Method for the treatment of bone space defects

专利摘要:

公开号:ES2667554T9
申请号:ES12746235.6T
申请日:2012-08-02
公开日:2018-10-01
发明作者:Xiaodong Li；Hua Zhu Ke
申请人:Amgen Inc；
IPC主号:

专利说明:

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DESCRIPTION
Method for the treatment of bone space defects Technical field of the invention
The invention generally relates to methods for using sclerostin inhibitors to enhance the healing of bone space defects.
Background of the invention
The bone tissue of mammals has a remarkable capacity for regeneration and, therefore, for the repair of lesions and other defects. For example, bone growth in general is sufficient to cause total recovery of the simplest fractures and small cracks. Unfortunately, there are many injuries, defects or conditions where bone growth is inadequate to achieve an acceptable result. For example, bone regeneration generally does not occur in large gaps or spaces. Therefore, fractures cannot be cured, unless the pieces are very close. If a considerable amount of bone tissue has been lost as a result of the injury, the healing process may be incomplete, resulting in unwanted cosmetic and / or mechanical consequences. This is often what happens with unconsolidated fractures or with bone lesions that result from massive trauma. Tissue growth is also generally inadequate in the gaps and segmental spaces in the bones, caused, for example, by surgical removal of tumors or cysts. In other cases, it may be desirable to stimulate bone growth where there is normally no bone, that is, ectopically. Vertebral fusion to relieve low back pain, where the fusion of two or more vertebrae is induced, is an example of desired ectopic bone formation.
WO 2009/079471 describes a method for improving the healing of bone fractures that involves the administration of a sclerostin inhibitor. Ominsky et al., Describes that the inhibition of sclerostin by a monoclonal antibody increases bone healing and improves bone density and resistance of non-fractured bones (Journal Of Bone And Mineral Research, 2011, vol. 26, no. 5 , 1012-1021). WO 2009/047356 refers to antibodies against sclerostin and to compositions and methods of using said antibodies to treat diseases related to bone abnormalities such as osteoporosis. Zhou et al. Describe the successful repair of a critical-sized bone defect in the rat femur with an external fixator (Tohoku J. Exp. Med., 2009, 219 (2), 115-120).
Summary of the invention
In one aspect, the invention provides an effective amount of an anti-sclerostin antibody for use in a method of treating a bone space defect in a mammal, said method comprising administering the antibody to the subject, optionally at a weekly dose of 1 mg. / kg at 50 mg / kg per week, in which the anti-sclerostin antibody is administered during a treatment period lasting at least 20 weeks, and in which the bone space defect comprises a space between two bone segments of at least 5 mm In one embodiment, the antibody is administered once a week during the treatment period. In another embodiment, the antibody is administered once every two weeks during the treatment period. Alternatively, the antibody is administered twice a week.
The treatment period can be at least about 5 months, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 6 months, 25 weeks, 26 weeks, 27 weeks 28 weeks, 7 months, 29 weeks, 30 weeks, 31 weeks or more (eg, 8 months, 9 months, 10 months, 11 months, 1 year, 15 months, 18 months or more). In some embodiments, the treatment period is about 20-32 weeks, or about 58 months. In some embodiments, the treatment period is not more than about 28 weeks. In some embodiments, the treatment period is about 1 year. In some cases, the treatment period is not more than about 18 months.
The bone space defect for treatment by the methods described herein includes any fracture that comprises a space between two bone segments (for example, a space that is at least 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, or at least about 1 cm or more, for example, the space is 5 mm to 1 cm.
Examples of bone space defects include, but are not limited to, a comminuted fracture, an unconsolidated fracture, a segmental skeletal defect, surgically created bone defects, surgically treated bone defects and bone defects created from traumatic bone injury or disease ( which includes, but is not limited to, arthritis, tumor removal (resection) or infection removal). In some or any of the embodiments, the bone space defect is caused by the removal of infected sections of bone or the removal of bone cancer due to bone cancers that include, but are not limited to, osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma and chordoma. In some or any of the
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embodiments, the bone space defect is a developmental deformation, for example, due to a genetic defect.
In some or any of the embodiments, the bone space defect is caused by the removal of bone sections that contain a benign tumor. Examples of benign bone tumors include, but are not limited to, osteoma, osteoid osteoma, osteoblastoma, osteochondroma, enchondroma, chondromyxoid fibroma, aneurysmal bone cyst, unicameral bone cyst, fibrous bone dysplasia and giant bone cell tumor.
The subject to whom the anti-sclerostin antibody is administered optionally suffers from a bone-related disorder that is selected from the group consisting of achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher's disease, hypophosphathic rickets, Marfan syndrome, exostosis hereditary multiple, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, bone loss induced by anti-epileptic drug, primary and secondary hyperparathyroidism syndromes, family-induced hyperparathyroidism, bone loss men, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative bone disorders, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, meloreostosis, metabolic bone diseases, mastocytosis, anemia / disease Sickle cells, bone loss related to organ transplantation, bone loss related to kidney transplant, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidosis, Fabry disease, Turner syndrome, Down syndrome, Klinefelter syndrome , leprosy, Perthe's disease, adolescent idiopathic scoliosis, early onset multisystem inflammatory disease, Winchester's syndrome, Menkes' disease, Wilson's disease, ischemic bone disease (such as Legg-Calve-Perthes disease and regional osteoporosis migratory), anemic states, conditions caused by steroids, glucocorticoid-induced bone loss, bone loss induced by heparin, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal state , chronic inflammatory conditions cas, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, thyroid gland disorders, parathyroid gland disorders, Cushing's disease, acromegaly, hypogonadism , immobilization or disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis, osteomalacia, bone loss associated with joint replacement, bone loss associated with HIV, bone loss associated with loss of growth hormone, bone loss associated with cystic fibrosis, Chemotherapy-associated bone loss, tumor-induced bone loss, cancer-related bone loss, hormonal ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, disease-associated facial bone loss, cranial bone loss associated with disease, bone loss of the jaw to associated with the disease, bone loss of the skull associated with the disease, bone loss associated with aging, facial bone loss associated with aging, bone loss cranial associated with aging, bone loss of the jaw associated with aging, bone loss of the skull associated with aging and bone loss associated with space travel. In one embodiment, the subject has undergone oral or maxillofacial surgery.
The administration of the anti-sclerostin antibody is also disclosed in combination with the use of materials that promote new bone growth, such as bone graft, bone dust, bone fragments, demineralized bone matrix, bone scaffolds, prostheses, metal stabilizers or bone scaffolding substances. comprising one or more of polymers, ceramic materials, cement and bone graft substitutes based on calcium phosphates. Many variations of such materials are known in the art.
In some or any of the embodiments, the anti-sclerostin antibody is administered together with a second bone strengthening therapeutic agent for the treatment of decreased bone mineral density or bone fracture. Many therapeutic agents of this type are known in the art. In some embodiments, the bone strengthening therapeutic agent is selected from the group consisting of an antiresorptive drug, a bone forming agent, an estrogen receptor antagonist (which includes, but is not limited to, raloxifene, bazedoxifene and lasofoxifene) and a drug that has an inhibitory effect on osteoclasts. In some embodiments, the antiresorptive drug includes, but is not limited to, parathyroid hormone, a bisphosphonate (which includes, but is not limited to, alendronate, risedronate, ibandronate and zoledronate), an estrogen or estrogen analogue, a selective modulator of estrogen receptor (SERM) and a source of calcium, tibolone, calcitonin, a calcitriol and hormone replacement therapy. In some embodiments, the bone strengthening agent includes, but is not limited to, parathyroid hormone (PTH) or a peptide fragment thereof, PTH-related protein (PTHrp), bone morphogenetic protein, osteogenin, NaF, a PGE2 agonist. , a statin, an anti-DKK1 antibody or inhibitor, an RANK anti-ligand antibody (RANKL) or RANKL inhibitor, strontium ranelate, vitamin D or a vitamin D derivative or imitation thereof. In some embodiments, the bone strengthening agent is Forteo® (teriparatide, or recombinant human parathyroid hormone 1-34) or Preotact® (parathyroid hormone). In some or any of the embodiments, the bone strengthening agent is Protelos®.
