Impact of dipeptide on ADC physicochemical properties and efficacy identifies Ala-Ala as the optimal dipeptide

Abstract

Side chains of natural occurring amino acids vary greatly in terms of charge state, polarity, size and hydrophobicity. Using a linear synthetic route, two amino acids were sequentially coupled to a potent glucocorticoid receptor modulator (GRM) to afford a library of dipeptide-GRM linker payloads with a range of in silico properties. The linker payloads were conjugated to a mouse anti-TNF antibody through interchain disulfide Cys. Impact of various dipeptide linkers on ADC physical properties, including solubility, hydrophobicity, and aggregation were evaluated and the in silico properties pI, Log P and tPSA of the linker drugs used to correlate with these properties. ADCs were screened in a GRE luciferase reporter assay to compare their in vitro efficacy. Data identified Ala-Ala as a superior dipeptide linker that allowed a maximum drug load of 10 while affording ADCs with low aggregation.

Fig. 1. Anti-TNF GRM ADC with a dipeptide linker.

Scheme 1. Synthetic route for the bromoacetamide–dipeptide-GRM. Regents and condition: (i) Boc-protected or Fmoc-protected amino acid in P2 position, BEP, DIEA, DMF; (ii) TFA (for Boc-protected amino acid) or piperidine (for Fmoc-protected amino acid), DCM; (iii) Boc-protected or Fmoc-protected amino acid in P1 position, BEP, DIEA, DMF; (iv) TFA (for Boc-protected amino acid) or piperidine (for Fmoc-protected amino acid), DCM; (v) 2-bromoacetic acid, BEP, DIEA, DMF.

Scheme 2. Generation of DAR4 (DPhPEA) and DAR10 (TCEP) ADCs.

Fig. 2. ADC HIC retention time vs. cLog P/cLog D of linker-payloads.

Fig. 3. cLog P/cLog D of linker-payload vs. ADC HIC retention time for ADCs with an Ala–X dipeptide linker.

Fig. 4. ADC aggregation vs. HIC retention time for DAR4 ADCs.

Fig. 5. ADC aggregation vs. HIC retention time for DAR10 ADCs.

Fig. 6. ADC aggregation vs. cLog P of linker-payload for DAR10 ADCs containing only neutral amino acids in the linker.