Rita Steeves

Disulfide-Linked Antibody−Maytansinoid Conjugates: Optimization of In Vivo Activity by Varying the Steric Hindrance at Carbon Atoms Adjacent to the Disulfide Linkage

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.

αv Integrin-Targeted Immunoconjugates Regress Established Human Tumors in Xenograft Models

Purpose: Targeted delivery of cytotoxic agents to solid tumors through cell surface antigens can potentially reduce systemic toxicity and increase the efficacy of the targeted compounds. The purpose of this study was to show the feasibility of treating solid tumors by targeting αv integrins with antibody-maytansinoid conjugates and to test the relative in vivo activities of several linker-maytansinoid chemistries. Experimental Design: CNTO 364, CNTO 365, and CNTO 366 are targeted cytotoxic agents created by conjugating the CNTO 95 anti-αv integrin antibody with three distinct maytansinoid-linker structures. These structures were designed to have varying degrees of chemical substitution surrounding the disulfide bond linking the cytotoxic agent to the antibody. A model conjugate was shown to be specifically cytotoxic in vitro and highly active against established human tumor xenografts in immunocompromised rats. The in vivo antitumor activities of CNTO 364, CNTO 365, and CNTO 366 were compared in rat xenograft models. Results: CNTO 365, with a linker chemistry of expected intermediate stability, was shown to be substantially more active than the other two conjugates with lesser or greater substitution around the disulfide linkage. Conclusion: CNTO 95–maytansinoid immunoconjugates are potent antitumor agents against αv integrin–expressing human carcinomas. These studies show for the first time the feasibility of targeting αv integrins on solid tumors with tumor-activated prodrugs. The DM4 linker-maytansinoid configuration of CNTO 365 was substantially more active in the models tested here when compared with alternative configurations with greater or lesser chemical substitution surrounding the linker.

The identification and isolation of reactive thiols in ricin A-chain and blocked ricin using 2-(4′-maleimidylanilino)naphathalene-6-sulfonic acid

Publisher Summary Identification of the affinity ligand attachment sites on the B-chain of proteins like blocked ricin is a matter of interest in recent studies. In general, the identification of reactive or chemically modified residues of proteins is extremely important for the characterization of proteins and their activity. Ricin is a heterodimeric protein composed of a toxic A-chain, which is responsible for inhibiting cellular protein synthesis, disulfide-linked to a B-chain, known to possess lectin activity. Identification of the residues of ricin involved in the covalent linkage to the affinity ligand is important for complete characterization of the cytotoxic effector moiety, and to ensure consistency of the immunoconjugate product. However, because of the intrinsic heterogeneity of the ligand, isolation of individual species of ligandbound B-chain peptides by traditional peptide mapping has not been possible. To minimize the effect of the ligand heterogeneity, a different approach was conceived exploiting the incorporated protected thiol at the peptide “end” of the ligand. The thiol-specific probe, 2-(4 ′ -maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) was used to label the ligand linked to B-chain in situ . MIANS is an attractive choice because its characteristic absorbance profile facilitates the identification of the peptides of interest. A monoclonal antibody recognizing MIANS is produced, such that affinity chromatography can be used to isolate only those peptides of the B-chain that are cross-linked to the MIANS-labeled ligand. This chapter discusses the results obtained using MIANS-labeling in conjunction with affinity chromatography to map free thiols in reduced, native ricin A-chain. It further describes the results obtained when utilizing this method to isolate ligand-bound ricin B-chain peptides.