The use of anti-sclerostin antibodies disclosed in U.S. Patent Publication No. 20070110747, for example, in any of the methods disclosed herein or for the preparation of medicaments for administration according to any of the methods disclosed herein are specifically contemplated. One or more doses of the antibody are administered in an amount and for a
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effective time to promote healing of the space defect at the fracture site. One or more doses of antibody may comprise between about 1 and about 50 milligrams (for example, between about 10 and about 50 milligrams), or about 1 to about 100 milligrams, of antibody per kilogram of body weight (mg / kg). For example, the dose may vary from at least about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 26 mg / kg, approximately 27 mg / kg, approximately 28 mg / kg, approximately 29 mg / kg, approximately 30 mg / kg, approximately 31 mg / kg, approximately 32 mg / kg, approximately 33 mg / kg, approximately 34 mg / kg, approximately 35 mg / kg, approximately 36 mg / kg, approximately 37 mg / kg, approximately 38 mg / kg, approximately 39 mg / kg, approximately 40 mg / kg, approximately 41 mg / kg, approximately 42 mg / kg, approximately 43 mg / kg, approximately 44 mg / kg, approximately 45 mg / kg, approximately 46 mg / kg, approximately 47 mg / kg, approximately 48 mg / kg, approximately 49 mg / kg, or approximately 50 mg / kg , approximately 55 mg / kg, approximately 60 mg / kg, approximately 65 mg / kg, approximately 70 mg / kg, approximately 75 mg / kg, approximately 80 mg / kg, approximately 85 mg / kg, approximately 90 mg / kg, approximately 95 mg / kg, or up to about 100 mg / kg. I also know
contemplate the intervals between any and all of these endpoints, eg, approximately 1 to
approximately 3 mg / kg, approximately 1 to approximately 5 mg / kg, approximately 1 to
approximately 10 mg / kg, approximately 1 to approximately 20 mg / kg, approximately 1 to
about 40 mg / kg, about 5 to about 30 mg / kg, or about 5 to about 20 mg / kg. In some embodiments, the antibody is administered shortly after the fracture (for example, at 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours or 24 hours after the fracture). In other embodiments, the antibody is administered 1 day after the fracture, in the 3 days following the fracture, in the 5 days following the fracture, in the 7 days following the fracture, in the two weeks following the fracture. , in which the antibody is administered for a period of time that is at least 11 weeks after the fracture (e.g., 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks 28 weeks, 29 weeks, 30 weeks, 31 weeks or more (e.g., 8 months, 9 months, 10 months, 11 months, 1 year, 18 months or more)).
Also described herein is the use of an effective amount of an anti-sclerostin antibody to treat a bone space defect in a subject, for example, in any of the amounts described above, such as about 1 mg / kg a approximately 100 mg / kg, in which one or more administrations of the sclerostin binding agent is carried out during a treatment period lasting at least 11 weeks (for example, any of the time periods described above, such as 12 weeks, 3 months, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 4 months, 17 weeks, 18 weeks 19 weeks, 20 weeks, 5 months, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 6 months, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 7 months, 29 weeks, 30 weeks, 31 weeks or more (e.g., 8 months, 9 months, 10 months, 11 months, 1 year, 15 months, 18 months or more)).
The anti-sclerostin antibody can also be used in the preparation of a medicament for administration to a subject with a bone space defect using any of the dosage and / or dosage regimens described herein. Optionally, the antibody is presented in a container, such as a single dose vial or a multidose vial. The description includes a container comprising an anti-sclerostin antibody or a fragment thereof and instructions for administering the antibody or fragment thereof to treat a bone space defect according to any of the dosage and / or dosage regimens described herein. memory.
In some embodiments, the anti-sclerostin antibody for use in the methods described herein binds to sclerostin of SEQ ID NO: 1, with an affinity (Kd) of less than or equal to 1 x 10-7 M (or less or equal to 1 x 10-8 M, or less than or equal to 1 x 10-9 M, or less than or equal to 1 x 10-10 M, or less than or equal to 1 x 10-11 M, or less than or equal to 1 x 10'12 M).
In various embodiments, the anti-sclerostin antibody binds to a sclerostin polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and binds to the sequence of SEQ ID NO: 6 (CGPARLLPNAIGRGKWWRPsGpDFRC; corresponding to amino acids 86- 111 of SEQ ID NO: 1). Alternatively or additionally, the anti-sclerostin antibody binds to a sclerostin polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 1 and binds to the sequence of at least one of SEQ ID NO: 2 (DVSEYSCRELHFTR; corresponding to amino acids 51-64 of SEQ ID NO: 1), SEQ ID NO: 3 (SAKPVTELVCSGQCGPAR; corresponding to amino acids 73-90 of SEQ ID NO: 1), SEQ ID NO: 4 (WWRPSGPDFRCIPDRYR; corresponding to amino acids 101-117 of SEQ ID NO: 1), SEQ ID NO: 5 (LVASCKCKRLTR; corresponding to amino acids 138-149 of SEQ ID NO: 1), SeQ ID NO: 70 (SAKPVTELVCSGQC; corresponding to amino acids 73-86 of SEQ ID NO: 1), SEQ ID NO: 71 (LVASCKC, which corresponds to amino acids 138-144 of SEQ ID NO: 1), SEQ ID NO: 72 (cReLHFTR, which corresponds to amino acids 57-64 of SEQ ID NO: 1) or SEQ ID NO: 73 (CiPdRYR, which corresponds to amino acids 111-117 of SEQ ID NO: 1) within SEQ ID NO: 1. For example, in one aspect, the antibody an ti-sclerostin binds to a
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Sclerostin subregion of SEQ ID NO: 1 comprising SEQ ID NOs: 2-5 (and / or SEQ ID NOs: 70-73), optionally in its native three-dimensional conformation. Optionally, the anti-sclerostin antibody binds to a peptide consisting of one or more of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID No: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73 (for example, a peptide consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 or a peptide consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73).
In several aspects, the anti-sclerostin antibody is capable of neutralizing human sclerostin in a mineralization assay based on MC3T3 cells when there is an excess of less than 6 times moles of sclerostin binding sites per well compared to the number of moles of sclerostin per well.
The anti-sclerostin antibody optionally has an IC50 of 100 nM or less, or 75 nM or less, or 50 nM or less, or 25 nM or less to neutralize human sclerostin in a cell-based assay, such as a phosphatase assay. bone specific alkaline. Alternatively or additionally, the anti-sclerostin antibody has an IC50 of 100 nM or less (eg, 75 nM or less, or 50 nM or less) to neutralize human sclerostin in a cell-based Wnt signaling assay HEK293 cell phones, such as the Wnt assay that involves induction of the STF-mediated reporter gene. Alternatively or additionally, the anti-sclerostin antibody has an IC50 of 500 nM or less (e.g., 250 nM or less, 150 nM or less, 100 nM or less, or 50 nM or less) to neutralize human sclerostin in a BMP2 induced mineralization assay in MC3T3 cells.
In one embodiment, the anti-sclerostin antibody has a cross-blocking effect of the binding of at least one of the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab -3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15 , Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin and / or suffer a cross-union blockage to sclerostin by at least one of the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab -7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19 , Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24. The heavy and light chain sequences of these antibodies are identified in Figure 1.
In some embodiments, the anti-sclerostin antibody comprises a CDR-H1 of SEQ ID NO: 245, a CDR-H2 of SEQ ID NO: 246, a CDR-H3 of SEQ ID NO: 247, a CDR-L1 of SEQ ID NO: 78, a CDR-L2 of SEQ ID NO: 79 and a CDR-L3 of SEQ ID NO: 80.
In one embodiment, the anti-sclerostin antibody comprises heavy chains comprising SEQ ID NO: 378 and light chains comprising SEQ ID NO: 376. In another embodiment, the anti-sclerostin antibody has heavy chains of sEq ID No: 145 or SEQ ID NO: 392 and light chains of SEQ ID NO: 141.
