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WO2001068812A2 - Procedes de stimulation de la formation de cartilage - Google Patents

Procedes de stimulation de la formation de cartilage Download PDF

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Publication number
WO2001068812A2
WO2001068812A2 PCT/US2001/005473 US0105473W WO0168812A2 WO 2001068812 A2 WO2001068812 A2 WO 2001068812A2 US 0105473 W US0105473 W US 0105473W WO 0168812 A2 WO0168812 A2 WO 0168812A2
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Prior art keywords
ror2
receptor
ror2 receptor
agent
cells
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PCT/US2001/005473
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English (en)
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WO2001068812A9 (fr
WO2001068812A3 (fr
Inventor
Thomas M. De Chiara
Robert Kimble
George D. Yancopoulos
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Regeneron Pharmaceuticals, Inc.
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Priority to US10/221,599 priority Critical patent/US6908744B1/en
Priority to AU2001238571A priority patent/AU2001238571A1/en
Publication of WO2001068812A2 publication Critical patent/WO2001068812A2/fr
Publication of WO2001068812A3 publication Critical patent/WO2001068812A3/fr
Publication of WO2001068812A9 publication Critical patent/WO2001068812A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders

Definitions

  • the field of this invention is methods of stimulating cartilage formation and methods of use.
  • cartilage formation is stimulated by stimulating chondrocyte growth, development and activity.
  • the present invention also provides for novel assay systems useful for identifying novel ligands capable of stimulating chondrocyte growth, development and activity.
  • Osteoarthritis also known as degenerative joint disease, is the most common form of arthritis. In addition to man, nearly all vertebrates suffer from osteoarthritis. Osteoarthritis is characterized by damage to and subsequent loss of cartilage in the joints. Cartilage, a slippery connective tissue that is located on the articular surfaces of bones, is necessary for flexibility, support and protection of the bone. When cartilage is damaged due to, for example, injury or stress related to obesity, the joint can no longer function properly and painlessly. When the cartilage deteriorates, osteoarthritis develops.
  • the surface of the cartilage swells and there is a loss of various tissue components, notably proteoglycans. Subsequently, fissures and pits appear on the cartilage and inflammation often occurs around the synovium. As the disease progresses further, the cartilage loses elasticity and becomes more and more susceptible to damage due to continued use and injury. Eventually, much of the cartilage is destroyed, resulting in unprotected bone surface, a condition which is extremely painful.
  • the body In an effort to repair the damage, the body often forms fluid-filled cysts around the bony areas or in the regions around the fissures. Local bone cells may respond to the damage by forming dense, misshapen plates around damaged and exposed areas, further limiting joint mobility.
  • Symptoms of osteoarthritis include pain, stiffness and loss of mobility in one or more joints. Severity of symptoms waxes and wanes with changing weather conditions, advancing disease, and following periods of inactivity.
  • Osteoarthritis is quite prevalent in the adult population, with over 85% of people over age 65 showing some evidence of the disease upon x-ray. Of these 35%-50% experience symptoms. The causes of osteoarthritis are not completely known, but clearly age, genetic factors, muscle disuse and weakness, trauma, obesity and anatomical abnormalities contribute to the development of the disease. Prevention has focused on weight loss, exercise, hormone therapy such as estrogen replacement in postmenopausal women, and diet supplements such as vitamin D and calcium.
  • Surgical alternatives include arthroscopy, resection arthroplasty, osteotomy, chondroplasty and joint replacement.
  • ROR2 a previously described orphan receptor termed ROR2 (see U.S. Patent No. 5,843,749) appears to play a critical role in cartilage formation and as such may be useful is developing therapeutic strategies to treat diseases of cartilage such as osteoarthritis.
  • Valenzuela D.M. et al. Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Neuron 15, 573-584 (1995). 10. DeChiara, T.M. et al. The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. Cell 85, 501-512 (1996).
  • the dopamine b-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice. Neuron 7, 703-716 (1991).
  • Receptor tyrosine kinases often play critical roles for particular cell lineages by initiating important signaling cascades in those lineages. Examples include the neural-specific Trk receptors ⁇ , the VEGF and Angiopoietin endothelial- specific receptors ⁇ ' S, a nd the muscle-specific MuSK receptor" " !. Many lineage-restricted receptor tyrosine kinases were initially identified as "orphans" homologous to known receptors, and only subsequently used to identify their unknown growth factors. There are few remaining receptor tyrosine kinase-like orphans still lacking identified ligands as well as clear-cut biological roles. Applicants describe herein a detailed characterization of one such orphan, termed ROR212 whose activation may play a role in treating diseases such as osteoarthritis which are characterized by destruction and loss of cartilage.
