First-in-Human Phase I Study of MP0250, a First-in-Class DARPin Drug Candidate Targeting VEGF and HGF, in Patients With Advanced Solid Tumors.

Purpose: A first-in-human study was performed with MP0250, a DARPin drug candidate. MP0250 specifically inhibits both vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) with the aim of disrupting the tumor microenvironment.

Patients And Methods: A multicenter, open-label, repeated-dose, phase I study was conducted to assess the safety, tolerability, and pharmacokinetics of MP0250 in 45 patients with advanced solid tumors.

Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins.

PD-1/PD-L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti-PD-1 therapy requires further improvements. Our aim was to explore the enhancement of T-cell function by using novel PD-1-blocking proteins and compare with clinically approved monoclonal antibodies (mAbs).

Targeting angiogenesis in multiple myeloma by the VEGF and HGF blocking DARPin protein MP0250: a preclinical study.

The investigational drug MP0250 is a multi-specific DARPin molecule that simultaneously binds and neutralizes VEGF and HGF with high specificity and affinity. Here we studied the antiangiogenic effects of the MP0250 in multiple myeloma (MM). In endothelial cells (EC) isolated from bone marrow (BM) of MM patients (MMEC) MP0250 reduces VEGFR2 and cMet phosphorylation and affects their downstream signaling cascades.

MP0250, a VEGF and HGF neutralizing DARPin molecule shows high anti-tumor efficacy in mouse xenograft and patient-derived tumor models.

Background: The VEGF/VEGFR and the HGF/cMET pathways are key mediators of the interplay of tumor cells and their microenvironment. However, inhibition of VEGF has been shown to produce only limited clinical benefit and inhibition of the activation of cMET by HGF has not translated into clinical benefit in pivotal trials. MP0250, a DARPin molecule that specifically inhibits both VEGF and HGF has been developed to explore the clinical potential of dual inhibition of these pathways.

Design and characterization of MP0250, a tri-specific anti-HGF/anti-VEGF DARPin® drug candidate.

MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains.

Novel ubiquitin-derived high affinity binding proteins with tumor targeting properties.

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range.

Angiopoietin-2 is critical for cytokine-induced vascular leakage.

Genetic experiments (loss-of-function and gain-of-function) have established the role of Angiopoietin/Tie ligand/receptor tyrosine kinase system as a regulator of vessel maturation and quiescence. Angiopoietin-2 (Ang-2) acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang-1/Tie-2 signaling. Ang-2 thereby controls the vascular response to inflammation-inducing as well as angiogenesis-inducing cytokines.

Treatment of diabetic macular edema with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study.

Purpose: To evaluate the safety and bioactivity of MP0112, a designed ankyrin repeat protein (DARPin) that specifically binds vascular endothelial growth factor (VEGF) in patients with diabetic macular edema (DME). DARPins are a novel class of proteins selected for specific, high-affinity binding to a target protein.

Design: Phase I/II, open-label, multicenter dose-escalation trial.

Efficacy of sequential treatment with sunitinib-everolimus in an orthotopic mouse model of renal cell carcinoma.

Background/aim: Sequential treatment with targeted agents is standard of care for patients with metastatic renal cell carcinoma (mRCC). However, clinical data directly comparing treatment outcomes with a mammalian target of rapamycin inhibitor or a vascular endothelial growth factor-targeted agent in the second-line setting are lacking. We evaluated sequential treatment in a syngeneic, orthotopic mouse model of mRCC.

Angiopoietin-1 mediates inhibition of hypertension-induced release of angiopoietin-2 from endothelial cells.

Aims: Adequate endothelial cell stimulation is a prerequisite for the adaptive remodelling of macro- and microvessels. A pivotal autocrine mechanism following endothelial cell activation is the release of angiopoietin-2 (Ang-2), which subsequently antagonizes the binding of Ang-1 to the Tie-2 receptor, thus sensitizing the endothelial cells to pro-angiogenic and/or pro-inflammatory stimuli. Based on the observation that hypertension in mice reduces the abundance of Ang-2 stored in arterial endothelial cells, this study was aimed at testing the hypothesis that an increase in wall stress (WS) or stretch-a hallmark of hypertension-is sufficient to release Ang-2 from endothelial cells.

Angiopoietin-2 stimulation of endothelial cells induces alphavbeta3 integrin internalization and degradation.

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence.

Workflow comparison for label-free, quantitative secretome proteomics for cancer biomarker discovery: method evaluation, differential analysis, and verification in serum.

