposedly resulted in attenuated turnover of extracellular matrix components permitting their structural organization. Thus, in this mouse model of a human squamous cell carcinoma cell line, blockade of VEGF signaling resulted in the reversion of the epithelial tumor phenotype through stromal normalization, further substantiating the crucial role of stromal microenvironment in regulating the tumor phenotype.

Induction of neoangiogenesis plays an important role in the pathogenesis of multiple myeloma. However, the mechanism by which expression of vascular endothelial growth factor (VEGF)-A and its receptors modulate the interaction of multiple myeloma cells with stromal cells is not known. Here, we describe a novel in vitro coculture system using fetal bone stromal cells as a feeder layer, which facilitates the survival and growth of human primary multiple myeloma cells. We show that stromal-dependent paracrine VEGF-A signaling promotes proliferation of human primary multiple myeloma cells. Primary multiple myeloma cells only expressed functional VEGF receptor (VEGFR)-1, but not VEGFR-2 or VEGFR-3. VEGFR-1 expression was detected in the cytoplasm and the nuclei of proliferating multiple myeloma cells. Inhibition of VEGFR-1 abrogated multiple myeloma cell proliferation and motility, suggesting that the functional interaction of VEGF-A with its cognate receptor is essential for the growth of primary multiple myeloma cells. Collectively, our results suggest that stromal-dependent paracrine and intracrine VEGF-A/VEGFR-1 signaling contributes to human primary multiple myeloma cell growth and therefore, VEGFR-1 blockade is a potential therapeutic strategy for the treatment of multiple myeloma.

Proc Amer Assoc Cancer Res, Volume 46, 2005 6181 The class III receptor tyrosine kinase FLT3 is an attractive therapeutic target as it is overexpressed in blasts of ∼90% of acute myelogenous leukemia (AML) and the majority of B-lymphoid leukemia patients. Internal tandem duplications (ITDs) in the juxtamembrane region and point mutations in the kinase domain of FLT3 are found in ∼37% of AML patients and are associated with a poor prognosis. We have recently developed a fully human, high affinity monoclonal antibody (EB10) which is readily internalized upon binding to FLT3 receptor on human leukemia cells. In the present study, a novel auristatin conjugate of the anti-FLT3 antibody (EB10-MMAF) was prepared using a dipeptide linker that allows for drug release inside the lysosomes of antigen-positive cells. The MMAF conjugates were stable in buffers and plasma. EB10-MMAF (drug/antibody ratio = 8) retained the binding affinity and internalizing capability of EB10. The conjugate was highly potent, and selectively inhibited the growth of FLT3-expressing leukemia cells with an IC50 of 0.19 nM and 0.08 nM for MV4;11 and BaF3-ITD cells (both positive for FLT3-ITD), 1.11 nM, 6.18 nM and 1.82 nM for REH, EOL-1, EM3 cells (all three positive for wild-type FLT3), and 135 nM for JM1 (negative for FLT3). An MMAF conjugate with a control antibody was not active in these cell lines (IC50s > 5.9 uM). Flow cytometric analysis with annexin V indicated that EB10-MMAF treatment induced apoptosis of leukemia cells in vitro . In vivo treatment with EB10-MMAF strongly inhibited leukemia growth and prolonged survival of mice in both EOL-1 (mean survival time ∼81.0 +/− 46.8 days for the 3 mg/kg group compared to 41.5+/−15 days for the untreated group) and BaF3-ITD (mean survival time ∼91.5 +/− 39.5 days for the 5 mg/kg group compared to 32.8 +/− 5.2 days for the untreated group) leukemia models. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other FLT3-positive leukemias. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other types of FLT3-positive leukemia.

Platelet-derived growth factor receptor α (PDGFRα) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRα, which did not cross-react with the β form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRα. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRα. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P = 0.0004) and SKLMS-1 (P < 0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRα.

