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 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.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Epidermal growth factor receptor (EGFR) is a type I receptor tyrosine kinase, the deregulation of which has been implicated in a variety of human carcinomas. EGFR signalling is preceded by receptor dimerization, typically thought to result from a ligand-induced conformational change in the ectodomain that exposes a loop (dimerization arm) required for receptor association. Ligand binding may also trigger allosteric changes in the cytoplasmic domain of the receptor that is crucial for signalling. Despite these insights, ensemble-averaging approaches have not determined the precise mechanism of receptor activation in situ. Using quantum-dot-based optical tracking of single molecules combined with a novel time-dependent diffusivity analysis, here we present the dimerization dynamics of individual EGFRs on living cells. Before ligand addition, EGFRs spontaneously formed finite-lifetime dimers kinetically stabilized by their dimerization arms. The dimers were primed both for ligand binding and for signalling, such that after EGF addition they rapidly showed a very slow diffusivity state that correlated with activation. Although the kinetic stability of unliganded dimers was in principle sufficient for EGF-independent activation, ligand binding was still required for signalling. Interestingly, dimers were enriched in the cell periphery in an actin- and receptor-expression-dependent fashion, resulting in a peripheral enhancement of EGF-induced signalling that may enable polarized responses to growth factors.