Intrinsic differences in the mechanisms of Tie2 binding to angiopoietins exploited by directed evolution to create an Ang2-selective ligand trap.

Angiopoietins Ang1 and Ang2 are secreted ligands for the endothelial receptor tyrosine kinase Tie2 essential for vascular development and maintenance. Ang1 acts as an agonist to maintain normal vessel function, whereas Ang2 acts as a Tie2 antagonist. Ang2 is increased in macular edema, sepsis, and other conditions, in which it blocks Ang1-mediated signaling, causing vascular dysfunction and contributing to disease pathology. Therefore, Ang2 is an attractive therapeutic target. Previously, we reported a Tie2 ectodomain variant that selectively binds Ang2 and acts as soluble ligand trap to sequester Ang2; however, the mechanism of Ang2-binding selectivity is unknown. In the present study, we used directed protein evolution to enhance Ang2-binding affinity of this Tie2 ectodomain trap. We examined contributions of individual residues in the ligand-binding interface of Tie2 to Ang1 and Ang2 binding. Surprisingly, different combinations of Tie2 residues were found to bind each ligand, with hydrophobic residues binding both ligands and polar residues contributing selectively to either Ang1 or Ang2 binding. Our analysis also identified a single Tie2 residue, His168, with a pivotal role in both Ang1 and Ang2 binding, enabling competition between binding ligands. In summary, this study reports an enhanced-affinity Ang2-selective ligand trap with potential for therapeutic development and reveals the mechanism behind its selectivity. It also provides the first analysis of contributions of individual Tie2 residues to Ang1 and Ang2 binding and identifies selectivity-determining residues that could be targeted in the future design of small molecule and other inhibitors of Ang2 for the treatment of vascular dysfunction.

Structural basis for angiopoietin-1-mediated signaling initiation.

Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.

Angularly resolved ellipsometric optical biosensing by means of Bloch surface waves.

In label-free biosensing, a continuous improvement of the limit of detection is necessary to resolve the small change of the surface refractive index produced by interacting biomolecules at a very small concentration. In the present work, optical sensors based on one-dimensional photonic crystals supporting Bloch surface waves are proposed and adopted for label-free optical biosensing. We describe the implementation of an angularly resolved ellipsometric optical sensing scheme based on Bloch surface waves sustained by tantala/silica multilayers. The angular operation is obtained using a focused beam at fixed wavelength and detection of the angular reflectance spectrum by means of an array detector. The results show that the experimental limit of detection for a particular photonic crystal design is 6.5 × 10(-7) refractive index units (RIU)/Hz(1/2) and further decrease could be obtained. For the first time, we report on the practical application of this technique to a cancer biomarker protocol that aims at the detection of a specific glycoprotein (angiopoietin 2) involved in angiogenesis and inflammation processes.

Ligand oligomerization state controls Tie2 receptor trafficking and angiopoietin-2-specific responses.

Angiopoietin 1 (Ang1) is an activating ligand for the endothelial receptor tyrosine kinase Tie2, whereas Ang2 acts as a context-dependent agonist or antagonist that has a destabilizing effect on the vasculature. The molecular mechanisms responsible for the versatile functions of Ang2 are poorly understood. We show here that Ang2, but not Ang1, induces Tie2 translocation to the specific cell-matrix contact sites located at the distal end of focal adhesions. The Ang2-specific Tie2 translocation was associated with distinct Tie2 activation and downstream signals which differed from those of Ang1, and led to impaired cell motility and weak cell-matrix adhesion. We demonstrate that the different oligomeric or multimeric forms of the angiopoietins induce distinct patterns of Tie2 trafficking; the lower oligomerization state of native Ang2 was crucial for the Ang2-specific Tie2 redistribution, whereas multimeric structures of Ang1 and Ang2 induced similar responses. The Ang2-specific Tie2 trafficking to cell-matrix contacts was also dependent on the cell substratum, α2ß1-integrin-containing cell-matrix adhesion sites and intact microtubules. Our data indicate that the different subcellular trafficking of Tie2-Ang2 and Tie2-Ang1 complexes generates ligand-specific responses in the angiopoietin-Tie signaling pathway, including modulation of cell-matrix interactions.

