The protective effect of anti-VEGF-A/Ang-2 bispecific antibody on retinal vein occlusion model mice.

(233/250) Retinal vein occlusion (RVO) causes macular edema and retinal ischemia resulting in visual field and vision loss. A bispecific antibody that blocks VEGF-A and angiopoietin-2 (Ang-2) has been recently launched and applied clinically to treat macular edema, but the role of Ang-2 in the pathogenesis of RVO is still unclear. In this study, we investigated the effects of the anti-VEGF-A/anti-Ang-2 bispecific antibody (BsAb) in a murine RVO model. By using RVO model mice, the expression of Ang-2 gene and protein was examined in the retina through real-time qPCR and Western blotting, respectively. A significant increase in Ang-2 was detected 1 day after occlusion. Immediately after occlusion, control IgG 400 µg/mL, anti-VEGF-A antibody 200 µg/mL, anti-Ang-2 antibody 200 µg/mL, and BsAb 400 µg/mL were intravitreally administered at 2 µL. Visual function was examined using electroretinograms, and apoptosis was examined using TUNEL staining. Interestingly, BsAb partially suppressed the decrease in amplitude of a and b waves compared to control IgG. Anti-Ang-2 antibody and BsAb reduced apoptosis-positive cells 1 day after occlusion. Comprehensive gene expression profiles were also examined using RNA sequencing analysis. RNA sequencing analysis of the retinal tissues showed that BsAb suppressed expression of gene groups associated with inflammatory response and vascular development compared to anti-VEGF-A antibody. Taken together, higher expression of Ang-2 contributes to the pathophysiology of RVO, providing a possible mechanism for the efficacy of BsAb in suppressing retinal dysfunction in RVO.

Angiogenic factors and the lectin pathway of complement in women with secondary recurrent pregnancy loss.

The poor remodeling of placental spiral arteries seen in preeclampsia is also discussed to contribute to recurrent pregnancy loss (RPL) preceded by abnormal angiogenesis and excessive complement activation. Low levels of Mannose-binding-lectin (MBL), a pattern recognition molecule (PRM) of the lectin pathway, have been found in women with RPL. We propose that pregnancy loss is connected to defective angiogenesis with reperfusion damage in the placenta and decreased levels of PRM in the lectin pathway in women with RPL. In this cohort study, we investigate the angiogenic factors and the lectin complement pathway in early pregnancy and their time-dependent relationship with pregnancy outcomes in 76 women with secondary RPL (sRPL) who have at least four prior pregnancy losses and a live birth. We evaluated levels of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Vascular Endothelial Growth Factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PRMs, MBL, ficolin-1, -2, -3 and an additional soluble PRM, Pentraxin-3, during the 5th, 6th, and 7th gestational weeks. Our results showed that, compared to live births, pregnancies that ended in loss were associated with elevated VEGF levels and decreased levels of the Ang-2/Ang-1 ratio. Also, increasing levels of ficolin-2 were significantly associated with pregnancy loss, with MBL showing no association. Our research suggests that women with sRPL may have inadequate placentation with impaired angiogenesis in pregnancies ending in a loss.

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.

Delayed angiopoietin-2 blockade reduces influenza-induced lung injury and improves survival in mice.

