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Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer. Up to 35% of USC may overexpress the HER2/neu oncogene at strong (i.e., 3+) levels by IHC while an additional 40% to 50% express HER2/neu at moderate (2+) or low (1+) levels. We investigated the efficacy of SYD985, (Synthon Biopharmaceuticals), a novel HER2-targeting antibody–drug conjugate (ADC) composed of the mAb trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin) in USC. We also compared the antitumor activity of SYD985 in head-to-head experiments to trastuzumab emtansine (T-DM1), a FDA-approved ADC, against multiple primary USC cell lines expressing different levels of HER2/neu in in vitro and in vivo experiments. Using antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability, and bystander killing assays as well as propidium iodide–based flow cytometry assays and multiple in vivo USC mouse xenograft models, we demonstrate for the first time that SYD985 is a novel ADC with activity against USC with strong (3+) as well as low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is 10- to 70-fold more potent than T-DM1 in comparative experiments and, unlike T-DM1, it is active against USC demonstrating moderate/low or heterogeneous HER2/neu expression. Clinical studies with SYD985 in patients harboring chemotherapy-resistant USC with low, moderate, and high HER2 expression are warranted. Mol Cancer Ther; 15(8); 1900–9. ©2016 AACR.

SYD985 is a novel anti-HER2 antibody-drug conjugate (ADC) in development for breast cancer. The ADC consists of three parts: i) the monoclonal antibody trastuzumab, ii) a linker that can be cleaved by tumor-resident proteases at the dipeptide valine-citrulline (vc) motif, and iii) the prodrug seco-DUocarmycin-hydroxyBenzamide-Azaindole. The average ‘drug to antibody ratio’ (DAR) of this ADC, also named trastuzumab vc-seco-DUBA, is ∼2.8. After antibody-mediated binding of SYD985 to its molecular target HER2 on the surface of tumor cells, the ADC is internalized. Subsequently, the active duocarmycin is released and binds to DNA in the minor groove, followed by alkylation of the DNA and killing of the tumor cells. In vitro and in vivo experiments were performed to (directly) compare various pharmacodynamic and pharmacokinetic properties of SYD985 with those of the most progressed (marketing-approved) HER2-targeting ADC, i.e. ado-trastuzumab emtansine (T-DM1). T-DM1 is currently indicated for second line treatment of breast cancer patients overexpressing HER2 tumor tissue. In in vitro experiments, SYD985 shows potencies similar to T-DM1 in cell lines expressing HER2 at high levels (IHC HER2 3+), whereas SYD985 is significantly more potent compared to T-DM1 in cell lines expressing low HER2 levels (IHC HER2 2+ or 1+). In line with these results, SYD985 has superior efficacy compared to T-DM1 in xenograft models in which patient-derived breast cancer tumor tissue with low HER2 levels (IHC HER2 2+ or 1+/FISH-) have been used. Pharmacokinetic experiments (in vitro and in vivo) have revealed that whereas in mice SYD985 has limited stability, in cynomolgus monkey SYD985 is very stable. Excellent stability in vitro is also observed in human plasma. Moreover, after i.v. administration of SYD985 in the cynomolgus monkey the amount of free toxin (DUBA) detected in the monkey plasma is many-fold lower than levels reported for the toxin (DM1) released from T-DM1. Resuming, the preclinical data of SYD985 support clinical studies to investigate whether SYD985 has benefit in cancer patients with moderate or even low HER2 levels of the tumor tissue. Citation Format: Gijs Verheijden, Patrick Beusker, Ruud Ubink, Miranda van der Lee, Patrick Groothuis, Peter Goedings, David Egging, Marco Timmers, Wim Dokter. Preclinical data of SYD985 support the clinical investigation of this novel anti-HER2 antibody-drug conjugate in breast cancer patients with low levels of HER2 expression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-14.

SYD985 is a HER2-targeting antibody–drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patient-derived 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 3+ 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 3+, 2+, or 1+ 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 3+, 2+, and 1+ models, whereas T-DM1 only showed significant antitumor activity in HER2 3+ 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. ©2015 AACR.

