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. Results MP0250 binding to VEGF and HGF inhibited downstream signalling through VEGFR2 and cMET resulting in inhibition of proliferation of VEGF- and HGF-dependent cells. Antitumor activity was demonstrated in VEGF- and HGF-dependent xenograft and syngeneic models with activity superior to that of individual VEGF- and HGF-blocking DARPin® molecules. Combination therapy studies showed potentiation of the antitumor activity of chemotherapy and immunotherapy agents, including an anti-PD1 antibody. Materials and Methods Potency of MP0250 was assessed in cellular models and in a variety of xenograft models as monotherapy or in combination with standard-of-care drugs. Conclusions Dual inhibition of VEGF and HGF by MP0250 produced powerful single agent and combination antitumor activity. This, together with increasing understanding of the role of the HGF/cMET pathway in resistance to VEGF (and other agents), supports testing of MP0250 in the clinic.

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 (R) domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin (R) drug candidate, little is known about DARPin (R) drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin (R) domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of similar to 30 hours in mouse and similar to 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin (R) proteins can be valuable next-generation drug candidates.

Abstract Background: The VEGF/VEGFR and HGF/cMet pathways are implicated in tumor survival, growth, angiogenesis, invasion and metastasis. DARPins (designed ankyrin repeat proteins) are small genetically engineered proteins that bind to specific targets with high affinity. MP0250 is a first-in-class, tri-specific DARPin with the ability to simultaneously neutralize the activities of VEGF and HGF and also to bind to human serum albumin (HSA) to give an increased plasma half-life and potentially enhanced tumor penetration. MP0250 is currently being explored in a Phase I study. Methods: A phase I, open-label, repeated-dose, dose escalation, multi-center study to assess safety, tolerability and pharmacokinetics of MP0250 is in progress in patients with advanced solid tumors who have progressed on at least one prior standard therapy. Using a 3+3 design, eligible patients are being enrolled into dose escalation cohorts receiving MP0250 by intravenous infusion every other week until disease progression or unacceptable toxicity. Results: Twelve patients have been enrolled in the first three cohorts dosed with MP0250 at 0.5 (n = 3), 1.5 (n = 3) and 4 mg/kg (n = 6). MP0250 has been well tolerated and a maximum tolerated dose has not been reached. A single dose-limiting toxicity was observed at 4mg/kg (significant reduction in cardiac ejection fraction after 1st infusion in a patient with multiple cardiac risk factors). The most frequent adverse events (AEs, CTC version 4.03) were transient hypertension (42%), diarrhea (33%), fatigue (25%) and nausea (25%). With the exception of hypertension (grade 3 in 33% of all patients), all AEs were grade 1 or 2. Interim pharmacokinetic analyses indicated linear behavior between doses 1.5 and 4 mg/kg with a mean half-life of approximately 11 days (range 9-15 days). Sustained exposure was observed for all patients throughout the treatment periods analyzed, the longest to-date being 9 months. Stable disease for 10 months (treatment ongoing) has been observed in one patient with a head and neck tumor and for 8 months in a patient with a cervical adenocarcinoma. Conclusion: Preliminary data from the Phase I study of the first-in-class, tri-specific VEGF, HGF and HSA binding DARPin MP0250 shows it to be well tolerated, to have a mean half-life around 11 days and to have sustained exposure on repeated dosing. In addition, there was disease stabilization exceeding 8 months in two patients suggestive of anti-tumor activity. Further dose escalation is ongoing. Citation Format: Jordi Rodon, Aurelius Omlin, Karin H. Herbschleb, Javier Garcia-Corbacho, Jan Steiner, Ignacio Dolado, Christof Zitt, Daniel Feurstein, Dascha Turner, Keith M. Dawson, Michael T. Stumpp, Patrick Gilboy, Andreas Harstrick, Analía Azaro, Christoph J. Ackermann, Mark R. Middleton, Richard D. Baird. First-in-human Phase I study to evaluate MP0250, a DARPin blocking HGF and VEGF, in patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B25.