In another embodiment, the anti-sclerostin antibody comprises CDR of SEQ ID NOs: 20-25 of International Patent Publication No. WO 2008/115732 (SEQ ID NOs: 416-421), CDR of SEQ ID NOs: 26- 31 of International Patent Publication No. WO 2008/115732 (SEQ ID NOs: 422-427), CDR of SEQ ID NOs: 32-37 of International Patent Publication No. WO 2008/115732 (SEQ ID NOs : 428-433) or CDR of SEQ ID NOs: 4, 15, 26, 37, 48 and 59 of International Patent Publication No. WO 2009/047356 (SEQ ID NOs: 443, 454, 465, 476, 487 and 498, respectively). In yet another embodiment, the anti-sclerostin antibody comprises an amino acid sequence of at least one of SEQ ID NOs: 135-143, 153-161 or 171-179 of International Patent Publication No. WO 2010/130830 ( SEQ ID NOs: 745-753, 763-771, 781-789, respectively).
The foregoing summary is not intended to define each aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. It is intended that the entire document be related as a unified disclosure, and it should be understood that all combinations of the features described herein are contemplated, even if the combination of features are not found together in the same sentence, paragraph or section of this document. document. With respect to the aspects of the invention described or claimed with "a" or "a," it should be understood that these terms mean "one or more" unless the context unequivocally requires a more restricted meaning. It should be understood that the term "or" encompasses alternative or joint elements, unless the context requires otherwise. If aspects of the invention are described as "comprising" a characteristic, the embodiments are also contemplated "consisting of" or "consisting essentially of" the characteristic.
Brief description of the figures
Figure 1 is a table listing the amino acid sequences and sequence identifiers for the amino acid sequences of various anti-sclerostin antibodies described herein. Sequence identifiers refer to the amino acid sequences provided in the Sequence Listing presented herewith. Amino acid sequences are also set forth in United States Patent Publication No. 2007/0110747 or in International Patent Publication Nos. WO 2008/115732, WO 2009/047356 or WO 2010/130830.
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Figures 2A and 2B provide graphs illustrating that administration of the anti-sclerostin antibody resulted in an increase in cortical area and cortical thickness in the femoral diaphysis of healthy cynomolgus monkeys after 28 weeks of treatment, compared to animals that They did not receive the antibody treatment.
Figures 3A and 3B provide graphs illustrating that administration of the anti-sclerostin antibody resulted in an increase in the rate of periodic and endocortical bone formation in the femoral diaphysis of healthy cynomolgus monkeys after 28 weeks of treatment, compared with animals that did not receive the antibody treatment.
Figure 4 provides a graph illustrating that administration of the anti-sclerostin antibody did not significantly increase cortical porosity in the femoral diaphysis of cynomolgus monkeys after 28 weeks of treatment, compared to animals that did not receive antibody treatment.
Detailed description of the invention
The invention is based, at least partially, on the discovery that sclerostin inhibitors enhance the healing of bone space defects. In this regard, the invention provides a method of treating a segmental skeletal defect or an unbound fracture. The treatment comprises administration to a subject (for example, a mammal, such as a human being) of one or more doses of an anti-sclerostin antibody for a treatment period of at least 20 weeks. The materials and methods of the invention are superior to existing therapies whose therapeutic efficacy is limited and require a longer recovery time.
The terms "bone space defect" and "segmental skeletal defect" are used herein as synonyms and refer to a space between two bone segments of at least 5 mm.
Administration of the anti-sclerostin antibody enhances or accelerates the healing of the bone space defect, thus "treating" the bone space defect. "Enhancing" bone healing means mediating a level of bone healing beyond (ie, higher) than the level of bone healing experienced by subjects (eg, mammals, such as humans) who were not given the antibody (i.e. control subjects). Bone healing is revealed, for example, by bridge bonding, greater bone volume, higher bone mineral content and density within the fracture space (i.e., formation of the bone that forms a bridge), mature bone callus, increased bone strength (optionally accompanied by an acceptable level in medical terms of bone stiffness) or improved use by the patient of the affected area. "Improvement" means an increase or decrease (as desired) in the measured parameter. The increase may be the total or partial return of the parameter measured at the reference level (for example, the level prior to the bone space defect), to the values provided in the normative databases used in the technique, or at the level contralateral function (for example, return, total or partial, to the functional capabilities of, for example, the contralateral member). In some cases, the increase may be an improvement beyond the reference level. If desired, the parameters measured in patients who were given one or more doses of the anti-sclerostin antibody can be compared with the same parameters in fractured patients (optionally with age and gender matching) to those who are not He administers the anti-sclerostin antibody to further analyze the efficacy of the methods described herein.
The formation of bridged bone, bone mineral content and bone density and / or mature bone callus at the site of bone defect can be measured by radiography (for example, radiographic densitometry), single and / or dual energy X-ray densitometry, tomography Quantitative computerized (QCT), ultrasonography, radiography (for example, radiographic densitometry) and magnetic resonance imaging. In some embodiments, the anti-sclerostin antibody can be administered at a dose and for an effective period of time to increase bone bridging formation, bone callus formation or bone density (or volume) at the site of the defect of at least about 5% (about 6%, about 7%, about 8%, or about 9%). In some embodiments, the formation of bone bridging, bone callus formation or bone density at the site of the defect increases by at least about 10% (for example, at least about 10%, at least about 12%, at least about 15 %, at least about 18%, at least about 20%, or at least about 22%). In other embodiments, the formation of bone bridging, bone callus formation or bone density at the site of the defect is increased by the antibody by at least about 25% (for example, at least about 26% or at least about 28%). In yet other embodiments, the formation of bone bridging, bone callus formation or bone density at the site of the defect increases by at least about 30% (for example, at least about 32%, at least about 35%, at least about 38%, or at least about 40%), or at least about 50%, (for example, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% ). The increase or restoration of bone bridging formation can be determined 1 week, 2 weeks, 3 weeks or 4 weeks after the initial administration of the antibody. Alternatively, the level of bone density can be determined after the end of the treatment period (for example, 1 week, 2 weeks, 3 weeks
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or 4 weeks after the end of the treatment period). In one aspect, the method reduces the amount of time required to establish a desired level of bone formation, bone volume, bone callus or bone density (eg, any increase in bone formation percentage, bone mineral density, bone callus or volume bone described herein), compared to patients with age and gender matching who do not receive the antibody, thus reducing recovery time for a subject. For example, in one embodiment, the antibody reduces the amount of time required to increase bone density or volume at the site of the defect by at least about 10% (for example, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50%).
Functional quality of life parameters that indicate bone healing include, but are not limited to, recovery of strength and load bearing capacity, pain reduction and use of pain medication and improvement of occupational status. The administration of one or more doses of the antibody, as described herein, accelerates the improvement of the functional parameters of quality of life associated with fractures in a statistically significant manner in the patient population analyzed. In certain aspects, the method reduces the recovery time in the patient who is administered one or more doses of antibody by at least 10% (for example, at least 20%, at least 30%, at least 40%, by minus 50%, or at least 65%), compared to the recovery time of patients who do not receive the antibody. "Recovery" can be estimated using any of several measures of rehabilitation results, such as the motor score of the FIM instrument for hip fractures (Munin et al., Arch. Phys. Med. Rehabil., 86: 367- 372 (2005)), the Olerud-Molander ankle scale (OMAS) and the SF-12 questionnaire for ankle fracture (Shah et al., Injury, 38 (11): 1308-1312 (2003)) and the Knee Society scale for knee arthroplasty (Insall et el., Clinical Orthopedics, 248: 13-14 10 (1989)).