BACKGROUND AND PURPOSE Graves' disease (GD) is an autoimmune disease in which the thyroid is overactive, producing excessive amounts of thyroid hormones, caused by thyroid-stimulating hormone (TSH) receptor-stimulating immunoglobulins (TSIs). Many GD patients also suffer from thyroid eye disease (Graves' ophthalmopathy or GO), as TSIs also activate TSH receptors in orbital tissue. We recently developed low molecular weight (LMW) TSH receptor antagonists as a novel therapeutic strategy for the treatment of GD and GO. Here, we determined the molecular pharmacology of a prototypic, nanomolar potent LMW TSH receptor antagonist, Org 274179-0. EXPERIMENTAL APPROACH Using CHO cells heterogeneously expressing human TSH receptors and rat FRTL-5 cells endogenously expressing rat TSH receptors, we determined the potency and efficacy of Org 274179-0 at antagonizing TSH- and TSI-induced TSH receptor signalling and its cross-reactivity at related follicle-stimulating hormone and luteinizing hormone receptors. We analysed the allosteric mode of interaction of Org 274179-0 and determined whether it is an inverse agonist at five naturally occurring, constitutively active TSH receptor mutants. KEY RESULTS Nanomolar concentrations of Org 274179-0 completely inhibited TSH (and TSI)-mediated TSH receptor activation with little effect on the potency of TSH, in accordance with an allosteric mechanism of action. Conversely, increasing levels of TSH receptor stimulation only marginally reduced the antagonist potency of Org 274179-0. Org 274179-0 fully blocked the increased basal activity of all the constitutively active TSH receptor mutants tested with nanomolar potencies. CONCLUSIONS AND IMPLICATIONS Nanomolar potent TSH receptor antagonists like Org 274179-0 have therapeutic potential for the treatment of GD and GO.

The TSH receptor (TSHR) on orbital fibroblasts (OF) is a proposed target of the autoimmune attack in Graves' ophthalmopathy. In the present study, we tested whether the novel low-molecular-weight (LMW) TSHR antagonist Org-274179-0 inhibits cAMP production induced by rhTSH, Graves' disease IgG (GD-IgG), or M22 (a potent human monoclonal TSHR stimulating antibody) in cultured and differentiated OF from Graves' ophthalmopathy patients. cAMP production significantly increased after incubation either with 10 mU/ml rhTSH (3-fold; P

Contains fulltext : 103595.pdf (Publisher’s version ) (Open Access) Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.

SYD985 is a HER2-targeting ADC based on trastuzumab and vc-seco-DUBA, Synthon's proprietary cleavable linker-duocarmycin payload. Vc-seco-DUBA was coupled to cysteines after partial reduction on the interchain disulfides of trastuzumab. SYD985 was obtained after purification by Hydrophobic Interaction Chromatography to yield a well-defined ADC consisting of predominantly DAR 2 and DAR 4 species. To evaluate the therapeutic potential of this new ADC, and to prepare clinical development, mechanistic in vitro studies and in vivo PDX studies were conducted to compare SYD985 head-to-head to T-DM1 (Kadcyla®), another trastuzumab-based ADC with a toxin of the maytansinoid class in combination with a non-cleavable linker. 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 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3 to 50-fold more potent than T-DM1. In contrast to T-DM1, SYD985 efficiently induced bystander killing in vitro of HER2 negative (HER2 0) cells when mixed with HER2 3+, 2+, or 1+ cells. SYD985 efficiently killed HER2 0 cells, even in the presence of only 20% of HER2 3+ cells. At pH conditions relevant for tumors, cathepsin B cleavage studies showed efficient release of the active toxin from SYD985 but not from T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2 expressing tumors in vivo, allowing the treatment of tumor tissues with low or heterogeneous membrane expression of HER2. In line with this, in vivo anti-tumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models whereas T-DM1 only showed significant anti-tumor activity in HER2 3+ breast cancer PDX models. We conclude that the properties of SYD985 may enable expansion of the target population to patients who have low HER2 expressing breast cancer, a patient population with unmet high medical need. Early phase clinical evaluation with SYD985 is ongoing. Citation Format: Willem Dokter, Miranda van der Lee, Patrick Groothuis, Ruud Ubink, Monique van der Vleuten, Tanja van Achterberg, Eline Loosveld, Desirée Damming, Myrthe Rouwette, David Egging, Diels van den Dobbelsteen, Patrick Beusker, peter goedings, Gijs Verheijden, Jacques Lemmens, Marco Timmers. The preclinical profile of the duocarmycin-based HER2-targeting ADC SYD985 predicts for clinical benefit in low HER2-expressing breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2015-5360

