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 murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (pl85"m), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "hum " antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant do was constructed. Light-and heavy-chain variable regions were simultaneously humned in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonudleotides, respectively. The protooncogene HER2 encodes a protein tyrosine kinase (pl85HER2) that is homologous to the human epidermal growth factor receptor (1-3). Amplification and/or overexpression of HER2 is associated with multiple human malignancies and appears to be integrally involved in progression of 25-30%o of human breast and ovarian cancers (4, 5).Furthermore, the extent of amplification is inversely correlated with the observed median patient survival time (5). The murine monoclonal antibody mumAb4D5 (6), directed against the extracellular domain (ECD) of p185HER2, specifically inhibits the growth of tumor cell lines overexpressing p185HER2 in monolayer culture or in soft agar (7,8).mumAb4D5 also has the potential of enhancing tumor cell sensitivity to tumor necrosis factor (7,9). Thus, mumAb4D5 has potential for clinical intervention in carcinomas involving the overexpression of p185HER2.A major limitation in the clinical use of rodent mAbs is an anti-globulin response during therapy (10,11). A partial solution to this problem is to construct chimeric antibodies by coupling the rodent antigen-binding variable (V) domains to human constant (C) domains (12)(13)(14). The isotype of the human C domains may be varied to tailor the chimeric antibody for participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (15). Such chimeric antibody molecules are still =30% rodent in sequence and are capable of eliciting a significant anti-globulin response.Winter and coworkers (16-18) pioneered the "humanization" of antibody V domains by transplanting the complementarity determining regions (CDRs), which are the hypervariable loops involved in antigen binding, from rodent antibodies into human V domains. The validity of this approach is supported by the clinical efficacy of a humanized antibody specific for the CAMPATH-1 antigen with two non-Hodgkin lymphoma patients, one of whom had previously developed an anti-globulin response to the parental rat antibody (17,19). In some cases, transplanting hypervariable loops from rodent antibodies into human frameworks is sufficient to transfer high antigen binding affinity (16, 18), whereas in other cases it has been necessary to also replace one (17) or several (20) framework region (FR) residues. For a given antibody, a small number of FR residues are ...

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.

Based on the knowledge that neutrophil elastase (NE) in cystic fibrosis (CF) epithelial lining fluid (ELF) can induce human bronchial epithelial cells to express the gene for interleukin 8 (IL-8), an 8.5-kD neutrophil chemoattractant, we have evaluated CF ELF for the presence of IL-8, and investigated the ability of aerosolized recombinant secretory leukoprotease inhibitor (rSLPI) to suppress NE, and hence IL-8, levels on the respiratory epithelial surface in CF. Enzyme-linked immunoassay revealed 21.9±4.8 nM IL-8 in CF ELF compared with none in normals. Active NE was detectable in ELF of all individuals with CF and was significantly decreased (P < 0.03) after aerosolization of rSLPI. Human bronchial epithelial cells exposed to CF ELF recovered before rSLPI therapy expressed IL-8 mRNA transcripts, but ELF recovered after rSLPI therapy induced far less bronchial epithelial cell IL-8 gene expression. Consistent with this, rSLPI aerosol therapy caused a marked reduction in CF ELF IL-8 levels (P < 0.05) and neutrophil number (P < 0.02). There was also a clear association between CF ELF active NE and IL-8 levels (r = 0.94). These data suggest that rSLPI therapy not only suppresses respiratory epithelial NE levels, but also breaks a cycle of inflammation on the CF epithelial surface. (J. Clin. Invest. 1992. 90:1296-1301.) Key words: aerosol therapy * antiprotease * cytokinev neutrophil chemoattractant * neutrophil elastase