In some embodiments, one or more doses of an anti-sclerostin antibody) are administered to a human being during a treatment period comprising 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31 weeks , 8 months, 9 months, 10 months, 11 months, 1 year, 18 months or more. A "treatment period" begins after administration of a first dose of anti-sclerostin antibody and ends after administration of a final dose of the antibody. An antibody dose may be administered several times a week, if desired. In some embodiments, the treatment period lasts 28 weeks. In other embodiments, the treatment period lasts 1 year. Alternatively or additionally, the treatment period lasts no more than 18 months. In fact, one or more administrations of a pharmaceutical composition comprising the antibody can be carried out during a therapeutic or treatment period lasting up to 18 months, less than 1 year, up to 8 months or up to 28 weeks. In one embodiment, the treatment period is approximately 28 weeks and provides a significant improvement in healing parameters, such as (not limited to) bone formation, bone strength (e.g., maximum load bearing capacity before experience pain), bone volume, without significant increase in cortical porosity, limb bypass function and / or recovery time, compared to untreated fractures. In addition, in one aspect, the treatment period begins just after a bone space defect has been detected, for example, at 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours or 24 hours from the detection of the default. In other embodiments, the antibody is administered 1 day later, in the 3 days following the bone defect, in the 5 days following the bone defect, in the 7 days following the bone defect or in the two weeks following the bone defect, in the that the sclerostin binding agent is administered for a period of time that is at least 11 weeks from the detection of the bone defect (for example, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks , 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks or more (for example , 8 months, 9 months, 10 months, 11 months, 1 year, 18 months or more)).
The anti-sclerostin antibody is administered in an amount that promotes, enhances or accelerates the healing of the bone space defect. The dose of the sclerostin binding agent that is administered to a subject (eg, a mammal, such as a human) may be in the range of about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to approximately 50 mg / kg body weight. For example, the antibody dose may be in the range of about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 25 mg / kg, about 26 mg / kg, about
27 mg / kg, approximately 28 mg / kg, approximately 29 mg / kg, approximately 30 mg / kg, approximately
31 mg / kg, approximately 32 mg / kg, approximately 33 mg / kg, approximately 34 mg / kg, approximately
35 mg / kg, approximately 36 mg / kg, approximately 37 mg / kg, approximately 38 mg / kg, approximately
39 mg / kg, approximately 40 mg / kg, approximately 41 mg / kg, approximately 42 mg / kg, approximately
43 mg / kg, approximately 44 mg / kg, approximately 45 mg / kg, approximately 46 mg / kg, approximately
47 mg / kg, approximately 48 mg / kg, approximately 49 mg / kg, or approximately 50 mg / kg, approximately 55 mg / kg, approximately 60 mg / kg, approximately 65 mg / kg, approximately 70 mg / kg, approximately 75 mg / kg, approximately 80 mg / kg, approximately 85 mg / kg, approximately 90 mg / kg, or approximately 95 mg / kg, to approximately 100 mg / kg body weight. In addition, it may be advantageous to administer multiple doses of the antibody or space dose administration, depending on the therapeutic regimen that is selected for a particular patient. For example, one dose of antibody can be administered every two weeks, once a week, twice a week, three times a week, four times a
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the week or more, depending on the severity of the defect, the age and physical health of the patient, and the like.
In some embodiments, the subject with the space defect optionally suffers from a bone-related disorder that is selected from the group consisting of achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher disease, hypophosphatemic rickets, Marfan syndrome, multiple exostosis hereditary, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, bone loss induced by anti-epileptic drug, primary and secondary hyperparathyroidism syndromes, familial hyperparathyroidism, bone loss in osteoporosis in bone loss , postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative bone disorders, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, melorreostosis, metabolic bone diseases, mastocytosis, anemia / cell disease Sickle cells, bone loss related to organ transplantation, bone loss related to kidney transplant, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidosis, Fabry disease, Turner syndrome, Down syndrome, Klinefelter syndrome , leprosy, Perthe disease, adolescent idiopathic scoliosis, early onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson disease, ischemic bone disease (such as Legg-Calve-Perthes disease and regional osteoporosis migratory), anemic states, conditions caused by steroids, glucocorticoid-induced bone loss, bone loss induced by heparin, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal state , chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, thyroid gland disorders, parathyroid gland disorders, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis, osteomalacia, bone loss associated with joint replacement, bone loss associated with HIV, bone loss associated with loss of growth hormone, bone loss associated with cystic fibrosis, bone loss associated with chemotherapy, tumor-induced bone loss, cancer-related bone loss, hormonal ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, facial bone loss associated with the disease, cranial bone loss associated with the disease , bone loss of the jaw asoc related to the disease, bone loss of the skull associated with the disease, bone loss associated with aging, facial bone loss associated with aging, bone loss cranial associated with aging, bone loss of the jaw associated with aging, bone loss of the skull associated with aging and bone loss associated with space travel.
In some embodiments, the subject optionally suffers (or has suffered) a cancer. The term "cancer" refers to a proliferative disorder associated with uncontrolled cell proliferation, uncontrolled cell growth and decreased cell death / apoptosis. Cancer includes, but is not limited to, breast cancer, prostate cancer, lung cancer, kidney cancer, thyroid cancer, melanoma, follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, which include, without limitation a, colon cancer, heart tumors, pancreatic cancer, retinoblastoma, glioblastoma, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, Kaposi's sarcoma, ovarian cancer, leukemia (which includes acute leukemia (for example, acute lymphocytic leukemia, acute myelocytic leukemia, including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemia (for example, chronic myelocytic leukemia (granulocytic) and leukemia chronic lymphocytic), myelodysplastic syndrome polycythemia vera, lymphomas (for example, Hodgkin's disease and disease non-Hodgkin disease), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain diseases and solid tumors that include, but are not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma , synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medicodenocarcinoma carcinoma , renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilm tumor, cervical cancer, microcytic lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pine Aloma, hemangioblastoma, acoustic neuroma, oligodendroglioma and menangioma. The term "metastasis" and the term "cancer metastasis" are used interchangeably herein to refer to the ability of a cancer cell to spread to other tissues. For example, "bone metastases" refers to the ability of certain types of cancer that include, but are not limited to, breast, prostate, lung, kidney, thyroid and melanoma, to metastasize to the bone.
In some embodiments, the subject optionally suffers from an osteolytic disorder. The term "osteolytic disorder", as used herein, refers to any condition caused by an increase in the activity of osteoclasts, which are cells responsible for bone resorption. The term "osteolysis" and the term "osteolytic bone loss" are used interchangeably to refer to osteoclast-mediated bone resorption or loss of bone mass associated with an osteolytic disorder. Osteolytic disorders occur in subjects with a predisposition to develop an osteolytic disorder, or occur in subjects with a disease that leads to or contributes to an osteolytic disorder by stimulating the activity of osteoclasts. In some
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embodiments, the osteolytic disorder is the loss of osteolytic bone mass. In other embodiments, the osteolytic disorder is the loss of osteolytic bone mass induced by cancer metastases. In further embodiments, osteolytic bone disorder is a metabolic bone disease, which includes, but is not limited to, endocrinopathies (eg, hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism and hyperthyroidism); dietary deficiency, which includes, without limitation, rickets, osteomalacia, scurvy and malnutrition; osteoporosis; use of drugs, including glucocorticoids (glucocorticoid-induced osteoporosis), heparin and alcohol; chronic disease, which include malabsorption syndromes; chronic renal failure, which includes renal osteodystrophy; chronic liver disease, which includes hepatic osteodystrophy; inherited disease, which includes osteogenesis imperfecta and homocystinuria; and bone inflammation associated with arthritis, rheumatoid arthritis, psoriatic arthritis, fibrous dysplasia, periodontal disease and Paget's disease.
The terms "osteolytic bone loss induced by metastases" and "osteolytic bone loss induced by cancer metastases" are used interchangeably herein to refer to osteolysis or loss of osteolytic bone mass caused by cancer cell metastases in cancer cells. bones. The term "osteoclast activation induced by cancer metastases" is used herein to refer to the ability of cancer cells that metastasized in the bone to induce osteoclast activation.
The anti-sclerostin antibody is preferably administered to a subject in a physiologically acceptable composition (eg, pharmaceutical), which may include vehicles, excipients or diluents. It should be noted that the anti-sclerostin antibody described herein can be used in the preparation of a medicament for administration using any of the dosage and dosage regimens disclosed herein. Pharmaceutical compositions and methods of treatment are disclosed in US Patent Publication No. 20050106683. "Physiologically acceptable" refers to molecular entities and compositions that do not produce a similar allergic or adverse reaction when administered to a being. human. In addition, the composition administered to a subject may contain more than one sclerostin inhibitor (for example, two anti-sclerostin antibodies, or an anti-sclerostin antibody and a synthetic chemical sclerostin inhibitor) or an anti-sclerostin antibody together with one or more therapeutic agents with different mechanisms of action.