Introduction: Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer which carries an extremely poor prognosis. Up to 35% of USC may overexpress the HER2/neu oncogene at strong (i.e., 3+) levels and and additional 45% of USC express HER2/Neu at moderate (2+) or low (1+) levels. For tumors with moderate/low HER2/neu expression, there is no sufficient targeted therapy. SYD985 (Synthon Biopharmaceuticals BV, Nijmegen, The Netherlands) is a novel HER2-targeting antibody-drug conjugate (ADC) composed of the mAb trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin). The objective of this study was to explore the anti-tumor activity of SYD985 and to compare it to trastuzumab emtansine (T-DM1) in USC in in vitro and in vivo studies. Methods: Nine primary USC cell lines were evaluated for HER2/neu surface expression by IHC and flow cytometry and gene amplification using FISH assays. The cytotoxicity of SYD985 and T-DM1 was evaluated using cell lines with differential HER2/Neu expression (i.e., 1+, 2+, and 3+). Proliferation and viability experiments were performed using propidium iodide-based, flow cytometry assays. In vivo activity of SYD985 in mouse xenograft models is ongoing. Results: SYD985 was 55 to 115 times more potent when compared to T-DM1 in primary USC cell lines with 1+ and 2+ HER2/neu expression. Specifically, in the HER2/neu 1+ the IC50's for SYD985 and T-DM1 were 0.065 μg/mL and 3.58 μg/mL, respectively (p = 0.004). In the HER2/neu 2+ cell lines the IC50's of SYD985 and T-DM1 were 0.016 μg/mL and 1.82 μg/mL, respectively (p = 0.005). In the HER2/neu 3+ cell lines a statistical trend was noted when comparing the cytotoxicity of SYD985 with T-DM1; the IC50's were 0.011 μg/mL and 0.035 μg/mL, respectively (p = 0.06). Conclusions: SYD985 is a novel ADC endowed with remarkable activity against not only USC with strong (3+) HER2/neu overexpression but also (as previously shown for SYD985 in breast cancer) versus USC with low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is significantly more potent than T-DM1 in comparative in vitro experiments. Currently, in vivo experiments in USC xenografts not responsive to T-DM1 are ongoing. This is a significant discovery as we do not currently have an adequate targeted therapy to HER2/neu 1+ and 2+ expressing tumors. Thus, clinical studies with SYD985 in patients with biologically aggressive endometrial cancer (e.g., USC) resistant to standard salvage chemotherapy are warranted. Citation Format: Jonathan D. Black, Salvatore Lopez, Emiliano Cocco, Stefania Bellone, Elena Bonazzoli, Carlton Schwab, Diana English, Peter Goedings, Patrick Beusker, Miranda van der Lee, Marco Timmers, Wim Dokter, Thomas Rutherfor, Peter Schwartz, Alessandro Santin. SYD985, a novel HER2-targeting antibody-drug conjugate, shows strong antitumor activity in primary USC cell lines with low (1+) and moderate (2+) HER2/Neu expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2461. doi:10.1158/1538-7445.AM2015-2461

e16527 Background: Uterine serous carcinoma (USC) is an aggressive endometrial cancer that carries a poor prognosis. Up to 35% of USC overexpress HER2/neu oncogene at strong (3+) levels while 45% d...

