ACCELERATING THE DEVELOPMENT OF NOVEL ANTIBODY-DRUG CONJUGATES THROUGH SITE-SPECIFIC CONJUGATION METHODS

Abstract Background Antibody–drug conjugates (ADC) typically consist of a monoclonal antibody (mAbs) attached to a cytotoxic payload via a chemical linker. ADC development requires careful consideration of each of its key components and development strategy since each element has the potential to affect the final therapeutic efficacy and safety. One important characteristic of an ADC drug is the drug-to-antibody ratio (DAR). This ratio elucidates the number of drug molecules bound onto a single antibody. Based on the conjugation strategy, the number of drug molecules that are bound to a single antibody varies. Low-drug loading reduces the overall potency, whereas high-drug loading can have higher cytotoxic effects but increased side-effects and altered pharmacokinetics (PK). As such, appropriate selection of conjugation strategy can affect the homogeneity of ADCs and resulting effectiveness. To increase the efficacy of ADCs, site-specific conjugation technologies, including engineered cysteine residues, unnatural amino acids, or enzymatic conjugation through glycosyltransferases, have been applied to obtain more homogeneous ADCs. This has proven to be clinically effective by improving ADC pharmacokinetics and therapeutic index. Furthermore, the increased control over conjugation site reduces the overall hydrophobicity of the linker–payload, preventing unintended payload release in blood. AGLink ADC site-specific conjugation kits were used to perform site-specific conjugation. The AGLink technology utilizes an enzymatic modification method (one-pot process) to reduce antibody N-glycans by fucosylation and enable site-specific and controllable conjugation. After conjugation, the resulting ADCs were evaluated for ADC homogeneity, immunoreactivity, and cytotoxicity. Methods and Results AGLink ADC site-specific conjugation were used and based on the conjugation platform YTConju™. To characterize the efficacy of AGLink, Trastuzumab and MMAE were used. N-glycans were identified to be at the asparagine 297 (N297) position of the CH2 domain on each heavy chain Fc fragment. The N-glycans were reduced to form reactive sites linked with payloads through glycosylation. The resulting glycosylation is predominantly composed of varied amounts of N-acetylglucosamine, fucose, galactose, mannose and N-acetylneuraminic acid (sialic acid) residues, which are assembled in different complex-type biantennary structures. The resulting ADCs (Trastuzumab-MMAE) with different DARs (2 or 4) were developed and characterized through varying studies. Conclusion Site-specific modifications are beginning to be used more frequently to meet the rapidly evolving applications of ADCs. Of these modification methods, we see that glycoengineering has been demonstrated as a useful approach for site-specific antibody conjugation methods. The AGLink site-specific conjugation kit utilizes glycoengineering by performing an enzymatic modification method of IgG Fc glycans to perform conjugation. AGLink does not require any prior engineering of the amino acid sequence, and results in stable antibody conjugates with minimal variation between batches. As a result, the AGLink technology used in the conjugation kit offers a unique site-specific conjugation method with potential applications for preclinical development of MMAE-ADCs.