The development of dosage and treatment regimens suitable for using the particular compositions described herein in a variety of treatment regimens, including, for example, administration and subcutaneous, oral, parenteral, intravenous, intranasal and intramuscular formulation and formulation are well known. in the art and are described in U.S. Patent Publication No. 20070110747. For example, in certain circumstances, it would be desirable to administer a pharmaceutical composition comprising an anti-sclerostin antibody subcutaneously, parenterally, intravenously, intramuscularly or even intraperitoneal. Such approaches are well known to the expert, some of which are described in more detail, for example, in US Pat. Nos. 5,543,158, US 5,641,515 and US 5,399,363. Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous dispersions or solutions and sterile powders for extemporaneous preparation of sterile injectable dispersions or solutions (for example, see US Patent 5,466,468). In all cases, the form must be sterile and fluid in order to easily load it into a syringe.
In one embodiment, for parenteral administration in an aqueous solution, the solution should be adequately buffered if necessary and first, the liquid diluent should be made isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, a dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermoclysis fluid or injected at the proposed infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th ed ., Mack Pub. Co., Easton, PA, pp. 1035-1038 and 1570-1580). There may be some variation of the dosage and the frequency of administration depending on the condition of the subject being treated, the age, height, weight and general health of the patient, and the existence of some side effect. In addition, a pharmaceutical composition comprising an anti-sclerostin antibody can be included in packages (eg, vials), together with the packaging material that provides instructions regarding the use of said pharmaceutical compositions. In general, said instructions will include a tangible expression describing the concentration of reagent, as well as in certain embodiments, relative amounts of excipient ingredients or diluents (for example, water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.
The methods described herein comprise administering an amount of an anti-sclerostin antibody. The antibody inhibits the biological activity of sclerostin in bones, as measured by changes in bone mineralization, bone density, effect on osteoblasts and / or osteoclasts, markers of bone formation, markers of bone resorption , markers of osteoblast activity and / or markers of osteoclast activity.
The anti-sclerostin antibody specifically binds to sclerostin or parts thereof to block or prevent the binding of human sclerostin to one or more ligands. Sclerostin, the product of the SOST gene, is absent in sclerosteosis, a skeletal disease characterized by excessive growth of strong dense bones and bones (Brunkow et al., Am. J. Hum. Genet., (58: 577-589 (2001); Balemans et al., Hum. Mol. Genet.,
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10: 537-543 (2001)). The amino acid sequence of human sclerostin has been described by Brunkow et al., And is disclosed in U.S. Patent Publication No. 20070110747 as SEQ ID NO: 1. Recombinant human sclerostin / SOST is marketed by R&D Systems (Minneapolis, Minn., USA; 2006, Catalog No. 1406-ST-025). Additionally, recombinant mouse sclerostin / SOST is marketed by R&D Systems (Minneapolis, Minn., USA; 2006 Catalog No. 1589-ST-025). Research grade sclerostin-binding monoclonal antibodies are marketed by R&D Systems (Minneapolis, Minn., USA; mouse monoclonal: 2006 Catalog No. MAB1406; rat monoclonal: 2006 Catalog No. MAB1589). US-6,395,511 and US-6,803,453, and U.S. Patent Publications No. 20040009535 and 20050106683 generally refer to anti-sclerostin antibodies. Examples of anti-sclerostin antibodies suitable for use in the context of the invention are also described in United States Patent Publications No. 20070110747 and 20070072797. Additional information regarding materials and methods for generating anti antibodies can be found. -Sclerostin in United States Patent Publication No. 20040158045.
The term "antibody" refers to an intact antibody, or a binding fragment thereof. An antibody may comprise a complete antibody molecule (immunoglobulin) (which includes the polyclonal, monoclonal, chimeric, humanized and / or human versions with heavy and / or light chains of full length) or may comprise an antigen-binding fragment of this . Antibody fragments include F (ab ') 2, Fab, Fab', Fv, Fc and Fd fragments, and can be incorporated into single domain antibodies (eg, nanobodies), single chain antibodies, maxybodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, for example, Hollinger and Hudson, Nature Biotechnology, 23 (9): 1126-1136 (2005)). Antibody polypeptides, which include fibronectin polypeptide monobodies, are also disclosed in U.S. Patent No. 6,703,199. Other antibody polypeptides are disclosed in United States Patent Publication No. 20050238646. Patents US-6,395,511 and US-6,803,453, and United States Patent Publications No. 20040009535 and 20050106683 are They generally refer to anti-sclerostin antibodies. The amino acid sequence of human sclerostin is set forth in SEQ ID No. 1 of the sequence listing and is provided as SEQ ID NO: 1 of U.S. Patent Publication No. 20070110747. Sclerostin is also described in Brunkow et al., Am. J. Hum. Genet., (68: 577-589 (2001); and in Balemans et al., Hum. Mol. Genet., 10: 537-543 (2001). Additional information regarding the materials and methods to generate can be found anti-sclerostin antibodies in U.S. Patent Publication No. 20040158045.
An antibody fragment can be any synthetic or genetically modified protein. For example, antibody fragments include isolated fragments consisting of the variable region of the light chain, "Fv" fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules where the variable regions Heavy and light are connected by a peptide linker (scFv proteins).
Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDR) of an antibody. CDRs (also called "minimum recognition units" or "hypervariable region") can be obtained by constructing polynucleotides encoding the CDR of interest. Such polynucleotides are prepared, for example, using the polymerase chain reaction to synthesize the variable region using mRNA from the cells that produce antibodies as template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology , 2: 106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et el., (Eds.), Page 166, Cambridge University Press (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies" in Monoclonal Antibodies: Principies and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)).
Anti-sclerostin antibodies may bind sclerostin of SEQ ID NO: 1, or a naturally occurring variant thereof, with an affinity (Kd) of less than or equal to 1 x 10-7 M, less than or equal at 1 x 10-8 M, less than or equal to 1 x 10 M, less than or equal to 1 x 10 M, less than or equal to 1 x 10 M or less than or equal to 1 x 10 M. Affinity It is determined using a variety of techniques, an example of which is an affinity ELISA. In several embodiments, affinity is determined by a BIAcore assay. In several embodiments, affinity is determined by a kinetic method. In several embodiments, affinity is determined by an equilibrium / solution method. US Patent Publication No. 20070110747 contains an additional description of the affinity assays suitable for determining the affinity (Kd) of an antibody for sclerostin.
Anti-sclerostin antibodies for use in the methods described herein preferably modulate the function of sclerostin in the cell-based assay described in U.S. Patent Publication No. 20070110747 and / or in the assay in vivo described in United States Patent Publication No. 20070110747 and / or bind to one or more epitopes described in United States Patent Publication No. 20070110747 and / or cross-block the binding of one of the antibodies described in U.S. Patent Publication No. 20070110747 and / or are cross-blocked at their binding to sclerostin by one of the antibodies described in U.S. Patent Publication No.
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20070110747.
In some or any of the embodiments, the anti-sclerostin antibody binds to a sclerostin polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 1 and binds to the sequence of SEQ ID NO: 6 (CGPARLLPNAIGRGKWWRPSGPDFRC; which corresponds to amino acids 86-111 of SEQ ID NO: 1). Alternatively or additionally, the anti-sclerostin antibody binds to a sclerostin polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 1 and binds to the sequence of at least one of SEQ ID NO: 2 (DVSEYSCRELHFTR; corresponding to amino acids 51-64 of SEQ ID NO: 1), SEQ ID NO: 3 (SAKPVTELVCSGQCGPAR; corresponding to amino acids 73-90 of SEQ ID NO: 1), SEQ ID NO: 4 (WWRPSGPDFRCIPDRYR; corresponding to amino acids 101-117 of SEQ ID NO: 1), SEQ ID NO: 5 (LVASCKCKRLTR; corresponding to amino acids 138-149 of SEQ ID NO: 1), SeQ ID NO: 70 (SAKPVTELVCSGQC; corresponding to amino acids 73-86 of SEQ ID NO: 1), SEQ ID NO: 71 (LVASCKC, which corresponds to amino acids 138-144 of SEQ ID NO: 1), SEQ ID NO: 72 (C1RELHFTR, which corresponds to amino acids 57 -64 of SEQ ID NO: 1), or SEQ ID NO: 73 (CiPdRYR, which corresponds to amino acids 111-117 of SEQ ID NO: 1) within SEQ ID NO: 1. For example, in one aspect, the antibody to nti-sclerostin binds to a sclerostin subregion of SEQ ID NO: 1 comprising SEQ ID NOs: 2-5 (and / or SEQ ID NOs: 70-73), optionally in its native three-dimensional conformation. Optionally, the anti-sclerostin antibody binds to a peptide consisting of one or more of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID No: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73 (for example, a peptide consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 or a peptide consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73).
