A single triggering event in the core of a dendrimer composed of two or more generations of multiple‐release monomeric building blocks can induce the release of all end groups at the termini of the dendrimer (see schematic representation). Such multiple‐release systems could find application, for example, in the field of (targeted) drug delivery, where a disease‐ or organ‐specific activation event could trigger simultaneous release of multiple biologically active molecules.

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.

SYD985 is a HER2-targeting antibody-drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linkerduocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patientderived xenograft (PDX) studies were conducted to compare SYD985 head-to-head with T-DM1 (Kadcyla), another trastuzumab-based ADC. SYD985 and T-DM1 had similar binding affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3þ cell lines, but in cell lines with low HER2 expression, SYD985 was 3-to 50-fold more potent than T-DM1. In contrast with T-DM1, SYD985 efficiently induced bystander killing in vitro in HER2-negative (HER2 0) cells mixed with HER2 3þ, 2þ, or 1þ cell lines. At pH conditions relevant for tumors, cathepsin-B cleavage studies showed efficient release of the active toxin by SYD985 but not by T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2-expressing tumors in vivo, especially in low HER2-expressing and/or in heterogeneous tumors. In line with this, in vivo antitumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3þ, 2þ, and 1þ models, whereas T-DM1 only showed significant antitumor activity in HER2 3þ breast cancer PDX models. These properties of SYD985 may enable expansion of the target population to patients who have low HER2-expressing breast cancer, a patient population with still unmet high medical need. Mol Cancer Ther; 14(3); 692-703. Ó2015 AACR.

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.

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To generate doxorubicin (Dox) specifically at the tumor site, the chemotherapeutic agent was incorporated into a prodrug by linkage to a peptide specifically recognized by plasmin, which is overproduced in many cancers. ST-9905, which contains an elongated self-elimination spacer, is activated more rapidly in vitro by plasmin than is ST-9802. Prodrug activation in vitro depended on the level of urokinase produced by tumor cells and was inhibited by aprotinin, a plasmin inhibitor. Comparison of equimolar concentrations of ST-9905, ST-9802, and Dox in EF43.fgf-4 and MCF7 models revealed that both prodrugs, in sharp contrast to Dox, displayed antiproliferative and antiangiogenic activities without discernible toxicity. Although MCF7 cells are poor urokinase producers in vitro, prodrug efficacy in this model may be explained by production of plasmin by tumor-infiltrating host cells. Mice treated with equitoxic concentrations (maximum tolerated doses) of prodrugs showed 100% survival and negligible body weight loss, in contrast to results after Dox treatment. ST-9905 was substantially more effective than ST-9802 and induced similar tumor growth inhibition as Dox but without apparent toxicity. This finding may be explained by the elongated spacer, which facilitates enzymatic prodrug activation. These data validate both the use of elongated spacers in vivo and the concept of targeting anticancer prodrugs to tumor-associated plasmin.

Engineering cysteines at specific sites in antibodies to create well-defined ADCs for the treatment of cancer is a promising approach to increase the therapeutic index and helps to streamline the manufacturing process. Here, we report the development of an in silico screening procedure to select for optimal sites in an antibody to which a hydrophobic linker−drug can be conjugated. Sites were identified inside the cavity that is naturally present in the Fab part of the antibody. Conjugating a linker−drug to these sites demonstrated the ability of the antibody to shield the hydrophobic character of the linker−drug while resulting ADCs maintained their cytotoxic potency in vitro. Comparison of site-specific ADCs versus randomly conjugated ADCs in an in vivo xenograft model revealed improved efficacy and exposure. We also report a selective reducing agent that is able to reduce the engineered cysteines while leaving the interchain disulfides in the oxidized state. This enables us to manufacture site-specific ADCs without introducing impurities associated with the conventional reduction/oxidation procedure for site-specific conjugation.

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