Peptide-Cleavable Self-immolative Maytansinoid Antibody–Drug Conjugates Designed To Provide Improved Bystander Killing

Abstract

A new type of antibody–drug conjugate (ADC) has been prepared that contains a sulfur-bearing maytansinoid attached to an antibody via a highly stable tripeptide linker. Once internalized by cells, proteases in catabolic vesicles cleave the peptide of the ADC’s linker causing self-immolation that releases a thiol-bearing metabolite, which is then S-methylated. Conjugates were prepared with peptide linkers containing only alanyl residues, which were all l isomers or had a single d residue in one of the three positions. A d-alanyl residue in the linker did not significantly impair a conjugate’s cytotoxicity or bystander killing unless it was directly attached to the immolative moiety. Increasing the number of methylene units in the maytansinoid side chain of a conjugate did not typically affect an ADC’s cytotoxicity to targeted cells but did increase bystander killing activity. ADCs with the highest in vitro bystander killing were then evaluated in vivo in mice, where they displayed improved efficacy compared to previously described types of maytansinoid conjugates.

Site-Specific Conjugation Strategy for Dual Antibody–Drug Conjugates Using Aerobic Formylglycine-Generating Enzymes

“…42,43 The discrepancy between these findings clearly arises from the lower cell permeability and slowed drug release of the DBCO-compound. 41 In contrast, Sc2-MMAE was able to reduce cell viability with an IC 50 of 0.05 nM for the highly EGFR-presenting A431 cells (Figure 4d), whereas for the MCF7 cells with a very low EGFR-level a higher dose tolerance (ca. 0.9 μM) was observed, indicating that the large protein reduces unspecific uptake of the toxin and further confirming selective EGFR-mediated uptake and lysosomal drug release.…”

“…As depicted in Figure c, both cell types respond similarly to free DBCO-MMAE (A431:84.53 nM; MCF7:111.50 nM). Pico- to nanomolar IC 50 values have been reported for the toxin MMAE. , The discrepancy between these findings clearly arises from the lower cell permeability and slowed drug release of the DBCO-compound . In contrast, Sc2-MMAE was able to reduce cell viability with an IC 50 of 0.05 nM for the highly EGFR-presenting A431 cells (Figure d), whereas for the MCF7 cells with a very low EGFR-level a higher dose tolerance (ca.…”

“…Monomethyl auristatin E (MMAE), an dolastatin-based cytotoxin that interferes with tubulin polymerization and shows high toxicity in his native form, was used . Commercially available DBCO-MMAE consists of the enzymatically cleavable dipeptide-linker comprising valine-citrulline (VC) and the self-immolative p -aminobenzyl group (PAB), which ensures intracellular drug release (DBCO-PEG-VC-PAB-MMAE, Figure a). , Fluorescent constructs like HIPS-CF (carboxyfluorescein) have been proven suitable for simple and fast detection of the conjugation by mass shift of protein bands or in-gel fluorescence. , …”

“…39 Commercially available DBCO-MMAE consists of the enzymatically cleavable dipeptide-linker comprising valinecitrulline (VC) and the self-immolative p-aminobenzyl group (PAB), which ensures intracellular drug release (DBCO-PEG-VC-PAB-MMAE, Figure 2a). 40,41 Fluorescent constructs like HIPS-CF (carboxyfluorescein) have been proven suitable for simple and fast detection of the conjugation by mass shift of protein bands or in-gel fluorescence. 9,10 Although no complete separation of conjugated and unconjugated material was achieved, the presence of the conjugate D1-MMAE was shown by hydrophobic interaction chromatography (HIC) (Figure 3a) and SDS-PAGE (Figure 3b).…”

The Chemistry Behind ADCs

“…For example, a Daratumumab (CD38 mAb approved by the FDA for the treatment of multiple myeloma) based ADC was shown to deliver DM4 specifically to CD38 overexpressing cancer cells [ 13 ]. ImmunoGen prepared recently a new type of ADC that contains a sulfur-bearing maytansinoid ( Figure 10 ) attached to an antibody via a highly stable tripeptide linker [ 14 ]. The attachment point is the same hydroxyl group as above.…”