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  • Antigen Target Selection

A major challenge in developing antibody-drug conjugates (ADCs) for cancer lies in the identification and validation of sufficient antigen targets for the monoclonal antibody component. Several factors need to be considered in antigen selection. To begin, to lower off-target toxicity and achieve an acceptable ADC therapeutic index, the ideal target antigen should exhibit high expression levels in tumors and minimal or absent expression in normal tissues, or at least be limited to specific tissue types. Secondly, the target antigen should be present on the cell surface for the circulating monoclonal antibody to interact with. Thirdly, it should be an internalizing antigen so that, upon binding, the ADC can be transported into the cell, allowing the cytotoxic drug to exert its effects. However, it has been reported that in some cases, non-internalizing ADCs can display significant toxicity, often inducing a strong “bystander effect”. For targets already validated with naked monoclonal antibodies, another consideration is whether to maintain extracellular mechanisms of action, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP). Future ADC design must account for the relative roles of cytotoxic drugs and antibodies throughout the anti-tumor activity and toxicity spectrum of the entire ADC.

  • Payloads for Clinical-Stage ADCs

ADCs currently in clinical trials use a limited family of cytotoxic drugs as payloads. These drugs mostly target DNA (exhibiting cytotoxicity for both proliferating and non-proliferating cells) or microtubules (exhibiting cytotoxicity for proliferating cells) and are optimized for potency. Due to the typically limited number of antigens on tumor cell surfaces and the average Drug-to-antibody ratio (DAR) of most clinical-stage ADCs restricted to 3.5–4, the amount of drug delivered to tumor cells is low. This is considered a primary reason for the clinical failure of ADCs using conventional cytotoxic drugs. Additionally, many cytotoxic drugs used for ADCs are hydrophobic and prone to inducing antibody aggregation, a situation that must be avoided to ensure long shelf life, rapid clearance, and reduced immunogenicity. The drug must also maintain its potency during conjugation, exhibit acceptable water solubility, and remain stable as a conjugate in aqueous formulations. Furthermore, the drug must be synthetically accessible and cost-effective under GMP conditions.

  • Linker Design and Optimization

Premature release of the drug in circulation can lead to systemic toxicity and a lower therapeutic index. Effective linker design must balance achieving good stability in circulation within days and effective cleavage upon delivery to target cells. Various strategies are currently under investigation to improve ADC solubility and DAR, as well as overcome protein-induced resistance to chemotherapy drugs being pumped out of cells. These strategies include drug release conditionally in the cytoplasm of target cells, enhancing bystander effects, and limiting bystander effects.

  • Antibody Selection and Optimization

Improving the uniformity and developability of antibodies is necessary for both naked antibodies and ADCs to reduce the attrition rate of candidate drugs. Over the past decade, hundreds of papers analyzing and structurally characterizing monoclonal antibodies have been published. Liquid chromatography, electrophoresis, and mass spectrometry are used for antibody discovery, preclinical development, and clinical development at various stages. These analytical techniques aid in selecting antibody-producing clones with suitable glycosylation profiles, allowing comprehensive structural characterization of research leads and potential clinical candidates.

  • Novel Conjugation Strategies

Most ADCs on the market and in clinical trials currently share common structural features, such as thioether bonds formed through maleimide-thiol reactions. This widely used strategy is favored because the reaction between maleimide and thiol is very rapid and quantitative under physiological conditions. However, thioether bond formation is slow and reversible under physiological conditions, leading to measurable drug loss during extended circulation. About two-thirds of ADCs in clinical development, including two approved ADCs, contain maleimide, potentially resulting in measurable drug loss during prolonged circulation. The pharmacological consequences of eliminating maleimide from ADCs include reduced anti-tumor activity due to decreased exposure of the antibody-drug conjugate form and increased toxicity from non-targeted release of the drug and linker. These issues might be addressed through site-specific conjugation and alternative conjugation chemistries, including cysteine, non-natural amino acid engineering, amino-terminal engineering of serine, conjugation to the Fab nucleotide binding site, natural cysteine re-bridging, and high-load ADCs, among other methods.

  • Enhancing ADC Potency

In addition to careful target, linker, and payload selection, several additional strategies have been employed to enhance ADC potency. These strategies are designed to avoid potential resistance to the payload, enhance tumor penetration by using smaller protein scaffolds, or improve efficacy by combining ADCs with recently approved monoclonal antibody-based immune checkpoint inhibitors. This includes overcoming resistance to ADCs, overcoming barriers to effective tumor penetration, and combining ADCs with immune-oncology antibodies.