We have built a linker-drug platform based on a cleavable linker-duocarmycin payload for the development of novel generation antibody-drug conjugates. SYD983, a lead ADC originating from that platform, is a cysteine-coupled HER2-targeting ADC based on trastuzumab. SYD983 is a heterogeneous product that consists of a mixture of naked Ab and ADCs with a drug-to-antibody ratio (DAR) of 2, 4, 6, the mean DAR being 2.0. We have shown that SYD983 binds to HER2, induces antibody-dependent cell-mediated cytotoxicity, and is efficiently internalized into HER2-expressing cells. SYD983 very potently kills HER2-expressing cells in vitro and is inactive in cells that don't express HER2. SYD983 is stable in human and cynomolgus monkey (CM) plasma in vitro but has relatively poor stability in mouse plasma. The latter is due to mouse-specific carboxylesterase (CES1c) since stability of SYD983 in plasma of CES1c knockout mice is similar to that in human and CM plasma. SYD983 could be dosed up to 30 mg/kg in CM in vivo with high exposure and stable kinetics and without severe toxic effects. The CM safety study showed no SYD983-induced thrombocytopenia and no induction of peripheral sensory neuropathy, which both commonly occur in studies with ADCs based on tubulin inhibitors. Finally, to reduce heterogeneity we have further fractionated SYD983 by hydrophobic interaction chromatography resulting in an ADC predominantly containing DAR2 and DAR4 species and we designated the resulting purified and more homogeneous ADC as SYD985. In line with its increased average DAR and the absence of naked Ab, SYD985 (average DAR of 2.7) shows increased potency in vitro compared to SYD983. In vivo, SYD985 showed high anti-tumor activity in two patient-derived xenograft models of HER2 positive metastatic breast cancers which underscores the potential for SYD985 in this indication. We have further initiated a program to directly compare SYD985 to T-DM1 for anti-tumor activity in a series of tumors with different HER2 status of which the results are published in a separate accompanying abstract. In conclusion, the data obtained indicates great potential for SYD985 to become an effective drug for patients with HER2-positive cancers. Clinical studies will further elucidate how the characteristics revealed in the preclinical studies, will translate into a benefit for patients. Taken more generally, we conclude that this novel generation duocarmycin-based LD technology could be used on other mAbs to create effective and safe ADCs to treat different forms of cancer. Citation Format: Willem Dokter, Ruud Ubink, Miranda van der Lee, Monique van der Vleuten, Tanja van Achterberg, Daniëlle Jacobs, Diels van den Dobbelsteen, David Egging, Ellen Mattaar, Patrick Groothuis, Patrick Beusker, Ruud Coumans, Ronald Elgersma, Michel Eppink, Guy de Roo, Gijs Verheijden, Marco Timmers. Impressive efficacy and safety profile of a novel generation duocarmycin-based HER2-targeting ADC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2651. doi:10.1158/1538-7445.AM2014-2651

