Stability and interactions of recombinant human nerve growth factor in different biological matrices: in vitro and in vivo studies.

The purpose of this investigation was to characterize the stability, activity, and interactions of recombinant human nerve growth factor (rhNGF) in various biological matrices in vitro and in vivo. rhNGF (10 microg/ml) remained stable in human plasma for up to 4 days at 37 degrees C. There was a decrease in the recovery of rhNGF after incubation at lower concentrations (20 ng/ml) and for longer time periods (3 and 5 days at 37 degrees C). Size exclusion HPLC analysis indicated that rhNGF forms high molecular weight (HMW) complexes after long incubation periods. We confirmed that alpha(2)-macroglobulin (alpha(2)M) is the major plasma component that binds to rhNGF. Furthermore, this interaction was considerably increased by treatment of plasma with primary amines such as CH(3)NH(2). Changes in the pH environment did not affect the interaction of rhNGF with alpha(2)M. We also determined that the binding of rhNGF to CH(3)NH(2)-treated pure alpha(2)M or alpha(2)M present in human plasma substantially diminished its immunoreactivity and bioactivity detection. The interaction of rhNGF with activated alpha(2)M was reversed and inhibited by coincubation with dimethyl sulfoxide. Released rhNGF under these conditions was fully bioactive. (125)I-rhNGF also binds to alpha(2)M by forming similar (125)I-rhNGF/HMW complexes in plasma after i.v. administration in rats and mice. Sixty minutes after dosing in rats, most of the labeled material was in the form of a (125)I-rhNGF/HMW complex. These studies have provided a better understanding of the nature of the interactions of rhNGF with plasma components as well as methods to enhance, reverse, and inhibit these interactions.

Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin).

HER2 is a ligand-less member of the human epidermal growth factor receptor or ErbB family of tyrosine kinases. In normal biological systems, HER2 functions as a co-receptor for a multitude of epidermal growth factor-like ligands that bind and activate other HER family members. HER2 overexpression is observed in a number of human adenocarcinomas and results in constitutive HER2 activation. Specific targeting of these tumors can be accomplished with antibodies directed against the extracellular domain of the HER2 protein. One of these antibodies, 4D5, has been fully humanized and is termed trastuzumab (Herceptin; Genentech, San Francisco, CA). Treatment of HER2-overexpressing breast cancer cell lines with trastuzumab results in induction of p27KIP1 and the Rb-related protein, p130, which in turn significantly reduces the number of cells undergoing S-phase. A number of other phenotypic changes are observed in vitro as a consequence of trastuzumab binding to HER2-overexpressing cells. These phenotypic changes include downmodulation of the HER2 receptor, inhibition of tumor cell growth, reversed cytokine resistance, restored E-cadherin expression levels, and reduced vascular endothelial growth factor production. Interaction of trastuzumab with the human immune system via its human immunoglobulin G1 Fc domain may potentiate its antitumor activities. In vitro studies demonstrate that trastuzumab is very effective in mediating antibody-dependent cell-mediated cytotoxicity against HER2-overexpressing tumor targets. Trastuzumab treatment of mouse xenograft models results in marked suppression of tumor growth. When given in combination with standard cytotoxic chemotherapeutic agents, trastuzumab treatment generally results in statistically superior antitumor efficacy compared with either agent given alone. Taken together, these studies suggest that the mechanism of action of trastuzumab includes antagonizing the constitutive growth-signaling properties of the HER2 system, enlisting immune cells to attack and kill the tumor target, and augmenting chemotherapy-induced cytotoxicity.

Tissue distribution and complex formation with IgE of an anti-IgE antibody after intravenous administration in cynomolgus monkeys.

A humanized antihuman IgE antibody, rhuMAb-E25, was designed to form complexes with free IgE, blocking its interaction with mast cells and basophils and thereby preventing the initiation of the allergic cascade. To characterize the rhuMAb-E25: IgE complexes formed in vivo and to examine the disposition of the antibody in a relevant animal model, 125I-rhuMAb-E25 was administered as an intravenous bolus dose to cynomolgus monkeys that have high levels of IgE. The pharmacokinetic values of unlabeled and radiolabeled antibody were similar, which indicated that the disposition of 125I-rhuMAb-E25 reflected that of rhuMAb-E25. Size-exclusion chromatography of serum samples showed that the rhuMAb-E25:IgE complexes were of limited size and were similar to the small complexes formed in vitro with human IgE. Pharmacokinetic analysis revealed that both rhuMAb-E25 and rhuMAb-E25:IgE complexes cleared the serum compartment, albeit slowly. No specific uptake of radioactivity was seen in any of the tissues collected from the cynomolgus monkeys at 1 hr and 96 hr postadministration; no association was observed between 125I-rhuMAb-E25, or the complexes, and blood cells. Urinary excretion was the primary route of elimination of radioactivity; > 90% of the radioactivity found in urine was not associated with protein. The lack of specific tissue uptake and blood cell association and the slow clearance of rhuMAb-E25:IgE complexes were consistent with low-avidity interaction of small complexes with Fc gamma receptors of leukocytes and the reticuloendothelial system.