Chemical generation of bispecific antibodies.

Bispecific antibodies (BsAbs) are regarded as promising therapeutic agents due to their ability to simultaneously bind two different antigens. Several bispecific modalities have been developed, but their utility is limited due to problems with stability and manufacturing complexity. Here we report a versatile technology, based on a scaffold antibody and pharmacophore peptide heterodimers, that enables rapid generation and chemical optimization of bispecific antibodies, which are termed bispecific CovX-Bodies. Two different peptides are joined together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. Whereas the pharmacophores are responsible for functional activities, the antibody scaffold imparts long half-life and Ig-like distribution. The pharmacophores can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. As a prototype, we developed a bispecific antibody that binds both vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) simultaneously, inhibits their function, shows efficacy in tumor xenograft studies, and greatly augments the antitumor effects of standard chemotherapy. This unique antiangiogenic bispecific antibody is in phase-1 clinical trials.

Inhibition of endothelial/smooth muscle cell contact loss by the investigational angiopoietin-2 antibody MEDI3617.

A tumor's dependence on angiogenesis for survival and growth has led to the advancement of a variety of blood vessel directed anticancer treatment strategies. Overexpression of angiopoietin-2 (Ang-2) in tumor vasculature and its crucial role in angiogenesis, i.e. the destabilization of endothelial/peri-endothelial cell interactions, now raises the possibility of additional novel anti-angiogenic therapeutics. The present study utilized a co-culture sphere model to (i) demonstrate the destabilizing effect of Ang-2 on endothelial/smooth muscle cell contact and (ii) evaluate the impact of the investigational Ang-2 antibody MEDI3617 on endothelial/smooth muscle cell dissociation. Real time imaging of spheres showed both exogenous Ang-2 and PMA induced endogenous Ang-2 secretion resulted in sphere destabilization (loss of endothelial cells from smooth muscle cell core). The presence of MEDI3617 inhibited this process. To assess the anti-angiogenic potential of MEDI3617 in vivo, nude mice were injected intradermally with human renal cell carcinoma cells (Caki-1, Caki-2) and the number of blood vessels induced over a 3 day period was scored. MEDI3617 (2, 10, 20 mg/kg) significantly reduced the initiation of blood vessels for both tumor models at all doses investigated. These data indicate that MEDI3617 treatment significantly impairs the initiation of angiogenesis by inhibiting the Ang-2 mediated disruption of endothelial/muscle cell interaction associated with blood vessel destabilization and thereby reduces tumor cell induced angiogenesis. The results support the notion that targeting the angiopoietin/Tie2 axis may offer novel anti-angiogenic strategies for cancer treatment.

Discovery of loperamide as an antagonist of angiopoietin1 and angiopoietin2 by virtual screening.

The angiopoietin-Tie2 binding and related signal transduction pathways are crucial for vascular angiogenesis, blood vessel integrity and maturation. In this study, we preformed a virtual screening of small molecules targeting to Tie2. The binding site was selected at the extracellular ligand binding region of Tie2, rather than its conventional endocellular ATP binding region. It was found that loperamide, a widely-used antidiarrhea drug, was among the top hits. The binding between loperamide and Tie2 was confirmed by surface plasmon resonance (SPR) assay. Loperamide competitively inhibited the binding of both angiopoietin1 and angiopoietin2. These results indicate that loperamide is an antagonist of angiopoietin1 and angiopoietin2.

Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex.

The Tie receptor tyrosine kinases and their angiopoietin (Ang) ligands play central roles in developmental and tumor-induced angiogenesis. Here we present the crystal structures of the Tie2 ligand-binding region alone and in complex with Ang2. In contrast to prediction, Tie2 contains not two but three immunoglobulin (Ig) domains, which fold together with the three epidermal growth factor domains into a compact, arrowhead-shaped structure. Ang2 binds at the tip of the arrowhead utilizing a lock-and-key mode of ligand recognition-unique for a receptor kinase-where two complementary surfaces interact with each other with no domain rearrangements and little conformational change in either molecule. Ang2-Tie2 recognition is similar to antibody-protein antigen recognition, including the location of the ligand-binding site within the Ig fold. Analysis of the structures and structure-based mutagenesis provide insight into the mechanism of receptor activation and support the hypothesis that all angiopoietins interact with Tie2 in a structurally similar manner.

