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Highlights • Angiogenesis is critical to the development and survival of solid tumors. • Vanucizumab inhibits both vascular endothelial growth factor and angiopoietin-2. • Vanucizumab exhibits anti-tumor, anti-angiogenic and anti-metastatic effects. • It markedly reduced blood-vessel markers in tumor/skin samples in cancer patients. • Skin biopsies are a valuable surrogate for studying angiogenesis-related mechanisms., Vanucizumab is a novel bispecific antibody inhibiting vascular endothelial growth factor (VEGF-A) and angiopoietin-2 (Ang-2) that demonstrated safety and anti-tumor activity in part I of a phase I study of 42 patients with advanced solid tumors. Part II evaluated the pharmacodynamic effects of vanucizumab 30 or 15 mg/kg every 2 weeks in 32 patients. Serial plasma samples, paired tumor, and skin-wound-healing biopsies were taken over 29 days to evaluate angiogenic markers. Vanucizumab was associated with marked post-infusion reductions in circulating unbound VEGF-A and Ang-2. By day 29, tumor samples revealed mean reductions in density of microvessels (−32.2%), proliferating vessels (−47.9%) and Ang-2 positive vessels (−62.5%). Skin biopsies showed a mean reduction in density of microvessels (−49.0%) and proliferating vessels (−25.7%). Gene expression profiling of tumor samples implied recruitment and potential activation of lymphocytes. Biopsies were safely conducted. Vanucizumab demonstrated a consistent biological effect on vascular-related biomarkers, confirming proof of concept. Skin-wound-healing biopsies were a valuable surrogate for studying angiogenesis-related mechanisms.

Angiogenesis is the process of formation of new blood vessels from existing ones. Vessels serve the purpose of providing oxygen, nutrients and removal of waste from the cells. The physiological angiogenesis is a normal process and is required in the embryonic development, wound healing, menstrual cycle. For homeostasis, balance of pro angiogenic factors and anti angiogenic factors like is important. Their imbalance causes a process known as "angiogenic switch" which leads to various pathological conditions like inflammation, tumor and restenosis. Like normal cells, tumor cells also require oxygen and nutrients to grow which is provided by tumor angiogenesis. Hence angiogenic process can be inhibited to prevent tumor growth. This gives rise to study of anti angiogenic drugs. Currently approved anti angiogenic drugs are mostly VEGF inhibitors, but VEGF inhibitors have certain limitations like toxicity, low progression free survival (PFS), and resistance to anti VEGF therapy. This article focuses on angiopoietins as alternative and potential targets for anti angiogenic therapy. Angiopoietins are ligands of Tie receptor and play a crucial role in angiogenesis, their inhibition can prevent many tumor growths even on later stages of development. We present current clinical and preclinical stages of angiopoietin inhibitors. Drugs studied in the article are selective as well as non-selective inhibitors of angiopoietin 2 like Trebananib (AMG 386), AMG 780, REGN 910, CVX 060, MEDI 3617 and dual inhibitors of angiopoietin 2 and VEGF like Vanucizumab and RG7716. The angiopoietin inhibitors show promising results alone and in combination with VEGF inhibitors in various malignancies.

