Comprehensive genomic profiling of epithelial ovarian cancer by next generation sequencing-based diagnostic assay reveals new routes to targeted therapies.

OBJECTIVE: Targeted next generation sequencing (NGS) was evaluated for its ability to identify unanticipated targetable genomic alterations (GA) for patients with relapsed ovarian epithelial carcinoma (OC). METHODS: DNA sequencing was performed for 3320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor ligated, hybridization-captured libraries using DNA isolated from FFPE sections from 48 histologically verified relapsed OC specimens. The original primary tumor was sequenced in 26 (54%) of the cases and recurrent/metastatic tumor site biopsies were sequenced in 22 (46%) of the cases. Actionability was defined as: GA that predict sensitivity or resistance to approved or standard therapies or are inclusion or exclusion criteria for specific experimental therapies in NCI registered clinical trials. RESULTS: There were 38 (80%) serous, 5 (10%) endometrioid, 3 (6%) clear cell, 1 mucinous (2%) and 1 (2%) undifferentiated carcinomas. 141 GA were identified with an average of 2.9 GA (range 0-8) per tumor, of which 67 were actionable for an average of 1.4 actionable GA per patient (range 0-5). 33/48 (69%) of OC patient samples harbored at least one actionable GA. Most common GA were TP53 (79%); MYC (25%); BRCA1/2 (23%); KRAS (16.6%) and NF1 (14.5%). One tumor featured an ERBB2 point mutation. One of 3 (33%) of clear cell tumors featured cMET amplification validated by both FISH and IHC. CONCLUSIONS: NGS assessment of therapy resistant OC identifies an unexpectedly high frequency of GA that could influence targeted therapy selection for the disease.

Pharmacokinetics and interspecies scaling of a novel VEGF receptor inhibitor, SU5416.

The pharmacokinetics and allometric relationships of SU5416, a novel small anti-angiogenesis agent, were studied. The pharmacokinetics of SU5416 were examined in mice, rats, dogs, and cancer patients. The in-vitro intrinsic clearance (CLint) was estimated from the in-vitro metabolism study in mouse, rat, dog, monkey and human liver microsomes. The parameters of interest were correlated across species as a function of bodyweight using an allometric approach. The steady-state volume of distribution (Vd(ss)), plasma clearance (CLs), and CLint of SU5416 were well correlated across species. The exponent of the allometric relationship (b) of the corresponding parameters was 0.92, 0.80 and 0.66, respectively. The elimination half-life (t1/2) was consistent across species and independent of bodyweight. The prediction of CLs, Vd(ss), CLint, and t1/2 in humans using the data from mouse, rat, and dog, and monkey (for CLint) was reasonably good (within 4-fold of the observed values). However, an improved prediction (within 2-fold of the observed values) of the corresponding parameters in humans was obtained when extrapolation from only the rodent data was performed, suggesting that the rodent data are sufficient for the scale-up of SU5416 pharmacokinetic parameters in humans. Using allometry, it was possible to achieve reasonable predictions of the pharmacokinetic parameters of SU5416 in cancer patients with various solid tumours.

Biotransformation of the anti-angiogenic compound SU5416.

SU5416 [3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1, 3-dihydro-indol-2-one], an inhibitor of VEGF (vascular endothelial growth factor) receptor tyrosine kinase, Flk-1/KDR (fetal liver kinase 1/kinase insert domain-containing receptor), also known as VEGF receptor 2 (VEGFR2) is in advanced clinical trials for treatment of AIDS-related Kaposi's sarcoma and colorectal and nonsmall cell lung cancers. Since this chemical class has not been studied previously with therapeutic intent, the present study was designed to investigate the in vitro metabolism of SU5416 by mouse, rat, dog, monkey, and human liver microsomes and to identify the major metabolites of SU5416. An HPLC procedure was developed and validated to resolve and quantify SU5416 and its metabolites. To evaluate the in vitro metabolism of SU5416, pooled liver microsomes from mice, rats, dogs, monkeys, and humans were incubated with SU5416 (25 microM) in the presence of an NADPH-generating system. In the presence of NADPH, mouse, rat, dog, monkey, and human liver microsomes converted SU5416 to at least 12, 9, 9, 7, and 6 polar metabolites, respectively. Microsomal metabolism of SU5416 showed marked species differences in the levels of different metabolites formed. The overall rate of SU5416 metabolism by liver microsomes from the species examined followed the rank order: monkey > or = mouse approximately rat > dog > human. Two major metabolites of SU5416 were identified, a hydroxymethyl derivative of SU5416 (M12) and a carboxylic acid derivative of SU5416 (M6), by spectroscopic methods and comparison with authentic compounds. Both of these oxidative metabolites were further metabolized in vivo through glucuronidation. The metabolic fate of SU5416 in microsomes from various species as well as data from in vivo biotransformation in the rat are discussed.

Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-alpha bigenic mice.

Overexpression of ErbB-2/Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)/Neu + MMTV/transforming growth factor alpha bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogen-activated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27(Kip1). In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1/2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.

SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors.

Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials.

Effects of SU101 in combination with cytotoxic agents on the growth of subcutaneous tumor xenografts.

SU101 (leflunomide, N-[4-(trifluoromethyl)-phenyl] 5-methylisoxazole-4-carboxamide), an inhibitor of platelet-derived growth factor receptor signaling, has shown promising clinical activity in Phase I and II studies. Currently, SU101 in combination with cytotoxic agents is in late-stage clinical development for the treatment of cancers. In previous reports, efficacy in vivo versus varied tumor xenografts was observed. As part of the preclinical development of SU101 as a cancer therapy, the combination of SU101 with cytotoxic agents was studied in athymic mice bearing small, established, s.c. human tumor cell xenografts of glioblastoma (SF763T cells), lung (Calu-6 cells), or head and neck (KB cells) origin. In the SF763T model, the combination of SU101 with carmustine resulted in a statistically significant growth inhibition of 74% compared with the vehicle control; this combination was more effective than either agent alone. In the Calu-6 model, the combination of SU101, cisplatin, and etoposide resulted in a growth inhibition of 75% that was statistically greater than that of the vehicle-treated control group and groups treated with one or two agents. In the KB model, the combination of SU101, 5-fluorouracil, and cisplatin resulted in a statistically significant growth inhibition of 69% compared with the vehicle control. Treatment with one or two agents did not significantly inhibit growth in this model. Importantly, in addition to enhanced efficacy resulting from combination therapies, the combination treatments tested were well tolerated, as evidenced by lack of mortality. These data suggest that SU101 in combination with cytotoxic agents may provide clinical benefit and warrant further clinical investigation.

Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent.

Angiogenesis, or the sprouting of new blood vessels, is a central process in the growth of solid tumors. For many cancers, the extent of vascularization of a tumor is a negative prognostic indicator signifying aggressive disease and increased potential for metastasis. Recent efforts to understand the molecular basis of tumor-associated angiogenesis have identified several potential therapeutic targets, including the receptor tyrosine kinases for the angiogenic factor vascular endothelial growth factor (VEGF). Here we review the approach taken at SUGEN, Inc. to discover and develop small molecule inhibitors of receptor tyrosine kinases as anti-angiogenic agents. We focus on SU5416, a selective inhibitor of VEGF receptors that is currently in clinical development for the treatment of advanced malignancies. Its biochemical, biological and pharmacological properties are reviewed and clinical implications discussed.

New paradigms for the treatment of cancer: the role of anti-angiogenesis agents.

Angiogenesis, the sprouting of new blood vessels, plays a role in diverse disease states including cancer, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis, atherosclerosis, and restenosis. With regard to cancer, the clinical association of tumor vascularity with tumor aggressiveness has been clearly demonstrated in numerous tumor types. The observation of increased microvessel density in tumors not only serves as an independent prognostic indicator, but also suggests that anti-angiogenic therapy may be an important component of treatment regimens for cancer patients. The complexity of the angiogenic process, which involves both positive and negative regulators, provides a number of targets for therapy. Many positive regulators, including growth factor receptors, matrix metalloproteinases, and integrins, have been correlated with increased vascularity of tumors and poor prognosis for patient survival. Thus, these serve as ideal targets for anti-angiogenesis therapy. Many inhibitors of these targets are currently undergoing clinical evaluation as potential anti-cancer agents. In this article, we discuss the role of positive regulators in angiogenesis and tumor growth and describe the anti-angiogenic agents under development.

Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity.

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.

The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function.

