Figure 5. Example illustrating that the mass measured is concentration independent. Top row is RIC (m/z 207.1394 (C 13 H 19 O 2 )) of OSPW NA standard, concentration range 10-100 mg/kg, bottom row is corresponding mass spectra in the range of m/z 207.10-207.30.
Antibody–drug conjugates (ADCs) have emerged as valuable targeted anticancer therapeutics with at least 11 approved therapies and over 80 advancing through clinical trials. Enediyne DNA-damaging payloads represented by the flagship of this family of antitumor agents, N-acetyl calicheamicin γ1I, have a proven success track record. However, they pose a significant synthetic challenge in the development and optimization of linker drugs. We have recently reported a streamlined total synthesis of uncialamycin, another representative of the enediyne class of compounds, with compelling synthetic accessibility. Here we report the synthesis and evaluation of uncialamycin ADCs featuring a variety of cleavable and noncleavable linkers. We have discovered that uncialamycin ADCs display a strong bystander killing effect and are highly selective and cytotoxic in vitro and in vivo.
Taking advantage of the C
2-symmetry
of the antitumor naturally occurring disorazole B1 molecule,
a symmetrical total synthesis was devised with a monomeric advanced
intermediate as the key building block, whose three-step conversion
to the natural product allowed for an expeditious entry to this family
of compounds. Application of the developed synthetic strategies and
methods provided a series of designed analogues of disorazole B1, whose biological evaluation led to the identification of
a number of potent antitumor agents and the first structure–activity
relationships (SARs) within this class of compounds. Specifically,
the substitutions of the epoxide units and lactone moieties with cyclopropyl
and lactam structural motifs, respectively, were found to be tolerable
for biological activities and beneficial with regard to chemical stability.
Antibody-drug conjugates (ADCs) are complex therapeutic agents that use the specific targeting properties of antibodies and the highly potent cytotoxicity of small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. Two critical quality attributes of ADCs are the purity and stability of the active small molecule drug linked to the ADC, but these are difficult to assess once the drug is conjugated to the antibody. In this study, we report a enzyme deconjugation approach to cleave small molecule drugs from ADCs, which allows the drugs to be subsequently characterized by reversed-phase high performance liquid chromatography. The model ADC we used in this study utilizes a valine-citrulline linker that is designed to be sensitive to endoproteases after internalization by tumor cells. We screened several proteases to determine the most effective enzyme. Among the 3 cysteine proteases evaluated, papain had the best efficiency in cleaving the small molecule drug from the model ADC. The deconjugation conditions were further optimized to achieve complete cleavage of the small molecule drug. This papain deconjugation approach demonstrated excellent specificity and precision. The purity and stability of the active drug on an ADC drug product was evaluated and the major degradation products of the active drug were identified. The papain deconjugation method was also applied to several other ADCs, with the results suggesting it could be applied generally to ADCs containing a valine-citrulline linker. Our results indicate that the papain deconjugation method is a powerful tool for characterizing the active small molecule drug conjugated to an ADC, and may be useful in ensuring the product quality, efficacy and the safety of ADCs.
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