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Introduction: Angiopoetin-2 (Ang-2) is a key mediator of tumour angiogenesis. When upregulated it is associated with tumour progression and poor prognosis. Anti-vascular endothelial growth factor (VEGF) therapy has been widely used in the treatment of metastatic colorectal cancer (mCRC). The potential benefit of combined inhibition of Ang-2 and VEGF-A in previously untreated patients with mCRC was evaluated in the phase II McCAVE study (NCT02141295), assessing vanucizumab versus bevacizumab (VEGF-A inhibitor), both in combination with mFOLFOX-6 (modified folinic acid [leucovorin], fluorouracil and oxaliplatin) chemotherapy. To date, there are no known predictors of outcome of antiangiogenic treatment in patients with mCRC. In this exploratory analysis, we investigate potential predictive biomarkers in baseline samples from McCAVE participants. Methods: Tumour tissue samples underwent immunohistochemistry staining for different biomarkers, including Ang-2. Biomarker densities were scored on the tissue images using dedicated machine learning algorithms. Ang-2 levels were additionally assessed in plasma. Patients were stratified by KRAS mutation status determined using next generation sequencing. Median progression-free survival (PFS) for each treatment group by biomarker and KRAS mutation was estimated using Kaplan-Meier plots. PFS hazard ratios (and 95% confidence intervals) were compared using Cox regression. Results: Overall low tissue baseline levels of Ang-2 were associated with longer PFS, especially in patients with wild-type KRAS status. In addition, our analysis identified a new subgroup of patients with KRAS wild-type mCRC and high levels of Ang-2 in whom vanucizumab/mFOLFOX-6 prolonged PFS significantly (logrank p=0.01) by ~5.5 months versus bevacizumab/mFOLFOX-6. Similar findings were seen in plasma samples. Discussion: This analysis demonstrates that additional Ang-2 inhibition provided by vanucizumab shows a greater effect than single VEGF-A inhibition in this subpopulation. These data suggest that Ang-2 may be both a prognostic biomarker in mCRC and a predictive biomarker for vanucizumab in KRAS wild-type mCRC. Thus, this evidence can potentially support the establishment of more tailored treatment approaches for patients with mCRC. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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.

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., Competing Interests: Declaration of Competing Interest The following authors are employees and shareholders of Roche Diagnostics GmbH: FH, GB, OK, SVH, KL; AJL is an employee of Soladis GmbH; CHO is an employee and shareholder of F. Hoffmann-La Roche AG; PG was an employee of Roche Innovation Center at the time of manuscript preparation, now retired; SR is an employee of F. Hoffmann-La Roche; AL and NW are employees of Roche Diagnostics GmbH; MH has received honoraria for acting as a speaker, consultant or advisory board member (Celgene, Pfizer, Novartis, MSD, EMD, Ipsem, Shire, SOBI, Champions Oncology, Agenus, Erytech, Pharmacyte, Bioline, BioOncotech, Oncomatrix, VCN, Bayer, BMS, Nelum, Eng T Cells), holds stock (Champions Oncology, Pharmacyte, BioOncotech, Nelum, Eng T Cell), and has received royalties (Myriad) and financial support paid to his institution for clinical trials or research grants (Berg, Bioline, Pfizer, EMD, Celgene, ASANA, Bycicle, Oncomatrix, BioExcell, Erytech); CM has acted as principal/sub-investigator of clinical trials (AbbVie, Aduro Biotech, Agios Pharmaceuticals, Amgen, Argen-X Bvba, Arno Therapeutics, Astex Pharmaceuticals, AstraZeneca, Aveo, Bayer Healthcare AG, Bbb Technologies Bv, Beigene, Bioalliance Pharma, Biontech AG, Blueprint Medicines, Boehringer Ingelheim, Bristol Myers Squibb, Ca, Celgene Corporation, Chugai Pharmaceutical Co., Clovis Oncology, Daiichi Sankyo, Debiopharm S.A., Eisai, Exelixis, Forma, Gamamabs, Genentech, Inc., Gilead Sciences, Inc, GlaxoSmithKline, Glenmark Pharmaceuticals, H3 Biomedicine, Inc, F. Hoffmann-La Roche AG, Incyte Corporation, Innate Pharma, Iris Servier, Janssen Cilag, Kura Oncology, Kyowa Kirin Pharm, Eli Lilly, Loxo Oncology, Lytix Biopharma AS, Medimmune, Menarini Ricerche, Merck Sharp & Dohme Chibret, Merrimack Pharmaceuticals, Merus, Millennium Pharmaceuticals, Nanobiotix, Nektar Therapeutics, Novartis Pharma, Octimet Oncology NV, Oncoethix, Oncomed, Oncopeptides, Onyx Therapeutics, Orion Pharma, Oryzon Genomics, Pfizer, Pharma Mar, Pierre Fabre, Rigontec GmbH, F. Hoffmann-La Roche, Sanofi Aventis, Sierra Oncology, Taiho Pharma, Tesaro, Inc., Tioma Therapeutics, Inc., Xencor), received research grants (AstraZeneca, BMS, Boehringer Ingelheim, Janssen Cilag, Merck, Novartis, Pfizer, F. Hoffmann-La Roche, Sanofi), received non-financial support (drug supplied) (AstraZeneca, Bayer, BMS, Boehringer Ingelheim, Johnson & Johnson, Eli Lilly, Medimmune, Merck, NH TherAGuiX, Pfizer, Roche) and received consultant/advisory fees (Amgen, Astellas, Astra Zeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Eli Lilly, MedImmune, Novartis, Pfizer, F. Hoffmann-La Roche, Sanofi, Orion); MMG has acted as consultant/advisory board member (F. Hoffmann-La Roche) and received travel expenses (F. Hoffmann-La Roche, Pfizer); ClT has participated in advisory boards (AstraZeneca, F. Hoffmann-La Roche, MSD, BMS, Merck Serono, GSK, Nanobiotix, Amgen); MK is a co-founder and shareholder of HistoGeneX N.V., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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.