Novel Peptide Linkers for Highly Potent Antibody−Auristatin Conjugate

Abstract

Auristatins are highly potent antimitotic agents that have received considerable attention because of their activities when targeted to tumor cells in the form of antibody-drug conjugates (ADCs). Our lead agent, SGN-35, consists of the cAC10 antibody linked to the N-terminal amino acid of monomethylauristatin E (MMAE) via a valine-citrulline p-aminobenzylcarbamate (val-cit-PABC) linker that is cleaved by intracellular proteases such as cathepsin B. More recently, we developed an auristatin F (AF) derivative monomethylauristatin F (MMAF), which unlike MMAE contains the amino acid phenylalanine at the C-terminal position. Because of the negatively charged C-terminal residue, the potency of AF and MMAF is impaired. However, their ability to kill target cells is greatly enhanced through facilitated cellular uptake by internalizing mAbs. Here, we explore the effects of linker technology on AF-based ADC potency, activity, and tolerability by generating a diverse set of dipeptide linkers between the C-terminal residue and the mAb carrier. The resulting ADCs differed widely in activity, with some having significantly improved therapeutic indices compared to the original mAb-Val-Cit-PABC-MMAF conjugate. The therapeutic index was increased yet further by generating dipeptide-based ADCs utilizing new auristatins with methionine or tryptophan as the C-terminal drug residue. These results demonstrate that manipulation of the C-terminal peptide sequence used to attach auristatins to the mAb carrier can lead to highly potent and specific conjugates with greatly improved therapeutic windows.

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids

“…We chose to use auristatin, a highly potent tubulin inhibitor, because it is well characterized and can be modified for facile coupling to an antibody (10,30). Although the first generation of auristatin-linker conjugates were coupled to the N terminus of the peptide and contained a cathepsin B protease cleavage site, Doronina et al synthesized C-terminally linked derivatives that were reported to be more cytotoxic and have improved pharmacological profiles (30 (31), presumably because of degradation of the ADC in the lysosome and release of drug. Therefore, we first chose to investigate ADCs containing a noncleavable linker (nAF) coupled to the C terminus of auristatin F (AF) (Fig.…”

“…AF was synthesized as previously reported (30). The noncleavable ethylene glycol linker derivatized with an alkoxy-amine was synthesized, as previously described (33), and attached to the C terminus of AF (SI Materials and Methods).…”

“…AF was synthesized as previously described (30). The noncleavable ethylene glycol linker was prepared as previously described (33) from commercial materials, and coupled to AF as described in SI Materials and Methods.…”

Antibody–Drug Conjugates for Tumor Targeting—Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

“…Although several disulfide-linked ADCs are currently under clinical investigation (Table 1), more stable cleavable linkers are engineered in the form of lysosomal protease substrates, e.g. containing a valine-citrulline dipeptide bond, which is specifically cleaved by cathepsin B [26][27][28] .…”

The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

“…[10][11][12][13][14] Appropriate linker technology will ensure the general stability of ADCs during in vivo circulation and efficient drug release once internalized. 4,10,15 Linkers are generally categorized into 2 groups: cleavable and non-cleavable.…”