The purpose of this work is to assess the heterogeneity across organs of response to treatment in metastatic colorectal patient based on longitudinal individual target lesion diameters (ILD) in comparison to sum of tumor lesion diameters (SLD). Data were from the McCAVE trial, in which 189 previously untreated patients with metastatic colorectal carcinoma (mCRC) received either bevacizumab (control, C) or vanucizumab (experimental, E), on top of standard chemotherapy. Bayesian hierarchical longitudinal non-linear mixed effect models were fitted to the data using Hamilton Monte Carlo algorithm to characterize the time dynamics of the tumor burden, and to obtain estimates of the tumor shrinkage and regrowth rates. The ILD model brought more nuanced results than to the SLD model. Besides substantial differences in tumor size at baseline (with lesions located in liver more than twice as large as the ones in lungs), it revealed a more durable response in lesions located in lymph nodes and ‘other organs’ compared to liver and lungs. Specifically, in lymph nodes and ‘other organs’, the projected time to nadir was doubled in group E (2.12 and 2.44 years respectively) compared to group C (1.07 and 1.20 years respectively). This long period of tumor shrinkage associated with a slightly larger change from baseline at nadir (− 51.4% in lymph nodes and − 62.6% in ‘other organs’ in the group E, compared to − 46.2% and − 46.9% in group C) resulted in a clinically meaningful difference in the tumor dynamics of patients in group E compared to the group C. The proportion of variance explained by the inter-lesion variability for each model parameter was large (ranging between 10 and 56%), reflecting the heterogeneity in tumor dynamics across organs. These findings suggest that there is value in understanding both within- and between-patient variability in tumor size’s time dynamics using an appropriate modeling framework, as this information may help in pairing the right treatment with individual patient profile.

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.

Vascular endothelial growth factor (VEGF) inhibition by the addition of bevacizumab to the chemotherapy regimen of metastatic colorectal cancer leads to an improved outcome. However, anti-angiogenic tumor therapy targeting a single factor may be limited by complementary mechanisms. Angiopoietin-2 (Ang-2, ANGPT2) is another important factor that cooperates with VEGF to drive tumor angiogenesis. It was shown that high Ang-2 levels are associated with a poor clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy. Therefore, combined inhibition of VEGF and Ang-2 was supposed to improve anti-angiogenic therapy. Here, we evaluated the efficacy of a bispecific antibody (CrossMab) co-targeting VEGF and Ang-2 in combination with chemotherapy in a chemoresistant colorectal carcinoma model. Antitumor activity was evaluated in athymic nude mice bearing subcutaneous DLD1 xenograft tumors and treated with anti-VEGF (B20), anti-Ang-2 (LC06) and anti-VEGF/Ang-2 (CrossMab) antibodies. Chemotherapy consisted of 5-FU and irinotecan. Resected tumors were analyzed immunohistochemically. First, an impact of targeting each single factor but also a clear advantage of co-targeting both factors could be demonstrated. Accordingly, tumor tissue showed strong staining for VEGF and Ang-2. Chemotherapy alone was less effective. Efficient tumor growth inhibition could be achieved by treatment with anti-VEGF/chemotherapy, single CrossMab and CrossMab/chemotherapy, which resulted in 3 out of 10, 6 out of 10 and 10 out of 10 complete responses, respectively, during seven weeks. Complete retarded tumors were characterized by massive intratumoral necrosis surrounded by layers of vital tumor cells and connective tissue with CD31-positive vessels at the periphery. In some cases, a distinct feature known as vessel co-option could be observed. In conclusion, the data from this model clearly support the strategy of co-targeting VEGF and Ang-2 and further demonstrate the beneficial impact of co-treatment with chemotherapy. The clear superiority of the CrossMab-containing regimen compared to clinical standard anti-VEGF/chemotherapy warrants further analyses in other models.

