Abstract 1545: Targeting anticancer agents to the tumor microenvironment with cathepsin B cleavable drug-linker conjugates of phosphatidylserine-binding proteins
Authors: Deng Yuling , Ford Jacob W. , VanNatta Jenny M. , Maguire Casey J. , Mondal Deboprosad , Phinney Natalie Z. , Brekken Rolf A. , Pinney Kevin G. , Trawick Mary Lynn 
Published: June 2023

Abstract
In healthy cells, the membrane lipid phosphatidylserine (PS) is confined to the inner leaflet of the plasma membrane but in cancer cells and in the tumor vasculature PS is prevalent in the outer leaflet where it is highly immunosuppressive. In this study engineered proteins, such as Bavituximab, a chimeric monoclonal antibody that binds to PS via the abundant serum protein β2-glycoprotein 1, and constructs containing the PS binding domain of β2-glycoprotein 1 (betabodies), are conjugated to anticancer drug-linkers designed to be cleavable by cathepsin B. Cathepsin B is a cysteine protease that is abundant in the lysosomes of normal cells but is upregulated and trafficked to the cell surface and is secreted into the extracellular parenchyma in solid tumors, including breast and pancreatic cancer. A series of drug-linker constructs were synthesized that incorporate unique small-molecule payloads with dual mechanism of action, functioning as both potent tubulin-binding antimitotic agents and vascular disrupting agents. The payloads include benzosuberene-based aniline KGP156 and its phenolic congener KGP18, dihydronaphthalene-based KGP05, and indole-based OXi8006. These drug-linker constructs were reacted with N-acetyl-L-cysteine (NAC) and evaluated for cathepsin B cleavage, and liberation of payload after spontaneous 1,6-fragmentation and decarboxylation. The drug linker construct, NAC-succinimidocaproyl-L-valine-L citrulline-p-aminobenzyl carbamate derivatized with aminobenzosuberene, KGP156, was cleaved by cathepsin B (260 nM) with release of payload at the rate of 12.7 μM/min and by cathepsin L (600 nM) at a rate of 11. 5 μM/min. Other drug-linker constructs in this series demonstrated slower release of payload upon cleavage with cathepsin B or cathepsin L. The drug-linker construct incorporating aminodihydronaphthalene-based KGP05 released KGP05 at a rate of 2.4 μM/min with a comparable rate upon exposure to cathepsin L. Payload release from drug-linker constructs was not observed, over 24 h in the absence of enzyme. Drug-linker constructs without the Val-Cit dipeptide were not cleaved by cathepsin B. The most efficient cathepsin B drug-linker substrate, Mc-Val-Cit-PABC-KGP156, was conjugated to Baxituximab to form the corresponding antibody-drug conjugate (ADC). Baxituximab was first reacted with 2-iminothiolane, a thiolating agent that reacts with accessible lysine residues. Reaction with Ellman’s reagent revealed an -SH/antibody ratio of 3.5. Conjugation of thiolated Bavituximab with Mc-Val-Cit-PABC-KGP156 gave a drug/antibody ratio (DAR) of 3.6/1 after cleavage by cathepsin B and determination of released KGP156 by LC-MS. These studies revealed 1) distinct differences in cleavage rates for drug-linkers, 2) cleavage by cathepsin L, and 3) efficient cathepsin B mediated release of payload from the ADC.
Citation Format: Yuling Deng, Jacob W. Ford, Jenny M. VanNatta, Casey J. Maguire, Deboprosad Mondal, Natalie Z. Phinney, Rolf A. Brekken, Kevin G. Pinney, Mary Lynn Trawick. Targeting anticancer agents to the tumor microenvironment with cathepsin B cleavable drug-linker conjugates of phosphatidylserine-binding proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1545.