Niina Veitonmäki

p130‐Angiomotin associates to actin and controls endothelial cell shape

Angiomotin, an 80 kDa protein expressed in endothelial cells, promotes cell migration and invasion, and stabilizes tube formation in vitro. Angiomotin belongs to a new protein family with two additional members, Amotl‐1 and Amotl‐2, which are characterized by conserved coiled‐coil domains and C‐terminal PDZ binding motifs. Here, we report the identification of a 130 kDa splice isoform of angiomotin that is expressed in different cell types including vascular endothelial cells, as well as cytotrophoblasts of the placenta. p130‐Angiomotin consists of a cytoplasmic N‐terminal extension that mediates its association with F‐actin. Transfection of p130‐angiomotin into endothelial cells induces actin fiber formation and changes cell shape. The p130‐angiomotin protein remained associated with actin after destabilization of actin fibers with cytochalasin B. In contrast to p80‐angiomotin, p130‐angiomotin does not promote cell migration and did not respond to angiostatin. We propose that p80‐ and p130‐angiomotin play coordinating roles in tube formation by affecting cell migration and cell shape, respectively.

Endothelial Cell Surface ATP Synthase-Triggered Caspase-Apoptotic Pathway Is Essential for K1-5-Induced Antiangiogenesis

We have recently reported the identification of kringle 1-5 (K1-5) of plasminogen as a potent and specific inhibitor of angiogenesis and tumor growth. Here, we show that K1-5 bound to endothelial cell surface ATP synthase and triggered caspase-mediated endothelial cell apoptosis. Induction of endothelial apoptosis involved sequential activation of caspases-8, -9, and -3. Administration of neutralizing antibodies directed against the α- and β-subunits of ATP synthase to endothelial cells attenuated activation of these caspases. Furthermore, inhibitors of caspases-3, -8, and -9 also remarkably blocked K1-5-induced endothelial cell apoptosis and antiangiogenic responses. In a mouse tumor model, we show that caspase-3 inhibitors abolished the antitumor activity of K1-5 by protecting the tumor vasculature undergoing apoptosis. These results suggest that the specificity of the antiendothelial effect of K1-5 is attributable, at least in part, to its interaction with the endothelial cell surface ATP synthase and that the caspase-mediated endothelial apoptosis is essential for the angiostatic activity of K1-5. Thus, our findings provide a mechanistic insight with respect to the angiostatic action and signaling pathway of K1-5 and angiostatin.

Differential roles of p80- and p130-angiomotin in the switch between migration and stabilization of endothelial cells

We have previously shown that angiomotin (Amot) plays an important role in growth factor-induced migration of endothelial cells in vitro. Genetic knock-down of Amot in zebrafish also results in inhibition of migration of intersegmental vessels in vivo. Amot is expressed as two different isoforms, p80-Amot and p130-Amot. Here we have analyzed the expression of the two Amot isoforms during retinal angiogenesis in vivo and demonstrate that p80-Amot is expressed during the migratory phase. In contrast, p130-Amot is expressed during the period of blood vessel stabilization and maturation. We also show that the N-terminal domain of p130-Amot serves as a targeting domain responsible for localization of p130-Amot to actin and tight junctions. We further show that the relative expression levels of p80-Amot and p130-Amot regulate a switch between a migratory and a non-migratory cell phenotype where the migratory function of p80-Amot is dominant over the stabilization and maturation function of p130-Amot. Our data indicates that homo-oligomerization of p80-Amot and hetero-oligomerization of both isoforms are critical for this regulation.

Therapeutic antibodies targeting angiomotin inhibit angiogenesis in vivo

We have previously shown that angiomotin (Amot) mediates angiostatin inhibition of endothelial migration and tube formation in vitro. A crucial role of angiomotin in regulating endothelial cell motility is indicated by the findings that knockdown of Amot in zebrafish reduces the number of filopodia of endothe‐lial tip cells and severely impairs the migration of intersegmental vessels. In addition, targeting angiomotin using DNA vaccination inhibits angiogenesis and tumor growth in vivo. In this report’ we have generated antibodies that’ similar to angiostatin’ bind to angiomo‐tin on the endothelial cell surface. These antibodies inhibited FGF‐2 and vascular endothelial growth factor (VEGF) ‐induced endothelial migration in the Boyden chamber assay. Furthermore, the anti‐Amot B06 antibody significantly reduced the number of endothelial filopodia and inhibited vessel migration during retinal angiogenesis in vivo. We also show that systemic or local treatment with this antibody inhibits pathological blood vessel formation associated with tumor growth or laser‐induced choroid neovascularization of the eye. These findings provide a rationale for using angiomotin antibodies for specifically targeting endothelial migration in angiogenesis‐dependent diseases.—Levchenko T., Veitonmaki N., Lundkvist, A., Gerhardt, H., Ming, Y., Berggren, K., Kvanta, A., Carlsson, R., Holmgren L. Therapeutic antibodies targeting angiomotin inhibit angiogenesis in vivo. FASEB J. 22, 880–889 (2008)