Background Bevacizumab, a VEGF‐A inhibitor, in combination with chemotherapy, has proven to increase progression‐free survival (PFS) and overall survival in multiple lines of therapy of metastatic colorectal cancer (mCRC). The angiogenic factor angiopoetin‐2 (Ang‐2) is associated with poor prognosis in many cancers, including mCRC. Preclinical models demonstrate improved activity when inhibiting both VEGF‐A and Ang‐2, suggesting that the dual VEGF‐A and Ang‐2 blocker vanucizumab (RO5520985 or RG‐7221) may improve clinical outcomes. This phase II trial evaluated the efficacy of vanucizumab plus modified (m)FOLFOX‐6 (folinic acid (leucovorin), fluorouracil (5‐FU) and oxaliplatin) versus bevacizumab/mFOLFOX‐6 for first‐line mCRC. Patients and Methods All patients received mFOLFOX‐6 and were randomized 1:1 to also receive vanucizumab 2,000 mg or bevacizumab 5 mg/kg every other week. Oxaliplatin was given for eight cycles; other agents were continued until disease progression or unacceptable toxicity for a maximum of 24 months. The primary endpoint was investigator‐assessed PFS. Results One hundred eighty‐nine patients were randomized (vanucizumab, n = 94; bevacizumab, n = 95). The number of PFS events was comparable (vanucizumab, n = 39; bevacizumab, n = 43). The hazard ratio was 1.00 (95% confidence interval, 0.64–1.58; p = .98) in a stratified analysis based on number of metastatic sites and region. Objective response rate was 52.1% and 57.9% in the vanucizumab and bevacizumab arm, respectively. Baseline plasma Ang‐2 levels were prognostic in both arms but not predictive for treatment effects on PFS of vanucizumab. The incidence of adverse events of grade ≥3 was similar between treatment arms (83.9% vs. 82.1%); gastrointestinal perforations (10.8% vs. 8.4%) exceeded previously reported rates in this setting. Hypertension and peripheral edema were more frequent in the vanucizumab arm. Conclusion Vanucizumab/mFOLFOX‐6 did not improve PFS and was associated with increased rates of antiangiogenic toxicity compared with bevacizumab/mFOLFOX‐6. Our results suggest that Ang‐2 is not a relevant therapeutic target in first‐line mCRC. Implications for Practice This randomized phase II study demonstrates that additional angiopoietin‐2 (Ang‐2) inhibition does not result in superior benefit over anti–VEGF‐A blockade alone when each added to standard chemotherapy. Moreover, the performed pharmacokinetic and pharmacodynamic analysis revealed that vanucizumab was bioavailable and affected its intended target, thereby strongly suggesting that Ang‐2 is not a relevant therapeutic target in the clinical setting of treatment‐naïve metastatic colorectal cancer. As a result, the further clinical development of the dual VEGF‐A and Ang‐2 inhibitor vanucizumab was discontinued., This phase II trial evaluated the efficacy of vanucizumab plus mFOLFOX‐6 versus bevacizumab/mFOLFOX‐6 in the first‐line setting of metastatic colorectal cancer.

Experimental evidence suggests that antiangiogenic therapy gives rise to a transient window of vessel normalization, within which the efficacy of radiotherapy and chemotherapy may be enhanced. Preclinical experiments that measure components of vessel normalization are invasive and expensive. We have developed a mathematical model of vascular tumor growth from preclinical time-course data in a breast cancer xenograft model. We used a mixed-effects approach for model parameterization, leveraging tumor size data to identify a period of enhanced tumor growth that could potentially correspond to the transient window of vessel normalization. We estimated the characteristics of the window for mice treated with an anti-VEGF antibody (bevacizumab) or with a bispecific anti-VEGF/anti-angiopoietin-2 antibody (vanucizumab). We show how the mathematical model could theoretically be used to predict how to coordinate antiangiogenic therapy with radiotherapy or chemotherapy to maximize therapeutic effect, reducing the need for preclinical experiments that directly measure vessel normalization parameters.

The major challenge in the generation of bispecific IgG antibodies is enforcement of the correct heavy and light chain association. The correct association of generic light chains can be enabled using immunoglobulin domain crossover, known as CrossMAb technology, which can be combined with approaches enabling correct heavy chain association such as knobs-into-holes (KiH) technology or electrostatic steering. Since its development, this technology has proven to be very versatile, allowing the generation of various bispecific antibody formats, not only heterodimeric/asymmetric bivalent 1+1 CrossMAbs, but also tri- (2+1), tetravalent (2+2) bispecific and multispecific antibodies. Numerous CrossMAbs have been evaluated in preclinical studies, and, so far, 4 different tailor-made bispecific antibodies based on the CrossMAb technology have entered clinical studies. Here, we review the properties and activities of bispecific CrossMAbs and give an overview of the variety of CrossMAb-enabled antibody formats that differ from heterodimeric 1+1 bispecific IgG antibodies.

