Abstract
Antibody-drug conjugates (ADCs) offer a combination of antibody therapy and specific delivery of potent small-molecule payloads to target cells. The properties of the ADC molecule are determined by the balance of its components. The efficacy of the payload component increases with higher drug-to-antibody ratio (DAR), while homogeneous DAR = 8 ADCs are easily prepared by conjugation to the four accessible antibody hinge cystines. However, use of hydrophobic payloads has permitted only DAR = 2–4, due to poor pharmacokinetics and aggregation problems. Here, we describe generation and characterization of homogeneous DAR = 8 ADCs carrying a novel auristatin β-D-glucuronide, MMAU. The glycoside payload contributed to overall hydrophilicity of the ADC reducing aggregation. Compared to standard DAR = 2–4 ADCs, cytotoxicity of the homogeneous DAR = 8 ADCs was improved to low-picomolar IC50 values against cancer cells in vitro. Bystander efficacy was restored after ADC internalization and subsequent cleavage of the glycoside, although unconjugated MMAU was relatively non-toxic to cells. DAR = 8 MMAU ADCs were effective against target antigen-expressing xenograft tumors. The ADCs were also studied in 3D in vitro patient-derived xenograft (PDX) assays where they outperformed clinically used ADC. In conclusion, increased hydrophilicity of the payload contributed to the ADC’s hydrophilicity, stability and safety to non-target cells, while significantly improving cytotoxicity and enabling bystander efficacy.
Highlights
There are currently four clinically approved antibody-drug conjugates (ADCs) in the market, directed against both hematologic malignancies and solid tumors
Standard compound mixture was generated by mixing trastuzumab and ADCs with different drug-to-antibody ratios; the ADCs were generated from reduced trastuzumab by adding varying amounts of the MC-Val-Cit-PABC-MMAE drug-linker compound until DAR = 8 was reached
Homogeneous DAR = 8 ADCs were successfully prepared from MMAU (Figure 1a), the β-D-glucuronic acid glycoside of MMAE (Figure 1b)
Summary
There are currently four clinically approved antibody-drug conjugates (ADCs) in the market, directed against both hematologic malignancies and solid tumors. Utilizing tumor-specific antibodies as targeting vehicles and development of sophisticated linker technologies has since enabled clinical use of these potent anticancer compounds [1]. The therapeutic window of ADCs is still quite narrow and many clinical programs have suffered from unwanted toxicity and insufficient efficacy against tumors. It has been established that ADCs with higher drug-to-antibody ratio (DAR) have greater in vitro potency than the current clinically approved ADCs with DAR of about four, and the higher DAR ADCs are especially effective against cells with low copy. 22 of especially effective against cells with low copy numbers of the target antigen [2].
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