In Vitro and In Vivo Evaluation of Cysteine Rebridged Trastuzumab–MMAE Antibody Drug Conjugates with Defined Drug-to-Antibody Ratios

Abstract

The conjugation of monomethyl auristatin E (MMAE) to trastuzumab using a reduction bis-alkylation approach that is capable of rebridging reduced (native) antibody interchain disulfide bonds has been previously shown to produce a homogeneous and stable conjugate with a drug-to-antibody ratio (DAR) of 4 as the major product. Here, we further investigate the potency of the DAR 4 conjugates prepared by bis-alkylation by comparing to lower drug loaded variants to maleimide linker based conjugates possessing typical mixed DAR profiles. Serum stability, HER2 receptor binding, internalization, in vitro potency, and in vivo efficacy were all evaluated. Greater stability compared with maleimide conjugation was observed with no significant decrease in receptor/FcRn binding. A clear dose-response was obtained based on drug loading (DAR) with the DAR 4 conjugate showing the highest potency in vitro and a much higher efficacy in vivo compared with the lower DAR conjugates. Finally, the DAR 4 conjugate demonstrated superior efficacy compared to trastuzumab-DM1 (T-DM1, Kadcyla), as evaluated in a low HER2 expressing JIMT-1 xenograft model.

Multiscale Modeling of Antibody-Drug Conjugates: Connecting Tissue and Cellular Distribution to Whole Animal Pharmacokinetics and Potential Implications for Efficacy

“…Bystander effects from more lipophilic payloads such as MMAE may explain why higher DAR can improve efficacy with the same antibody dose (e.g. (83)), while in the examples above with a hydrophilic payload, it had little benefit. The diffusion of the metabolite in the tissue could be incorporated into this model to predict the additional penetration of the metabolite into tumor tissue as a function of lipophilicity(36) and whether this reaches therapeutic concentrations far from the original site of antibody degradation.…”

Site‐Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome

“…The drug‐to‐antibody ratio (DAR) ratio can be manipulated through variation of the concentration of reductant and the stoichiometries of reactants . In addition, the site‐specific disulfide rebridging by applying the bis‐sulfones has been also demonstrated by the successful modification of antibody fragments and therapeutic proteins while retaining their activities …”

“…The extensive work carried out with bis‐sulfones and next‐generation maleimides clearly underlines the viability of the disulfide rebridging strategy to achieve site‐selective modifications that are beyond what the genome, proteome and post‐translational modifications found in organisms can provide. In addition, the specific number of functional groups introduced per protein can be varied in proteins with more than one accessible disulfide bond through stoichiometric control of the reagent or choice of reductant . Nevertheless, these rebridging reagents also exhibit certain limitations.…”

Antibody-Drug Conjugates in Head and Neck Cancer

“…Cysteine rebridging is another strategy that was recently developed to better control DAR and heterogeneity of ADCs. Dibromomaleimide (Behrens et al, 2015;Bryden et al, 2014), dibromopyridazinediones (Maruani et al, 2015), and a 1,3-bis(p-toluenesulfonyl)propane-based core (Bryant et al, 2015) can accept two reduced cysteines derived from interchain disulfide bonds to afford a rebridged antibody (Fig. 4B).…”