In some or any of the embodiments, the anti-sclerostin antibody binds to a sclerostin polypeptide having the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SeQ ID NO: 5, in which SEQ ID NO: 2 and 4 are linked by a disulfide bond at amino acid positions 57 and 111 with reference to SEQ ID NO: 1, and SEQ ID NO: 3 and 5 are joined by at least one of (a) a disulfide bond at amino acid positions 82 and 142 with reference to SEQ ID NO: 1, and (b) a disulfide bond at amino acid positions 86 and 144 with reference to SEQ ID NO: 1 ; the polypeptide can retain the tertiary structure of the corresponding polypeptide region of human sclerostin of SEQ ID NO: 1. Alternatively or additionally, the sclerostin binding agent (eg, anti-sclerostin antibody) binds to a polypeptide which has the amino acid sequences of SEQ ID NO: 70, SEQ ID No: 71, SEQ ID NO: 72 and SEQ ID NO: 73, in which SEQ ID NO: 72 and 73 are linked by a disulfide bond in the amino acid positions 57 and 111 with reference to SEQ ID NO: 1, and SEQ ID NO: 70 and 71 are joined by at least one of (a) a disulfide bond at amino acid positions 82 and 142 with reference to SEQ ID NO: 1, and (b) a disulfide bond at amino acid positions 86 and 144 with reference to SEQ ID NO: 1.
In several aspects, the anti-sclerostin antibody is capable of neutralizing human sclerostin in a mineralization assay based on MC3T3 cells when there is less than a 6-fold molar excess of sclerostin binding sites per well compared to the amount of moles of sclerostin per well. Mineralization by cells of the osteoblast lineage in the culture, either of primary cells or cell lines, is used as an in vitro model of bone formation. An example of a cell-based mineralization assay is described in US Patent Publication No. 20070110747 in, for example, Example 8. MC3T3-E1 cells (Sudo et al., J. Cell Biol., 96: 191-198 (1983)) and subclones of the original cell line can form minerals in the cultures by growing in the presence of differentiation agents. Such subclones include MC3T3-E1-BF (Smith et al., J. Biol. Chem., 215: 19992-20001 (2000)). Sclerostin can inhibit one or more of the sequences of events that lead to, and include, a mineral deposition (i.e., sclerostin inhibits mineralization), both for the MC3T3-E1-BF subclone and for the original MC3T3-E1 cells . Antisclerostin antibodies that are capable of neutralizing sclerostin inhibitory activity allow the mineralization of the culture in the presence of sclerostin so that there is a statistically significant increase in, for example, calcium phosphate deposition (measured as calcium ) compared to the amount of calcium measured in the sclerostin-only treatment group (i.e., without antibody).
When the assay is carried out for the purpose of determining whether a particular anti-sclerostin antibody can neutralize sclerostin, the amount of sclerostin used in the assay is desirably the minimum amount of sclerostin that causes at least a 70% reduction, Statistically significant, in the deposit of calcium phosphate (measured as calcium) in the sclerostin-only group, compared to the amount of calcium measured in the group without sclerostin. A neutralizing anti-sclerostin antibody is defined as one that causes a statistically significant increase in calcium phosphate deposition (measured as calcium) compared to the amount of calcium measured in the sclerostin-only treatment group (i.e., without antibody ). To determine whether an anti-sclerostin antibody is neutralizing or not, the amount of anti-sclerostin antibody used in the assay needs to be such that there is a molar excess of sclerostin binding site per well compared to the number of moles of sclerostin per well. Depending on the potency of the antibody, the excess that may be needed may be 24, 18, 12, 6, 3, or 1.5 times, and one skilled in the art is familiar with the routine practice of testing more than one concentration of the binding agent (antibody). For example, a very potent anti-sclerostin neutralizing antibody will neutralize sclerostin when there is less than a 6-fold molar excess of sclerostin binding sites per well compared to the number of
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moles of sclerostin per well. A less potent anti-sclerostin neutralizing antibody will neutralize sclerostin only in an excess of 12, 18 or 24 times.
The anti-sclerostin antibody optionally has an IC50 of 100 nM or less, or 75 nM or less, or 50 nM or less, or 25 nM or less to neutralize human sclerostin in a cell-based assay, such as a phosphatase assay. bone-specific alkaline, for example, the bone-specific alkaline phosphatase assay described in International Patent Publication No. WO 2008/115732 and US-7,744,874. The bone-specific alkaline phosphatase assay is based on the ability of sclerostin to decrease levels of alkaline phosphatase stimulated by BMP-4 and Wnt3a in the multipotential murine cell line, C2C12. According to WO 2008/115732, a neutralizing anti-sclerostin antibody mediates a dose-dependent increase in alkaline phosphatase activity in this assay. Examples of protocols from cell-based assays are given in Example 1.
Alternatively or additionally, the anti-sclerostin antibody has an IC50 of 100 nM or less (eg, 75 nM or less, or 50 nM or less) to neutralize human sclerostin in a Wnt signaling assay based on cells in HEK293 cell lines, such as the Wnt assay involving a Wntl-mediated induction of the STF reporter gene described in, for example, International Patent Publication No. WO 2009/047356. Alternatively or additionally, the anti-sclerostin antibody has an IC50 of 500 nM or less (e.g., 250 nM or less, 150 nM or less, 100 nM or less, or 50 nM or less) to neutralize human sclerostin in a BMP2 induced mineralization test in MC3T3 cells, such as the mineralization test described in, for example, International Patent Publication No. WO 2009/047356. An example protocol is provided in Example 1.
Examples of anti-sclerostin antibodies suitable for use in the context of the invention are described in US Patent Publication No. 20070110747 and 20070072797. In one embodiment of the invention, the anti-sclerostin antibody cross-blocks the binding of at least one of the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab- 7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 (all of which are described in United States Patent Publication 20070110747) to sclerostin. Alternatively or additionally, the anti-sclerostin antibody is cross-blocked at its binding to sclerostin by at least one of the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab- 2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23 and Ab-24 (all of which are described in the Patent Publication of the United States No. 20070110747). The terms "cross-block" and "cross-block" are used interchangeably herein to indicate the ability of an antibody to interfere with the binding of other antibodies to sclerostin. The extent to which one antibody is capable of interfering with the binding of another to sclerostin can be determined and therefore, if it can be said to cross-block, using competitive binding assays. In some aspects of the invention, an antibody or cross-blocking fragment thereof reduces the binding of the sclerostin of a reference antibody between about 40% and about 100%, such as about 60% and about 100%, specifically between 70% and 100%, and more specifically between 80% and 100%. A quantitative assay particularly suitable for detecting cross-block uses uses a Biacore machine that measures the extent of interactions using surface plasmon resonance technology. Another suitable quantitative blocking assay uses an ELISA-based approach to measure competitiveness among antibodies in terms of their binding to sclerostin.
Examples of suitable anti-sclerostin antibodies and fragments thereof include antibodies and antibody fragments having one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and cDr-l3 specifically described in US Patent Publication No. 20070110747. At least one of the regions of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 may have at least one substitution of amino acid, as long as the antibody retains the binding specificity of the unsubstituted CDR. Preferably, the anti-sclerostin antibody is Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab- 7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, or Ab-24 of United States Patent Publication No. 20070110747.