SU5416, a selective inhibitor of the tyrosine kinase activity of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR, is currently in Phase III clinical trials for the treatment of advanced malignancies. In cellular assays, SU5416 inhibits the VEGF-dependent mitogenic/proliferative response of human umbilical vein endothelial cells (HUVECs). In tumor xenograft models, SU5416 inhibits the growth of tumors from a variety of origins by inhibiting tumor angiogenesis. In three different human tumor xenograft models, infrequent (once or twice a week) administration of SU5416 is efficacious despite the fact that it has a short plasma half-life (30 min), which suggests that SU5416 has long-lasting inhibitory activity in vivo. The goal of the present study was to determine the basis for the prolonged activity of SU5416. The results indicate that a short (3 h) exposure to 5 microM SU5416 (to mimic plasma levels of the compound as measured in patients who were receiving SU5416 therapy) produced long-lasting (at least 72 h) inhibition of the VEGF-dependent proliferation of HUVECs in culture, which indicate that SU5416 has long-lasting inhibitory activity in vitro as well as in vivo. SU5416 treatment of HUVECs did not affect surface expression of Flk-1/KDR or the affinity of the receptor for VEGF. Instead, the durability of the in vitro activity of SU5416 was shown to be attributable to its long-lasting ability to specifically inhibit VEGF-dependent phosphorylation of Flk-1/KDR and subsequent downstream signaling, although SU5416 is not an irreversible inhibitor of Flk-1/KDR tyrosine kinase activity. The long-lasting inhibition of cellular responses to VEGF was attributable to the accumulation of SU5416 in cells, as shown using radiolabeled compound, such that inhibitory cellular concentrations of SU5416 are maintained long after the removal of the compound from the medium. The long-lasting inhibitory activity of SU5416 in vitro is consistent with the finding that SU5416 has demonstrated evidence of biological activity in clinical studies when administered twice a week despite a short plasma half-life.

Design, synthesis, and evaluations of substituted 3-[(3- or 4-carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of VEGF, FGF, and PDGF receptor tyrosine kinases.

Receptor tyrosine kinases (RTKs) have been implicated as therapeutic targets for the treatment of human diseases including cancers, inflammatory diseases, cardiovascular diseases including arterial restenosis, and fibrotic diseases of the lung, liver, and kidney. Three classes of 3-substituted indolin-2-ones containing propionic acid functionality attached to the pyrrole ring at the C-3 position of the core have been identified as catalytic inhibitors of the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) RTKs. Some of the compounds were found to inhibit the tyrosine kinase activity associated with isolated vascular endothelial growth factor receptor 2 (VEGF-R2) [fetal liver tyrosine kinase 1 (Flk-1)/kinase insert domain-containing receptor (KDR)], fibroblast growth factor receptor (FGF-R), and platelet-derived growth factor receptor (PDGF-R) tyrosine kinase with IC(50) values at nanomolar level. Thus, compound 1 showed inhibition against VEGF-R2 (Flk-1/KDR) and FGF-R1 tyrosine kinase activity with IC(50) values of 20 and 30 nM, respectively, while compound 16f inhibited the PDGF-R tyrosine kinase activity with IC(50) value of 10 nM. Structural models and structure-activity relationship analysis of these compounds for the target receptors are discussed. The cellular activities of these compounds were profiled using cellular proliferation assays as measured by bromodeoxyuridine (BrdU) incorporation. Specific and potent inhibition of cell growth was observed for some of these compounds. These data provide evidence that these compounds can be used to inhibit the function of these target receptors.

Recombinant immunotoxins directed against the c-erb-2/HER2/neu oncogene product: in vitro cytotoxicity, pharmacokinetics, and in vivo efficacy studies in xenograft models.

TAB-250 and BACH-250 are murine and human chimeric antibodies directed at the extracellular domain of the gp185c-erb-2 (HER2/neu) growth factor receptor overexpressed in a variety of tumor types, including ovarian and breast carcinoma. The ribosome-inhibiting plant toxin gelonin (rGel) was chemically coupled to both antibodies, and the resulting immunotoxins were purified and tested in vitro against human tumor cells expressing various levels of HER-2/neu and in vivo against human tumor xenograft models. The binding of both BACH-250 and BACH-250/rGel conjugate to target cells was essentially equivalent. Against SKOV-3 cells, the IC50 of BACH-250/rGel was 97 pM (17 ng/ml), whereas BACH-250 and rGel alone showed no cytotoxic effects. There was a clear correlation between expression levels of HER-2/neu and cytoimmunotoxin. Tissue distribution studies showed that the antibody and immunotoxin both concentrate 2-10-fold higher in tumors than in normal tissues, with optimal tumor uptake occurring 48-96 h after administration. Plasma clearance curves for BACH-250 and BACH-250/rGel showed terminal-phase half-lives of 26 and 72 h, respectively. In athymic mice bearing s.c. or i.p. SKOV-3 tumors, immunotoxin treatment slowed tumor growth by 99 and 94 % at days 35 and 49 after implantation, respectively, and lengthened the median survival by 40% (from 30 to 50 days) in mice bearing lethal i.p. tumors. We conclude that clinical development of BACH-250/rGel may be warranted in patients with HER2/neu-expressing malignancies.

SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types.

SU5416, a novel synthetic compound, is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast, systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals, supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types.

Tyrosine kinases in disease: overview of kinase inhibitors as therapeutic agents and current drugs in clinical trials.

Tyrosine kinases, first described as oncogenes, have been shown to play a role in normal cellular processes. Aberrations in tyrosine kinase activity lead to disease states. For fifteen years it has been postulated that the inhibition of tyrosine kinases may have therapeutic utility and the design and testing of inhibitors have been major focuses of research and development in both academic institutions and pharmaceutical companies. While early research focused on developing chemical entities that mimic phosphotyrosine, later research has focused on developing competitive adenosine triphosphate (ATP) inhibitors with various levels of selectivity on kinase targets. This review focuses on a discussion of tyrosine kinases thought to be important in disease, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF) receptors, HER-2 and Src. In addition, the classes of inhibitors designed to affect these targets and that have overcome research and development challenges and entered clinical trials are discussed. These include isoxazole, quinazoline, substituted pyrimidines and indolinone compounds, all of which are in clinical trials or near clinical development by SUGEN, Zeneca, Novartis, Pfizer and Parke-Davis. A summary of the chemistry and activity of these agents is provided.

Unliganded epidermal growth factor receptor dimerization induced by direct interaction of quinazolines with the ATP binding site.

Receptor dimerization is critical for signaling by the epidermal growth factor receptor (EGFR) tyrosine kinase. This occurs after binding of the receptor's extracellular domain by ligand or bivalent antibodies. The role of other receptor domains in dimerization is less clear, and there are no examples of dimers induced by direct perturbation of the EGFR kinase domain. Submicromolar concentrations of AG-1478 and AG-1517, quinazolines specific for inhibition of the EGFR kinase, induced reversible receptor dimerization in vitro and in intact A431 cells. Consistent with the inhibitory effect of quinazolines on receptor kinase activity, the dimers formed lacked a detectable Tyr(P) signal. Quinazoline-induced EGFR dimerization was abrogated in vitro by ATP and the ATP analog adenyl-5'-yl imidodiphosphate. Receptors with a single-point mutation in the ATP binding site as well as wild-type EGFR with a covalent modification of the ATP site failed to dimerize in response to AG-1478 and AG-1517. These data suggest that EGFR dimerization can be induced by the interaction of quinazolines at the ATP site in the absence of receptor ligand binding. In SKBR-3 cells, the quinazolines induced the formation of inactive EGFR/ErbB-2 heterodimers, potentially sequestering ErbB-2 from interacting with other coreceptors of the ErbB family. Structural studies of the quinazoline interaction with the EGFR tyrosine kinase domain should allow for an analysis of receptor-specific chemical features required for binding to the ATP site and disruption of signaling, a strategy that can be perhaps applied to other tumor cell receptor systems.

Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide.

