Gijs Verheijden

Human cartilage gp-39+,CD16+ monocytes in peripheral blood and synovium: correlation with joint destruction in rheumatoid arthritis.

OBJECTIVE To investigate the expression of human cartilage (HC) gp-39, a possible autoantigen in rheumatoid arthritis (RA), in peripheral blood and synovium, to characterize its cellular source, and to analyze correlations with clinical features. METHODS The expression of HC gp-39 in synovium and peripheral blood mononuclear cells (PBMC) was assessed by immunohistochemistry and flow cytometry. Synthesis and secretion were investigated by both reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS PBMC expressing HC gp-39 were increased in RA patients compared with spondylarthropathy patients (P = 0.0029) and with healthy control subjects (P = 0.0013). HC gp-39+ cells were also slightly overrepresented in RA synovium (P = 0.01). In both blood and synovium, HC gp-39+ cells were identified as CD14dim,CD16+ monocytes, a phenotype which can differentiate from classic CD14++ monocytes by maturation in vitro. HC gp-39 messenger RNA was detected in RA synovium and PBMC, and PBMC secreted HC gp-39 in vitro. The number of HC gp-39+ PBMC correlated with serum levels of C-reactive protein (r = 0.39, P = 0.003) and HC gp-39 (r = 0.52, P = 0.014). HC gp-39 expression in RA synovial lining correlated with joint destruction (r = 0.77, P < 0.001). CONCLUSION CD16+ monocytes, a cellular source of HC gp-39 in vivo, are overrepresented in both RA peripheral blood and synovial tissue. The presence of HC gp-39+ cells in RA synovium is correlated with the degree of joint destruction. These data support a role of these cells in the local autoimmune response that leads to chronic inflammation and joint destruction.

Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985.

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.

The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers

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.

Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263–275) epitope: an MHC anchor variant peptide for immune modulation

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263–275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263–275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.

Abstract 5360: The preclinical profile of the duocarmycin-based HER2-targeting ADC SYD985 predicts for clinical benefit in low HER2-expressing breast cancers

SYD985 is a HER2-targeting ADC based on trastuzumab and vc-seco-DUBA, Synthon9s 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, Desiree 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