In addition, the anti-sclerostin antibody may comprise at least one CDR sequence that has at least 75% identity (e.g., 100% identity) with a CDR selected from SEQ iD NOs: 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 78, 79, 80, 81, 99 , 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,
247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,
270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,
293, 294, 295, 296, 297, 298, 351, 352, 353, 358, 359, and 360 provided in the Sequence Listing and disclosed in No. 20070110747. Preferably, the anti-sclerostin antibody comprises at least one sequence of CDRs that have at least 75% identity with a CDR selected from SEQ ID NOs: 245, 246, 247, 78, 79, 80, 269, 270, 271, 239, 240, and 241, all of which are provided in the Sequence Listing and are described in United States Patent Publication No. 20070110747. As described in United States Patent Publication No. 20070110747, the anti-sclerostin antibody may comprise:
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a) CDR sequences of SEQ ID NOs: 54, 55 and 56 and CDR sequences of SEQ ID NOs: 51, 52 and 53; b) CDR sequences of SEQ ID NOs: 60, 61 and 62 and CDR sequences of SEQ ID NOs: 57, 58 and 59; c) CDR sequences of SEQ ID NOs: 48, 49 and 50 and CDR sequences of SEQ ID NOs: 45, 46 and 47; d) CDR sequences of SEQ ID NOs: 42, 43 and 44 and CDR sequences of SEQ ID NOs: 39, 40 and 41; e) CDR sequences of SEQ ID NOs: 275, 276 and 277 and CDR sequences of SEQ ID NOs: 287, 288 and 289; f) CDR sequences of SEQ ID NOs: 278, 279 and 280 and CDR sequences of SEQ ID NOs: 290, 291 and 292; g) CDR sequences of SEQ ID NOs: 78, 79 and 80 and CDR sequences of SEQ ID NOs: 245, 246 and 247; h) CDR sequences of SEQ ID NOs: 81, 99 and 100
and CDR sequences of SEQ ID NOs: 248, 249 and 250; i) CDR sequences of SEQ ID NOs:
CDR sequences of SEQ ID NOs: 251 CDR sequences of SEQ ID NOs: 254, 255 CDR sequences of SEQ ID NOs: 257, 258 CDR sequences of SEQ ID NOs: 260, 261 CDR sequences of SEQ ID NOs: 293, 294 CDR sequences of SEQ ID NOs: 263 CDR sequences of SEQ ID NOs: 296 CDR sequences of SEQ ID NOs: 266 CDR sequences of SEQ ID NOs: 269 CDR sequences of SEQ ID NOs: 272,
j) CDR sequences of SEQ ID NOs:
k) CDR sequences of SEQ ID NOs:
l) CDR sequences of SEQ ID NOs:
m) CDR sequences of SEQ ID NOs:
n) CDR sequences of SEQ ID NOs:
o) CDR sequences of SEQ ID NOs:
p) CDR sequences of SEQ ID NOs:
q) CDR sequences of SEQ ID NOs:
270 and 271; r) CDR sequences of SEQ ID NOs: 242;
273 and 274; or s) CDR sequences of SEQ ID NOs: 351
252 and 253; and 256; and 259; and 262; and 295 and 265 and 298 and 268
264
297
267
101,
104,
107,
110,
281,
113,
284,
116,
239,
102
105
108
111
282
114
285
237
240
243
352
103
106
109
112
283
115
286
238
241
244
and 353
CDR sequences of SEQ ID NOs: 358, 359 and 360.
The anti-sclerostin antibody may also comprise at least one CDR sequence that has at least 75% identity (eg, 100% identical) to a CDR selected from CDR-H1, CDR-H2, CDR-H3, CDR- L1, CDR-L2, and CDR-L3 in which CDR-H1 has the sequence given in SEQ ID NO: 245, CDR-H2 has the sequence given in SeQ ID NO: 246, CDR-H3 has the sequence given in SEQ ID NO: 247, CDR-L1 has the sequence given in SEQ ID NO: 78, CDR-L2 has the sequence given in SEQ ID NO: 79 and CDR-L3 has the sequence given in SEQ ID NO: 80, all of which they are provided in the Sequence Listing and are described in United States Patent Publication No. 20070110747. The anti-sclerostin antibody, in several respects, comprises two of the CDRs or six of the CDRs. Optionally, the anti-sclerostin antibody comprises heavy chains comprising SEQ ID NO: 378 and light chains comprising SEQ ID NO 376.
The anti-sclerostin antibody may also comprise at least one CDR sequence that has at least 75% identity (eg, 100% identical) to a CDR selected from CDR-H1, CDR-H2, CDR-H3, CDR- L1, CDR-L2, and CDR-L3 in which CDR-H1 has the sequence given in SEQ ID NO: 269, CDR-H2 has the sequence given in SEQ ID NO: 270, CDR-H3 has the sequence given in SEQ ID NO: 271, CDR-L1 has the sequence given in SEQ ID NO: 239, CDR-L2 has the sequence given in SEQ ID NO: 240 and CDR-L3 has the sequence given in SEQ ID NO 241, all of which provided in the Sequence Listing and are described in United States Patent Publication No. 20070110747. The anti-sclerostin antibody, in various aspects, comprises at least two of the CDRs or six of the CDRs.
Alternatively, the anti-sclerostin antibody may have a heavy chain comprising H1, H2 and H3 of the CDR and comprising a polypeptide having the sequence provided in SEQ ID NO: 137 or a variant thereof in which said CDRs are at least 75% identical (eg, 100% identical) to SEQ ID N °: 245, 246 and 247, respectively, and a light chain comprising L1, l2 and L3 of the CDR and comprising a polypeptide having the sequence provided in SEQ ID NO: 133 or a variant thereof in which said CDRs are at least 75% identical (eg, 100% identical) to SEQ ID NO: 78, 79 and 80, respectively (as described in United States Patent Publication No. 20070110747).
The anti-sclerostin antibody may have a heavy chain comprising H1, H2 and H3 of the CDR and comprising a polypeptide having the sequence provided in SEQ ID NO: 145 or 392 or a variant thereof in which said CDR they are at least 75% identical (e.g., 100% identical) to SEQ ID N °: 245, 246 and 247, respectively, and a light chain comprising L1, L2 and L3 of the CRD and comprising a polypeptide that has the sequence provided in SEQ ID NO: 141 or a variant thereof in which said CDRs are at least 75% identical (eg, 100% identical) to SEQ ID NO: 78, 79 and 80, respectively (as described in United States Patent Publication No. 20070110747).
The anti-sclerostin antibody may have a heavy chain comprising H1, H2 and H3 of the CDR and comprising a polypeptide having the sequence provided in SEQ ID NO: 335, 331, 345 or 396 or a variant of any of the above in which said CDRs are at least 75% (for example, 100% identical) identical to SEQ ID NO: 269, 270 and 271, respectively, and a light chain comprising L1, L2 and L3 of CRD and comprising a polypeptide having the sequence provided in SEQ ID NO: 334 or 341 or a variant of any of the above in which said CDRs are at least 75% identical (eg, 100% identical) to SEQ ID NO: 239 , 240 and 241, respectively (as described in US Patent Publication No. 2007011074). All combinations of the heavy and light chain sequences are contemplated (for example, heavy chains comprising SEQ ID NO: 335 and light chains comprising SEQ ID NO: 334; heavy chains comprising SEQ ID NO: 331 and light chains comprising SEQ ID NO: 334 or 341; and heavy chains comprising SEQ ID NO: 345 or 396 and light chains comprising SEQ ID
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NO: 341).
Alternatively, the anti-sclerostin antibody has a heavy chain comprising a polypeptide having the sequence provided in SEQ ID NO: 137, and a light chain comprising a polypeptide that
it has the sequence provided in SEQ ID NO: 133; a heavy chain comprising a polypeptide that
it has the sequence provided in SEQ ID NO: 145 or 392, and a light chain comprising a polypeptide having the sequence provided in SEQ ID NO: 141; a heavy chain comprising a polypeptide having the sequence provided in SEQ ID NO: 335, and a light chain comprising a polypeptide that
it has the sequence provided in SEQ ID NO: 334; a heavy chain comprising a polypeptide that
it has the sequence provided in SEQ ID NO: 331, and a light chain comprising a polypeptide that
it has the sequence provided in SEQ ID NO: 341; or a heavy chain comprising a polypeptide having the sequence provided in SEQ ID NO: 345 or 396, and a light chain comprising a polypeptide having the sequence provided in SEQ ID NO: 341 (as described in Publication U.S. Patent No. 2007011074).
