In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.

Maytansine, a highly cytotoxic natural product, failed as an anticancer agent in human clinical trials because of unacceptable systemic toxicity. The potent cell killing ability of maytansine can be used in a targeted delivery approach for the selective destruction of cancer cells. A series of new maytansinoids, bearing a disulfide or thiol substituent were synthesized. The chain length of the ester side chain and the degree of steric hindrance on the carbon atom bearing the thiol substituent were varied. Several of these maytansinoids were found to be even more potent in vitro than maytansine. The targeted delivery of these maytansinoids, using monoclonal antibodies, resulted in a high, specific killing of the targeted cells in vitro and remarkable antitumor activity in vivo.

1429 Several anti-tumor antibody-maytansinoid conjugates, commonly called TAPs (tumor-activated prodrugs) are currently under clinical evaluation. In particular, cantuzumab mertansine (huC242-DM1), has been evaluated in three Phase I clinical trials (A.W. Tolcher et al., J Clin Oncol 2003, 21:211-22; P.R. Helft et al., Clin Cancer Res 2004, 10:4363-4368) using different dosing schedules to treat patients with tumors expressing the CanAg target antigen. The immunoconjugate could be safely administered at pharmacologically meaningful doses and evidence of anti-tumor activity was seen in several patients, which led to a recommendation for broad clinical development of the agent (A.W. Tolcher et al., J Clin Oncol 2003, 21:211-22). During the clinical evaluation, further advancement in the TAP technology led to the development of TAPs with disulfide-containing linkers with increased stability under physiological conditions. One of these new TAPs that demonstrated improved pharmacokinetic behavior and superior anti-tumor activity in murine test models is huC242-DM4, which contains the same antibody as cantuzumab mertansine and targets therefore the same CanAg-expressing tumors. HuC242-DM4 caused complete regressions in several tumor xenograft models even with single i.v. administration at doses far below its maximal tolerated dose (MTD). For example, treatment of SCID mice bearing measurable N87 human gastric tumor xenografts with a single dose of 3.4 mg/kg caused complete regression of the tumors, with significant tumor regression observed at a dose below 2 mg/kg. With a MTD in SCID mice of greater than 40 mg/kg when given as a single i.v. bolus injection, a therapeutic index of over 20 was achieved. HuC42-DM 4 was also curative in mice bearing human colon tumor xenografts of HT29 and COLO 205 cells, at doses that were non-toxic. The disulfide linkage of huC242-DM4 was approximately 2-fold more stable during circulation in mice than that of cantuzumab mertansine. While the huC242-DM4 conjugate demonstrated increased stability and efficacy in vivo compared to cantuzumab mertansine, preliminary acute toxicity studies suggest that the two conjugates have similar tolerability in normal mice. The resulting increased therapeutic window for huC242-DM4 provides a preclinical rationale for its clinical evaluation in patients with CanAg-positive tumors, such as colorectal, pancreatic, gastric, and esophageal carcinomas.

We conducted an expression analysis of prostate stem cell antigen (PSCA) in normal urogenital tissues, benign prostatic hyperplasia (n = 21), prostatic intraepithelial neoplasia (n = 33), and primary, (n = 137) and metastatic (n = 42) prostate adenocarcinoma, using isotopic in situ hybridization on tissue microarrays. In normal prostate, we observe PSCA expression in the terminally differentiated, secretory epithelium; strong expression was also seen in normal urothelium. Forty-eight percent of primary and 64% of metastatic prostatic adenocarcinomas expressed PSCA RNA. Our studies did not confirm a positive correlation between level of PSCA RNA expression and high Gleason grade. We characterized monoclonal anti-PSCA antibodies that recognize PSCA expressed on the surface of live cells, are efficiently internalized after antigen recognition, and kill tumor cells in vitro in an antigen-specific fashion upon conjugation with maytansinoid. Unconjugated anti-PSCA antibodies demonstrated efficacy against PSCA-positive tumors by delaying progressive tumor growth in vivo. Maytansinoid-conjugated antibodies caused complete regression of established tumors in a large proportion of animals. Our results strongly suggest that maytansinoid-conjugated anti-PSCA monoclonal antibodies should be evaluated as a therapeutic modality for patients with advanced prostate cancer.