Structural insights into the clustering and activation of Tie2 receptor mediated by Tie2 agonistic antibody.

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.

Non-genetic cell-surface modification with a self-assembling molecular glue.

This report describes the development of a non-genetic cell-surface modification method, in which a self-assembling small molecule is combined with Halo-tag proteins. Cell-surface functionalization with cancer-linked extracellular proteins led to enhanced cell motility, angiogenesis, and immune shielding of the cells, paving the way for translational opportunities for cell therapy.

A designed angiopoietin-2 variant, pentameric COMP-Ang2, strongly activates Tie2 receptor and stimulates angiogenesis.

Despite that angiopoietin-2 (Ang2) produces more versatile and dynamic functions than angiopoietin-1 (Ang1) in angiogenesis and inflammation, the molecular mechanism that underlies this difference is still unknown. To define the role of oligomerization of Ang2 in activation of its receptor, Tie2, we designed and generated different oligomeric forms of Ang2 by replacement of the amino-terminal domains of Ang2 with dimeric, tetrameric, and pentameric short coiled-coil domains derived from GCN4, matrillin-1, and COMP. COMP-Ang2 strongly binds and activates Tie2, whereas GCN4-Ang2 and MAT-Ang2 weakly to moderately bind and activate Tie2. Although native Ang2 strongly binds to Tie2, it does not activate Tie2. Accordingly, COMP-Ang2 strongly promotes endothelial cell survival, migration, and tube formation in a Tie2-dependent manner, and the potency of COMP-Ang2 is almost identical to that of COMP-Ang1. Furthermore, the potency of COMP-Ang2-induced enhanced angiogenesis in the wound healing region is almost identical to the potency of COMP-Ang1-induced enhanced angiogenesis. Overall, there is no obvious difference between COMP-Ang2 and COMP-Ang1 in in vitro and in vivo angiogenesis. Our results provide compelling evidence that proper oligomerization of Ang2 is a critical determinant of its binding and activation of Tie2.

Assessing the Quality of in Silico Produced Biomolecules: The Discovery of a New Conformer.

The computational procedures for predicting the 3D structure of aptamers interacting with different biological molecules have gained increasing attention in recent years. The information acquired through these methods represents a crucial input for research, especially when relevant crystallographic data are not available. A number of software programs able to perform macromolecular docking are currently accessible, leading to the prediction of the quaternary structure of complexes formed by two or more interacting biological macromolecules. Nevertheless, the scoring protocols employed for ranking the candidate structures do not always produce satisfactory results, making difficult the identification of structures that are most likely to occur in nature. In this paper, we propose a novel procedure to improve the predictive performances of computational scoring protocols, using a maximum likelihood estimate based on topological and electrical properties of interacting biomolecules. The reliability of the new computational approach, enabling the ranking of aptamer-protein configurations produced by an open source docking program, has been assessed by its successful application to a set of antiangiopoietin aptamers, for which experimental data highlighting the sequence-dependent affinity toward the target protein are available. The procedure led to the identification of two main types of aptamer conformers involved in angiopoietin binding. Interestingly, one of these reproduces the arrangement of angiopoietin with its natural target, tyrosine kinase, while the other one is completely unexpected. The possible scenarios related to these results have been discussed. The methodology here described can be used to refine the outcomes of different computational procedures and can be applied to a wide range of biological molecules, thus representing a new tool for guiding the design of bioinspired sensors with enhanced selectivity.

Beyond conventional dose-response curves: Sensorgram comparison in SPR allows single concentration activity and similarity assessment.

