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.

Validation of a capillary isoelectric focusing method for the recombinant monoclonal antibody C2B8.

A capillary isoelectric focusing (cIEF) method has been developed for the purpose of determining the identity and charge distribution of mouse/human chimeric antibody to human CD20 antigen (C2B8). The assay was validated in accordance with ICH guidelines in order to demonstrate that it is suitable for its intended purpose and so that it may be performed as a lot release test for bulk and final product. As a result of the validation process the assay was found to be linear over the concentration range of 2-356 micrograms ml-1 with recovery of 125I-labeled C2B8 at the target sample concentration of 125 micrograms ml-1 equal to 99%. The repeatability and intermediate precision relative standard deviations of the four major peaks for migration time, peak area, and peak area percent ranged from 0.9-4.4%. The specificity of the assay was demonstrated by baseline resolution of the C2B8 main peak from product excipients, and other Genentech monoclonal antibodies. The results of this validation demonstrate that the cIEF assay for the determination of identity and charge distribution of C2B8 is accurate, precise, linear, and highly specific. The assay is rapid and suitably rugged.

A simple, inexpensive, disposable electrochemical sensor for clinical and immuno-assay.

An inexpensive, rapid, disposable electrochemical system has been developed for both clinical and immunochemical assays. The three-electrode planar configuration is produced by silk screening graphite paint on to cardboard. Assays are carried out amperometrically using constant voltage and monitoring current produced by the oxidation of an electron mediator. In the case of immuno-assay the system described uses the enzyme glucose oxidase with 1,4-benzoquinone as the mediator. The assay is carried out by effecting a separation of the bound and free fractions of the immunoreactants via immobilizing the antibodies on to magnetic particles which are magnetically concentrated on the electrode surface for analysis. This concentration of the bound antigen-antibody complex with the glucose oxidase label effectively increases the concentration of the reduced mediator in the electrode vicinity, thus increasing the amperometric response to the bound fraction of the label.

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.

X-ray structures of fragments from binding and nonbinding versions of a humanized anti-CD18 antibody: structural indications of the key role of VH residues 59 to 65.

X-ray crystal structures of fragments from two different humanized anti-CD18 antibodies are reported. The Fv fragment of the nonbinding version has been refined in space group C2 with a = 64.2 A, b = 61.3 A, c = 51.8 A, and beta = 99 degrees to an R-value of 18.0% at 1.9 A, and the Fab fragment of the tight-binding version has been refined in space group P3 with a = 101. A and c = 45.5 A to an R-value of 17.8% at 3.0 A resolution. The very large difference in their binding affinity (> 1000-fold) is attributed to large and local structural differences in the C-terminal part of CDR-H2, and from this we conclude there is direct contact between this region and antigen when they combine. X-ray structures of antibody-antigen complexes available in the literature have yet to show this part of CDR-H2 in contact with antigen, despite its hypervariable sequence. Implications of this result for antibody humanization are discussed.

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.

Observation of optical pumping of mesospheric sodium.

We have observed a large variation with laser polarization in the amount of laser light resonantly backscattered from the Earth's mesospheric sodium layer located at a 90-km altitude. This variation is evidence of optical pumping of mesospheric sodium atoms.

Humanization of an anti-lymphocyte function-associated antigen (LFA)-1 monoclonal antibody and reengineering of the humanized antibody for binding to rhesus LFA-1.

Lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is involved in leukocyte adhesion during cellular interactions essential for immunologic responses and inflammation. mAbs against LFA-1 have been shown to inhibit several T cell-dependent immune functions in vitro and prevent graft failure after bone marrow transplantation in vivo. A murine anti-human CD11a mAb, MHM24, has been humanized and shown to prevent adhesion of human T cells to human keratinocytes and the proliferation of T cells in response to nonautologous leukocytes in the mixed lymphocyte response assay. However, of the nonhuman primate CD11a that we tested, the murine and humanized mAbs cross-reacted only with chimpanzee CD11a. To have a mAb available for preclinical studies in rhesus monkeys, the humanized mAb was reengineered to bind to rhesus CD11a by changing four residues in one of the complementarity-determining regions, CDR-H2, in the variable heavy domain. Cloning and molecular modeling of rhesus CD11a I-domain suggested that the changes from Lys197 and/or Arg189 in human CD11a I-domain to Glu197 and Gln189 in rhesus CD11a I-domain may be the reason that rhesus CD11a does not bind to the murine and humanized mAbs.

Secretion of glycosylation site mutants can be rescued by the signal/pro sequence of tissue plasminogen activator.

Strategies that prevent the attachment of N-linked carbohydrates to nascent glycoproteins often impair intracellular transport and secretion. In the present study, we describe a method to rescue the intracellular transport and secretion of glycoproteins mutagenized to delete N-linked glycosylation sites. Site-directed mutagenesis was used to delete N-linked glycosylation sites from a chimeric protein, TNFR-IgG1. Deletion of any of the three glycosylation sites in the TNFR portion of the molecule, alone or in combination, resulted in a moderate or near total blockade of TNFR-IgG1 intracellular transport and secretion. Pulse chase experiments suggested that the glycosylation site mutants accumulated in the endoplasmic reticulum (ER) and were inefficiently exported to the Golgi apparatus (GA). Replacement of the TNFR signal sequence with the signal/pro sequence of human tissue plasminogen activator (tPA) overcame the blockade to intracellular transport, and restored secretion to levels comparable to those achieved with the fully glycosylated molecule. Ligand binding studies suggested that the secreted glycosylation variants possessed binding characteristics similar to the fully glycosylated protein. This study demonstrates that N-terminal sequences of tPA are unexpectedly efficient in facilitating transport from the ER to the GA and suggests that these sequences contain a previously unrecognized structural element that promotes intracellular transport.