Many reports have cited coexpression of platelet-derived growth factor (PDGF) and its receptors by tumor cells or cells supporting tumor growth, suggesting both autocrine and paracrine mechanisms for PDGF-mediated tumor growth. We found that a small organic molecule, N-[4-(trifluoromethyl)phenyl] 5-methylisoxazole-4-carboxamide (SU101, leflunomide), inhibited PDGF-mediated signaling events, including receptor tyrosine phosphorylation, DNA synthesis, cell cycle progression, and cell proliferation. SU101 inhibited PDGF-stimulated tyrosine phosphorylation of PDGF receptor (PDGFR) beta in C6 (rat glioma) and NIH3T3 cells engineered to overexpress human PDGFRbeta (3T3-PDGFRbeta). SU101 blocked both PDGF- and epidermal growth factor (EGF)-stimulated DNA synthesis. Previously, this compound was shown to inhibit pyrimidine biosynthesis by interfering with the enzymatic activity of dihydroorotate dehydrogenase. In the current study, EGF-stimulated DNA synthesis was restored by the addition of saturating quantities of uridine, whereas PDGF-induced DNA synthesis was not, suggesting that the compound demonstrated some selectivity for the PDGFR pathway that was independent of pyrimidine biosynthesis. Selectivity was further demonstrated by the ability of the compound to block the entry of PDGF-stimulated cells into the S phase of the cell cycle, without affecting cell cycle progression of EGF-stimulated cells. In cell growth assays, SU101 selectively inhibited the growth of PDGFRbeta-expressing cell lines more efficiently than it inhibited the growth of PDGFRbeta-negative cell lines. SU101 inhibited the s.c., i.p., and intracerebral growth of a panel of cell lines including cells from glioma, ovarian, and prostate origin. In contrast, SU101 failed to inhibit the in vitro or s.c. growth of A431 and KB tumor cells, both of which express EGF receptor but not PDGFRbeta. SU101 also inhibited the growth of D1B and L1210 (murine leukemia) cells in syngeneic immunocompetent mice, without causing adverse effects on the immune response of the animals. In an i.p. model of tumor growth in syngeneic immunocompetent mice, SU101 prevented tumor growth and induced long-term survivors in animals implanted with 7TD1 (murine B-cell hybridoma) tumor cells. Because PDGFRbeta was detected on most of the tumor cell lines in which in vivo growth was inhibited by SU101, these data suggest that SU101 is an effective inhibitor of PDGF-driven tumor growth in vivo.

Flk-1 as a target for tumor growth inhibition.

A number of growth factor receptor tyrosine kinases have been implicated in angiogenesis, including epidermal growth factor receptor, fibroblast growth factor receptor, platelet-derived growth factor receptor, Flk-1/KDR, Flt-1, Tie-1, and Tek/Tie-2. Flk-1/KDR, a receptor for vascular endothelial growth factor (VEGF), is expressed exclusively in endothelial cells. Using dominant-negative methods, Flk-1 was shown to play a role in angiogenesis and the growth of a variety of tumor types. Because of this, a drug discovery effort was established to identify Flk-1 kinase inhibitors. For initial screening, an ELISA in, a 96-well format was used to measure VEGF-induced Flk-1 tyrosine phosphorylation in whole cells. Compounds that inhibited ligand-induced receptor autophosphorylation were confirmed by antiphosphotyrosine immunoblotting. Inhibition of VEGF-stimulated DNA synthesis in human endothelial cells was also assessed. Inhibitors were further evaluated for their effects on vessel formation using the chorioallantoic membrane assay. Using these methods, antiangiogenesis compounds that inhibit Flk-1 tyrosine kinase activity, endothelial cell mitogenesis, and blood vessel formation in the chorioallantoic membrane assay have been found.

Dominant-negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo.

Angiogenesis, the sprouting of new blood vessels from existing vessels, occurs in many physiological and pathological processes, including embryonic development, wound healing, and tumor growth. It is required for tumor growth because new blood vessel formation is necessary for tumors to expand beyond a minimum volume. Several growth factor receptor tyrosine kinases have been implicated in angiogenesis, including receptors for epidermal, fibroblast, and platelet-derived growth factors, as well as the receptors Flk-1/KDR, Flt-1 Tek/Tie-2, and Tie-1. Endothelial cells in the vessels of tumors express Flk-1/KDR, a receptor for vascular endothelial growth factor. Flk-1 was previously shown to play a role in angiogenesis and tumor formation of s.c. xenografts of C6 glioma cells using dominant-negative methodology. We now demonstrate that Flk-1 seems to be generally involved in the growth of a wide range of solid tumors, including mammary, ovarian, and lung carcinoma, as well as glioblastoma. Furthermore, survival times in rats bearing intracerebral tumors were prolonged using the same dominant-negative methodology. The involvement of Flk-1 in a variety of tumor types suggests an important role for Flk-1 in tumor angiogenesis.

Construction of a chimeric antibody with therapeutic potential for cancers which overexpress c-erbB-2.

We describe the chimerization of a monoclonal antibody directed against the c-erbB-2 protein using a novel PCR method for cloning immunoglobulin variable region genes. We also describe the characterization of the chimera and show its potential use for treating cancers which overexpress the c-erbB-2 protein. The genomic DNA fragments of heavy and light chain variable genes were cloned by PCR using uniquely designed primers which allowed for isolation of genes containing functional promoters, signal and coding sequences. The chimeric genes were then constructed by linking variable regions of murine genes to human constant gamma 1 and kappa genes. Expression of the chimeric immunoglobulin genes resulted in production of properly assembled chimeric antibody with improved biological properties.