Background: To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A). Methods: Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg−1 dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 106 mm2 s−1), vascular perfusion/permeability (Ktrans, min−1) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed. Results: Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P

VAN is a bispecific IgG-like antibody targeting both VEGF-A and Ang-2, two key factors in tumor angiogenesis pathway that was investigated as monotherapy in a single arm study in platinum-resistant ovarian cancer (PROC) patients. A modeling approach working with tumor size data was applied to predict and compare overall survival (OS) of VAN + chemotherapy (CT) against BEV + CT. For VAN, individual tumor size data from 41 patients enrolled in study NCT01688206 were available. Primary endpoint of the study was objective response rate; survival data were not collected. For CT and BEV +CT, historical tumor size data were obtained from AURELIA, a randomized phase 3 study designed to compare progression free survival (PFS) in patients treated with CT alone or in combination with BEV. In AURELIA, patients were followed for OS after treatment discontinuation. In both studies, sum of longest diameters (SLD) were measured on CT/MRI scans collected every 6 to 8 weeks, as per RECIST 1.1. Non-linear tumor kinetics (TK) models accounting for the dynamics of tumor growth, drug effect and resistance to the drug effect were used to fit SLD following each treatment (VAN, CT and BEV+CT). Several tumor metrics summarizing the individual TK were derived: early tumor shrinkage at week 6 (ETS6), tumor size at baseline (TS0), maximum shrinkage (MaxSh) and time to tumor growth (TTG). As no VAN+CT data were available, individual tumor responses were simulated assuming an additive drug effect from CT on VAN. The median TTG was prolonged by 4 weeks and median MaxSh was increased by 7% for V+CT as compared to BEV+CT. To correlate tumor metrics to OS, a time-to-event model was fitted to the OS data from the AURELIA study. Covariates including ECOG, FIGO score at baseline, histological grade and subtype, presence of ascites, CA-125 at baseline, TS0, ETS6 and TTG were tested as prognostic factors in the survival model. In the survival model, two sets of covariates were found significant: those related to disease severity (ECOG, FIGO, presence of ascites) and those describing the key features of TK (ETS6, TTG, TS0). Treatment group was not retained in the final model making the model drug independent. Assuming the same survival model and a similar mechanism for VAN and BEV, the BEV+CT TK metrics were replaced by the VAN+CT TK metrics in the survival model. Based on this assumption, the median OS with VAN+CT was predicted to be 2 months longer compared to BEV+CT. This approach shows the benefit of using TK modeling, when drugs show similar mechanism of action, in early phase to predict potential outcome of drug combination, not yet tested in patients. Leveraging historical data for the development of survival model integrating TK metrics can be used to inform the expected OS outcome in phase 3 and can provide a quantitative tool to evaluate the chance of drug success. Citation Format: Alexandre Sostelly, Kevin Smart, Felix Jaminion, Christophe Boetsch, Francois Mercier. Leveraging tumor size and time to death from bevacizumab (BEV) historical data to predict overall survival in ovarian cancer patients treated with vanucizumab (VAN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1643.