VEGF acting through VEGFR-2 is of critical importance for physiologic folliculogenesis by regulating intraovarian angiogenic events. The role of VEGFR-1 and its binding protein, soluble VEGFR-1, in ovarian folliculogenesis is less understood. This receptor and its binding protein might be involved in the regulation of VEGF availability locally in the ovary and in blood (“decoy function”). We tested for such a possibility by studying the effect of a blocking antibody on ovarian function. This antibody prevents binding of VEGF toVEGFR-1 and the binding protein s-Flt. We hypothesized that this antibody would increase VEGF availability, which in turn would enhance folliculogenesis. Prospective Follicles of interest were stained with an antibody against VEGFR-1 (R&D). Experiment 1: Hypophysectomized (HX) mouse were injected i.p. with an anti-VEGF-R1 blocking antibody (treatment group; N=5) (MF 1, ImClone Systems; 2 mg/animal) or with saline (control group; N=5), and sacrificed 72h later. Angiogenesis and folliculogenesis were evaluated by staining for endothelial cells with PECAM (Pharmingen) and proliferation with BrDU (Zymed), and by counting the total number of the most developed preantral follicles per ovary. Experiment 2: HX mouse were injected with 20 IU of PMSG, and sacrificed 72h later. Treatment group animals received 2 doses of MF1 24h before and 48 after PMSG, while control animals were given saline injections. The total number of preovulatory follicles per ovary was counted, and follicular vasculature was evaluated. VEGF plasma levels were measured in all animals at the time of sacrifice. Endothelial VEGFR-1 staining was present in preantral and antral follicles. Anti-VEGFR-1 antibody increased plasma VEGF levels four-fold from a baseline level of 53±2.1 pg/ml (p<0.01). In the absence of gonadotropin (GT) vascular endowment of the most advanced follicles increased by about 30% when compared to control (p<0.05). Proliferating endothelial cells were more prominent in treatment group follicles (control 1 to 2 cells; treatment 5-7 cells). The total number of advanced preantral follicles increased from 8.5± 1.3 to 12.4±2.1 (p<0.05). No formation of antral follicles was observed in the absence of GT. Treatment with PMSG in the presence of anti-VEGFR-1 antibody increased the number of preovulatory follicles from 9.1±1.5 to 11.8±2.1 (p<0.05). Vascular density and number of proliferating endothelial cells was similar in both groups. Administration of anti-VEGFR-1 antibody increased plasma VEGF levels indicating that this antibody through its action on the extracellular domain of the VGFR-1 molecule augments availability of VEGF in the ovary. Enhanced availability of VEGF locally in the ovary in turn might stimulate follicular angiogenesis, which allows the development of an increased number of advanced preantral follicles, which are responsive to GT stimulation. Enhanced vascular endowment of developing follicles, in turn increases the delivery of substances required for follicle development, which might also help in the maintenance of function. Stimulation of animals exposed to anti-VEGFR-1 antibody with GT allows an increased number of follicles to reach the preovulatory stage. Therefore strategies, which increase intra-ovarian availability of VEGF, might be helpful in increasing the number of follicles responsive to GT treatment.

The molecular and cellular pathways that support the maintenance and stability of tumor neovessels are not well defined. The efficacy of microtubule-disrupting agents, such as combretastatin A4 phosphate (CA4P), in inducing rapid regression of specific subsets of tumor neovessels has opened up new avenues of research to identify factors that support tumor neoangiogenesis. Herein, we show that CA4P selectively targeted endothelial cells, but not smooth muscle cells, and induced regression of unstable nascent tumor neovessels by rapidly disrupting the molecular engagement of the endothelial cell-specific junctional molecule vascular endothelial-cadherin (VE-cadherin) in vitro and in vivo in mice. CA4P increases endothelial cell permeability, while inhibiting endothelial cell migration and capillary tube formation predominantly through disruption of VE-cadherin/beta-catenin/Akt signaling pathway, thereby leading to rapid vascular collapse and tumor necrosis. Remarkably, stabilization of VE-cadherin signaling in endothelial cells with adenovirus E4 gene or ensheathment with smooth muscle cells confers resistance to CA4P. CA4P synergizes with low and nontoxic doses of neutralizing mAbs to VE-cadherin by blocking assembly of neovessels, thereby inhibiting tumor growth. These data suggest that the microtubule-targeting agent CA4P selectively induces regression of unstable tumor neovessels, in part through disruption of VE-cadherin signaling. Combined treatment with anti-VE-cadherin agents in conjunction with microtubule-disrupting agents provides a novel synergistic strategy to selectively disrupt assembly and induce regression of nascent tumor neovessels, with minimal toxicity and without affecting normal stabilized vasculature.