Influenza remains a major cause of death and disability with limited treatment options. Studies of acute lung injury have identified angiopoietin-2 (Ang-2) as a key prognostic marker and a potential mediator of Acute respiratory distress syndrome. However, the role of Ang-2 in viral pneumonia remains poorly defined. This study characterized the time course of lung Ang-2 expression in severe influenza pneumonia and tested the therapeutic potential of Ang-2 inhibition. We inoculated adult mice with influenza A (PR8 strain) and measured angiopoietin-1 (Ang-1), Ang-2, and Tie2 expressions during the evolution of inflammatory lung injury over the first 7 days post-infection (dpi). We tested a peptide-antibody inhibitor of Ang-2, L1-7, administered at 2, 4, and 6 dpi and measured arterial oxygen saturation, survival, pulmonary edema, inflammatory cytokines, and viral load. Finally, we infected primary human alveolar type II epithelial (AT2) cells grown in air-liquid interface culture with influenza and measured Ang-2 RNA expression. Influenza caused severe lung injury between 5 and 7 dpi in association with increased Ang-2 lung RNA and a dramatic increase in Ang-2 protein in bronchoalveolar lavage. Inhibition of Ang-2 improved oxygenation and survival and reduced pulmonary edema and alveolar-capillary barrier permeability to protein without major effects on inflammation or viral load. Finally, influenza increased the expression of Ang-2 RNA in human AT2 cells. The increased Ang-2 levels in the airspaces during severe influenza pneumonia and the improvement in clinically relevant outcomes after Ang-2 antagonism suggest that the Ang-1/Ang-2 Tie-2 signaling axis is a promising therapeutic target in influenza and potentially other causes of viral pneumonia.

ARID1A knockdown in human endothelial cells directly induces angiogenesis by regulating angiopoietin-2 secretion and endothelial cell activity.

AT-rich interactive domain 1A (ARID1A) is a novel tumor suppressor gene found in several human cells and its loss/defect is commonly observed in many cancers. However, its roles in angiogenesis, which is one of the hallmarks for tumor progression, remained unclear. Herein, we demonstrated the direct effects of ARID1A knockdown in human endothelial cells by lentivirus-based short-hairpin RNA (shRNA) (shARID1A) on angiogenesis. Functional assays revealed that shARID1A significantly enhanced cell proliferation and migration/invasion and endothelial tube formation compared with the control cells transfected with scramble shRNA (shControl). Additionally, the shARID1A-transfected cells had significantly increased podosome formation and secretion of angiopoietin-2 (ANG2), a key angiogenic factor. Moreover, neutralization of ANG2 with monoclonal anti-ANG2 antibody strongly reduced cell proliferation and migration/invasion and endothelial tube formation in the shARID1A-transfected cells. These findings indicate that down-regulation of ARID1A in human endothelial cells directly induces angiogenesis by regulating angiopoietin-2 secretion and endothelial cell activity.

Targeting Angiopoietin in Retinal Vascular Diseases: A Literature Review and Summary of Clinical Trials Involving Faricimab.

This review summarizes the latest findings in the literature of Angiopoietin-2 (Ang-2), Tyrosine-protein kinase receptor (Tie-2) complex, and faricimab along with their involvement for the treatment of retinal vascular diseases in various clinical trials. In ischemic diseases, such as diabetic retinopathy, Ang-2 is upregulated, deactivating Tie-2, resulting in vascular leakage, pericyte loss, and inflammation. Recombinant Angiopeotin-1 (Ang-1), Ang-2-blocking molecules, and inhibitors of vascular endothelial protein tyrosine phosphatase (VE-PTP) decrease inflammation-associated vascular leakage, showing therapeutic effects in diabetes, atherosclerosis, and ocular neovascular diseases. In addition, novel studies show that angiopoietin-like proteins may play an important role in cellular metabolism leading to retinal vascular diseases. Current therapeutic focus combines Ang-Tie targeted drugs with other anti-angiogenic or immune therapies. Clinical studies have identified faricimab, a novel bispecific antibody designed for intravitreal use, to simultaneously bind and neutralize Ang-2 and VEGF-A for treatment of diabetic eye disease. By targeting both Ang-2 and vascular endothelial growth factor-A (VEGF-A), faricimab displays an improved and sustained efficacy over longer treatment intervals, delivering superior vision outcomes for patients with diabetic macular edema and reducing the treatment burden for patients with neovascular age-related macular degeneration and diabetic macular edema. Phase 2 results have produced promising outcomes with regard to efficacy and durability. Faricimab is currently being evaluated in global Phase 3 studies.

Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS.

