Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

In vitro and in vivo activity of cabozantinib (XL184), an inhibitor of RET, MET, and VEGFR2, in a model of medullary thyroid cancer.

BACKGROUND: A limited number of approved therapeutic options are available to metastatic medullary thyroid cancer (MTC) patients, and the response to conventional chemotherapy and/or radiotherapy strategies is inadequate. Sporadic and inherited mutations in the tyrosine kinase RET result in oncogenic activation that is associated with the pathogenesis of MTC. Cabozantinib is a potent inhibitor of MET, RET, and vascular endothelial factor receptor 2 (VEGFR2), as well as other tyrosine kinases that have been implicated in tumor development and progression. The object of this study was to determine the in vitro biochemical and cellular inhibitory profile of cabozantinib against RET, and in vivo antitumor efficacy using a xenograft model of MTC. METHODS: Cabozantinib was evaluated in biochemical and cell-based assays that determined the potency of the compound against wild type and activating mutant forms of RET. Additionally, the pharmacodynamic modulation of RET and MET and in vivo antitumor activity of cabozantinib was examined in a MTC tumor model following subchronic oral administration. RESULTS: In biochemical assays, cabozantinib inhibited multiple forms of oncogenic RET kinase activity, including M918T and Y791F mutants. Additionally, it inhibited proliferation of TT tumor cells that harbor a C634W activating mutation of RET that is most often associated with MEN2A and familial MTC. In these same cells grown as xenograft tumors in nude mice, oral administration of cabozantinib resulted in dose-dependent tumor growth inhibition that correlated with a reduction in circulating plasma calcitonin levels. Moreover, immunohistochemical analyses of tumors revealed that cabozantinib reduced levels of phosphorylated MET and RET, and decreased tumor cellularity, proliferation, and vascularization. CONCLUSIONS: Cabozantinib is a potent inhibitor of RET and prevalent mutationally activated forms of RET known to be associated with MTC, and effectively inhibits the growth of a MTC tumor cell model in vitro and in vivo.

Discovery of a novel series of potent and orally bioavailable phosphoinositide 3-kinase γ inhibitors.

The phosphoinositide 3-kinases (PI3Ks) have been linked to an extraordinarily diversified group of cellular functions making these enzymes compelling targets for the treatment of disease. A large body of evidence has linked PI3Kγ to the modulation of autoimmune and inflammatory processes making it an intriguing target for drug discovery. Our high-throughput screening (HTS) campaign revealed two hits that were nominated for further optimization studies. The in vitro activity of the first HTS hit, designated as the sulfonylpiperazine scaffold, was optimized utilizing structure-based design. However, nonoptimal pharmacokinetic properties precluded this series from further studies. An overlay of the X-ray structures of the sulfonylpiperazine scaffold and the second HTS hit within their complexes with PI3Kγ revealed a high degree of overlap. This feature was utilized to design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability.

Discovery of a novel class of potent and orally bioavailable sphingosine 1-phosphate receptor 1 antagonists.

A series of subtype selective sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists are disclosed. Our high-throughput screening campaign revealed hit 1 for which an increase in potency and mouse oral exposure was achieved with minor modifications to the chemical scaffold. In vivo efficacy revealed that at high doses compounds 12 and 15 inhibited tumor growth. Further optimization of our lead series led to the discovery of proline derivatives 37 (XL541) and 38 which had similar efficacy as our first generation analogues at significantly lower doses. Analogue 37 displayed excellent pharmacokinetics and oral exposure in multiple species.