(452f) Investigating the Effect of Cross-Linker Branching and Conjugation Site on the Stability and Efficacy of Antibody-Drug Conjugates

Antibody-drug conjugates are a class of anti-cancer therapeutics that combine the potency of cytotoxic drugs with the antigen specificity of antibodies via a chemical cross-linker. To date, the FDA has approved two ADCs (brentuximab vedotin and ado-trastuzumab emtansine) with many more compounds in pre-clinical and clinical trials. Despite these success stories, there are still many challenges in the design of highly potent and selective ADCs. Chief among these challenges is improving the plasma stability of ADCs.

The hydrophobicity of drug payloads has been identified as a driving factor for the aggregation and subsequent plasma clearance of ADCs. This inherent property of many cytotoxic drugs limits the potency of ADCs with a high drug-to-antibody ratio (DAR). Accordingly, researchers have turned to hydrophilic cross-linkers such as polyethylene glycol (PEG)-based cross-linkers to conjugate and solubilize hydrophobic drug payloads. Many of these systems have employed bifunctional cross-linkers that position the hydrophilic PEG chain between the antibody and hydrophobic drug payload. Comparatively fewer systems have employed hydrophilicity in the form of branched PEG chains. We have sought to explore the effect of cross-linker sequence on ADC stability and efficacy. To this end, we have employed the sequence-defined oligothioetheramide (oligoTEA) synthesis methodology developed in our lab to design a new class of branched chemical cross-linkers. These constitutionally isomeric cross-linkers enable precise control of the relative placement of hydrophilic PEG chains and hydrophobic drug payload. In turn, this allows us to modulate the local cross-linker hydrophilicity.

In addition to cross-linker structure, antibody conjugation site has been shown to influence ADC stability. Our collaborators at Catalent Biologics have developed a novel chemoenzymatic method based on the hydrazino-iso-Pictet-Spengler (HIPS) ligation to site-specifically modify any protein of interest. Using this technology, Catalent Biologics has synthesized variants of trastuzumab modified at the C-terminus (CT), the light chain (LC), and the CH1 domain (CH1). Utilizing these site-specifically modified antibodies, we have investigated the coupled effects of cross-linker hydrophobicity and antibody conjugation site. We present the biophysical characterization of this library of ADCs. Specifically, the effects of cross-linker hydrophilicity and conjugation site have been correlated to antigen binding, serum stability, and in vitro efficacy. Further studies will explore the correlation between these biophysical properties and in vivo efficacy.