Effective Inhibition of Bone Morphogenetic ProteinFunctionbyHighlySpeci fi cLlama-Derived Antibodies

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4þ malignancies. Mol Cancer Ther; 14(11); 2527–40. 2015 AACR. Introduction Bone morphogenetic proteins (BMP) are growth factors that belong to the TGFb superfamily. They consist of around 20 members, classified into distinct subfamilies, depending on their sequence homology and functionality. BMP2, BMP4 and BMP5, BMP6, BMP7 form two subgroups that have been extensively studied (1). They mediate their function by binding to two molecules of type 1 (BMPR1a, BMPR1b, or ACVR1) and two molecules of type 2 (BMPR2, ACVRL1, ActRII, or ActRIIB) serine/threonine kinase receptors (2). Depending on the presence or absence of preformed BMPR complexes, the signaling is mediated via the SMAD family of transcription factors following a defined canonical pathway or by less defined noncanonical intracellular effectors (3, 4). Translocation to the nucleus of these transcription factors initiates transcription of BMP target genes (5, 6). BMPs are emerging as attractive therapeutic targets due to their multiple roles in a variety of diseases, in particular cancer. For many years, the BMP field has been dogged by the assumption that many BMPs are functionally interchangeable. However, there is increasing evidence indicating the disparate actions of BMPs (7). For instance, whereas BMP4 and BMP2 seem to facilitate metastasis and invasion in several cancers, BMP6 and BMP7 present a suppressive role in metastatic breast cancer and melanomas (8). Most intriguing is the role of BMP4, which presents opposing roles depending on the neoplasm (7, 9). In certain malignancies, such as glioblastoma (10) and myeloma (11), high levels of BMP4 are associated with less malignant features while in breast (12), ovarian (13), lung (14), and prostate (15), BMP4 is involved in epithelial–mesenchymal transition (EMT) and metastatic behavior. In gastrointestinal cancers such as pancreas (16), colon (17), and gastric cancer (18), BMP4 has been associated with tumor progression and chemotherapy resistance. Therefore, to resolve and inhibit the individual action of BMP4 in tumors, the generation of potent and specific BMP4 inhibitors is warranted. Targeting and regulating BMP signaling at the extracellular receptor level is clinically highly attractive (19). Neutralization of BMP by several natural antagonists and chemical inhibitors has already been proven successful for the suppression of metastasis in prostate (15), lung (14), as well as breast cancer (20). Albeit significant progress has been made in generating highly specific and less toxic small-molecule inhibitors that Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, the Netherlands. AIMM Therapeutics, Academic Medical Center, Amsterdam, the Netherlands. QVQ BV, Utrecht, the Netherlands. Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. DepartmentofGastroenterologyandHepatology,AcademicMedical Center, Amsterdam, the Netherlands. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Kausilia K. Krishnadath, Academic Medical Center, Meibergdreef 9, Amsterdam 1105AZ, the Netherlands. Phone: 31-0205666703; Fax: 31-0-206917033; E-mail: k.k. krishnadath@amc.uva. nl doi: 10.1158/1535-7163.MCT-14-0956 2015 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals. org 2527 on June 25, 2017. © 2015 American Association for Cancer Research. mct.aacrjournals. org Downloaded from Published OnlineFirst September 8, 2015; DOI: 10.1158/1535-7163.MCT-14-0956