Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates:  Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity

Abstract

The anticancer drug doxorubicin (DOX) has been linked to chimeric BR96, an internalizing monoclonal antibody that binds to a Lewis(y)-related, tumor-associated antigen, through two lysosomally cleavable dipeptides, Phe-Lys and Val-Cit, giving immunoconjugates 72 and 73. A self-immolative p-aminobenzyloxycarbonyl (PABC) spacer between the dipeptides and the DOX was required for rapid and quantitative generation of free drug. DOX release from model substrate Z-Phe-Lys-PABC-DOX 49 was 30-fold faster than from Z-Val-Cit-PABC-DOX 42 with the cysteine protease cathepsin B alone, but rates were identical in a rat liver lysosomal preparation suggesting the participation of more than one enzyme. Conjugates 72 and 73 showed rapid and near quantitative drug release with cathepsin B and in a lysosomal preparation, while demonstrating excellent stability in human plasma. Against tumor cell lines with varying levels of BR96 expression, both conjugates showed potent, antigen-specific cytotoxic activity, suggesting that they will be effective in delivering DOX selectively to antigen-expressing carcinomas.

Linkage with cathepsin B-sensitive dipeptide promotes the in vitro and in vivo anticancer activity of PEGylated tumor necrosis factor-alpha (TNF-α) against murine fibrosarcoma

“…We showed that PEG-vcTNF-α was cleaved by cysteine proteases, but not other proteases, to release TNF-α, and this release is dependent upon pH. Our finding that the valine-citrulline linker peptide is specifically targeted by cysteine proteases to release the peptide drug from the PEGylated conjugate is in agreement with previous reports [20][21][22][23][24].…”

“…Previous reports have demonstrated the success of conjugating a cytotoxic agent to a tumor-selective monoclonal antibody (mAb) using a protease-sensitive dipeptide linker. These antibody-drug conjugates include cBR96-valine-citrulline-MMAE and BR96-valine-citrulline-DOX (specific to Lewis Y antigen on carcinomas), cAC10-valine-citrulline-MMAE (specific to CD30 on hematological malignancies), and huPr1-valine-citrulline-MMAE (specific to hormone-resistant prostate cancer), and have high stability in plasma and efficient release of drug in tumor tissues [20][21][22][23][24][25].…”

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

“…The removal of the acetazolamide moiety from the linker-payload combinations abrogated therapeutic activity in all experimental systems, confirming that the ligand-based delivery of cytotoxic drugs to the extracellular tumor environment represents a strict requirement for anticancer efficacy. Both compounds 4a and 5a described in this article feature a linker containing a valine-citrulline moiety, which was previously believed to be particularly suited for the intracellular release of drug payloads, due to the action of lysosomal cathepsin B [41]. This protein is a primarily intracellular protease that can be secreted extracellularly by dying cells, but also by living tumor cells to initiate extracellular proteolytic cascades and to enable tumor cell proliferation [42].…”

CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

“…Additionally we explored antibody-drug conjugates (ADCs) as a therapeutic modality for the selective targeting of CD133-expressing tumours. Recent advances with ADCs using peptide linkers showed enhanced efficacy and better specificity to antigen-expressing cells (Dubowchik et al, 2002;Doronina et al, 2003Doronina et al, , 2006. Specifically, an anti-human CD133 monoclonal antibody (MAb), AC133, was directly conjugated to the potent cytotoxic drug, monomethyl auristatin F (MMAF) , using a valine-citrulline dipeptide linker that is cleavable by proteases (Doronina et al, 2003).…”