e19574 Background: In multiple myeloma, tumor cells and stroma exert a reciprocal effect on each other that leads to tumor expansion in the bone marrow and bone destruction. Hepatocyte growth factor (HGF) is a main player in osteolytic bone destruction and also stimulates myeloma cell proliferation, migration and adhesion in the bone marrow. Additionally, VEGF plays a key neovascularization role in multiple myeloma. Therefore, we investigated whether MP0250, a bispecific DARPin simultaneously inhibiting tumor stroma HGF and VEGF, could potentiate the anti-tumor effect of the standard of care proteasome inhibitor bortezomib. Methods: MP0250 was tested as monotherapy and in combination with 0.6 mg/kg of bortezomib in an orthotopic murine model, where human multiple myeloma cells are implanted in the murine bone marrow. Tumor growth and invasion into the muscle were monitored by X-ray, whereas bone destruction was analyzed by microCT. Results: MP0250 significantly inhibited bone lysis and tumor invasion both...

11039 Background: The interplay of tumor cells and their microenvironment is crucial in the growth of solid tumors. Key mediators of such interaction are the VEGF/VEGFR and the HGF/cMet pathways, which drive tumor survival, growth, angiogenesis, invasion and metastasis. It is anticipated that simultaneous inhibition of the VEGF/VEGFR and HGF/cMet pathways will interfere with crucial steps of tumor growth as well as with the onset of treatment resistance. MP0250 is a bispecific DARPin targeting both pathways simultaneously by specifically neutralizing VEGF and HGF. Its low toxicity offers the potential for combination with chemotherapy and other targeted therapies. Methods: Antitumor activity of MP0250 was tested in kidney, liver, lung and gastric patient-derived xenograft (PDX) models and compared to standard of care (SoC) therapies. Results: MP0250 showed consistent antitumor activity in 6 out of 7 investigated models. Its potency was superior to SoC in renal and gastric carcinoma models, and similar to SoC in liver and one lung cancer (NSCLC) model. Of note, neither MP0250 nor SoC (5-FU) showed potency in one SCLC lung cancer model. Tumor regression was induced by MP0250 in one liver and one renal model with optimal T/C values of 6.9% and 8.9%. Tumor growth inhibition was recorded in the other liver and one lung model (T/C values of 37% and 26%) whereas moderate efficacy was observed in the second renal cancer model (T/C value of 56%). Interestingly, MP0250 increased the potency of the SoC drug paclitaxel in the gastric cancer model (T/C values: MP0250 37%; paclitaxel 46%, MP0250/paclitaxel 21%). No toxicity of MP0250 was observed as monotherapy or in combination with SoC. Conclusions: MP0250, a bispecific DARPin blocking HGF and VEGF, is a potent inhibitor of tumor growth in PDX models, often surpassing the efficacy of SoC therapy. In a gastric cancer model, MP0250 increased potency of SoC. These results indicate that MP0250 has the potential to be used as monotherapy in a range of solid tumors and, as a result of its good safety profile, the possibility exists that it may be also combined with SoC and other tumor targeting molecules, i.e. TKIs. A phase I clinical trial is in preparation.