  • ROR2 drives chondrocyte growth, development and activity. Because of this, Applicants contend that activating ROR2 may be useful in treating diseases and disorders wherein cartilage is damaged or destroyed. Stimulation of the growth, development and activity of chondrocytes would result in formation of new cartilage or repair of damaged cartilage.
  • one embodiment of the invention is a method of increasing chondrocyte growth, development and activity comprising contacting chondrocytes expressing a ROR2 receptor with an agent capable of activating the ROR2 receptor.
  • Another embodiment of the invention is a method of increasing cartilage formation comprising contacting chondrocytes expressing a ROR2 receptor with an agent capable of stimulating chondrocyte growth, development and activity.
  • a preferred embodiment of the invention is a method of treating a patient with damaged or diseased cartilage comprising administering to the patient an agent capable of activating a ROR2 receptor.
  • Another embodiment of the invention is the method of increasing chondrocyte growth, development and activity comprising contacting chondrocytes expressing a ROR2 receptor with an agent capable of activating the ROR2 receptor, the method of treating a patient with damaged or diseased cartilage comprising administering to the patient an agent capable of activating a ROR2 receptor, or the method of treating a patient with damaged or diseased cartilage comprising administering to the patient an agent capable of activating a ROR2 receptor wherein the agent is an activating antibody.
  • the method of increasing chondrocyte growth, development and activity comprising contacting chondrocytes expressing a ROR2 receptor with an agent capable of activating the ROR2 receptor, the method of treating a patient with damaged or diseased cartilage comprising administering to the patient an agent capable of activating a ROR2 receptor, or the method of treating a patient with damaged or diseased cartilage comprising administering to the patient an agent capable of activating a ROR2 receptor is where the agent is a monoclonal antibody, a wholly human monoclonal antibody, a ligand of the ROR2 receptor, a naturally occurring ligand of the ROR2 receptor, or a small molecule.
  • a preferred embodiment of the invention is a method of identifying an agent capable of activating the ROR2 receptor comprising (a) obtaining cells expressing the ROR2 receptor; (b) subjecting the cells to a test agent; (c) determining whether the test agent has activated the ROR2 receptor.
  • the cells are obtained from an animal, are chondrocytes or are obtained by transfecting cells that normally do not express the ROR2 receptor with the ROR2 receptor nucleic acid under conditions in which the cell expresses the ROR2 receptor protein on the cell surface.
  • a further embodiment of the invention involves determining whether the ROR2 receptor has been activated by measuring phosphorylation of the receptor or by measuring chondrocyte growth, development or activity.
  • the ROR2 receptor is used in assays designed to identify natural ligands of the receptor or small molecules or antibodies capable of activiating the receptor and stimulating chondrocyte growth, development and activity.
  • a preferred embodiment of the invention is a method of preventing chondrocyte growth, development and activity comprising contacting chondrocytes expressing a ROR2 receptor with an agent capable of blocking activation of the ROR2 receptor.
  • Another embodiment of the invention is a method of preventing cartilage formation comprising contacting chondrocytes expressing a ROR2 receptor with an agent capable of preventing chondrocyte growth, development and activity by binding to but not activating the ROR2 receptor wherein the agent is a neutralizing antibody, a monoclonal antibody, a wholly human monoclonal antibody or wherein the agent is a ligand of the ROR2 receptor including a naturally occurring ligand of the ROR2 receptor. Also contemplated in an embodiment wherein the agent is a small molecule.
  • a further embodiment is a method of identifying an agent capable of blocking activation of the ROR2 receptor comprising (a) obtaining cells expressing the ROR2 receptor; (b) subjecting the cells to a test agent; (c) determining whether the test agent has blocked activation of the ROR2 receptor.
  • the cells are obtained from an animal and in another embodiment, the cells are chondrocytes.
  • the cells are obtained by transfecting cells that normally do not express the ROR2 receptor with the ROR2 receptor nucleic acid under conditions in which the cell subsequently expresses the ROR2 receptor protein on the cell surface.
  • the method of determining whether the ROR2 receptor activation has been blocked is accomplished by measuring phosphorylation of the receptor and in another preferred embodiment, the method of determining whether the ROR2 receptor activation has been blocked is accomplished by measuring chondrocyte growth, development or activity.
  • a further preferred embodiment of the invention is an agent identified by any of the above-described methods.
  • the present invention also provides for antibodies to the ROR2 receptor.
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497) as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in 'Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc. pp. 77-96) and the like are within the scope of the present invention.
  • the monoclonal antibodies for diagnostic or therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al, 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16).
