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.

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for

Targeting cytotoxic drugs to cancer cells using antibody-drug conjugates (ADCs), particularly those with stable linkers between the drug and the antibody, could be an effective cancer treatment with low toxicity. However, for stable-linker ADCs to be effective, they must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes. CD79a and CD79b

Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-

MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences (''mucin repeats'') in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique, nonrepeating epitope in the ECD and another (3A5) that recognizes the repeats and binds multiple sites on each MUC16 protein. 3A5 conjugated to cytotoxic drugs exhibited superior toxicity against tumor cells in vitro and in tumor xenograft models compared with antibody-drug conjugates of 11D10. Importantly, drug conjugates of 3A5 were well tolerated in primates at levels in excess of therapeutic doses. Additionally, the presence of circulating CA125 in a rat model did not exacerbate the toxicity of 3A5 drug conjugates. We conclude that targeting the repeat MUC16 domains, thereby increasing cell-associated levels of drug-conjugated antibody, provides superior efficacy in vitro and in vivo without compromising safety. [Cancer Res 2007;67(10):4924-32]