A re-engineered immunotoxin shows promising preclinical activity in ovarian cancer.

RG7787 is a re-engineered mesothelin-targeted immunotoxin with reduced immunogenicity composed of a humanized anti-mesothelin Fab fragment and a B-cell epitope silenced 24 kD fragment of Pseudomonas exotoxin A. High prevalence of mesothelin-positive cases and a large unmet medical need make ovarian cancer a promising indication for the clinical development of RG7787. However, ovarian cancer patients also frequently have elevated serum levels of the cancer antigen 125 (CA-125). In principle this could pose a problem, since the binding sites for CA-125 and RG7787 on mesothelin were reported to overlap. However, we show here that RG7787 can readily displace even excess amounts of CA-125 in different cellular assays. Moreover when tested in-vitro on a panel of 12 ovarian cancer cell lines, RG7787 had high cytotoxic activity on COV644, Caov-4, and SNU-119 cells and fully inhibited growth of EFO-21, KURAMOCHI, OVSAHO, and Caov-3 cells with potency values ranging from 1 to 86 pM. Finally, we evaluated the in-vivo efficacy of RG7787 in OvCa6668, a patient-derived ovarian cancer model with high levels of CA-125 expression. RG7787 had moderate monotherapy efficacy but in combination with standard chemotherapies (cisplatin, paclitaxel) achieved pronounced tumor regressions. In summary our data support clinical testing of RG7787 in ovarian cancer.

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains.

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-ß family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy.

Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation. In vitro RG7155 treatment results in cell death of CSF-1-differentiated macrophages. In animal models, CSF-1R inhibition strongly reduces F4/80(+) tumor-associated macrophages accompanied by an increase of the CD8(+)/CD4(+) T cell ratio. Administration of RG7155 to patients led to striking reductions of CSF-1R(+)CD163(+) macrophages in tumor tissues, which translated into clinical objective responses in diffuse-type giant cell tumor (Dt-GCT) patients.

Quantitative chemical proteomics profiling differentiates erlotinib from gefitinib in EGFR wild-type non-small cell lung carcinoma cell lines.

Although both erlotinib and gefitinib target the EGF receptor (EGFR), erlotinib is effective in patients with EGFR wild-type or mutated tumors, whereas gefitinib is only beneficial for patients with activating mutations. To determine whether these differences in clinical outcomes can be attributed to their respective protein interaction profiles, a label-free, quantitative chemical proteomics study was conducted. Using this method, 24 proteins were highlighted in the binding profiles of erlotinib and gefitinib. Unlike gefinitib, erlotinib displaced the ternary complex formed by integrin-linked kinase (ILK), α-parvin, and PINCH (IPP). The docking of erlotinib in the three-dimensional structure of ILK showed that erlotinib has the ability to bind to the ATP-binding site, whereas gefitinib is unlikely to bind with high affinity. As the IPP complex has been shown to be involved in epithelial-to-mesenchymal transition (EMT) and erlotinib sensitivity has been correlated with EMT status, we used a cellular model of inducible transition and observed that erlotinib prevented EMT in a more efficient way than gefitinib by acting on E-cadherin expression as well as on IPP levels. A retrospective analysis of the MERIT trial indicated that, besides a high level of E-cadherin, a low level of ILK could be linked to clinical benefit with erlotinib. In conclusion, we propose that, in an EGFR wild-type context, erlotinib may have a complementary mode of action by inhibiting IPP complex activities, resulting in the slowing down of the metastatic process of epithelial tumors.

Crystal structures of active SRC kinase domain complexes.

c-Src was the first proto-oncoprotein to be identified, and has become the focus of many drug discovery programs. Src structures of a major inactive form have shown how the protein kinase is rigidified by several interdomain interactions; active configurations of Src are generated by release from this "assembled" or "bundled" form. Despite the importance of Src as a drug target, there is relatively little structural information available regarding the presumably more flexible active forms. Here we report three crystal structures of a dimeric active c-Src kinase domain, in an apo and two ligand complexed forms, with resolutions ranging from 2.9A to 1.95A. The structures show how the kinase domain, in the absence of the rigidifying interdomain interactions of the inactivation state, adopts a more open and flexible conformation. The ATP site inhibitor CGP77675 binds to the protein kinase with canonical hinge hydrogen bonds and also to the c-Src specific threonine 340. In contrast to purvalanol B binding in CDK2, purvalanol A binds in c-Src with a conformational change in a more open ATP pocket.