Martyn K. Robinson

Mechanism of action of certolizumab pegol (CDP870): In vitro comparison with other anti-tumor necrosis factor α agents

Background: Inhibitors of tumor necrosis factor &agr; (TNF&agr;) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohns disease (CD). To further elucidate the mechanisms of action of these agents, we compared the anti‐TNF&agr; agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems. Methods: The ability of each anti‐TNF&agr; agent to neutralize soluble and membrane‐bound TNF&agr;; mediate cytotoxicity, affect apoptosis of activated human peripheral blood lymphocytes and monocytes; induce degranulation of human peripheral blood granulocytes, and modulate lipopolysaccharide (LPS)‐induced interleukin (IL)‐1&bgr; production by human monocytes was measured in vitro. Results: All 4 agents neutralized soluble TNF&agr; and bound to and neutralized membrane TNF&agr;. Infliximab and adalimumab were comparable in their ability to mediate complement‐dependent cytotoxicity and antibody‐dependent cell‐mediated cytotoxicity, and to increase the proportion of cells undergoing apoptosis and the level of granulocyte degranulation. Etanercept generally mediated these effects to a lesser degree, while certolizumab pegol gave similar results to the control reagents. LPS‐induced IL‐1&bgr; production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept. Conclusions: In contrast to the other anti‐TNF&agr; agents tested, certolizumab pegol did not mediate increased levels of apoptosis in any of the in vitro assays used, suggesting that these mechanisms are not essential for the efficacy of anti‐TNF&agr; agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS‐induced IL‐1&bgr; release from monocytes, inhibition of cytokine production may be important for efficacy of anti‐TNF&agr; agents in CD.

Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength

The development of bone‐rebuilding anabolic agents for treating bone‐related conditions has been a long‐standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin‐neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin‐neutralizing monoclonal antibody (Scl‐AbIV) to gonad‐intact female cynomolgus monkeys. Two once‐monthly subcutaneous injections of Scl‐AbIV were administered at three dose levels (3, 10, and 30u2009mg/kg), with study termination at 2 months. Scl‐AbIV treatment had clear anabolic effects, with marked dose‐dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl‐AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle‐treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest‐dose group. Taken together, the marked bone‐building effects achieved in this short‐term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis.

Characterization of the Structural Features and Interactions of Sclerostin MOLECULAR INSIGHT INTO A KEY REGULATOR OF Wnt-MEDIATED BONE FORMATION

The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N- and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.

Sclerostin: A gem from the genome leads to bone-building antibodies

Discovery of Sclerostin databases using either homology-based programs (eg, BLAST) or a special CxGxC-class cystine-knot search pattern Genomics technologies and DNA sequencing have had a transformative impact on biological research, giving us unprecedented access to the genomes of numerous organisms and ushering in such new fields as systems biology and, more recently, synthetic biology. In the 1990s, the advent of highthroughput sequencing spurred application of this powerful new technology to the challenging endeavor of trying to understand the genetic basis of various inherited human diseases. One such disease was sclerosteosis, a very rare, recessively inherited highbone-mass disorder that was thought to be caused primarily by excessive osteoblast-mediated bone formation rather than by defective osteoclast-mediated bone resorption. Within the realm of inherited high-bone-mass disorders, it was the hope for a discovery in the area of osteoblast biology that made sclerosteosis particularly interesting. Indeed, the identification of new bone-building pathways that might yield novel anabolic agents had been a long-standing goal in bone biology, with hopes for therapeutic application in fracture healing, orthopedic procedures, and low-bone-mass conditions such as osteoporosis. Against this backdrop came the exciting news, independently from Mary Brunkow’s group at Celltech R&D, Inc., and from Wim Van Hul’s group at the University of Antwerp, that sclerosteosis was caused by mutations in a single gene (SOST) encoding a novel secreted protein. Because these were inactivating mutations, it was clear that this protein, aptly given the name sclerostin, functioned either directly or indirectly as an inhibitor of bone formation. During this same general time period, large-scale cDNA/ expressed sequence tag (EST) and genomic DNA sequencing efforts were beingmade in academia and industry to rapidly identify new human genes. A novel secreted protein of unknown function, which turned out to be sclerostin, was discovered by computational mining of large DNA sequence

