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.

Objective. The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7␣-hydroxy-DHEA (7␣-OH-DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collageninduced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7␣-OH-DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).Methods. The presence of 7␣-OH-DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7␣-OH-DHEA with the use of high-performance liquid chromatography.Results. The CYP7B enzyme product 7␣-OH-DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor ␣ (TNF␣), interleukin-1␣ (IL-1␣), IL-1␤, and IL-17 up-regulated CYP7B activity in an FLS cell line 2-10-fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor ␤ inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNF␣ increased CYP7B activity in all cell lines tested.Conclusion. Exposure to the proinflammatory cytokines TNF␣, IL-1␣, IL-1␤, and IL-17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7␣-OH-DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.

Carboxylesterase 1c (CES1c) is responsible for linker-drug instability and poor pharmacokinetics (PK) of several antibody-drug conjugates (ADC) in mice, but not in monkeys or humans. Preclinical development of these ADCs could be improved if the PK in mice would more closely resemble that of humans and is not affected by an enzyme that is irrelevant for humans. SYD985, a HER2-targeting ADC based on trastuzumab and linker-drug vc--DUBA, is also sensitive to CES1c. In the present studies, we first focused on the interaction between CES1c and SYD985 by size- exclusion chromatography, Western blotting, and LC/MS-MS analysis, using recombinant CES1c and plasma samples. Intriguingly, CES1c activity not only results in release of the active toxin DUBA but also in formation of a covalent bond between CES1c and the linker of vc--DUBA. Mass spectrometric studies enabled identification of the CES1c cleavage site on the linker-drug and the structure of the CES1c adduct. To assess the impact, CES1c SCID mice were generated that showed stable PK for SYD985, comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showed enhanced efficacy compared with PDX studies in CES1c mice and provided a more accurate prediction of clinical efficacy of SYD985, hence delivering better quality data. It seems reasonable to assume that CES1c SCID mice can increase quality in ADC development much broader for all ADCs that carry linker-drugs susceptible to CES1c, without the need of chemically modifying the linker-drug to specifically increase PK in mice. .

Prednisolone and other glucocorticoids (GCs) are potent anti-inflammatory drugs, but chronic use is hampered by metabolic side effects. Therefore, there is an urgent medical need for improved GCs that are as effective as classical GCs but have a better safety profile. A well-established model to assess anti-inflammatory efficacy is the chronic collagen-induced arthritis (CIA) model in mice, a model with features resembling rheumatoid arthritis. Models to quantify undesired effects of glucocorticoids on glucose kinetics are less well-established. Recently, we have described a model to quantify basal blood glucose kinetics using stably-labeled glucose. In the present study, we have integrated this blood glucose kinetic model in the CIA model to enable quantification of both efficacy and adverse effects in one animal model. Arthritis scores were decreased after treatment with prednisolone, confirming the anti-inflammatory properties of GCs. Both inflammation and prednisolone induced insulin resistance as insulin secretion was strongly increased whereas blood glucose concentrations and hepatic glucose production were only slightly decreased. This insulin resistance did not directly resulted in hyperglycemia, indicating a highly adaptive compensatory mechanism in these mice. In conclusion, this ‘all-in-one’ model allows for studying effects of (novel) GC compounds on the development of arthritis and glucose kinetics in a single animal. This integrative model provides a valuable tool for investigating (drug-induced) metabolic dysregulation in an inflammatory setting.

<p>Table S1. Summary of status of tumors used in cell line and patient-derived xenograft models. Table S2. SYD985 and T-DM1 IC50, 95% confidence interval and % efficacy values from the in vitro cytotoxicity assays as depicted in Figure 1B. Table S3. Pharmacokinetic parameters for ADC (SYD985 and T-DM1) in BT-474-tumor-bearing mice after a single i.v. bolus injection of ADC. Figure S1. In vitro cytotoxicity. Figure S2. A-G HER2 expression in BT-474. Figure S3. HER2 cell surface expression quantified on a panel of 8 cell lines using the DAKO Qifikit. Figure S4. In vitro cytotoxicity. Figure S5. In vitro bystander killing. Figure S6. Mean ADC (SYD985 conjugated antibody) concentrations in plasma in carboxylesterase CES1c knock-out (-/-) , CES1c heterozygeous (+/-) and CES1c wild type (+/+) mice, after single i.v. bolus injection of SYD985 at 5 mg/kg (+/- SEM, n = 3).</p>