Angiopoietin-2 (Ang2), a ligand of the endothelial Tie2 tyrosine kinase, is involved in vascular inflammation and leakage in critically ill patients. However, the role of Ang2 in demyelinating central nervous system (CNS) autoimmune diseases is unknown. Here, we report that Ang2 is critically involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis. Ang2 expression was induced in CNS autoimmunity, and transgenic mice overexpressing Ang2 specifically in endothelial cells (ECs) developed a significantly more severe EAE. In contrast, treatment with Ang2-blocking Abs ameliorated neuroinflammation and decreased spinal cord demyelination and leukocyte infiltration into the CNS. Similarly, Ang2-binding and Tie2-activating Ab attenuated the development of CNS autoimmune disease. Ang2 blockade inhibited expression of EC adhesion molecules, improved blood-brain barrier integrity, and decreased expression of genes involved in antigen presentation and proinflammatory responses of microglia and macrophages, which was accompanied by inhibition of α5ß1 integrin activation in microglia. Taken together, our data suggest that Ang2 provides a target for increasing Tie2 activation in ECs and inhibiting proinflammatory polarization of CNS myeloid cells via α5ß1 integrin in neuroinflammation. Thus, Ang2 targeting may serve as a therapeutic option for the treatment of CNS autoimmune disease.

Antiangiogenic immunotherapy suppresses desmoplastic and chemoresistant intestinal tumors in mice.

Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.

Hereditary angioedema attack: what happens to vasoactive mediators?

Hereditary angioedema is a disabling, life-threatening condition caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) leading to bradykinin accumulation and recurrent episodes of edema attack. Vascular leakage is a complex process sustained by the coordinated production of several permeabilizing factors including vascular endothelial growth factors (VEGFs), angiopoietins (ANGPTs) and phospholipase A2 enzymes (PLA2). We previously reported that patients with C1-INH-HAE in remission have increased plasma levels of VEGFs, ANGPTs and secreted PLA2. In this study, we sought to analyze plasma levels of these mediators in 15 patients with C1-INH-HAE during the acute attack compared to remission. Plasma concentrations of VEGF-A, VEGF-C and VEGF-D were not altered during attack compared to remission. Moreover, VEGF-D concentrations were not altered also in remission phase compared to controls. Concentrations of ANGPT1, a vascular stabilizer, were increased during attacks compared to symptoms-free periods, whereas ANGPT2 levels were not altered. The ANGPT2/ANGPT1 ratio was decreased during angioedema attacks. Platelet activating factor acetylhydrolase activity was increased in patients with C1-INH-HAE in remission compared to controls and was decreased during angioedema attacks. Our results emphasize the complexity by which several vasoactive mediators are involved not only in the pathophysiology of C1-INH-HAE, but also during angioedema attacks and its resolution.

Immunoregulation and Clinical Implications of ANGPT2/TIE2+ M-MDSC Signature in Non-Small Cell Lung Cancer.

Myeloid-derived suppressor cells (MDSC) promote immunosuppression and are a target in the field of immuno-oncology. Accumulation of MDSCs is associated with poor prognosis and resistance to immunotherapy for several cancers. Here, we describe an accumulation of a subset of circulating monocytic MDSCs (M-MDSC) overexpressing TIE2, the receptor for angiopoietin-2 (ANGPT2), in patients with non-small cell lung cancer (NSCLC). Greater numbers of circulating TIE2+ M-MDSCs were detected in patients with NSCLC compared with healthy subjects, and this accumulation correlated with ANGPT2 concentration in blood. The presence of an ANGPT2-rich environment was associated with impairment of preexisting T-cell responses against tumor-associated antigens (TAA) in patients with NSCLC. We demonstrated that ANGPT2 sensitizes TIE2+ M-MDSCs such that these cells suppress TAA-specific T cells. In patients with NSCLC, upregulation of the ANGPT2/TIE2+ M-MDSC signature in blood was associated with a poor prognosis. Our results identify the ANGPT2/TIE2+ M-MDSC axis as a participant in tumor immune evasion that should be taken into account in future cancer immunotherapy.

