When epidermal growth factor and its relatives bind the ErbB family of receptors, they trigger a rich network of signalling pathways, culminating in responses ranging from cell division to death, motility to adhesion. The network is often dysregulated in cancer and lends credence to the mantra that molecular understanding yields clinical benefit: over 25,000 women with breast cancer have now been treated with trastuzumab (Herceptin), a recombinant antibody designed to block the receptor ErbB2. Likewise, small-molecule enzyme inhibitors and monoclonal antibodies to ErbB1 are in advanced phases of clinical testing. What can this pathway teach us about translating basic science into clinical use?
HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease. A variation on antibody-targeted therapy is utilization of antibodies to deliver cytotoxic agents specifically to antigenexpressing tumors. We determined in vitro and in vivo efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid (microtubule-depolymerizing agents) conjugates using disulfide and thioether linkers. Antiproliferative effects of trastuzumab-maytansinoid conjugates were evaluated on cultured normal and tumor cells. In vivo activity was determined in mouse breast cancer models, and toxicity was assessed in rats as measured by body weight loss. Surprisingly, trastuzumab linked to DM1 through a nonreducible thioether linkage (SMCC), displayed superior activity compared with unconjugated trastuzumab or trastuzumab linked to other maytansinoids through disulfide linkers. Serum concentrations of trastuzumab-MCC-DM1 remained elevated compared with other conjugates, and toxicity in rats was negligible compared with free DM1 or trastuzumab linked to DM1 through a reducible linker. Potent activity was observed on all HER2-overexpressing tumor cells, whereas nontransformed cells and tumor cell lines with normal HER2 expression were unaffected. In addition, trastuzumab-DM1 was active on HER2-overexpressing, trastuzumab-refractory tumors. In summary, trastuzumab-DM1 shows greater activity compared with nonconjugated trastuzumab while maintaining selectivity for HER2-overexpressing tumor cells. Because trastuzumab linked to DM1 through a nonreducible linker offers improved efficacy and pharmacokinetics and reduced toxicity over the reducible disulfide linkers evaluated, trastuzumab-MCC-DM1 was selected for clinical development. [Cancer Res 2008;68(22):9280-90]
Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-Å resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, Tarceva TM ). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization.Growth factor interactions with cell surface receptors influence proliferation, survival, differentiation, and metabolism (1). The loss of control over these vital cellular processes is a hallmark of oncogenesis (2). For instance, aberrant signaling from overexpressed growth factor receptor ErbB2 is causal in approximately 30% of invasive breast cancers (3). Growth factors bind to a cognate membrane-bound receptor system and mediate changes in the intracellular portion of the receptor, often through the formation of dimers or oligomers of receptors that initiate signal transduction cascades. The epidermal growth factor receptor (EGFR, 1 also ErbB1 or HER1) and its ligands, epidermal growth factor (EGF) and transforming growth factor-␣, are among the earliest characterized members of the growth factor/receptor tyrosine kinase (RTK) family. In contrast to the widely applicable ligand-induced receptor dimerization paradigm, there is evidence that EGFR family members exist as preformed dimers (4) and form higher oligomer signaling complexes (5). Normal signaling in the EGFR system involves ligand-induced homo-oligomerization or hetero-oligomerization with the closely related RTKs ErbB2 (HER2), ErbB3 (HER3) and/or ErbB4 (HER4) (6). Autophosphorylation of key tyrosine residues within the carboxyl-terminal portion of the receptor provides sites for direct interaction with SH2-containing proteins, leading to subsequent signal transduction events.The EGFR system, including receptor homologues and relevant ligands, is complex. There are at least 12 different ligands that bind to the EGF receptor family with partially redundant specificity for certain receptors. Several of the ligands including EGF, transforming growth factor-␣, heparin-binding EGF, and betacellulin are reported to bind to EGFR with nanomolar dissociation constants (7). Betacellulin also binds ErbB4 with high affinity. Similarly, heregulin binds to ErbB3 or ErbB4 with dissociation constants in the nanomolar range. So ...
The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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