Previously we have introduced two SPR-based assay principles (dual-binding assay and bridging assay), which allow the determination of two out of three possible interaction parameters for bispecific molecules within one assay setup: two individual interactions to both targets, and/or one simultaneous/overall interaction, which potentially reflects the inter-dependency of both individual binding events. However, activity and similarity are determined by comparing report points over a concentration range, which also mirrors the way data is generated by conventional ELISA-based methods So far, binding kinetics have not been specifically considered in generic approaches for activity assessment. Here, we introduce an improved slope-ratio model which, together with a sensorgram comparison based similarity assessment, allows the development of a detailed, USP-conformal ligand binding assay using only a single sample concentration. We compare this novel analysis method to the usual concentration-range approach for both SPR-based assay principles and discuss its impact on data quality and increased sample throughput.

Validation of a human angiopoietin-2 ELISA for measurement of angiopoietin-2 concentrations in canine plasma samples and supernatant of primary canine aortic endothelial cell cultures.

OBJECTIVE To assess 2 human ELISA kits for measurement of angiopoietin-1 and -2 concentrations in canine plasma samples, determine whether plasma angiopoeitin-2 concentration differed between septic and healthy dogs, and determine the effect of tumor necrosis factor-α (TNF-α) stimulation on angiopoeitin-2 release from primary canine aortic endothelial cells (pCAECs) in vitro. ANIMALS 10 healthy dogs and 10 septic dogs. PROCEDURES Human angiopoietin-1 and -2 ELISAs were used to detect recombinant canine angiopoietins-1 and -2 in canine plasma samples. The angiopoietin-2 ELISA was further validated by use of plasma samples from healthy and septic dogs and supernatants of pCAEC cultures. Associations between plasma angiopoeitin-2 and C-reactive protein (CRP) concentrations were examined. RESULTS Angiopoeitin-2 but not angiopoeitin-1 was detected in canine plasma samples by the respective ELISAs. The angiopoeitin-2 ELISA had excellent dilutional linearity, parallelism, accuracy, precision, and reproducibility for measurements in canine plasma samples and pCAEC supernatants. Plasma angiopoeitin-2 concentration was significantly higher in septic dogs (median, 25.5 ng/mL) than in healthy dogs (median, 6.7 ng/mL) and was positively correlated with plasma CRP concentration (R2 = 0.60). Stimulation of pCAECs with TNF-α resulted in a significant increase in supernatant angiopoietin-2 concentration. CONCLUSIONS AND CLINICAL RELEVANCE The tested human angiopoietin-2 ELISA kit was useful for measuring angiopoietin-2 concentrations in canine plasma samples and pCAEC supernatants. Sepsis appeared to increase angiopoietin-2 concentration in dogs in vivo, whereas TNF-α stimulation caused release of angiopoietin-2 from pCAECs in vitro. These findings support the use of angiopoietin-2 as a marker of endothelial cell activation and inflammation in dogs.

SPR-based assays enable the full functional analysis of bispecific molecules.

The increasing complexity of novel biotherapeutics such as bispecific antibodies or fusion proteins raises new challenges for functional characterization. When compared to standard antibodies, two individual interactions and the inter-dependency of binding events need to be considered for bispecific antibodies. We have previously described an SPR-based assay setup, which enables us to assess the binding activity of a bivalent-bispecific molecule to both targets simultaneously and - in addition to one individual target - in a single setup. However, there might be some pitfalls when applying the bridging assay, e.g. change of antigen activity upon immobilization. Therefore, we have developed an alternative SPR-based assay principle, which allows the individual assessment of both targets in solution. Comparison of data between the assays showed that simultaneous binding can be calculated based on both individual readouts, and revealed a good correlation. Hence, both SPR-based assay principles allow a "full" functional analysis of a bispecific CrossMab in only one assay. The assay principles can be qualified and enable an efficient drug development.

Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy.

In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.

Biological characterization of angiopoietin-3 and angiopoietin-4.