Recent developments in the research on biomarkers of cholangiocarcinoma in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by a chronic inflammatory process of the bile ducts of unclear aetiology. It is often complicated by cholangiocarcinoma (CCA) with a dismal prognosis. Early detection of CCA is important because treatment options for advanced disease are limited. Besides the established markers, like CA19-9, recent developments have been made using latest technologies. This review summarizes the recent advances and remaining limitations of biomarkers of CCA in PSC.

Improving immunotherapy outcomes with anti-angiogenic treatments and vice versa.

Immunotherapies have revolutionized medical oncology following the remarkable and, in some cases, unprecedented outcomes observed in certain groups of patients with cancer. Combination with other therapeutic modalities, including anti-angiogenic agents, is one of the many strategies currently under investigation to improve the response rates and duration of immunotherapies. Such a strategy might seem counterintuitive given that anti-angiogenic agents can increase tumour hypoxia and reduce the number of blood vessels within tumours. Herein, we review the additional effects mediated by drugs targeting VEGF-dependent signalling and other pathways, such as those mediated by angiopoietin 2 or HGF, which might increase the efficacy of immunotherapies. In addition, we discuss the seldom considered possibility that immunotherapies, and immune-checkpoint inhibitors in particular, might increase the efficacy of anti-angiogenic or other types of antivascular therapies and/or promote changes in the tumour vasculature. In short, we propose that interactions between both therapeutic modalities could be considered a 'two-way street'.

Reprogramming Tumor Blood Vessels for Enhancing Immunotherapy.

Angiogenic blood vessels contribute to generating an immunosuppressive tumor microenvironment, in part by limiting the extravasation of T cells. Functional reprogramming of angiogenic blood vessels, for example through angiopoietin-2 blockade, may improve T cell trafficking in tumors and the efficacy of immune checkpoint blockade (ICB) and other cancer immunotherapies.

Angiopoietin-2 Blockade Promotes Survival of Corneal Transplants.

Purpose: Corneal transplantation remains the last hope for vision restoration, and lymphangiogenesis (LG) is a primary mediator of transplant rejection. This study was to investigate the specific role of angiopoietin-2 (Ang-2) in transplantation-associated LG and graft rejection. Methods: Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice to assess the effects of Ang-2 blockade via neutralizing antibody. Grafts were evaluated in vivo by ophthalmic slit-lamp biomicroscopy and anterior segment optical coherence tomography (OCT) up to 8 weeks after surgery. Additionally, whole-mount corneas were analyzed for lymphatic and blood vessels and macrophages by immunofluorescent microscopy, and draining lymph nodes were assessed for donor-derived cells by flow cytometry. Results: Anti-Ang-2 treatment significantly suppressed LG and graft rejection. In this study, we achieved 75% suppression of LG and 80% graft survival. Our approach also inhibited donor-derived cell trafficking to draining lymph nodes and affected macrophage morphologic phenotypes in the grafted corneas. Additionally, Ang-2 blockade also reduced central corneal thickening, a parameter strongly associated with graft rejection. Conclusions: Ang-2 is critically involved in corneal transplant rejection and anti-Ang-2 treatment significantly improves the outcomes of corneal grafts. Moreover, we have shown that anterior segment OCT offers a new tool to monitor murine corneal grafts in vivo. This study not only reveals new mechanisms for transplant rejection, but also offers a novel strategy to treat it.

Acute tumour response to a bispecific Ang-2-VEGF-A antibody: insights from multiparametric MRI and gene expression profiling.

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), × 10(6) mm(2) s(-1)), vascular perfusion/permeability (K(trans), 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<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour K(trans) (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis. CONCLUSIONS: Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.

A bispecific antibody (ScBsAbAgn-2/TSPO) target for Ang-2 and TSPO resulted in therapeutic effects against glioblastomas.

