Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies

Abstract

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 BMP4 þ malignancies.

Comparison of newly developed anti-bone morphogenetic protein 4 llama-derived antibodies with commercially available BMP4 inhibitors

“…We recently generated 2 llama-derived BMP4-specific antibodies or VHHs, C4 and C8. 33 VHHs are single domain antibodies only composed by the variable domain of the heavy chain of a llama-derived antibody, which is around 15 kDa in size. Due to their unique structure and small size, VHHs can specifically bind with high affinity to their antigens.…”

“… 28 Surprisingly, the concentrations at which those antibodies were used in those and in other publications 29-31 ranged between 1-10 μg/ml, in contrast to the nanomolar range of which the VHH were shown to be biologically active. 33 To test whether these disparities are due to differences in experimental conditions or not, we compared the activity of C4C4 and C8C8 with current anti-BMP4 antibodies in the luciferase C2C12 system. These experiments confirmed that these antibodies are BMP4-specific, as only C8C8 inhibited BMP2-mediated signals ( Fig.…”

“… 32 We recently developed 2 selective anti-BMP4 VHH: C4C4 and C8C8. 33 C4C4 showed remarkable BMP4 specificity, whereas C8C8 could bind and inhibit both BMP2 and BMP4 signals. Both antibodies were shown to bind to the BMPR1-binding area of BMP4.…”

Autocrine BMP-4 Signaling Is a Therapeutic Target in Colorectal Cancer

“…Calpe and colleagues (37) showed that neutralizing antibodies for BMP-4 increase the chemosensitivity of HT29 cells. Dorsomorphin was identified as a potent BMP inhibitor, and its analogue, DMH-1, Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001, as determined by Student t test (A and C, bottom) or two-way ANOVA (C, top).…”

Single Domain Antibodies and Bioengineering Drugs on Their Basis: New Opportunities for Diagnostics and Therapy

“…Для терапии онкологических процессов также были разработаны высокоселективные наноан-титела, связывающие и блокирующие активность костных морфогенетических белков разных ти-пов, которые участвуют как в физиологических, так и патологических процессах, в том числе кан-церогенезе и опухолевой прогрессии [19].…”