Generation and Selection of Novel Fully Human Monoclonal Antibodies That Neutralize Dickkopf-1 (DKK1) Inhibitory Function in Vitro and Increase Bone Mass in Vivo

Abstract

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.The Wnt signaling pathway plays a key role in embryonic differentiation, adult tissue maintenance, stem cell biology, oncogenesis, and the etiology of degenerative diseases and has been directly linked to regulation of bone metabolism (1-5). Loss of function mutations in the Wnt coreceptor LRP5 results in osteoporosis-pseudoglioma syndrome in humans and mice characterized by low bone mass with reduced bone formation (6). Consistently, allelic LRP5 variants have been associated with altered bone mineral densities (BMD) 3 and fracture risk in human populations (7). Importantly, discrete LRP5 gain-of-function mutations lead to a high bone mass (HBM) phenotype in humans and mice (8 -11). Interestingly, HBM LRP5 mutations have been shown to be refractory to the inhibitory effects of Dickkopf-1 (DKK1) on canonical Wnt-signaling (9, 12), thus, strongly suggesting that the impairment of DKK1 signaling plays a role in the etiology of the HBM phenotype (9, 12). Recently this mechanism has been expanded to include other inhibitors of Wnt signaling such as sclerostin (13). Analyses of genetically DKK1 insufficient mice provides compelling evidence for an inhibitory role of DKK1 on new bone formation and a titratable effect on bone mass accrual resulting in a HBM phenotype that is maintained throughout adulthood (14, 15). DKK1 is a high affinity ligand for LRP...

Kremen1 and Dickkopf1 control cell survival in a Wnt-independent manner

“…It is noteworthy that multiple studies provided proof of concept that Dkk1 targeting by immunotherapy can be used efficiently in both protective and therapeutic manners against cancer. [42][43][44][45][46][47] Sato et al 46 found that anti-Dkk1 treatment was able to induce apoptosis of A549 lung cancer cells (which express high levels of Dkk1) by an unknown mechanism, which we believe could be the one we describe here. Furthermore, tumors formed in nude mice following A549 cells injection were significantly smaller in animals treated with anti-Dkk1 compared with controls, demonstrating that Dkk1 targeting by antibodies can efficiently inhibit tumor growth in vivo.…”

Nano-formulated curcumin accelerates acute wound healing through Dkk-1-mediated fibroblast mobilization and MCP-1-mediated anti-inflammation

“…25 Interestingly, however, Koch et al 26 found that wounding of epithelial monolayers returned the leading edge cells to a nonquiescent state with activated β-catenin signal but subsequent induction and secretion of Dkk-1. Given that the Wnt signaling cascade with its antagonist Dkk-1 has been proposed to have crucial roles for maintaining homeostasis of a variety of tissues including the skin, 27 and that the physiologic Dkk-1 level is of vital importance to maintaining its biological functions, 28 we speculate that curcumin could precisely control the Dkk-1 level in a temporospatial manner to regulate the downstream Wnt signaling pathway, as Dkk-1 has been shown to participate in a negative feedback loop in Wnt signaling as well. 29 However, more studies are required to fully elucidate its less well-understood role in wound healing.…”

Genetics in Endocrinology: Autosomal dominant osteopetrosis revisited: lessons from recent studies

“…Recent studies indicated that these antibodies induce bone formation, reduce bone resorption, and thereby lead to an increase in bone volume in rodent and monkey models (171,172,177). Thus, anti-DKK may have a potential as treatment of osteoporosis.…”

“…As the Wnt ligands, as well as the downstream signaling molecules GSK3b and b-catenin, are rather ubiquitous and have been implicated in cancer progression, these are less attractive targets despite their obvious anabolic potential (169,170). On the other hand, the identification of the soluble inhibitors of Wnt signaling, such as sclerostin, DKK1-4, WIF, and sFRPs, identified a series of interesting targets for antibody and small-molecule inhibitor therapy (169), and especially sclerostin that appears rather bone specific and DKK1 have been explored extensively (171,172,173,174).…”