Oliver Krieter

Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

Significance Improving survival of patients with glioblastoma (GBM) using antiangiogenic therapy remains a challenge. In this study we show that dual blockade of angiopoietin-2 and vascular endothelial growth factor delays tumor growth and enhances survival benefits through reprogramming of tumor-associated macrophages toward an antitumor phenotype as well as by pruning immature tumor vessels. The antitumor immunomodulatory potential of this dual blockade supports clinical testing of this approach for GBM with other immunotherapeutic approaches such as checkpoint blockers. Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.

Phase 1 dose-escalation study of the antiplacental growth factor monoclonal antibody RO5323441 combined with bevacizumab in patients with recurrent glioblastoma

BACKGROUND We conducted a phase 1 dose-escalation study of RO5323441, a novel antiplacental growth factor (PlGF) monoclonal antibody, to establish the recommended dose for use with bevacizumab and to investigate the pharmacokinetics, pharmacodynamics, safety/tolerability, and preliminary clinical efficacy of the combination. METHODS Twenty-two participants with histologically confirmed glioblastoma in first relapse were treated every 2 weeks with RO5323441 (625 mg, 1250 mg, or 2500 mg) plus bevacizumab (10 mg/kg). A standard 3 + 3 dose-escalation trial design was used. RESULTS RO5323441 combined with bevacizumab was generally well tolerated, and the maximum tolerated dose was not reached. Two participants experienced dose-limiting toxicities (grade 3 meningitis associated with spinal fluid leak [1250 mg] and grade 3 cerebral infarction [2500 mg]). Common adverse events included hypertension (14 participants, 64%), headache (12 participants, 55%), dysphonia (11 participants, 50%) and fatigue (6 participants, 27%).The pharmacokinetics of RO5323441 were linear, over-the-dose range, and bevacizumab exposure was unaffected by RO5323441 coadministration. Modulation of plasmatic angiogenic proteins, with increases in VEGFA and decreases in FLT4, was observed. Dynamic contrast-enhanced/diffusion-weighted MRI revealed large decreases in vascular parameters that were maintained through the dosing period. Combination therapy achieved an overall response rate of 22.7%, including one complete response, and median progression-free and overall survival of 3.5 and 8.5 months, respectively. CONCLUSION The toxicity profile of RO5323441 plus bevacizumab was acceptable and manageable. The observed clinical activity of the combination does not appear to improve on that obtained with single-agent bevacizumab in patients with recurrent glioblastoma.

Integrated Spatio-Temporal Segmentation of Longitudinal Brain Tumor Imaging Studies

Consistent longitudinal segmentation of brain tumor images is a critical issue in treatment monitoring and in clinical trials. Fully automatic segmentation methods are a good candidate for reliably detecting changes of tumor volume over time. We propose an integrated 4D spatio-temporal brain tumor segmentation method, which combines supervised classification with conditional random field regularization in an energy minimization scheme. Promising results and improvements over classic 3D methods for monitoring the temporal volumetric evolution of necrotic, active and edema tumor compartments are demonstrated on a longitudinal dataset of glioma patient images from a multi-center clinical trial. Thanks to its speed and simplicity the approach is a good candidate for standard clinical use.

First-in-Human Phase I Study of Single-agent Vanucizumab, A First-in-Class Bispecific Anti-Angiopoietin-2/Anti-VEGF-A Antibody, in Adult Patients with Advanced Solid Tumors

Purpose: Vanucizumab is an investigational antiangiogenic, first-in-class, bispecific mAb targeting VEGF-A and angiopoietin-2 (Ang-2). This first-in-human study evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of vanucizumab in adults with advanced solid tumors refractory to standard therapies. Experimental Design: Patients received escalating biweekly (3–30 mg/kg) or weekly (10–30 mg/kg) intravenous doses guided by a Bayesian logistic regression model with overdose control. Results: Forty-two patients were treated. One dose-limiting toxicity, a fatal pulmonary hemorrhage from a large centrally located mediastinal mass judged possibly related to vanucizumab, occurred with the 19 mg/kg biweekly dose. Arterial hypertension (59.5%), asthenia (42.9%), and headache (31%) were the most common toxicities. Seventeen (41%) patients experienced treatment-related grade ≥3 toxicities. Toxicity was generally higher with weekly than biweekly dosing. A MTD of vanucizumab was not reached in either schedule. Pharmacokinetics were dose-linear with an elimination half-life of 6–9 days. All patients had reduced plasma levels of free VEGF-A and Ang-2; most had reductions in KTRANS (measured by dynamic contrast-enhanced MRI). Two patients (renal cell and colon cancer) treated with 30 mg/kg achieved confirmed partial responses. Ten patients were without disease progression for ≥6 months. A flat-fixed 2,000 mg biweekly dose (phamacokinetically equivalent to 30 mg/kg biweekly) was recommended for further investigation. Conclusions: Biweekly vanucizumab had an acceptable safety and tolerability profile consistent with single-agent use of selective inhibitors of the VEGF-A and Ang/Tie2 pathway. Vanucizumab modulated its angiogenic targets, impacted tumor vascularity, and demonstrated encouraging antitumor activity in this heterogeneous population. Clin Cancer Res; 24(7); 1536–45. ©2017 AACR.

