Protease-Cleavable Linkers Modulate the Anticancer Activity of Noninternalizing Antibody–Drug Conjugates

Abstract

Antibody-drug conjugates (ADCs) represent an attractive class of biopharmaceutical agents, with the potential to selectively deliver potent cytotoxic agents to tumors. It is generally assumed that ADC products should preferably bind and internalize into cancer cells in order to liberate their toxic payload, but a growing body of evidence indicates that also ADCs based on noninternalizing antibodies may be potently active. In this Communication, we investigated dipeptide-based linkers (frequently used for internalizing ADC products) in the context of the noninternalizing F16 antibody, specific to a splice isoform of tenascin-C. Using monomethyl auristatin E (MMAE) as potent cytotoxic drug, we observed that a single amino acid substitution of the Val-Cit dipeptide linker can substantially modulate the in vivo stability of the corresponding ADC products, as well as the anticancer activity in mice bearing the human epidermoid A431 carcinoma. In these settings, the linker based on the Val-Ala dipeptide exhibited better performances, compared to Val-Cit, Val-Lys, and Val-Arg analogues. Mass spectrometric analysis revealed that the four linkers displayed not only different stability in vivo but also differences in cleavage sites. Moreover, the absence of anticancer activity for a F16-MMAE conjugate featuring a noncleavable linker indicated that drug release modalities, based on proteolytic degradation of the immunoglobulin moiety, cannot be exploited with noninternalizing antibodies. ADC products based on the noninternalizing F16 antibody may be useful for the treatment of several human malignancies, as the cognate antigen is abundantly expressed in the extracellular matrix of several tumors, while being virtually undetectable in most normal adult tissues.

Cathepsin B Is Dispensable for Cellular Processing of Cathepsin B-Cleavable Antibody–Drug Conjugates

“…Dorywalska and colleagues demonstrated that carboxylesterase 1c present in rodent serum could also cleave the VC linker, leading to increased toxicities of the ADCs in rodents (30). A noninternalizing ADC, coupled with various protease cleavable linkers, including the VC(S) linker, was also recently shown to still inhibit tumor growth in mouse xenografts (31). Aside from carboxylesterase 1c, extracellular cleavage may be carried out by secreted cathepsins and other degradative proteases in the tumor microenvironment (32)(33)(34).…”

Protease‐activated prodrugs: strategies, challenges, and future directions

“…Recently, noninternalizing Ab–drug conjugates have gained substantial attention in the field of targeted drug delivery. These conjugates contain noninternalizing antibodies that are not transported into the lysosomes; thus, their activation primarily relies on enzymatic activity in the extracellular space . In 2017, the Neri laboratory reported the development of five noninternalizing ADCs containing the F16 Ab targeting a splice isoform of tenascin‐C .…”

“…These conjugates contain noninternalizing antibodies that are not transported into the lysosomes; thus, their activation primarily relies on enzymatic activity in the extracellular space . In 2017, the Neri laboratory reported the development of five noninternalizing ADCs containing the F16 Ab targeting a splice isoform of tenascin‐C . Four of these ADCs contained dipeptide linkers: Val–Cit, Val–Arg, Val–Lys, and Val–Ala, and one contained a NCs (Fig.…”

2019 FDA TIDES (Peptides and Oligonucleotides) Harvest

“…Second, the programable release of cargo should be easily triggered once the conjugate reaches its target. Thus, a suitable linker should successfully combine serum stability and in-target lability without adversely affecting the stability of the antibody itself upon conjugation [64,65].…”

“…Among the four known types of linkers, namely hydrazones, disulfides, peptides, and thioethers [57], short peptidyl linkers, such as Val-Cit dipeptide, fulfill the requirements for this critical function and even outperform the tetra-peptidyl linkers (Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu) previously used and that showed some aggregation issues upon conjugation [57,64,65]. The premature release of the payload in the case of hydrazone (due to pH changes) and disulfide linkers (due to exchange with other thiols, such as glutathione) may influence the potency of the treatment, while the delayed release of cargo in the case of thioether linkers (payload is released only after total degradation of the antibody) may cause the loss of anticancer activity [65,66]. In Padcev TM and Polivy TM , a maleimidocarpoyl moiety is added to the N-terminal of the dipeptide Val-Cit to facilitate conjugation to the antibody.…”