  • Chimeric antibody molecules may be prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).
  • ROR2 receptor Various procedures known in the art may be used for the production of polyclonal antibodies to the ROR2 receptor described herein.
  • various host animals can be immunized by injection with the ROR2 receptor, or fragments or derivatives thereof, including but not limited to rabbits, mice and rats.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • a molecular clone of an antibody to a selected ROR2 receptor epitope can be prepared by known techniques. Recombinant DNA methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • fragments include, but are not limited to, the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • Antibody molecules may be purified by known techniques including, but not limited to, immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.
  • HPLC high performance liquid chromatography
  • FIG. 1 Disruption of the endogenous murine ROR2 allele, resulting in the encoding of a novel product in which the ecto- and transmembrane-domains of ROR2 are fused to LacZ (which replaces the normal tyrosine kinase-like cytodomain).
  • a Top schematic depicts the murine genomic region spanning the exon encoding the entire tyrosine kinase-like domain of ROR2, as well as the following ST1/PR/ST2 motifs (exon in black and designated "TK domain").
  • Middle schematic shows the targeting vector, with the indicated regions of homology to the above endogenous allele;
  • LacZ signifies the beta galactosidase coding region which was fused in frame to the beginning of the TK domain,
  • neo signifies the subsequent neo resistance gene used for positive selection, and
  • tk signifies the herpes virus thymidine kinase gene used for negative selection.
  • Lower schematic represents the predicted disrupted allele after targeting.
  • b Schematic depicting how the LacZ allele was fused in frame to the ROR2 coding region, just after the transmembrane domain; the usual initiating methionine used by LacZ is indicated (see Methods),
  • c Southern analysis depicting genomic analysis which distinguishes ROR2+/+ mice, ROR2+/- mice, and ROR2-/- mice; tail DNA from these mice was digested with Sacl and probed with the fragment indicated at bottom right of panel A, which detects a 3.9 kb Sacl fragment from the normal allele but a 6.2kb fragment from the disrupted allele (see panel A),
  • d Northern analysis demonstrating loss of endogenous ROR2 transcript in total E15.5 limb RNA prepared from ROR2-/- mice; embryonic RNA from both ROR2+/- mice and ROR2-/- mice express a novel ROR2/LacZ chimeric transcript, hybridizing with both a ROR2 ectodomain probe (shown) and a LacZ probe (not
  • ROR2 is expressed in chondrocytes of early cartilaginous anlagen in embryos, as well as in chondrocytes of articular cartilage and growth plates in the adult, a, In sagittal section of entire E12 ROR2+/- embryo, ROR2 expression (visualized by LacZ staining) is notable in developing rib and vertebral anlagen, though also noted in telencephalic neuroepithelium of forebrain and snout region; surrounding mesenchyme and stroma are largely negative, b, High-power view of ROR2 staining (visualized by LacZ staining) in rib anlagen from embryo shown in previous panel, showing lack of expression in surrounding mesenchyme and stroma.
  • FIG. 3 Disruption of murine ROR2 allele results in widespread skeletal abnormalities (in bones formed by endochondral but not intramembranous ossification), though distal long bones in limb are more dramatically affected than proximal long bones, a, Wild-type pup compared to ROR2-/- littermate shortly after birth, b, Comparison of newborn pups after staining with alcian blue and alizarin red S to visualize the cartilaginous (in blue) and ossified (in red) portions of their skeletons; inset shows normal appearance in ROR2-/- embryos of frontal (F) and parietal (P) skull bones forming by intramembranous ossification, c and d, Close-up views of forelimbs from skeletons depicted in previous panel, showing more dramatic abnormalities in distal long bones (radius and ulna) as compared to proximal long bone (humerus); arrowheads point to ossification
  • Figure 4 Developmental progression of abnormalities in cartilaginous anlagen of ROR2-/- mice, a-d, Alcian blue stains of whole limbs from E13 and E14 embryos reveal that cartilaginous anlagen in ROR2-/- embryos are shorter than their wild-type littermates, but not obviously abnormal in shape at these early stages except for the missing anlagen for the middle phalanges (indicated by arrow in wild-type paw in panel c).
  • ROR2-/- mice develop a delayed and eccentric hypertrophic region that juts out from the side of the developing strom, covered by an abnormally positioned thin collar of bone, i-k, higher-power views focusing on progression of abnormal eccentric hypertrophic region, and associated abnormal peripheral ossification that forms the bony collar, in developing bones from E15.5 to E17.5 ROR2-/- embryos.
  • FIG. 5 Schematic summary of comparison between forelimb long bones in normal and ROR2-/- embryos at E17.5.
  • the E17.5 radius shows a profound decrease of 56% in the length of the bone, as well as an abnormal eccentric orientation of the proliferative and hypertrophic zones in the ROR2-/- bone compared with the proximo-distal orientation of these zones in the normal radius
  • b Summary of E17.5 humerus data showing the decrease in the width of the proliferative zones and the increase in the width of the hypertrophic zones in both the proximal and distal growth plates of the ROR2-/- embryo, with this abnormal interplay of these growth plate zones presumably leading to the overall reduction of 19.5% in total humerus bone length.