3539 Background: VEGF-A and ANG-2 have complementary roles in regulation of tumor angiogenesis. Targeting VEGF-A with BEV in combination chemotherapy (CT) in mCRC has proven to increase PFS and OS. ANG-2 is overexpressed and associated with poor outcome of mCRC pts receiving BEVcontaining treatment. Hence, dual blockade of VEGF-A and ANG-2 by the bispecific mAb VAN with standard CT may improve clinical activity in mCRC. Methods: All pts received mFOLFOX-6 and were randomized 1:1 to also receive intravenous VAN 2000 mg every other week (Q2W) (Arm A) or BEV 5 mg/kg Q2W (Arm B). The primary end point was investigator assessed progression-free survival (PFS). Key eligibility criteria included pts with non-resectable mCRC, no prior therapy for advanced disease, PS 0-1, adequate organ functions, and no history of GI fistula/perforation or intraabdominal abscess within the last 6 months. Results: 192 pts were randomized (Arms A/B, n = 95/97) by 39 sites in 7 countries, between Oct 2014 and May 2016. Median follow-up was 17.6 months (range 2.8 – 20.7). In the ITT population (n = 189; Arms A/B, n = 94/95), median PFS in Arms A and B was 11.3 and 11.0 months (stratified hazard ratio (HR) 1.00 (95%CI 0.64-1.58; p = 0.985)), respectively. Objective response rate was 52.1% vs 57.9%. Relevant prognostic factors incl. RAS/BRAF status and tumor sidedness were balanced between arms and did not significantly influence outcome. Baseline plasma ANG-2 levels were prognostic in both arms but not predictive for response to VAN. The overall incidence of adverse events (AEs) grade ≥ 3 was similar (Arms A/B, 83.9%/82.1%); AEs grade ≥ 3 attributed to the mode of action of VAN/BEV included hypertension (37.6%/18.9%), hemorrhage (2.2%/1.1%), thromboembolic events (venous 6.5%/2.1%; arterial 1.1%/3.2%) and GI perforations incl. GI fistula & abdominal abscess (10.6%/8.4%). Conclusions: The combination of VAN and FOLFOX did not improve PFS and was associated with a marked increase in hypertension compared with BEV plus FOLFOX. Our results strongly suggest that ANG-2 is not a relevant therapeutic target in the setting of first line mCRC. Clinical trial information: NCT02141295.

e15108 Background: VAN is a novel bispecific mAb targeting VEGF & ANG-2. A randomized phase 2 study (McCAVE) investigating VAN & BEV, each combined with chemotherapy (mFOLFOX-6), in 1L metastatic colorectal cancer patients was completed. Reported gastrointestinal perforation (GIP) events (including GI fistula & abdominal abscess) in both arms (approx. 10%) exceeded those expected with BEV+CTx. The present work focused on identifying the explicit molecular pathways mediating VAN/BEV associated GIP. Methods: Clinical data from McCave (safety outcomes & gene expression profiling) was integrated with data from other clinical/preclinical VAN/BEV studies. Machine learning was applied to the dataset to computationally infer GIP mechanisms. The methodology was constrained by relevant biological & clinical knowledge. Literature reviews & database queries mapped 194 unique proteins to 8 biological processes previously hypothesized to play a role in GIP. Identified proteins guided construction of a GIP protein interaction network which was transformed into an artificial neural network and was fitted to the dataset to predict molecular pathways/biological processes most likely linking VEGF-A and/or ANG-2 inhibition to GIP. Results: VAN/BEV were predicted to enhance GIP risk via 4 out of 8 previously suggested biological effects: (i) reduced GI mucosal integrity; (ii) impaired wound healing; (iii) inhibition of angiogenesis (iv) increased thromboembolic risk. The specific proteins & associated interactions predicted to mediate these 4 effects were identified as solely VEGF-A inhibition driven. These effects may drive increased susceptibility to GIP triggered by secondary insults, e.g. preclinical ulcer & intramural lesion. Candidate GIP biomarkers for monitoring these effects were identified, e.g., aPTT, citrulline & I-FABP. Conclusions: The molecular pathways mediating GIP in VAN/BEV treated patients are likely driven by VEGF inhibition with no contribution from ANG-2 inhibition. It is proposed, that in addition to VAN/BEV, one or more secondary insults to the GI tract wall are required to drive perforation.

e17042Background: Vanucizumab is a novel bi-specific IgG-like antibody which is directed against both VEGF-A and ANG2, two key factors in tumor angiogenesis. As such, vanucizumab represents an enco...

5549 Background: Vanucizumab is a bi-specific human IgG1 antibody, simultaneously blocking the complementary roles of Ang-2 and VEGF-A induced tumor angiogenesis. Both, VEGF-A and Ang-2 inhibitors ...