Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.

A linker-drug platform was built on the basis of a cleavable linker-duocarmycin payload for the development of new-generation antibody-drug conjugates (ADC). A leading ADC originating from that platform is SYD983, a HER2-targeting ADC based on trastuzumab. HER2-binding, antibody-dependent cell-mediated cytotoxicity and HER2-mediated internalization are similar for SYD983 as compared with trastuzumab. HER2-expressing cells in vitro are very potently killed by SYD983, but SYD983 is inactive in cells that do not express HER2. SYD983 dose dependently reduces tumor growth in a BT-474 mouse xenograft in vivo. The ADC is stable in human and cynomolgus monkey plasma in vitro but shows relatively poor stability in mouse plasma due to mouse-specific carboxylesterase. SYD983 could be dosed up to 30 mg/kg in cynomolgus monkeys with high exposure, excellent stability in blood, and without severe toxic effects. The monkey safety study showed no SYD983-induced thrombocytopenia and no induction of peripheral sensory neuropathy, both commonly observed in trials and studies with ADCs based on tubulin inhibitors. Finally, to improve homogeneity, SYD983 was further purified by hydrophobic interaction chromatography resulting in an ADC (designated SYD985) predominantly containing DAR2 and DAR4 species. SYD985 showed high antitumor activity in two patient-derived xenograft models of HER2-positive metastatic breast cancers. In conclusion, the data obtained indicate great potential for this new HER2-targeting ADC to become an effective drug for patients with HER2-positive cancers with a favorable safety profile. More generally, this new-generation duocarmycin-based linker-drug technology could be used with other mAbs to serve more indications in oncology. Mol Cancer Ther; 13(11); 2618-29. Ó2014 AACR.

<p>Details are given of the synthesis of SYD983 and the non-binding control, preparation of fluorescently-labeled SYD983, bioanalysis of SYD983 using SEC, HIC, and RP-HPLC, functional assays like an ADCC assay and internalization, and details on the PK analysis of SYD983.</p>

<p>Supplementary tables describe a comparison of half-lives of SYD983 in the different species (Table S1), PK details in mice (Table S2), PK details in tumor-bearing mice (Table S3), PK details in cynomolgus monkeys (Tables S4 and S5), Flow rates of SYD983 in RP-HPLC, HIC profile of SYD983 and SYD985 (Figure S1), Cytotoxicity of SYD983 versus SYD985 (Figure S2), and control staining for IHC of the tumors used in PDX (Figure S3).</p>

<p>Details are given of the synthesis of SYD983 and the non-binding control, preparation of fluorescently-labeled SYD983, bioanalysis of SYD983 using SEC, HIC, and RP-HPLC, functional assays like an ADCC assay and internalization, and details on the PK analysis of SYD983.</p>

<p>Supplementary tables describe a comparison of half-lives of SYD983 in the different species (Table S1), PK details in mice (Table S2), PK details in tumor-bearing mice (Table S3), PK details in cynomolgus monkeys (Tables S4 and S5), Flow rates of SYD983 in RP-HPLC, HIC profile of SYD983 and SYD985 (Figure S1), Cytotoxicity of SYD983 versus SYD985 (Figure S2), and control staining for IHC of the tumors used in PDX (Figure S3).</p>