The angiopoietin (Ang) family of growth factors includes Ang1, Ang2, Ang3, and Ang4, all of which bind to the endothelial receptor tyrosine kinase Tie2. Ang3 (mouse) and Ang4 (human) are interspecies orthologs. In experiments with human endothelial cell lines, Ang3 was identified as an antagonist of Tie2 and Ang4 was identified as an agonist of Tie2. However, the biological roles of Ang3 and Ang4 are unknown. We examined the biological effect of recombinant Ang3 and Ang4 proteins in primary cultured endothelial cells and in vivo in mice. Recombinant Ang3 and Ang4 formed disulfide-linked dimers. Ang4 (400 ng/mL) markedly increased Tie2 and Akt phosphorylation in primary cultured HUVECs whereas Ang3 (400 ng/mL) did not produce significant changes. Accordingly, Ang4, but not Ang3, induced survival and migration in primary cultured HUVECs. Unexpectedly, intravenously administered Ang3 (30 microg) was more potent than Ang4 (30 microg) in phosphorylating the Tie2 receptor in lung tissue from mice in vivo. Accordingly, Ang3 was more potent than Ang4 in phosphorylating Akt in primary cultured mouse lung microvascular endothelial cells. Ang3 and Ang4 both produced potent corneal angiogenesis extending from the limbus across the mouse cornea in vivo. Thus, Ang3 and Ang4 are agonists of Tie2, but mouse Ang3 has strong activity only on endothelial cells of its own species.

Computational selection of RNA aptamer against angiopoietin-2 and experimental evaluation.

Angiogenesis plays a decisive role in the growth and spread of cancer and angiopoietin-2 (Ang2) is in the spotlight of studies for its unique role in modulating angiogenesis. The aim of this study was to introduce a computational simulation approach to screen aptamers with high binding ability for Ang2. We carried out computational simulations of aptamer-protein interactions by using ZDOCK and ZRANK functions in Discovery Studio 3.5 starting from the available information of aptamers generated through the systematic evolution of ligands by exponential enrichment (SELEX) in the literature. From the best of three aptamers on the basis of ZRANK scores, 189 sequences with two-point mutations were created and simulated with Ang2. Then, we used a surface plasmon resonance (SPR) biosensor to test 3 mutant sequences of high ZRANK scores along with a high and a low affinity binding sequence as reported in the literature. We found a selected RNA aptamer has a higher binding affinity and SPR response than a reported sequence with the highest affinity. This is the first study of in silico selection of aptamers against Ang2 by using the ZRANK scoring function, which should help to increase the efficiency of selecting aptamers with high target-binding ability.

Accelerated healing of cutaneous wounds using phytochemically stabilized gold nanoparticle deposited hydrocolloid membranes.

Rapid healing of dermatological wounds is of vital importance in preventing infection and reducing post-treatment side-effects. Here we report the therapeutic effects of phytochemically stabilized gold nanoparticles (pAuNPs) coated on a hydrocolloid membrane (HCM) for curing cutaneous wounds. Furthermore, the remedial effects of pAuNPs on skin regeneration and angiogenesis were examined using Sprague Dawley® (SD) rats with skin injuries after a pAuNP-deposited hydrocolloid membrane (pAuNP-HCM) had been applied for 15 days. The rate of wound closure was 4 times faster in the pAuNP-HCM-treated group than in the gauze (GZ)- or HCM-treated groups in the first 5 days. Moreover, wound widths in the pAuNP-HCM-treated group were significantly reduced after 5-15 days of treatment following the injury, compared with the other groups. In addition, a significant increase in collagen expression and a decrease in matrix metalloproteinase (MMP)-1 expression and transforming growth factor (TGF-ß1) concentration were observed in the pAuNP-HCM-treated group on day 5. Wound tissue applied with the pAuNP-HCM showed enhancement of vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang-1), and angiopoietin 2 (Ang-2) expression. Furthermore, the activity of superoxide dismutases (SODs) was significantly increased in the skin tissue of the pAuNP-HCM-treated group, compared with the GZ- or HCM-treated groups. It is probable that the accelerated process of wound healing in the injured skin of SD rats via pAuNP-HCM results from the synergistic regulation of angiogenesis and connective tissue formation, as well as the stimulation of antioxidant effects.

Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides.

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvß3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.