  • ROR2 Disruption of the murine ROR2 gene leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened and /or misshapen, albeit to differing degrees.
  • ROR2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it plays a critical role during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion.
  • LacZ beta-galactosidase
  • ROR2 continues to play a key role in chondrocytes later in development, ROR2 is expressed post-natally in articular cartilage, perichondrium (as well as periosteum), and a particular subset of growth plate chondrocytes - reserve and proliferating but not hypertrophic chondrocytes (Fig. 2e,f,g); ROR2 was absent in bone tissue itself.
  • ROR2-/- anlagen displayed delayed eccentric hypertrophy covered by a bony collar, forming a characteristic abnormal knob on one side of the developing bone (Figs 4f,h,i-k and 5a), and apparently leading to the gross abnormalities eventually noted in the older limb bones (Fig. 41,m).
  • the initially shortened limb anlagen in ROR2-/- embryos suggest that ROR2 is required for proper growth, development and activity of chondrocytes normally expressing this receptor, while the subsequent gross abnormalities in strom and growth plate organization suggest that ROR2 function is required for proper patterning of developing anlagen.
  • ROR2 plays a profound role in the initial growth and patterning of developing cartilage anlagen and their growth plates, as noted in the more distal limb bones, and that it continues to be required for the normal interplay and expansion of proliferative and hypertrophic zones in maturing growth plates, as noted in proximal limb bones.
  • Detailed characterization of ROR2 expression patterns is consistent with a direct role for ROR2 in regulating chondrocyte growth, development and activity, as ROR2 is selectively expressed in the chondrocytes of the early anlagen as well as in resting and proliferative (but not hypertrophic) chondrocytes in older growth plates.
  • ROR2 mutations have been shown as the cause of inherited limb malformations in man! 3.
  • the phenotype in humans is substantially milder than in ROR2-/- mice, probably because the ROR2-/- mice are homozygous for mutations removing the entire ROR2 tyrosine kinase-like domain, while the human mutations are found in heterozygotes and appear to act as partial dominant negatives retaining most of the ROR2 TK-like domains ⁇ .
  • Remaining questions include the varying severity of the ROR2-/- phenotype in different bones, and the role of ROR2 in the context of all the other pathways acting to regulate cartilage growth, development and activity.
  • ROR2 seems somewhat analogous to that of the Indian Hedgehog (Ihh)/ parathyroid hormone-related peptide (PTHrP) loop, since disruption of either depletes the proliferative zone ⁇ - 0_ Conversely, ROR2 appears to oppose the actions of the FGFR3 pathway at the growth plate, since FGFR3 disruption leads to expansion of the proliferative zone21 ⁇ 25_ Thus, the two receptor tyrosine kinases now known to be required for normal growth plate expansion may push chondrocytes in opposite directions, with ROR2 promoting chondrocyte growth, development and activity and FGFR3 limiting such growth, development and activity.
  • Ihh Indian Hedgehog
  • PTHrP parathyroid hormone-related peptide
  • ROR2 is especially important for the chondrocyte lineage.
  • ROR2 is also expressed by other chondrocytes in the adult, such as those in the articular cartilage surface of joints and the fibrocartilaginous menisci of the knee. Osteoarthritis results from damage to the articular surface in joints, coupled with the inability of this surface to regenerate. Since ROR2 apparently drives chondrocyte growth, development and activity, promoting ROR2 function could be of benefit in this and other clinical settings.
  • the ROR2 gene targeting vector was constructed from mouse genomic DNA fragments isolated from a lambda FIX II phage library prepared with 129 strain mouse DNA (Stratagene).
  • the 3.5 kb Xhol-Accl fragment depicted in Fig. la is comprised mostly of intron sequence except for the 75 nucleotides of coding sequence that terminated in the Accl site.
  • the Accl site was filled-in using Klenow DNA Polymerase in the presence of dATP and dGTP and was blunt- end ligated to a promoter-less LacZ gene cassette ⁇ that possessed a short upstream bluescript polylinker sequence terminating in a 5' Clal site that was similarly filled-in using dCTP and dGTP to created an in-frame fusion at the 25 th amino acid from the start of the tyrosine kinase domain of ROR2 with the LacZ coding region as shown in Figure IB.
  • Embryos derived from crosses between ROR2 +/- matings were subjected to skeletal stains to visualize cartilage and bone as described ⁇ .
  • Embryos derived from crosses between ROR2 +/- matings were subjected to skeletal stains to visualize cartilage and bone as described ⁇ .
  • For LacZ staining embryos were fixed in 4% paraformaldehyde for 12 hours (E12-15.5) or 24 hours (E17.5 and PI), frozen in OCT or embedded in paraffin (for bone morphology), sectioned and stained for LacZ as described ⁇ or stained with safranin O/fast green to visualize cartilage and bone.
  • Example 3 Quantitation of Bone Lengths and Growth Plate Zones Bone lengths and various zones ⁇ O in the growth plate were measured from histological sections using the OsteoMeasure morphometry system (OsteoMetrics Inc., Atlanta, Ga.)