Antibody-based targeted therapy of cancers requires the antibody targeting of specific molecules inducing tumor cells apoptosis or death. Angiopoietin-2 (Agn-2) and translocator protein (TSPO) are identified as potential target molecules for glioblastoma therapy. The single chain anti-Agn-2 antibody (Anag-2) and anti-TSPO antibody (ATSPO) were obtained by monoclonal antibody screening. In the present study, for specific targeting and killing, we generated a recombinant bispecific antibody comprising a single-chain Fragment variable (ScFv) of anti-human Agn-2 and anti-human TSPO (ScBsAbAgn-2/TSPO), which is the mediator for mitochondrial apoptosis and tumor angiogenesis. In vitro, ScBsAbAgn-2/TSPO simultaneously bounded to both targets with a high antigen-binding affinity to Anag-2 and TSPO compared to the individual antibody. The higher expression of Ang-2 and TSPO was observed in bevacizumab-treated glioblastoma compared to normal rat brain endothelium. We also observed apoptosis-mediated cytotoxicity was improved, which resulted in the elimination of up to 90% of the target cells within 72 h. ScBsAbAgn-2/TSPO inhibited tumor growth, decreased vascular permeability, led to extended survival, improved pericyte coverage, depletion of tumor-associated macrophages, and increased numbers of intratumoral T lymphocytes infiltration in a murine bevacizumab-treated glioblastoma model. These findings were also confirmed ex vivo using glioblastoma cells from bevacizumab-treated rats with glioblastoma. We conclude that ScBsAbAgn-2/TSPO targeting of glioblastoma cell lines can be achieved in vitro and in vivo that the efficient elimination of glioblastoma cells supports the potential of ScBsAbAgn-2/TSPO as a potent, novel immunotherapeutic agent.

Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-ß in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-ß promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.

Effect of Ang-2-VEGF-A Bispecific Antibody in Renal Cell Carcinoma.

The blockade of VEGF pathway has been clinically validated as an initial treatment for renal cell carcinoma (RCC). Angiopoietin-2 (Ang-2) has been indicated as a key regulator for angiogenesis escape. The effect of a novel bispecific antibody (A2V CrossMab) against both Ang-2 and VEGF was investigated in comparison with either factor. A2V CrossMab significantly reduced tumor volume, vessel density, and interstitial fluid pressure compared to either monotherapy of anti-VEGF or anti-Ang-2. Host-derived angiogenesis-related genes have been significantly down-regulated in A2V CrossMab group. These data demonstrate that A2V CrossMab has additive anti-tumor effect for the treatment of RCC.

Angiopoietin-2 blocking antibodies reduce early atherosclerotic plaque development in mice.

OBJECTIVE: Angiopoietin-2 (Ang-2) blocking agents are currently undergoing clinical trials for use in cancer treatment. Ang-2 has also been associated with rupture-prone atherosclerotic plaques in humans, suggesting a role for Ang-2 in plaque stability. Despite the availability of Ang-2 blocking agents, their clinical use is still lacking. Our aim was to establish if Ang-2 has a role in atheroma development and in the transition of subclinical to clinically relevant atherosclerosis. We investigated the effect of antibody-mediated Ang-2 blockage on atherogenesis after in a mouse model of atherosclerosis. METHODS: Hypercholesterolemic (low-density lipoprotein receptor(-/-) apolipoprotein B(100/100)) mice were subjected to high-cholesterol diet for eight weeks, one group with and one group without Ang-2 blocking antibody treatment during weeks 4-8.To enhance plaque development, a peri-adventitial collar was placed around the carotid arteries at the start of antibody treatment. Aortic root, carotid arteries and brachiocephalic arteries were analyzed to evaluate the effect of Ang-2 blockage on atherosclerotic plaque size and stable plaque characteristics. RESULTS: Anti-Ang-2 treatment reduced the size of fatty streaks in the brachiocephalic artery (-72%, p < 0.05). In addition, antibody-mediated Ang-2 blockage reduced plasma triglycerides (-27%, p < 0.05). In contrast, Ang-2 blockage did not have any effect on the size or composition (collagen content, macrophage percentage, adventitial microvessel density) of pre-existing plaques in the aortic root or collar-induced plaques in the carotid artery. CONCLUSIONS: Ang-2 blockage was beneficial as it decreased fatty streak formation and plasma triglyceride levels, but had no adverse effect on pre-existing atherosclerosis in hypercholesterolemic mice.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.