AMG 706, an oral, multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis and induces regression in tumor xenografts.

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies.

A polyclonal antibody to the prepore loop of transient receptor potential vanilloid type 1 blocks channel activation.

Transient receptor potential vanilloid type 1 (TRPV1) can be activated by multiple chemical and physical stimuli such as capsaicin, anandamide, protons, and heat. Capsaicin interacts with the binding pocket constituted by transmembrane regions 3 and 4, whereas protons act through residues in the prepore loop of TRPV1. Here, we report on characterization of polyclonal and monoclonal antibodies to the prepore loop of TRPV1. A rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) acted as a full antagonist of proton activation (IC(50) values for pH 5 and 5.5 were 364.68 +/- 29.78 and 28.31 +/- 6.30 nM, respectively) and as a partial antagonist of capsaicin, heat, and pH 6 potentiated chemical ligand (anandamide and capsaicin) activation (50-79% inhibition). Ab-156H antagonism of TRPV1 is not affected by the conformation of the capsaicin-binding pocket because it is equally potent at wild-type (capsaicin-sensitive) rat TRPV1 and its T550I mutant (capsaicin-insensitive). With the goal of generating monoclonal antagonist antibodies to the prepore region of human TRPV1, we used a recently developed rabbit immunization protocol. Although rabbit polyclonal antiserum blocked human TRPV1 activation, rabbit monoclonal antibodies (identified on the basis of selective binding to Chinese hamster ovary cells expressing human TRPV1) did not block activation by either capsaicin or protons. Thus, rabbit polyclonal antibodies against rat and human TRPV1 prepore region seem to partially lock or stabilize the channel in the closed state, whereas rabbit anti-human TRPV1 monoclonal antibodies bind to the prepore region but do not lock or stabilize the channel conformation.

Generation and characterization of monoclonal antibodies to human keratinocyte growth factor receptor.

Keratinocyte growth factor receptor (KGFR) and fibroblast growth factor receptor (FGFR) 2c share identical amino acid sequences, except for a 46-amino acid domain in the extracellular region. Monoclonal antibodies (MAbs) specific to KGFR have not been reported nor are commercially available. In this study, we generated murine MAbs specific to KGFR in non-obese diabetic (NOD) mice using a modified Repeated Immunizations at Multiple Sites (RIMMS) technology. Stable cell lines expressing the full-length human KGFR or FGFR2c were produced to facilitate the identification of KGFR-specific MAbs. Following the initial screening of hybridoma clones with a fluorescence-based, confocal cell detection method and ELISA, KGFR-specific MAbs were selected and confirmed by flow cytometry and Western blot analyses. Antagonistic MAbs were identified using a cell-based functional assay. These KGFR MAbs will be important reagents for studying the biological function and tissue distribution of this receptor in normal and pathological conditions.

Epratuzumab, a humanized monoclonal antibody targeting CD22: characterization of in vitro properties.

PURPOSE: Epratuzumab is a novel humanized antihuman CD22 IgG1 antibody that has recently shown promising clinical activity, both as a single agent and in combination with rituximab, in patients with non-Hodgkin's lymphomas (NHL). In an attempt to better understand the mode of action of epratuzumab, the antibody was tested in vitro in a variety of cell-based assays similar to those used to evaluate the biological activity of other therapeutic monoclonal antibodies, including rituximab. In this report, we present epratuzumab activities as they relate to binding, signaling, and internalization of the receptor CD22. METHODS: Chinese hamster ovary-expressed CD22 extracellular domain was used to measure epratuzumab affinity on Biacore. CD22 receptor density and internalization rate were measured indirectly using a monovalently labeled, noncompeting (with epratuzumab) anti-CD22 antibody on Burkitt lymphoma cell lines, primary B cells derived from fresh tonsils, and B cells separated from peripheral blood samples obtained from patients with chronic lymphocytic leukemia or healthy volunteers. Epratuzumab-induced CD22 phosphorylation was measured by immunoprecipitation/Western blot and compared with that induced by anti-IgM stimulation. RESULTS: Epratuzumab binds to CD22-extracellular domain, with an affinity of K(D) = 0.7 nM. Binding of epratuzumab to B cell lines, or primary B cells from healthy individuals and patients with NHL, results in rapid internalization of the CD22/antibody complex. Internalization appears to be faster at early time points in cell lines than in primary B cells and NHL patient-derived B cells, but the maximum internalization reached is comparable for all B cell populations after several hours of treatment and appears to reach saturation at antibody concentrations of 1-5 micro g/ml. Finally, epratuzumab binding results in modest but significant CD22 phosphorylation. CONCLUSIONS: Epratuzumab represents an excellent anti-CD22 ligating agent, highly efficacious in inducing CD22 internalization, and can induce phosphorylation. Although we cannot unequivocally demonstrate here that epratuzumab-induced internalization and signaling of CD22 directly contribute to its therapeutic efficacy, these properties are the fundamental characteristics of the target CD22 and its interaction with epratuzumab. Similar results were observed when epratuzumab was tested in vitro on Burkitt B cell lines as well as on primary normal B cells and neoplastic B cells separated from fresh peripheral blood samples from patients with chronic lymphocytic leukemia.

Polyglutamine repeat length-dependent proteolysis of huntingtin.

Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntington's disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than full-length huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeat-containing huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.

Changes in protein conformation and dynamics upon complex formation of brain-derived neurotrophic factor and its receptor: investigation by isotope-edited Fourier transform IR spectroscopy.

The interactions of brain-derived neurotrophic factor (BDNF) with the extracellular domain of its receptor (trkB) are investigated by employing isotope-edited Fourier transform IR (FTIR) spectroscopy. The protein secondary structures of individual BDNF and trkB in solutions are compared with those in their complex. The temperature dependence of the secondary structures of BDNF, trkB, and their complex is also investigated. Consistent with the crystal structure, we observe by FTIR spectroscopy that BDNF in solution contains predominantly beta strands (approximately 53%) and relatively low contents of other secondary structures including beta turns (approximately 16%), disordered structures (approximately 12%), and loops (approximately 18%) and is deficient in alpha helix. We also observe that trkB in solution contains mostly beta strands (52%) and little alpha helix. Conformational changes in both BDNF and trkB are observed upon complex formation. Specifically, upon binding of BDNF, the conformational changes in trkB appear to involve mostly beta turns and disordered structures while the majority of the beta-strand conformation remains unchanged. The IR data indicate that some of the disordered structures in the loop regions are likely converted to beta strands upon complex formation. The FTIR spectral data of BDNF, trkB, and their complex indicate that more amide NH groups of trkB undergo H-D exchange within the complex than those of the ligand-free receptor and that the thermal stability of trkB is decreased slightly upon binding of BDNF. The FT-Raman spectra of BDNF, trkB, and their complex show that the six intramolecular disulfide bonds of trkB undergo significant conformational changes upon binding of BDNF as a result of changes in the tertiary structure of trkB. Taken together, the FTIR and Raman data are consistent with the loosening of the tertiary structure of trkB upon binding of BDNF, which leads to more solvent exposure of the amide NH group and decreased thermal stability of trkB. This finding reveals an intriguing structural property of the neurotrophin ligand-receptor complex that is in contrast to other ligand-receptor complexes such as a cytokine-receptor complex that usually shows protection of the amide NH group and increased thermal stability upon complex formation.