Abstract Introduction: DARPins are genetically engineered small-sized proteins exhibiting high affinity for target proteins. The epithelial growth factor receptor 2 (Her2/neu) is a target of interest for targeted therapies as it is overexpressed in epithelial tumors such as breast and gastric cancers. Antitumor activity and biodistribution of novel Her2-targeting DARPins were evaluated in mice bearing Her2-positive human breast tumors. Materials and methods: Antitumor activity and biodistribution studies were performed in female Balb/c Nude mice bearing subcutaneous BT-474 human breast tumors. Mice were treated intravenously (IV) every 3 days with five different DARPins at 20 or 35 mg/kg and antitumor activity was evaluated by measuring tumor growth twice weekly. For the biodistribution study, DARPins were radiolabelled with technetium-99m (99mTc) using the IsoLink procedure. Quality control of the radiolabelled DARPins was performed by size exclusion chromatography, ELISA assay, and binding to tumor cells. 99mTc-radiolabelled DARPins were injected IV to mice at 23-25 MBq/mouse. 4 and 24 hours post-injection, animals were euthanized for blood, tumor and organ collection. Radioactivity in each sample was measured and normalized to sample weight and injected dose (% ID/g). The depth of tumor penetration of 99mTc-radiolabelled DARPins was also assessed by planar autoradiography imaging. Results: All five DARPins displayed a significant antitumor activity. Four DARPins induced BT-474 tumor regression while the fifth led to BT-474 tumor stasis. All DARPins were efficiently radiolabelled with 99mTc to radiochemical purities within 50% to 90%, and they all retained the ability to fully bind Her2. The five DARPins displayed a comparable distribution pattern. Radioactivity uptake in blood ranged from 25-30% and 8-10 % ID/g at 4 and 24 hours post-injection, respectively. Accumulation in tumors increased from 6-10% ID/g at 4 hours to 12-20% ID/g at 24 hours. All 99mTc-radiolabelled DARPins were excreted through the liver and kidneys and radioactivity uptake in both organs, respectively, ranged from 7-14% ID/g and 4-8% ID/g at 24 hours post-injection. Planar autoradiography imaging revealed that the depth of penetration of DARPinsinto the tumor was > 2 mm as soon as 4 hours post-injection. Conclusions:The five anti-Her2 DARPins investigated efficiently targeted BT-474 tumors and significantly inhibited their growth, making them attractive as new therapeutic agents for the treatment of Her2-positive cancers. Their easy radiolabelling with 99mTc also makes them attractive tools for imaging with diagnostic and predictive purposes, for instance to follow the modulation of Her2 expression during therapy. Citation Format: Olivier Raguin, Marie Leblanc, Bertrand Collin, Alexandra Oudot, Jean-François Mirjolet, Ulrike Fiedler, Ignacio Dolado. Biodistribution and antitumor efficacy study of novel Her2 targeting DARPins. [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 5442. doi:10.1158/1538-7445.AM2014-5442

The mechanisms that allow colon cancer cells to form liver and lung metastases, and whether KRAS mutation influences where and when metastasis occurs, are unknown. We provide clinical and molecular evidence showing that different MAPK signalling pathways are implicated in this process. Whereas ERK2 activation provides colon cancer cells with the ability to seed and colonize the liver, reduced p38 MAPK signalling endows cancer cells with the ability to form lung metastasis from previously established liver lesions. Downregulation of p38 MAPK signalling results in increased expression of the cytokine PTHLH, which contributes to colon cancer cell extravasation to the lung by inducing caspase-independent death in endothelial cells of the lung microvasculature. The concerted acquisition of metastatic traits in the colon cancer cells together with the sequential colonization of liver and lung highlights the importance of metastatic lesions as a platform for further dissemination.

The p38 MAPK pathway is an important regulator of many cellular responses. It is well established that p38 MAPK signalling negatively regulates epithelial cell transformation, but enhanced p38 MAPK activity has been also correlated with bad clinical prognosis in some tumour types. Here, we provide genetic and pharmacological evidence showing that p38 MAPK inhibition cooperates with the chemotherapeutic agent cisplatin to kill tumour cells. We show that p38 MAPK inhibition results in ROS upregulation, which in turn activates the JNK pathway via inactivation of phosphatases, sensitizing human tumour cells to cisplatin-induced apoptosis. Using a mouse model for breast cancer, we confirm that inhibition of p38 MAPK cooperates with cisplatin treatment to reduce tumour size and malignancy in vivo. Taken together, our results illustrate a new function of p38 MAPK that helps tumour cells to survive chemotherapeutic drug treatments, and reveal that the combination of p38 MAPK inhibitors with cisplatin can be potentially exploited for cancer therapy.

