Antibody Format and Drug Release Rate Determine the Therapeutic Activity of Noninternalizing Antibody–Drug Conjugates

Abstract

The development of antibody-drug conjugates (ADC), a promising class of anticancer agents, has traditionally relied on the use of antibodies capable of selective internalization in tumor cells. We have recently shown that also noninternalizing antibodies, coupled to cytotoxic drugs by means of disulfide linkers that can be cleaved in the tumor extracellular environment, can display a potent therapeutic activity. Here, we have compared the tumor-targeting properties, drug release rates, and therapeutic performance of two ADCs, based on the maytansinoid DM1 thiol drug and on the F8 antibody, directed against the alternatively spliced Extra Domain A (EDA) domain of fibronectin.The antibody was used in IgG or in small immune protein (SIP) format. In both cases, DM1 was coupled to unpaired cysteine residues, resulting in a drug-antibody ratio of 2. In biodistribution studies, SIP(F8)-SS-DM1 accumulated in the tumor and cleared from circulation more rapidly than IgG(F8)-SS-DM1. However, the ADC based on the IgG format exhibited a higher tumor uptake at later time points (e.g., 33%IA/g against 8%IA/g at 24 hours after intravenous administration). In mouse plasma, surprisingly, the ADC products in IgG format were substantially more stable compared with the SIP format (half-lives >48 hours and <3 hours at 37 C, respectively), revealing a novel mechanism for the control of disulfide-based drug release rates. Therapy experiments in immunocompetent mice bearing murine F9 tumors revealed that SIP(F8)-SS-DM1 was more efficacious than IgG(F8)-SS-DM1 when the two products were compared either in an equimolar basis or at equal milligram doses.

Non‐internalizing antibody–drug conjugates display potent anti‐cancer activity upon proteolytic release of monomethyl auristatin E in the subendothelial extracellular matrix

“…Upon incubation with mouse serum at 37°C, the SIP-based ADC product exhibited a half-life of approximately 9 hours, while the corresponding IgG derivative was completely stable for three days [Figure 5A]. A similar difference in stability for ADC products based on IgG or SIP antibodies had previously been reported for conjugates featuring disulfide-bonded drugs 17. It is reasonable to assume that the C-terminal extremity of the SIP antibody may be less sterically hindered, compared to the C-terminal end of the light chain in intact immunoglobulin formats.…”

“…In analogy to previous studies 17, 35, the full immunoglobulin format was engineered (through Cys -> Ser mutations) to display a single reactive cysteine residue at the C-terminus of the light chain [Figure 1]. In addition, we observed that the Asn-88 residue at the beginning of the CDR3 loop in the VL domain of the antibody was heavily glycosylated in the IgG format [Figure 2], but not in the SIP format.…”

“…We have reported that non-internalizing ADC products, based on monoclonal antibodies specific to splice-isoforms of fibronectin, could liberate their payload in the tumor sub-endothelial extracellular space and display a potent therapeutic activity 15–17. In those cases, cytotoxic drugs were coupled to the antibody via disulfide linkers, which could be cleaved at the site of disease by reducing agents.…”

“…The progressive death of tumor cells releases increasing amounts of glutathione (the most abundant thiol in the intracellular space) 18, 19, leading to an amplification of the drug release process 20. Interestingly, a high variability in the stability of disulfide-linked payloads, which impacted on therapeutic performance in vivo , was observed when comparing ADC products based on intact antibodies and smaller antibody fragments 17.…”

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

“…This concept, however, has recently been challenged, as potent and selective anticancer activity has been observed with ADCs and SMDCs specific to antigens, which do not internalize [19,20,[28][29][30][31].…”