Site-specific antibody drug conjugates for cancer therapy

Abstract

Antibody therapeutics have revolutionized the treatment of cancer over the past two decades. Antibodies that specifically bind tumor surface antigens can be effective therapeutics; however, many unmodified antibodies lack therapeutic activity. These antibodies can instead be applied successfully as guided missiles to deliver potent cytotoxic drugs in the form of antibody drug conjugates (ADCs). The success of ADCs is dependent on four factors--target antigen, antibody, linker, and payload. The field has made great progress in these areas, marked by the recent approval by the US Food and Drug Administration of two ADCs, brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). However, the therapeutic window for many ADCs that are currently in pre-clinical or clinical development remains narrow and further improvements may be required to enhance the therapeutic potential of these ADCs. Production of ADCs is an area where improvement is needed because current methods yield heterogeneous mixtures that may include 0-8 drug species per antibody molecule. Site-specific conjugation has been recently shown to eliminate heterogeneity, improve conjugate stability, and increase the therapeutic window. Here, we review and describe various site-specific conjugation strategies that are currently used for the production of ADCs, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases. In addition, we also summarize differences among these methods and highlight critical considerations when building next-generation ADC therapeutics.

Keywords: THIOMAB; antibody drug conjugate; cytotoxic drug; internalization; linker; site-specific conjugation; tumor antigen.

Figure 1. ADCs expand the therapeutic window. ADC therapeutics can increase efficacy and decrease toxicity in comparison to traditional chemotherapeutic cancer treatments. Select delivery of drugs to cancer cells increases the percent of dosed drug reaching the tumor, thus lowering the minimum effective dose (MED). The maximum tolerated dose (MTD) is increased, as less drug reaches normal, non-target tissue due to targeted delivery by the antibody. Taken together, the therapeutic window is improved by the use of ADCs.

Figure 2. Delivery of cytotoxic drugs to cancer cells by ADCs. The monoclonal antibody component of an ADC selectively binds a cell-surface tumor antigen, resulting in internalization of the ADC-antigen complex through the process of receptor-mediated endocytosis. The ADC-antigen complex then traffics to lysosomal compartments and is degraded, releasing active cytotoxic drug inside the cell. Free drug causes cell death through either tubulin polymerization inhibition or DNA binding/damage depending on the drugs mechanism of action.

Figure 3. Critical factors that influence ADC therapeutics. ADCs consist of a cytotoxic drug conjugated to a monoclonal antibody bu means of a select linker. These components all affect ADC performance and their optimization is essential for development of successful conjugates.

Figure 4. ADC metabolism in vivo. The therapeutic window of an ADC depends on the optimization of the delicate balance between efficacy and toxicity. The desired effect of ADCs is the target-dependent killing of tumor cells expressing high levels of target antigen (A). Side effects can be caused by target-dependent toxicity and killing of normal cells expressing low levels of target antigen (B), or by target-independent toxicity caused by entry of free drug into normal cells (C). Free drug can be released by ADC catabolism or by unstable labile linkers in the plasma.

Figure 5. Conjugation methods for ADC development. ADC production using traditional conjugation through lysine residues or reduction of inter-chain disulfide bonds results in high heterogeneity in both drug to antibody ratio (DAR) and location of conjugation site. Site-specific conjugation greatly decreased this heterogeneity. (A) Lysine conjugation results in a DAR of 0–8 and potential conjugation at ~40 lysine residues/mAb. (B) Conjugation through reduced inter-chain disulfide bonds results in a DAR of 0–8 and potential conjugation at eight cysteine residues per mAb. (C) Site-specific conjugation utilizing two engineered cysteine residues results in a DAR of 0–2 and conjugation at two sites/mAb. DAR can be doubled by engineering four sites if desired. Data displayed in graphs were re-plotted from previous publications.^,,

Figure 6. Applications of site-specific antibody conjugates. The site-specific conjugation of molecules to monoclonal antibodies has a wide range of applications. Site-specific conjugation decreases conjugate heterogeneity and improves stability and function. A number of possible antibody conjugates are represented here and include antibody-drug conjugates (ADCs) for cancer treatment, radionuclide-antibody conjugates (RACs) for imaging, antibody-antibiotic conjugates (AACs) to fight infectious disease, and antibody fluorophore conjugates (AFCs) for imaging and detection.