p38α MAPK (mitogen-activated protein kinase) plays an important tumour suppressor role, which is mediated by both its negative effect on cell proliferation and its pro-apoptotic activity. Surprisingly, most tumour suppressor mechanisms co-ordinated by p38α have been reported to occur at the post-translational level. This contrasts with the important role of p38α in the regulation of transcription and the profound changes in gene expression that normally occur during tumorigenesis. We have analysed whole-genome expression profiles of Ras-transformed wild-type and p38α-deficient cells and have identified 202 genes that are potentially regulated by p38α in transformed cells. Expression analysis has confirmed the regulation of these genes by p38α in tumours, and functional validation has identified several of them as probable mediators of the tumour suppressor effect of p38α on Ras-induced transformation. Interestingly, approx. 10% of the genes that are negatively regulated by p38α in transformed cells contribute to EGF (epidermal growth factor) receptor signalling. Our results suggest that inhibition of EGF receptor signalling by transcriptional targets of p38α is an important function of this signalling pathway in the context of tumour suppression.

Contact inhibition is a fundamental process in multicellular organisms aimed at inhibiting proliferation at high cellular densities through poorly characterized intracellular signals, despite availability of growth factors. We have previously identified the protein kinase p38 alpha as a novel regulator of contact inhibition, as p38 alpha is activated upon cell-cell contacts and p38 alpha-deficient cells are impaired in both confluence-induced proliferation arrest and p27(Kip1) accumulation. Here, we establish that p27(Kip1) plays a key role downstream of p38 alpha to arrest proliferation at high cellular densities. Surprisingly, p38 alpha does not directly regulate p27(Kip1) expression levels but leads indirectly to confluent upregulation of p27(Kip1) and cell cycle arrest via the inhibition of mitogenic signals originating from the epidermal growth factor receptor (EGFR). Hence, confluent activation of p38 alpha uncouples cell proliferation from mitogenic stimulation by inducing EGFR degradation through downregulation of the EGFR-stabilizing protein Sprouty2 (Spry2). Accordingly, confluent p38 alpha-deficient cells fail to downregulate Spry2, providing them in turn with sustained EGFR signaling that facilitates cell overgrowth and oncogenic transformation. Our results provide novel mechanistic insight into the role of p38 alpha as a sensor of cell density, which induces confluent cell cycle arrest via the Spry2-EGFR-p27(Kip1) network.

AKT, a protein kinase frequently hyperactivated in cancer, plays an important role in cell survival and contributes to tumor cell resistance to cytotoxic therapies. A new study in this issue of Cancer Cell shows that AKT also induces the accumulation of oxygen radicals, which can be exploited to selectively kill cancer cells containing high levels of AKT activity.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in stress-induced cell-fate decisions by orchestrating responses that go from cell-cycle arrest to apoptosis. We have identified a new p38 MAPK-regulated protein that we named p18(Hamlet), which becomes stabilized and accumulates in response to certain genotoxic stresses such as UV or cisplatin treatment. Overexpression of p18(Hamlet) is sufficient to induce apoptosis, whereas its downregulation reduces the apoptotic response to these DNA damage-inducing agents. We show that p18(Hamlet) interacts with p53 and stimulates the transcription of several proapoptotic p53 target genes such as PUMA and NOXA. This correlates with enhanced p18(Hamlet)-induced recruitment of p53 to the promoters. In proliferating cells, low steady-state levels of p18(Hamlet) are probably maintained by a p53-dependent negative feedback loop. Therefore, p18(Hamlet) is a new cell-fate regulator that links the p38 MAPK and p53 pathways and contributes to the establishment of p53-regulated stress responses.

p38alpha is a stress-activated protein kinase that negatively regulates malignant transformation induced by oncogenic H-Ras, although the mechanisms involved are not fully understood. Here, we show that p38alpha is not a general inhibitor of oncogenic signaling, but that it specifically modulates transformation induced by oncogenes that produce reactive oxygen species (ROS). This inhibitory effect is due to the ROS-induced activation of p38alpha early in the process of transformation, which induces apoptosis and prevents the accumulation of ROS and their carcinogenic effects. Accordingly, highly tumorigenic cancer cell lines have developed a mechanism to uncouple p38alpha activation from ROS production. Our results indicate that oxidative stress sensing plays a key role in the inhibition of tumor initiation by p38alpha.