Abstract 1803: Combining anti-Ang-2/VEGF-A therapy with radio- and chemotherapy in glioma

Angiogenesis is a biological hallmark of malignant gliomas, but antiangiogenic strategies especially targeting the VEGF axis did not show striking antitumor activities in the majority of patients so far. Other pathways may be more relevant in selected tumor entities or patients. Further, it remains unresolved which antiangiogenic combination regimen with standard radio- and/or chemotherapy is most effective. Therefore, we compared the therapeutic effects of anti-VEGF-A, anti-Ang-2, and bispecific anti-Ang-2/VEGF-A antibodies, alone and in combination with radio- or temozolomide (TMZ) chemotherapy in a malignant glioma model using multi-parameter two-photon in vivo microscopy in mice. We demonstrate that anti-Ang-2/VEGF-A leads to strongest vascular changes including vascular normalization, both as monotherapy and when combined with chemotherapy. The latter combination regimen was accompanied by most effective chemotherapy-induced death of cancer cells independent of blood vessel proximity, indicative of a better distribution of TMZ throughout the tumor. Furthermore, the combination of anti-Ang-2/VEGF-A plus TMZ consistently resulted in decreased tumor cell motility, and decreased formation of resistance-associated tumor microtubes (TMs), which finally lead to best tumor growth inhibition. Remarkably, all these parameters were just reverted when radiotherapy was chosen as combination partner. In contrast, when anti-VEGF-A was combined with radiotherapy, vascular normalization was highest, and TM length, nuclear motility and tumor growth were concordantly reduced. In conclusion, while TMZ chemotherapy benefits most from combination with anti-Ang-2/VEGF-A, radiotherapy does from anti-VEGF-A. The findings imply that unexpected, even divergent effects can occur when a specific antiangiogenic therapy is added to chemo- or radiotherapy in glioma, and that uninformed combination regimens should be avoided. Note: This abstract was not presented at the meeting. Citation Format: Gergely Solecki, Matthias Osswald, Weber Daniel, Malte Glock, Miriam Ratliff, Hans-Joachim Muller, Oliver Krieter, Yvonne Kienast, Wolfgang Wick, Frank Winkler. Combining anti-Ang-2/VEGF-A therapy with radio- and chemotherapy in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2017-1803

An apparent clinical pharmacokinetic drug–drug interaction between bevacizumab and the anti-placental growth factor monoclonal antibody RO5323441 via a target-trapping mechanism

PurposeRO5323441 is a humanized anti-placental growth factor (PlGF) monoclonal antibody that has shown preclinical activity in several cancer models. The objective of this analysis is to examine the pharmacokinetic (PK) results from four Phase I studies that have been conducted with RO5323441 (n = 61) and to report an apparent drug–drug interaction observed when RO5323441 was administered in combination with bevacizumab.MethodsThe four Phase I studies were a multiple-ascending dose study in 23 patients with solid tumors (Study 1), an open-label study in seven patients with colorectal/ovarian cancer (Study 2), a sorafenib combination study in nine patients with hepatocellular carcinoma (Study 3), and a bevacizumab combination study in 22 patients with recurrent glioblastoma (Study 4). A two-compartment linear population PK model was developed from these four studies to characterize the PK of RO5323441 in patients with cancer.ResultsThe PK properties of RO5323441 were similar in the first three studies. However, substantially higher RO5323441 exposures were observed in Study 4 when RO5323441 was administered in combination with bevacizumab. A linear two-compartmental population PK model indicated that the co-administration of bevacizumab would decrease the clearance of RO5323441 by 53%. Clinical data suggested that the decrease in RO5323441 clearance was inversely associated with bevacizumab exposure.ConclusionsThe exact reason for the increase in RO5323441 exposure following bevacizumab co-administration is not currently known. One possibility is a drug–drug interaction via a target-trapping mechanism that is mediated by the vascular endothelial growth factor receptor-1 (VEGFR-1).

Abstract C125: Preclinical pharmacodynamic biomarker and combination strategy of RG7386, a novel FAP-DR5 bispecific antibody for targeting solid tumors

