Antibody–Drug Conjugate Payloads; Study of Auristatin Derivatives

Abstract

Auristatins are important payloads used in antibody drug conjugates (ADCs), and the most well-known compound family member, monomethyl auristatin (MMAE), is used in two Food and Drug Administration (FDA)-approved ADCs, Adcetris ® and Polivy ®. Multiple other auristatin-based ADCs are currently being evaluated in human clinical trials and further studies on this class of molecule are underway by several academic and industrial research groups. Our group's main focus is to investigate the structure-activity relationships (SAR) of novel auristatins with the goal of applying these to next generation ADCs. Modifications of the auristatin backbone scaffold have been widely reported in the chemical literature focusing on the terminal subunits: P1 (N-terminus) and P5 (C-terminus). Our approach was to modulate the activity and hydrophilic character through modifications of the central subunits P2-P3-P4 and thorough SAR study on the P5 subunit. Novel hydrophilic auristatins were observed to have greater potency in vitro and displayed enhanced in vivo antitumor activity when conjugated via protease-cleavable linkers and delivered intracellularly. Analysis of ADC aggregation also indicated that novel hydrophilic payloads enabled the synthesis of high-drug-to-antibody ratio (DAR) ADCs that were resistant to aggregation. Modification of the central peptide subunits also resulted in auristatins with potent cytotoxic activity in vitro and these azide-modified auristatins contain a handle for linker attachment from the central portion of the auristatin backbone.

Anti-tumor efficacy of an MMAE conjugated antibody targeting cell surface TACE/ADAM17-cleaved Amphiregulin in breast cancer

“…HIC-HPLC of 1A3-MMAE (red) exhibited increased retention time relative to the unconjugated 1A3 (blue), resulting in a calculated average drug antibody ratio (DAR) of 4.4 (Fig 3C). This placed our ADC within the range of several previously investigated ADCs with DARS between 2-6 that was strikes a balance between ADC solubility with an effective degree of payload delivery (Akaiwa et al, 2020;Hamblett et al, 2004;Lyon et al, 2015;Sun et al, 2017).…”

Antibody–Drug Conjugates—A Tutorial Review

“…This family of tubulin-inhibiting cytotoxins binds at the tubulin vinca alkaloid binding domain, causing cells to accumulate in metaphase arrest [ 70 ]. The structure of dolastatin 10 was used as the basis for derivatives monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), both of which possess an N-terminal secondary amine (rather than the tertiary amine present in dolastatin 10), allowing for straightforward linker attachment [ 71 ]. MMAF is less able to cross cell membranes than MMAE due to its C-terminal carboxylic acid group, but MMAF is also more hydrophilic, has a lesser tendency to aggregate and shows lower systemic toxicity than MMAE [ 72 ].…”

The Chemistry Behind ADCs

“…Recently Agensys modulated the central subunits P2-P3-P4 and generated novel hydrophilic derivatives with improved in vitro and in vivo potency when conjugated with protease-cleavable linkers [ 9 ] Introduction of azide groups into P2 and P4 subunits opened the way to serve as handles for linker attachment. Thus, novel drug–linker conjugates were prepared using click chemistry ( Figure 5 ) and the payloads incorporating azide groups could serve as attachment sites for other non-cleavable or cleavable linkers.…”