The humanized monoclonal antibody Abegrin TM , currently in phase II trials for treatment of solid tumors, specifically recognizes the integrin A v B 3 . Due to its high expression on mature osteoclasts, angiogenic endothelial cells, and tumor cells, integrin A v B 3 functions in several pathologic processes important to tumor growth and metastasis. Targeting of this integrin with Abegrin TM results in antitumor, antiangiogenic, and antiosteolytic activities. Here, we exploit the species specificity of Abegrin TM to evaluate the effects of direct targeting of tumor cells (independent of targeting of endothelia or osteoclasts). Flow cytometry analysis of human tumor cell lines shows high levels of A v B 3 on many solid tumors, including cancers of the prostate, skin, ovary, kidney, lung, and breast. We also show that tumor growth of A v B 3 -expressing tumor cells is inhibited by Abegrin TM in a dose-dependent manner. We present a novel finding that high-dose administration can actively impair the antitumor activity of Abegrin TM . We also provide evidence that antibody-dependent cellular cytotoxicity contributes to in vitro and in vivo antitumor activity. Finally, it was observed that peak biological activity of Abegrin TM arises at serum levels that are consistent with those achieved in clinical trials. These results support a concept that Abegrin TM can be used to achieve selective targeting of the many tumor cells that express A v B 3 integrin. In combination with the well-established concept that A v B 3 plays a key role in cancer-associated angiogenesis and osteolytic activities, this triad of activity could provide new opportunities for therapeutic targeting of cancer.

A new series of naphthalene analogs of medetomidine have been prepared and evaluated for their alpha-adrenergic activities. The methylnaphthyl analog 5a showed significant selectivity for alpha 2-adrenoceptors and behaved as a partial alpha 1-agonist in rat aorta preparations. In contrast, the Z-ethylene analog 8c was alpha 1-selective and behaved as a potent alpha 1-antagonist. Two rigid analogs (6 and 7) exhibited large differences in binding affinities at alpha 1-VS alpha 2-receptors, indicating that the conformational flexibility of 5a is important for the fulfillment of the alpha-adrenergic activities. Molecular modeling studies began with conformational analysis of classical phenethylamines and medetomidine analogs. Superimposition of medetomidine conformations with those of phenethylamines provided a tentative explanation for the alpha 2-adrenergic activity of the new imidazoles. A common binding mode for phenethylamines and imidazoles with alpha 2-adrenoceptors is proposed. Knowledge of the biological properties of the 4-substituted imidazoles, integrated with the information derived from computer-assisted molecular modeling, has provided new insights for the structural and conformational requirements of this class as new adrenergic drugs.

Biotherapeutic proteins are commonly dosed at high concentrations into the blood, which is an inherently complex, crowded solution with substantial protein content. The effects of macromolecular crowding may lead to an appreciable level of non-specific hetero-association in this physiological environment. Therefore, developing a method to characterize the diverse consequences of nonspecific interactions between proteins under such non-ideal, crowded conditions, which deviate substantially from those commonly employed for in vitro characterization, is vital to achieving a more complete picture of antibody function in a biological context. In this study, we investigated non-specific interactions between human serum albumin (HSA) and two monoclonal antibodies (mAbs) by static light scattering and determined these interactions are both ionic strength-dependent and mAbdependent. Using biolayer interferometry (BLI), we assessed the effect of HSA on antigen binding by mAbs, demonstrating that these non-specific interactions have a functional impact on mAb:antigen interactions, particularly at low ionic strength. While this effect is mitigated at physiological ionic strength, our in vitro data support the notion that HSA in the blood may lead to non-specific interactions with mAbs in vivo, with a potential impact on their interactions with antigen. Furthermore, the BLI method offers a high-throughput advantage compared to orthogonal techniques such as analytical ultracentrifugation and is amenable to a greater variety of solution conditions compared to nuclear magnetic resonance spectroscopy. Our study demonstrates that BLI is a viable technology for examining the impact of non-specific interactions on specific biologically relevant interactions, providing a direct method to assess binding events in crowded conditions.