The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3).

Therapeutic interference with EphrinB2 signalling inhibits oxygen-induced angioproliferative retinopathy.

Purpose: To investigate whether EphrinB2 (EfnB2) or EphB4 influence retinal angiogenesis under physiological or pathological conditions.

Methods: Using the mouse model of oxygen-induced proliferative retinopathy (OIR), the expression of EfnB2, EphB4, vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 was quantified by quantitative polymerase chain reaction (qPCR) and localized in EfnB2- and EphB4-lacZ mice. Angioproliferative retinopathy was manipulated by intravitreal injection of dimeric EfnB2 and monomeric or dimeric EphB4.

Affilin molecules selected against the human papillomavirus E7 protein inhibit the proliferation of target cells.

Intracellular binding proteins can be applied as research tools for target validation and study of protein function in cells and potentially as therapeutics. The success of intracellular binding reagents depends on their affinity and specificity for target molecules, although their stability and functionality in the intracellular environment actually determine their usefulness for such application. Alternative binding proteins derived from scaffolds devoid of disulfide bonds are well suited for intracellular use, as their folding and stability are usually not impaired under reducing conditions.

Host-derived angiopoietin-2 affects early stages of tumor development and vessel maturation but is dispensable for later stages of tumor growth.

The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu.

Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein.

The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses.

High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells.

Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering.

Angiopoietins: a link between angiogenesis and inflammation.

The angiopoietin (Ang)-Tie ligand-receptor system has a key regulatory role in regulating vascular integrity and quiescence. Besides its role in angiogenesis, it is an important regulator in numerous diseases including inflammation. Ang-1-mediated Tie2 activation is required to maintain the quiescent resting state of the endothelium.

Emerging roles of the Angiopoietin-Tie and the ephrin-Eph systems as regulators of cell trafficking.

Vascular receptor tyrosine kinases (RTK) have been identified as critical regulatory signaling molecules of developmental and adult vascular morphogenic processes [vascular endothelial growth factor (VEGF) receptors=sprouting; EphB receptors=assembly; Tie2 receptor=maturation and quiescence]. It is intriguing that the same molecules that control the growth of blood and lymphatic vessels play critical roles in the adult to regulate maintenance functions related to vascular homeostasis. VEGF is among the most potent inducers of vascular permeability.

The sialomucin CD34 is a marker of lymphatic endothelial cells in human tumors.

The mechanisms of lymphangiogenesis have been increasingly understood in recent years. Yet, the contribution of lymphangiogenesis versus lymphatic cooption in human tumors and the functionality of tumor lymphatics are still controversial. Furthermore, despite the identification of lymphatic endothelial cell (LEC) markers such as Prox1, podoplanin, LYVE-1, and VEGFR-3, no activation marker for tumor-associated LECs has been identified.

Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence.

A single local injection of recombinant VEGF receptor 2 but not of Tie2 inhibits retinal neovascularization in the mouse.

Purpose: The purpose of this study was to develop pharmacological therapeutic alternatives for ischemia-induced proliferative retinopathy.

Methods: C57BL/6J mice were placed in 76% oxygen on postnatal day 7 (P7) for 5 days. On P12 recombinant, chimeric vascular endothelial growth factor (sVEGF-R2) or sTie2 was injected intravitreally in one eye.

The Tie-2 ligand angiopoietin-2 destabilizes quiescent endothelium through an internal autocrine loop mechanism.

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the endothelial receptor tyrosine kinase Tie-2, which controls vascular assembly and endothelial quiescence. The largely complementary phenotypes of Ang-1-deficient mice and Ang-2-overexpressing mice have led to an antagonistic model in which Ang-1 acts as Tie-2-activating agonist and Ang-2 acts as a Tie-2-inhibiting antagonist. To date, no mechanistic equivalent of the antagonistic Ang-1/Ang-2 model has been established and the mechanisms of Ang-2 function in particular remain mysterious.

Expression of angiopoietin-2 in endothelial cells is controlled by positive and negative regulatory promoter elements.

Objective: Angiopoietin-2 (Ang-2) is a non-signal transducing ligand of the endothelial receptor tyrosine kinase Tie-2. Ang-2 is produced by endothelial cells and acts as an autocrine regulator mediating vascular destabilization by inhibiting Angiopoietin-1-mediated Tie-2 activation. To examine the transcriptional regulation of Ang-2, we studied the Ang-2 promoter in endothelial cells and nonendothelial cells.