A Short Treatment With an Antibody to Sclerostin Can Inhibit Bone Loss in an Ongoing Model of Colitis

Chronic inflammation leads to bone loss, and increased fracture rates have been reported in a number of human chronic inflammatory conditions. The study reported here investigates the skeletal effects of dosing a neutralizing antibody to the bone regulatory protein sclerostin in a mouse model of chronic colitis. When dosed prophylactically, an antibody to sclerostin (Scl‐AbI) did not reduce the weight loss or histological changes associated with colitis but did prevent inflammation‐induced bone loss. At the end of the experiment, Scl‐AbI–treated animals had a significantly higher femoral BMD (+27%, p < 0.05) than control antibody (Cntrl‐Ab)‐treated animals. In a second experiment, treatment with Scl‐AbI was delayed until colitis had developed, by which time the mechanical properties of femurs in colitic animals were significantly worse than those of healthy age‐matched control mice (maximum load, −26%, p < 0.05; energy, −37%, p < 0.05; ultimate strength, −33%, p < 0.05; elastic modulus, −17%, p < 0.05). A short treatment with Scl‐AbI halted bone loss and reversed the decline of both intrinsic and extrinsic mechanical properties of the femur such that, after 19 days of treatment, the bone mechanical properties in the Scl‐AbI–treated animals were not significantly different from those of noncolitic age‐matched controls. Serum markers of bone formation and resorption suggested that the antibody to sclerostin stimulated osteoblast activity and inhibited osteoclast‐mediated bone resorption.

A tumor-associated β1 integrin mutation that abrogates epithelial differentiation control

SCC4 human keratinocytes are derived from a squamous cell carcinoma of the tongue and undergo very little spontaneous differentiation. Introduction of a wild-type β1 integrin subunit into SCC4 cells stimulates differentiation, suggesting either that the cells have a defect in the integrin signaling pathways that control differentiation or that the β1 subunit itself is defective. Here we describe a heterozygous mutation in the SCC4 β1 subunit. The mutation, T188I, maps to the I-like domain. It results in constitutive activation of ligand binding, irrespective of the partner α subunit, in solid phase assays with recombinant protein and in living cells. The mutation promotes cell spreading, but not proliferation, motility, or invasiveness. It results in sustained activation of Erk MAPK independent of cell spreading. When introduced into SCC4 keratinocytes, the wild-type β1 integrin stimulates differentiation, whereas the mutant is inactive. Activation of β1 integrins in normal keratinocytes also suppresses differentiation. These results establish, for the first time, mutation as a mechanism by which integrins can contribute to neoplasia, because the degree of differentiation in epithelial cancers is inversely correlated with prognosis. They also provide new insights into how integrins regulate keratinocyte differentiation.

Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors

Background: Sclerostin, an inhibitor of Wnt signaling, binds to the β-propeller domain-containing Wnt co-receptors LRP6 and LRP4. Results: An NXI motif in sclerostin mediates interactions with LRP6 (but not LRP4) and blocks Wnt1 signaling. Conclusion: The sclerostin/LRP6 interaction shares features with the well characterized nidogen/laminin interaction. Significance: NXI motifs are important in mediating interactions with β-propeller containing proteins. LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the β-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.

CDP7657, an anti-CD40L antibody lacking an Fc domain, inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study

IntroductionCD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG1 antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8.MethodsThe potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE.ResultsCDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria.ConclusionsThese findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.

Discovery and evaluation of N-(triazin-1,3,5-yl) phenylalanine derivatives as VLA-4 integrin antagonists

SAR studies aimed at improving the rate of clearance of a series of VLA-4 integrin antagonists by the introduction of a 1,3,5-triazine as an amide isostere are described.

Squaric acid derivatives as VLA-4 integrin antagonists.

SAR studies aimed at improving the rate of clearance by the incorporation of a 3,4-diamino-3-cyclobutene-1,2-dione group as an amino acid isostere in a series of VLA-4 integrin antagonists are described.