Cell migration is critical for many processes, such as angiogenesis, inflammation, development and wound healing, and is also involved in tumour progression and metastasis. Here we show that CXCL12, complement factor 5a (C5a), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF)-BB, which stimulate cell migration, also activate p38α MAPK. Pharmacological inhibition of this protein kinase with SB 203580 or BIRB 0796, or the genetic ablation of p38α MAPK, blocked cell migration induced by the aforementioned chemo-attractants. Macrophages from mice lacking one or more of the other p38 MAPK isoforms showed normal cell migration in response to C5a. We also show that the activation of p38α MAPK in response to CXCL12 requires the p21-activated protein kinases (PAK)-1 and PAK-2. MAPKAP-K2 is a protein kinase that is activated by p38α MAPK. Reducing its expression using RNA interference blocked CXCL12-induced HeLa cell migration, while macrophages from mice that do not express MAPKAP-K2 failed to migrate in response to C5a. Moreover, RNA interference against the small heat shock protein 27 (HSP27), a physiological substrate of MAPKAP-K2, blocked the CXCL12-induced cell migration. These results demonstrate a general and essential role of the PAK-p38α MAPK-MAPKAP-K2-HSP27 signalling pathway in mediating the effects of chemotactic stimuli on cell migration.

Oncogenic Ras signaling has been long known to play an important role in tumorigenesis and human cancer. In this report, we have used the sensitive 2-D-DIGE coupled to MS for the identification of proteins differentially expressed at the cell membrane level between oncogenic H-RasV12-transformed wild-type and p38alpha-deficient mouse embryo fibroblasts (MEFs). Following trifluoroethanol solubilization, 76 proteins were found to be differentially regulated. After PMF, 63 spots containing 42 different proteins were unequivocally identified by MALDI-TOF MS coupled with database interrogation. As expected, many of them were membrane proteins. Six proteins were selected for further validation studies based on their potential functional link with malignant transformation and signal transduction. These were prohibitin (PHB), protein disulfide isomerase 3 (PDIA3), focal adhesion kinase 2 (FAK2), c-GMP dependent protein kinase 2 (KGP2), NADH-ubiquinone oxidoreductase 30 kDa subunit (NUGM) and translationally controlled tumor protein (TCTP). All these proteins were up-regulated in the membranes of H-RasV12-transformed p38alpha-/-cells, except for prohibitin, which was down-regulated. An excellent correlation was found between DIGE results and Western blot studies, indicating the reliability of the 2-D-DIGE analysis. The available evidence about the putative function of the identified proteins supports the emerging role of p38alpha as a negative regulator of tumorigenesis. Further studies are in progress to elucidate the implications of these findings in the regulation of H-Ras-induced transformation by p38alpha signaling.

Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.

Proliferation of nontransformed cells is regulated by cell–cell contacts, which are referred to as contact-inhibition. Despite its generally accepted importance for cell cycle control, knowledge about the intracellular signalling pathways involved in contact inhibition is scarce. In the present work we show that p38α mitogen-activated protein kinase (MAPK) is involved in the growth-inhibitory signalling cascade of contact inhibition in fibroblasts. p38α activity is increased in confluent cultures of human fibroblasts compared to proliferating cultures. Time course studies show a sustained activation of p38α in response to cell–cell contacts in contrast to a transient activation after serum stimulation. The induction of contact inhibition by addition of glutaraldehyde-fixed cells is impaired by pharmacological inhibition of p38 as well as in p38α−/− fibroblasts. Further evidence for a central role of p38α in contact inhibition comes from the observation that p38α−/− fibroblasts show a higher saturation density compared to wild-type (wt) fibroblasts, which is reversed by reconstituted expression of p38α. In agreement with a defect in contact inhibition, p27Kip1 accumulation is impaired in p38α−/− fibroblasts compared to wt fibroblasts. Hence, our work shows a new role for p38α in contact inhibition and provides a mechanistic basis for the recently proposed tumour suppressive function of this MAPK pathway.