Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1999-09-15
2003-11-11
Kemmerer, Elizabeth (Department: 1647)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S198100, C424S192100, C424S201100, C530S350000, C514S002600
Reexamination Certificate
active
06645500
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to improvements in therapy and prevention of osteoporosis and other diseases characterized by continued loss of bone tissue. More specifically, the present invention provides a method for down-regulating osteoprotege-rin ligand (OPGL) by enabling the production of antibodies against OPGL in subjects suffering from or in danger of suffering from osteoporosis. The invention also provides for methods of producing modified OPGL useful in this method as well as for the modified OPGL as such. Also encompassed by the present invention are nucleic acid fragments encoding modified OPGL as well as vectors incorporating these nucleic acid fragments and host cells and cell lines transformed therewith. The invention also provides for a method for the identification of OPGL analogues which are useful in the method of the invention as well as for compositions comprising modified OPGL or comprising nucleic acids encoding the OPGL analogues.
FIELD OF THE INVENTION
Osteoporosis is a major and growing health problem worldwide. It affects an estimated 75 million people in the United States of America, Europe and Japan combined. Thus, it is the most common systemic bone disorder in the industrialised part of the world.
Osteoporosis affects one in four postmenopausal women and a majority of the elderly, including a substantial number of men. The cost of osteoporosis in the United States of America with 15 million affected people was estimated to be 3.8 billion USD annually in 1984. This translates by extrapolation to a worldwide cost of something in the order of at least 20 billion USD.
Osteoporosis is a systemic skeletal disease characterised by low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fractures. Although all bones are affected, fractures of the spine, wrist and hip are typical and the most common. The risk of developing osteoporosis increases with age and is higher in women than in men. Its etiology appears to reside in the mechanisms underlying an accentuation of the normal loss of bone mass, which follows the menopause in women and occurs in all individuals with advancing age.
Peak bone mass is achieved at about 35 years of age. After reaching its peak, bone mass declines throughout life due to an imbalance in remodelling. Bones lose both mineral and organic matrix but retain their basic organisation.
Bone consists of a mineralised extracellular matrix composed of a variety of proteins and proteoglycans; the principal component being type I collagen. The mineral encrusting the extracellular matrix is hydroxyapatite (Ca
3
(PO
4
)
2
·Ca(OH)
2
). Bone is continuously modelled during growth and development and remodelled throughout life in response to physical and chemical signals.
The growth, development and maintenance of bone are highly regulated processes, which at the cellular level involves the co-ordinate regulation of bone-forming cells (osteoblasts) and bone-resorbing cells (osteoclasts). The level of bone mass reflects the balance of bone formation and resorption.
Osteoblasts arise from mesenchymal stem cells and produce bone matrix during development, after bone injury, and during the normal bone remodelling that occurs throughout life. Osteoclasts differentiate from hematopoietic precursors of the monocyte-macrophage lineage and resorb bone matrix.
An imbalance of osteoblast and osteoclast functions can result in the skeletal abnormalities characterised by increased bone mass (osteopetrosis) or by decreased bone mass (osteoporosis).
Studies of osteopetrosis in mutant mice have shown that genetic defects in osteoclast development, maturation, and/or activation lead to decreased bone resorption and uniformly result in severe osteopetrosis (Marks, 1989). Nevertheless, relatively little has so far been known about the soluble factors that act physiologically to regulate osteoclast development.
Recently, however, two proteins that take part in this regulation have been described and characterized (Simonet et al., 1997; Lacey et al., 1998). These two proteins are osteoprotegerin and osteoprotegerin ligand.
Osteoprotegerin is a novel secreted member of the tumour necrosis factor receptor family. In vitro, osteoprotegerin blocks osteoclastogenesis in a dose dependent manner. Transgenic mice expressing osteoprotegerin exhibit a generalized increase in bone density (osteopetrosis) associated with a decrease in osteoclasts. Administration of recombinant osteoprotegerin produces similar effects in normal mice and protects against ovariectomy-associated bone loss in rats (Simonet et al., 1997). In addition, osteoprotegerin-deficient mice (knock out mice) while normal at birth develop early onset osteoporosis and arterial calcification (Bucay et al., 1998). These observations strongly point to the possibility that osteoprotegerin blocks the differentiation of osteoclasts, the principal if not sole bone-resorbing cell type, suggesting that it can act as a humoral regulator of bone resorption. Osteoprotegerin is the subject matter of WO 97/23614. It was hypothesized that osteoprotegerin may exert its effect by binding to and neutralising a factor that stimulates osteoclast development, thus inhibiting osteoclast maturation (Simonet et al., 1997).
Osteoprotegerin ligand (OPGL) is a novel member of the tumour necrosis factor family of cytokines that exists in both a membrane-bound and a soluble form. OPGL binds to osteoprotegerin with a binding affinity of 4 nM. In vitro, OPGL activates mature osteoclasts and modulates osteoclast formation from bone marrow precursors in the presence of CSF-1. It has also been demonstrated that OPGL binds to the surface of osteoclast progenitors in CSF-1-treated bone marrow. The receptor for OPGL on these hematopoeitic progenitor cells is, however, unknown. Recombinant soluble OPGL is a potent inducer of bone resorption in vivo (Lacey et al., 1998).
Description of OPGL
OPGL is synthesised as a type II transmembrane protein consisting of 317 amino acid residues (human, cf. SEQ ID NO: 2) or 316 amino acid residues (murine, cf. SEQ ID NOs: 4 and 6). Alignment of the two amino acid sequences show that identical amino acid residues are found at 87% of the homologous positions.
The OPGL amino acid sequence contains a short cytoplasmic domain in the N-terminus followed by the putative transmembrane region between amino acid residues 49 and 69. Based on its homology to tumour necrosis factor alpha, the extracellular part of OPGL has been suggested to be comprised by two domains: a stalk region extending from amino acid residue 70 to 157, and the active ligand moiety extending from amino acid residue 158 to the C-terminus.
The most closely related protein to OPGL appears to be the apoptosis inducing cytokine TRAIL with less that 25% identical amino acid residues. OPGL has also very recently been cloned in other contexts and was called TRANCE (Wong et al., 1997, J. Biol. Chem. 272: 25190-25194) and RANKL, respectively (Anderson et al., 1997, Nature 390: 175-179. The protein is also known as osteoclast differentiation factor (ODF).
Several N-terminal deletion variants of murine OPGL have been expressed in
E. coli
and purified. These variants consisted of amino acid residues 75-316, 128-316, 137-316, and 158-316, respectively. The three shortest variants had similar &bgr;-sheet structure based on circular dichroism studies, and all were able to bind to osteoprotegerin. More important, though, is that the three variants were active in in vitro assays (Lacey et al., 1998).
The shortest variant was studied further. Like tumour necrosis factor alpha, this variant OPGL exists as a trimer in solution and forms 3:3 complexes when incubated with osteoprotegerin. The binding affinity was found to be 4 nM. This variant induces duces significant increases in blood ionized calcium (hypercalcemia) in mice in vivo. Co-administration of osteoprotegerin significantly reduced this hypercalcemic effect of OPGL.
The longest variant (amino acid residues 75
Haaning Jesper
Halkier Torben
Birch & Stewart Kolasch & Birch, LLP
DeBerry Regina M.
Kemmerer Elizabeth
M & E Biotech A/S
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