**2.4 The cytotoxic payload**

The novel design of ADCs allows for use of potent cytotoxic agents with halfmaximal inhibitory concentrations in nano and picomolar range [26]. High potency of the payload is required since only a small fraction of the ADC reaches the tumor site [27]. As previously mentioned, internalization of the ADC is the first step required for release of the cytotoxic payload followed by linker or mAb degradation in cleavable and non-cleavable linkers-based ADCs, respectively. Other desired characteristics of the cytotoxic payload include stability in physiological conditions, ability to chemically conjugate with the antibody and cell membrane permeability [28]. Currently, the majority of approved ADCs utilize one of three pharmacologic categories of payloads including tubulin inhibitors, DNA damaging agents, and immunomodulators [29].

Tubulin inhibitors can be classified either as tubulin polymerization promoters (e.g., auristatin derivatives monomethyl auristatin E and monomethyl auristatin F) or tubulin polymerization inhibitors (e.g., maytansinoid derivatives DM1 and DM4) [30, 31]. Tubulin inhibitors halt cell division by interfering with mitosis and are considered cell-cycle specific [30]. T-DM1 was the first FDA approved ADC with a maytansinoid derivative cytotoxic payload [14].

The mechanism of action of DNA damaging agents include: DNA alkylation (e.g., duocarmycins), DNA double strand break (e.g., calicheamicins), DNA crosslink (e.g., pyrrolobenzodiazepines), and DNA intercalation (e.g., topoisomerase I inhibitors) [32]. DNA damaging agents are not cell-cycle specific and can be relatively more potent than tubulin inhibitors [32]. The payloads utilized in T-Dxd and SG are topoisomerase I inhibitors [33, 34]. The cytotoxic payload of SG is SN-38, which is the active metabolite of irinotecan. Dxd is the paylaod of T-Dxd, which is an exatecan derivative [33, 34]. It is reported that Dxd has potency that is 10-fold higher than SN-38 [5].

Drug-to-antibody ratio (DAR) is another important characteristic of ADCs that impacts efficacy and safety. DAR refers to the average number of cytotoxic molecules conjugated to the mAb [35]. Low DAR can negatively impact efficacy and high DAR can affect stability, antigen binding ability, and clearance [36]. DAR is also used to determine the therapeutic index of ADCs [35]. DAR values vary among ADCs, and low values can result in reduced potency and efficacy. Initially developed ADCs have a DAR average of 2–4 [37]. T-Dxd and SG have higher DAR values at 8:1 and 7.6:1, respectively compared to T-DM1 that has DAR of 3.5:1 [38].