Introduction RG7386 is a novel bispecific antibody, binding with high affinity to fibroblast activation protein (FAP) and with low affinity to death receptor 5 (DR5). Avidity-driven binding of the bispecific antibody mediates hyper-clustering of DR5 thus triggering tumor cell death. Induction of FAP dependent apoptosis translated into strong efficacy in vivo using patient derived xenografts thereby proposing RG7386 as an attractive therapeutic approach for the treatment of stroma rich FAP-positive solid tumors as well as FAP positive sarcomas. However, thus far DR5 targeting strategies failed to show clinical efficacy likely due to lack of hyperclustering and intrinsic resistance mechanisms. Preclinical translational studies were conducted to demonstrate the on-target mode of action, to ensure maximal activity and to guide pharmacodynamic (PD) analysis to unravel potential resistance mechanisms. Material and Methods Based on strong anti-tumor efficacy of RG7386 alone and in combination with irinotecan in a colorectal cancer (CRC) cell line based xenograft model (DLD-1) co-injected with fibroblasts a kinetic study was designed. Tumors were explanted 6, 16, 72 and 168 hours after RG7386 single agent treatment and harvested for immunohistochemical (IHC) and ELISA based protein analysis of apoptosis markers, such as cleaved caspase 3 (cc3), cleaved PARP and activated caspase 8 and 9. Additionally, PD effects of RG7386 treatment as single agent and in combination with doxorubicin were investigated in a FAP positive desmoplastic melanoma cell line derived model (LOX-IMVI). Results We observed significant time-dependent induction of apoptosis upon treatment with RG7386 by IHC and ELISA in xenograft tumors expressing FAP in stroma or on tumor cells directly. High, transient levels of apoptosis markers such as cc3 were observed by IHC early after treatment compared to vehicle control. Analysis of equivalent tissue lysates by ELISA revealed also rapid induction of cc3, cleaved PARP and activated caspase 8 and 9 in monotherapy in the DLD-1 CRC xenograft model which was superior when given together with doxorubicin in the LOX-IMVI desmoplastic melanoma model. Conclusion We identified that RG7386 strongly induces tumor cell apoptosis in vivo shortly after injection independently if FAP was expressed on tumor stroma or at tumor cells and discovered optimal pharmacodynamic markers and time points for sampling and analysis. As a result, early clinical trials of RG7386 will be designed to increase the therapeutic potential by choosing the right combination partner and to ensure demonstration of the postulated mode of action by pharmacodynamic data. Citation Format: Thomas Friess, Ann-Marie Broeske, Stefanie Lechner, Esther Abraham, Gabriele Hoelzlwimmer, Hadassa Sade, Peter Bruenker, Oliver Krieter. Preclinical pharmacodynamic biomarker and combination strategy of RG7386, a novel FAP-DR5 bispecific antibody for targeting 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 C125.

Abstract 952: Induction of avidity-driven hyperclustering of DR5 by a new FAP-DR5 bispecific antibody (RG7386) leads to strong anti-tumor efficacy

Background: Activation of the extrinsic apoptotic pathway by TRAIL is dependent on clustering of death receptors (DR) on the surface of cells. However, current TRAIL-based strategies have proven ineffective in clustering death receptors and failed to demonstrate robust therapeutic activity in clinical trials. More potent DR agonist therapies could help to overcome insufficient pathway activation and resistance to TRAIL activation. RG7386 is a novel bispecific FAP-DR5 antibody, binding with high affinity to fibroblast activation protein (FAP) and with low affinity to DR5. FAP is expressed at high prevalence on cancer associated fibroblasts (CAFs) in various tumor types as well as on tumors of mesenchymal origin, such as sarcomas. Avidity-driven binding of the bispecific antibody induces hyperclustering of DR5, which leads to potent induction of extrinsic apoptosis pathway signaling and tumor cell death. Biomarkers will be crucial in predicting sensitivity to DR5 activation and apoptosis induction and for selection of patients most likely to benefit from treatment with RG7386. Aim: The aim of the study was to explore the efficacy of RG7386 in vitro and in vivo. CRC and PDAC xenograft models expressing FAP on tumor stroma as well as sarcoma models were used to explore in vivo efficacy. Molecular profiling of sensitive and resistant tumors was also performed to identify response prediction markers. Results: RG7386 demonstrated additive efficacy in vitro with clinically relevant combinations (e.g. irinotecan, paclitaxel) in a variety of CRC and PDAC cell lines. In a xenograft model, where CRC cells (DLD-1) were co-injected with fibroblasts, RG7386 showed strong anti-tumor efficacy in combination with irinotecan. Remarkably, in a patient-derived CRC xenograft model (Co5896), the efficacy of RG7386 in combination with irinotecan induced complete tumor remission in all mice (n = 10/10). Furthermore, the combination of RG7386 with doxorubicin generated complete remissions in FAP+ sarcoma patient and desmoplastic melanoma cell line derived xenograft models such as Sarc4605 and LOX-IMVI. Finally, extensive molecular profiling of sensitive and resistance models in vitro revealed a distinct response prediction signature of DR5 sensitivity. Conclusion: RG7386 is a novel bispecific antibody inducing avidity-driven DR5 crosslinking by binding to FAP. This induces potent apoptosis of tumor cells, making it an attractive therapeutic approach for treatment of FAP+ solid tumors. Encouraging data indicate the high potential of RG7386 to treat FAP positive sarcomas. A comprehensive biomarker program will be employed in the early clinical development of RG7386 to enable selection of patients likely to benefit and to corroborate the mode of action, anti-tumor activity and potential response prediction markers. Citation Format: Thomas Friess, Stefanie Lechner, Esther Abraham, Ann-Marie Broeske, Sabine Bader, Andreas Roller, Meher Majety, Katharina Wartha, Suzana Vega-Harring, Hadassah Sade, Oliver Krieter, Peter Bruenker. Induction of avidity-driven hyperclustering of DR5 by a new FAP-DR5 bispecific antibody (RG7386) leads to strong anti-tumor efficacy. [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 952. doi:10.1158/1538-7445.AM2015-952