Published on January 21, 2020
slide 1: ADCs Linker Source: https://www.bocsci.com/tag/adcs-linker-488.html Clinically useful unconjugated monoclonal antibodies mAbs selectively recognize antigens that are preferentially expressed on or near tumor cells and exert their cytotoxic effects through mechanisms such as cell signaling antibody- dependent cellular cytotoxicity antibody-dependent cellular phagocytosis and complement-dependent cytotoxicity. However the majority of these mAbs are used in combination with chemotherapy and many others have demonstrated insufficient clinical activity. Therefore significant effort has been devoted to empowering mAbs through various modifications. One approach by which the activities of these mAbs have been enhanced is through conjugation with cytotoxic drugs generating antibody-drug conjugates ADCs capable of antigen-specific delivery of highly potent cytotoxic drugs to tumor cells. In order to improve efficacy without compromising safety an ADC must preferentially deliver the cytotoxic payload to tumor cells expressing the target antigen rather than to healthy tissue. This is accomplished by exploiting the specificity of a mAb targeting an antigen that is highly expressed on the surface of malignant cells. After binding the target antigen the ADC-antigen complex is typically internalized and transported to intracellular organelles where release of the attached drug can occur. Upon release the cytotoxic drug can interfere with various cellular mechanisms leading to cell death. The development of ADCs has relied on several key areas of research including the choice of an appropriate antigen target development of novel highly potent cytotoxic drugs development of stable linkers that can release the cytotoxic payload upon internalization and conjugation technology. Linker The linker that connects the cytotoxic drug to the mAb is a key determinant of ADC activity. These linkers covalently couple the cytotoxic drug to the antibody producing an ADC that should be relatively stable in circulation. Stability of the linker is important because it prevents damage to nontarget tissue through spontaneous release of the cytotoxic drug while maximizing tumor drug exposure. Upon internalization however the linkers should facilitate efficient drug release. Intracellular conditions such as the low-pH environment in lysosomes or the reducing environment of the cytosol can destabilize acid-labile hydrazone linkers or disulfide-based linkers respectively resulting in drug release. ADCs have entered the clinic with a variety of different linkers that all effectively balance these attributes. The properties of a successful linker can be slide 2: split up into different modules that have been combined to form an effective bridge between the cytotoxic payload and the carrier antibody. Connection to the antibody The two most common naturally occurring amino acids that are used to attach the linker drug to the antibody are cysteines and lysines. Attachment chemistry is limited to those reactions that are either selective for a primary thiol or a primary amine unless unnatural amino acids are incorporated into the antibody. Lysine residues are the attachment point for trastuzumab emtansine T-DM1 that is currently in Phase III clinical testing for breast cancer. The ADC is composed of the antibody trastuzumab connected to a cytotoxic maytansine derivative DM1. In this case the first step of the conjugation involves reacting succinimidyl-4-N- maleimidomethylcyclohexane-1-carboxylate SMCC with a lysine residue. The N-hydroxysuccinimide ester enables formation of a stable amide bond with the primary amine of one of the 84 available lysine residues on the antibody at pH 7- 9. The maleimide moiety undergoes a Michael addition with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds that are used to attach the drug to the antibody. The process results in the formation of a mixture of conjugates with drug-antibody ratios DARs ranging from 0 to 9. The average DAR for trastuzumab emtansine is 3.5. Polarity The overall polarity of the linker plays a large role in the pharmacokinetics potency DAR and efficacy of the ADC. Many of the commonly used linkers and cytotoxic agents used with ADCs are inherently hydrophobic. Drug loads are normally on the order of 3-4 drugs per antibody. Increasing the drug load of the ADC may be desired in cases where the target antigen density on the cell surface is low and the antibody needs more payloads to deliver a cytotoxic level of drug. Efforts to increase the drug load on the antibody have often failed because the resulting ADC suffers from increased aggregation higher systemic clearance or loss of affinity for the antigen. In the case of T-DM1 attempts to increase the drug load to an average DAR 5 resulted in high levels of aggregation and subsequent precipitation of the ADC. A polyethylene glycol or sulfate group was introduced into the linker portion of the ADC and DAR ratios of up to 9 were formed without appreciable aggregation or loss of affinity to the target antigen. Reference: Flygare J. A. Pillow T. H. Aristoff P. 2013. Antibody-drug conjugates for the treatment of cancer. Chemical biology drug design 811 113-121.