SYD985 is a HER2-targeting ADC based on trastuzumab and Synthon's proprietary cleavable linker-duocarmycin payload. The preclinical profile, including mechanism of action studies, PK, efficacy, and safety studies are described in the accompanying abstract. We have further compared SYD985 to T-DM1 (Kadcyla) and have focused on the in vitro and in vivo efficacies of both of these HER2-targeting ADCs. SYD985 and T-DM1 were compared head-to-head in a panel of 10 cell lines expressing different levels of HER2, including levels characterized as HER2 0, 1+, 2+, or 3+. SYD985 was 3 to 5 times more potent in one HER2 2+ and two HER2 3+ cell lines, while similar potencies were obtained on two other HER2 3+ cell lines. In cell lines with lower HER2 membrane expression (HER2 1+), SYD985 was a factor 7 to 300-fold more potent. These data show that the difference in potencies between SYD985 and T-DM1 becomes more pronounced at lower HER2 expression levels. Subsequently, we next studied how the in vitro data correlate with in vivo anti-tumor activities. In a xenograft with a breast BT-474 cell line (IHC HER2 3+) both SYD985 and T-DM1 dose-dependently reduced tumor growth after single dose (SD) i.v. administration. SYD985 was approximately 5-fold more potent than T-DM1 (tumor stasis at 1 mg/kg vs 5 mg/kg respectively). Subsequently, both ADCs were evaluated (SD i.v.) in a series of patient-derived xenografts (PDX) using HER2 FISH negative tumors from breast cancer patients. We have tested both ADCs in two models classified as Triple Negative Breast Cancer (TNBC; ER negative/PR negative/HER2 negative) with IHC HER2 1+ staining, two TNBC models with IHC HER2 2+ staining, and two Hormone Positive tumor models with IHC HER2 2+ staining. One model (TNBC IHC HER2 2+) did not respond to either SYD985 or T-DM1. In the other five models, SYD985 was more active than T-DM1. Corroborating with the in vitro data, the improved anti-tumor activity of SYD985 versus T-DM1 is most prominent in the PDX models with lowest HER2 expression. In the two TNBC IHC HER2 1+ models, SYD985 induced CR at 1-3 mg/kg, whereas T-DM1 was inactive at all dosages tested, up to 30 mg/kg. We conclude that i) SYD985 is more potent than T-DM1, both in vitro and in vivo in all responsive models; ii) In these studies, the potency advantage of SYD985 over T-DM1 is enhanced even further in the tumor cell lines and PDX models with low HER2 expression (i.e. IHC HER2 1+); iii) The preclinical profile of SYD985 enables extending the target population of BC patients that may respond to this treatment modality to include IHC HER2 1+ and 2+ / FISH negative patients. Early phase clinical trials with SYD985 in patients with this tumor profile, besides refractory BC patients who are IHC HER2 3+ or FISH positive, appear justified and warranted. Citation Format: Willem H.A. Dokter, Miranda van der Lee, Patrick Groothuis, Tanja van Achterberg, Eline Loosveld, Daniëlle Jacobs, Monique van der Vleuten, Patrick H. Beusker, Leon Hooftman, Peter Goedings, Gijs Verheijden, Marco Timmers. In vitro and in vivo antitumor activity of SYD985, a novel HER2-targeting ADC: a comparison with T-DM1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2652. doi:10.1158/1538-7445.AM2014-2652

We have generated cleavable linker-duocarmycin payloads that previously have been shown to be highly stable in human plasma and to cause excellent in vitro and in vivo efficacy when conjugated to antibodies directed against multiple targets in different models. In our HER2 program, we have used these linker-duocarmycin payloads to generate HER2-targeting antibody-drug conjugates (ADCS) and prepare these for FIM studies. HER2 is a validated target in breast (BC) and gastric cancer (GC) that is currently used to target trastuzumab towards HER2-positive BC and GC tumors. We have generated a set of cysteine-coupled ADCs using newly developed duocarmycins conjugated to a therapeutic HER2/neu antibody. In vitro studies with the anti-Her2 ADCs showed that compared to the naked Ab, the ADCs tested have gained a more than 100-fold potency and more than two-fold efficacy in cell killing capacity. Studies in HER2 positive and negative cell lines indicated that cytotoxicity was HER2- mediated. HER2 binding and ADCC activity was not affected by conjugation. Single dose and multiple dose xenograft studies in mice using a human HER2-positive breast cancer cell line showed dose-dependent anti-tumor activity of the ADCs in vivo. Patient-derived xenografts (PDx) using primary tumors from HER2-positive breast cancer and NSCLC patients demonstrated the ability of the ADC to induce complete remission of tumors that remained unresponsive to the naked Ab trastuzumab. Also taken into account toxicology studies in rodents and cynomolgous monkeys, we conclude that we have generated a new class of HER2-based ADCs that have great potential to become effective treatment for HER2-positive tumors, including breast cancer and NSCLC. From a series of compounds, a preclinical candidate was selected that is currently being prepared for Phase I clinical trials that will start in 2014. Citation Format: Wim Dokter, Patrick Beusker, Gijs Verheijden, Ruud Ubink, Miranda van der Lee, Diels van den Dobbelsteen, Peter Goedings, Jacques Lemmens, Vincent de Groot, Marco Timmers. Novel HER2 targeting antibody-drug conjugates based on DNA-interacting duocarmycin and an unique linker technology with great potential in breast cancer and NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4329. doi:10.1158/1538-7445.AM2013-4329