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Abstract

La rupture du gène murin ROR2 conduit à de profondes anomalies du squelette, telles que des raccourcissements et/ou des déformations observés dans presque tous les os d'origine endochondrale, bien qu'à des degrés différents. ROR2 est exprimé sélectivement dans les chondrocytes de tout primordium de cartilage, où il joue un rôle critique au cours de la croissance initiale et de la structuration fonctionnelle, ainsi qu'ultérieurement dans les chondrocytes proliférants de cartilages de conjugaison à maturité, où il est nécessaire à un développement normal. ROR2 semble jouer un rôle critique dans la formation du cartilage et il peut s'avérer utile au développement de stratégies thérapeutiques destinées au traitement de maladies du cartilage telles que l'arthrose.
PCT/US2001/005473 2000-03-14 2001-02-20 Procedes de stimulation de la formation de cartilage WO2001068812A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094641A3 (fr) * 2003-04-16 2005-03-10 Wyeth Corp Nouveau procede de modulation de l'activite liee a l'os
WO2007098198A3 (fr) * 2006-02-17 2008-03-13 Wyeth Corp Modulation de formation d'os
US11730755B2 (en) 2017-11-16 2023-08-22 Queen Mary University Of London ROR2 inhibitors and use thereof in treating and/or preventing cartilage loss

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5843749A (en) * 1991-07-26 1998-12-01 Regeneron Pharmaceuticals, Inc. Ehk and Ror tyrosine kinases

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094641A3 (fr) * 2003-04-16 2005-03-10 Wyeth Corp Nouveau procede de modulation de l'activite liee a l'os
JP2006524508A (ja) * 2003-04-16 2006-11-02 ワイス 骨関連の活性を調節する新規な方法
WO2007098198A3 (fr) * 2006-02-17 2008-03-13 Wyeth Corp Modulation de formation d'os
US11730755B2 (en) 2017-11-16 2023-08-22 Queen Mary University Of London ROR2 inhibitors and use thereof in treating and/or preventing cartilage loss

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