Monodisperse polysarcosine-based highly-loaded antibody-drug conjugates

Abstract

Antibody-drug conjugates (ADCs) convey highly potent anticancer drugs to antigen-expressing tumor cells, thereby sparing healthy tissues throughout the body. Pharmacokinetics and tolerability of ADCs are predominantly influenced by the drug-antibody ratio (DAR) of the conjugates, which is to-date limited to a value of 3-4 drugs per antibody in ADCs under clinical investigations. Here, we report the synthesis of monodisperse (i.e. discrete) polysarcosine compounds and their use as a hydrophobicity masking entity for the construction of highly-loaded homogeneous β-glucuronidase-responsive antibody-drug conjugates (ADCs). The highly hydrophilic drug-linker platform described herein improves drug-loading, physicochemical properties, pharmacokinetics and in vivo antitumor efficacy of the resulting conjugates.

Fig. 1. Chemical structure of the polysarcosine-based ADC drug-linker platform and schematic representation of homogeneous ADCs with a Drug-Antibody-Ratio (DAR) of 8. See ESI for detailed chemical synthetic and bioconjugation procedures.

Scheme 1. On-resin synthesis of monodisperse side-functionalized polysarcosine oligomers. (a) Fmoc-Sar-Sar-OH, HATU, DIPEA, DMF then piperidine/DMF. (b) Bromoacetic acid (BAA), diisopropylcarbodiimide (DIC), DMF then methylamine in water. This step is repeated until the desired oligomer length is obtained. (c) BAA, DIC, DMF then 2-azidoethan-1-amine, DMF. (d) 4-Maleimidophenylacetic acid, COMU, DIPEA, DMF then TFA, CH₂Cl₂.

Fig. 2. (A) Structures of the negative control drug-linkers used in the study. See ESI for detailed chemical synthetic procedures. (B) Hydrophobic Interaction Chromatography (HIC) profiles of ADCs.

Fig. 3. (A) Total ADC pharmacokinetic study in SCID mice after a single intravenous ADC dose of 3 mg kg^–1. Clearance was 15.8 and 37.6 mL per day per kg respectively for ADC-PSAR12 and ADC-PSAR0, as calculated by two-compartmental model analysis. (B) Antitumor activity in SCID/BT-474 breast cancer model following a single intravenous ADC dose of 3 mg kg^–1. No body-weight changes were observed during the study. CR = complete remission.

Fig. 4. (A) Total ADC pharmacokinetic study in Sprague-Dawley rats after a single intravenous ADC dose of 3 mg kg^–1 and (B) associated clearance rates (two-compartmental model analysis) as a function of the hydrophobicity masking entity length (monomer units). (C) Antitumor activity in SCID/BT-474 breast cancer model following a single intravenous ADC dose of 2.5 mg kg^–1. No body-weight changes were observed during the study.