Bispecific antibodies in cancer immunotherapy

Following the clinical success of immune checkpoint antibodies targeting CTLA-4, PD-1 or PD-L1 in cancer treatment, bispecific antibodies are now emerging as a growing class of immunotherapies with potential to further improve clinical efficacy and safety. We describe three classes of immunotherapeutic bispecific antibodies: (a) cytotoxic effector cell redirectors; (b) tumor-targeted immunomodulators; and (c) dual immunomodulators. Cytotoxic effector cell redirectors are dominated by T-cell redirecting compounds, bispecific compounds engaging a tumor-associated antigen and the T-cell receptor/CD3 complex, thereby redirecting T-cell cytotoxicity to malignant cells. This is the most established class of bispecific immunotherapies, with two compounds having reached the market and numerous compounds in clinical development. Tumor-targeted immunomodulators are bispecific compounds binding to a tumor-associated antigen and an immunomodulating receptor, such as CD40 or 4-1BB. Such compounds are usually designed to be inactive until binding the tumor antigen, thereby localizing immune stimulation to the tumor environment, while minimizing immune activation elsewhere. This is expected to induce powerful activation of tumor-specific T cells with reduced risk of immune-related adverse events. Finally, dual immunomodulators are bispecific compounds that bind two distinct immunomodulating targets, often combining targeting of PD-1 or PD-L1 with that of LAG-3 or TIM-3. The rationale is to induce superior tumor immunity compared to monospecific antibodies to the same targets. In this review, we describe each of these classes of bispecific antibodies, and present examples of compounds in development.

Abstract 4569: Cell death-inducing ICAM-1 antibody has broad and potent anti-myeloma activity in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We previously developed combined target and drug discovery methodology enabling isolation of tumor cell death-inducing antibodies from the human antibody library n-CoDeR®. Among several antibodies targeting different tumor cell associated receptors with documented cell death-inducing properties, antibodies specific for intercellular adhesion molecule-1 (ICAM-1) – a receptor not previously associated with tumor cell death- were obtained. We here characterize the ICAM-1 specific antibody BI-505 with respect to in vivo anti-tumor activity in different well-established tumor disease models, and explore mechanisms underlying its anti-tumor activity. We demonstrate in different tumor xenograft models that BI-505 is equally or more efficacious in preventing CD20 positive B cell tumor growth and in pro-longing tumor-bearing mouse survival compared to CD20 mAb Rituximab in vivo. Moreover, BI-505 showed significant anti-tumor activity against a panel of CD20 negative myeloma tumors in vivo, and BI-505 conferred enhanced mouse survival compared to the proteasome inhibitor bortesomib – approved for treatment of relapsed refractory multiple myeloma – in an experimental model of advanced multiple myeloma. Preliminary data indicate that the BI-505 epitope is highly expressed on myeloma cells in patients with multiple myeloma. The differential in vitro and in vivo anti-tumor activities of BI-505 Fc-variants correlated with binding to recombinant human FcγRIIIa and mouse FcγRIV receptors, respectively. These differences could not be explained by altered antigen-binding properties as all Fc-variants retained near identical EC50 values for binding to recombinant or cell surface expressed ICAM-1 protein. These observations are consistent with cell death and FcγR-dependent anti-tumor immunity being important mechanisms underlying BI-505 therapeutic activity. Collectively, our results demonstrate proof-of-principle for our human antibody based combined target and drug discovery methodology, and provide a rationale for further pre-clinical and clinical evaluation of BI-505 in the treatment of MM. An open multicenter phase I dose-escalation clinical trial with BI-505 in relapsed/refractory MM has recently started in the United States and Sweden. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4569. doi:10.1158/1538-7445.AM2011-4569