5288 It was recently suggested that epidermal growth factor receptor (EGFR) mutations may predict sensitivity to small molecule tyrosine kinase inhibitors (TKIs), as several somatic mutations in the EGFR gene have been identified in certain patients with non-small-cell lung cancer (NSCLC). We characterized the activation state of four different EGFR receptors bearing mutations in the kinase domain (G719C, L858R, L861Q, del747-753) and studied their sensitivity to small molecule TKIs. We found two mutants (L858R, L861Q) to be ligand-independent, constitutively activate kinases. Activation of the G719 mutant was found to be ligand-dependent, and the deletion mutant (del747-753) exhibited reduced kinase activity (20-fold less as compare to wild type EGFR). In the present study we determined the inhibitory activity of gefitinib and IM092214 against three EGFR mutants (G719C, L858R, L861Q) and wild type EGFR using enzymatic and cell-based phosphorylation assays. IM092214 is a structurally novel benzoxazepine that potently inhibits the EGFR kinase activity (cell-based IC50 = 60 nM). We found that gefitinib and IM092214 inhibited wild-type and mutant EGFR activity to similar extent in both the enzymatic assay (gefitinib IC50 = 15 nM, IM092214 IC50 = 60 nM) and the cell-based phosphorylation assay (gefitinib IC50 = 20 nM, IM092214 IC50 = 50 nM). Importantly, we did not observe hypersensitivity of any of the tested mutants to gefitinib or IM092214. Our data suggest that there may be other mechanisms responsible for the observed favorable response of certain lung cancer patients with EGFR mutations to TKIs.

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of cancers. Here we propose that simultaneous targeting of both receptors with a bispecific antibody would lead to enhanced antitumor activity. To this end, we produced a recombinant human IgG-like bispecific antibody, a Di-diabody, using the variable regions from two antagonistic antibodies: IMC-11F8 to EGFR and IMC-A12 to IGFR. The Di-diabody binds to both EGFR and IGFR and effectively blocked both EGF- and IGF-stimulated receptor activation and tumor cell proliferation. The Di-diabody also inherited the biological properties from both of its parent antibodies; it triggers rapid and significant IGFR internalization and degradation and mediates effective antibody-dependent cellular cytotoxicity in a variety of tumor cells. Finally, the Di-diabody strongly inhibited the growth of two different human tumor xenografts in vivo. Our results underscore the benefits of simultaneous targeting of two tumor targets with bispecific antibodies.

Purpose: Inhibition of angiogenesis can influence tumor cell invasion and metastasis. We previously showed that blockade of vascular endothelial growth factor receptor-2 (VEGFR-2) with the monoclonal antibody DC101 inhibited intracerebral glioblastoma growth but caused increased tumor cell invasion along the preexistent vasculature. In the present study, we attempted to inhibit glioma cell invasion using a monoclonal antibody against the epidermal growth factor receptor (EGFR), which in the context of human glioblastomas, has been implicated in tumor cell invasion. In addition, we analyzed whether blockade of vascular endothelial (VE)-cadherin as a different antiangiogenic target could also inhibit glioblastoma angiogenesis and growth. Experimental Designs: Nude mice who received intracerebral glioblastoma xenografts were treated using monoclonal antibodies against VEGFR-2 (DC101), EGFR (C225), and VE-cadherin (E4G10) either alone or in different combinations. Results: Increased tumor cell invasion provoked by DC101 monotherapy was inhibited by 50% to 66% by combined treatment with C225 and DC101. C225 inhibited glioblastoma cell migration in vitro, but had no effect on the volume of the main tumor mass or on tumor cell proliferation or apoptosis in vivo, either alone or in combination with DC101. The anti-VE-cadherin monoclonal antibody E4G10 was a weaker inhibitor of tumor angiogenesis and growth than DC101, and also caused a weaker increase in tumor cell invasion. Conclusions: Inhibition of angiogenesis achieved by blocking either VEGFR-2 or VE-cadherin can cause increased glioma cell invasion in an orthotopic model. Increased tumor cell invasion induced by potent inhibition of angiogenesis with DC101 could be inhibited by simultaneous blockade of EGFR.

Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and blockade of VEGF receptor 2 (VEGFR-2), with the monoclonal antibody DC101, inhibits angiogenesis and tumor growth. To examine the short-term effects of DC101, we surface transplanted the squamous cell carcinoma cell line A5-RT3 onto nude mice. After short-term treatment with DC101, we observed rapid reduction in vascularization and reversion of the tumor phenotype. Beginning 24 hours after treatment, VEGFR-2 inhibition resulted in decreased vessel density within the tenascin-c-staining tumor-associated stroma and reduced endothelial cell proliferation. Stromal expression of matrix metalloproteinase-9 and -13 was drastically reduced 96 hours after VEGFR-2 inhibition as detected by in situ hybridization and in situ zymography. Moreover, the morphology of the tumor-stroma border changed from a highly invasive carcinoma to a well-demarcated, premalignant phenotype. The latter was characterized by the appearance of a regular basement membrane in immunostaining and ultrastructural analyses. These findings suggest that VEGFR-2 inhibition by DC101 evokes very rapid reduction of preformed vessels and decreases both stromal protease expression and gelatinolytic activity, resulting in the modulation of the tumor-stroma border zone and reversion of the tumor phenotype. Thus, short-term inhibition of VEGF signaling results in complex stromal alterations with crucial consequences for the tumor phenotype.