Examples of anti-sclerostin antibodies also include, but are not limited to, the anti-sclerostin antibodies disclosed in International Patent Publications Nos. WO 2008/092894, WO 2008/115732, WO 2009/056634, WO 2009/047356, WO 2010/100200, WO 2010/100179, WO 2010/115932 and WO 2010/130830, such as an anti-sclerostin antibody comprising the CDRs of SEQ ID NOs: 20-25 of International Patent Publication No. WO 2008 / 115732 (SEQ iD NOs: 416-421 herein), an anti-sclerostin antibody comprising the CDRs of SEQ ID NOs: 26-31 of International Patent Publication No. WO 2008/115732 (SEQ iD NOs: 422-427 herein), an anti-sclerostin antibody comprising the CDRs of SEQ ID NOs: 32-37 of International Patent Publication No. WO 2008/115732 (SEQ ID NOs: 428-433 herein) memory), an anti-sclerostin antibody comprising the CDRs of SEQ ID NOs: 4, 15, 26, 37, 48 and 59 of the Publication of Pa International Patent No. WO 2009/047356 (SEQ ID NOs: 443, 454, 465, 476, 487 and 498, respectively, herein), or an anti-sclerostin antibody comprising the amino acid sequence of at least one of SeQ ID NOs: 135-143, 153-161, or 171-179 of International Patent Publication No. WO 2010/130830 (SEQ ID NOs: 745-753, 763-771, 781-789, respectively, In the present memory).
The activity of an anti-sclerostin antibody for use in the methods described herein can be measured in several ways, including the methods described above to detect increases in bone mineral content or bone density. The ability of an anti-sclerostin antibody to modulate bone mass can be calculated from body weights or using other methods (see Guinness-Hey, Metab. Bone Dis. Relat. Res., 5: 177-181 (1984)) . Animals and models of particular animals are used in the art to evaluate the effect of pharmaceutical compositions and methods for, for example, the parameters of bone loss, bone resorption, bone formation, bone resistance or bone mineralization. Examples of such models include the ovariectomized rat model (Kalu, Bone and Mineral, 15: 175-192 (1991); Frost and Jee, Bone and Mineral, 18: 227-236 (1992); and Jee and Yao, J. Musculoskel. Neuron. Interact., 1: 193-207 (2001)).
Alternatively, an anti-sclerostin antibody can be selected based on its ability to modulate bone marker levels. Bone markers are products created during the bone remodeling process and are released by bones, osteoblasts and / or osteoclasts. Fluctuations in bone resorption and / or "marker" levels of bone formation imply changes in bone remodeling / modeling. The International Osteoporosis Foundation (IOF) recommends using bone markers to control bone density therapies (see, for example, Delmas et al., Osteoporos Int., Suppl. 6: S2-17 (2000). Indicative markers of bone resorption (or osteoclast activity) includes, for example, C-telopeptide (eg, terminal C terminal telopeptide of type 1 collagen (CTX) or serum bound C-telopeptide), N-telopeptide (telopeptide of the N-terminal end of type 1 collagen (NTX)), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline, galactosyl hydroxylysine and tartrate resistant acid phosphatase (eg, tartrate-resistant serum acid phosphatase, isoform 5b). Bone formation / mineralization include, but is not limited to, bone-specific alkaline phosphatase (BSAP), type I procollagen extension peptides released from the N and C end (PlNP, PICP), and osteocalcin (OstCa). Several kits have been used to detect and quantify markers in clinical samples, such as urine and blood.
Several routes of administration of an anti-sclerostin antibody to a subject are known in the art, and are described, for example, in US Patent Publication No. 20070110747. For example, in several embodiments, it is desirable to administer a pharmaceutical composition comprising the anti-sclerostin antibody subcutaneously, parenterally, intravenously, intramuscularly or even intraperitoneally. Such approaches are known to the expert, some of which are described in more detail, for example, in patents US 5,543,158, US 5,641,515 and US 5,399,363. Physiologically acceptable (for example, pharmaceutical) forms for use include sterile aqueous dispersions or solutions and sterile powders for the extemporaneous preparation of sterile injectable dispersions or solutions (for example, see US Patent 5,466,468). The shape must be sterile and is desirably fluid so that it can be easily included in a syringe (i.e., it is not excessively viscous to prevent passage through a syringe). A pharmaceutical composition comprising the anti-sclerostin antibody can be included in packages (for example,
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vials or syringes), together with the packaging material that provides instructions regarding the use of said pharmaceutical compositions. In general, said instructions will include a tangible expression describing the concentration of reagent, as well as in certain embodiments, relative amounts of excipient or diluent ingredients (eg, water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.
The treatment of a pathology by combining two or more agents that target the same pathogen or biochemical pathway sometimes results in greater efficacy and fewer side effects with respect to the use of the therapeutically relevant dose of each agent separately. In some cases, the effectiveness of the drug combination is additive (the effectiveness of the combination is approximately equal to the sum of the effects of each drug separately), but in other cases the effect may be synergistic (the effectiveness of the combination is greater than the sum of the effects of each drug administered separately). As used herein, the term "combination therapy" means that the two components can be administered simultaneously, for example, concurrently or in which one of the compounds is administered first, followed by the second agent, for example, sequentially. The desired result may be either a subjective relief of one or more symptoms or an objectively identifiable improvement in the recipient of the dosage.
According to this disclosure, the anti-sclerostin antibody is administered in combination with the use of materials that promote new bone growth, such as bone graft, bone dust, bone fragments, demineralized bone matrix, bone scaffolds, prostheses, metal stabilizers or Bone scaffolding substances comprising one or more polymers, ceramic materials, cement and graft substitutes based on calcium phosphates. Many variations of such materials are known in the art.
In some embodiments, the anti-sclerostin antibody is administered together with a second bone strengthening therapeutic agent, useful for the treatment of decreased bone mineral density or bone defect. In some embodiments, the bone strengthening therapeutic agent is selected from the group consisting of an antiresorptive drug, a bone-forming agent, an estrogen receptor antagonist (including, but not limited to, raloxifene, bazedoxifene and lasofoxifene) and a drug that has a inhibitory effect on osteoclasts. In some embodiments, the antiresorptive drug includes, but is not limited to, a bisphosphonate (which includes, but is not limited to, alendronate, risedronate, ibandronate and zoledronate), an estrogen or estrogen analog, a RANK antiligand antibody (RANKL) or RANKL inhibitor. , vitamin D or a vitamin D derivative or mimic thereof, a selective estrogen receptor modulator (SERM) and a source of calcium, tibolone, calcitonin, a calcitriol and hormone replacement therapy. In some embodiments, the bone strengthening agent includes, but is not limited to, parathyroid hormone (PTH) or a peptide fragment thereof, PTH-related protein (PTHrp), bone morphogenic protein, osteogenin, NaF, a PGE2 agonist, a statin, strontium ranelate, an antibody or anti-DKKl inhibitor. In some embodiments, the bone strengthening agent is Forteo® (teriparatide), Preotact®, or Protelos®.
The invention is also described in the following examples. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
Examples
Example 1
This Example describes several cell-based neutralization assays that are useful for characterizing the neutralization activity of an anti-sclerostin antibody.
Mineralization test based on MC3T3 cells - Ascorbic acid and B-glycerophosphate are used to induce differentiation of MC3T3-E1-BF cells that leads to mineral deposition. An example of a detection protocol, in a 96-well format, involves sowing cells on day 1, followed by seven media changes over a period of 12 days, with the majority of mineral deposition taking place in the last eighteen hours. The specific time and extent of the mineral deposition may vary depending, in part, on the particular serum lot number used. Control experiments will allow such variables to be taken into account, as is generally known in the cell culture experimentation technique. For statistical analyzes (using MS Excel and JMP) a 1-way ANOVA can be used followed by Dunnett's comparison to determine the differences between the groups. The group means for each data set are considered significantly different when the P value is less than 0.05 (P <0.05).
Cell culture for the expansion of MC3T3-E1-BF cells is performed as follows. The cell culture is carried out at 37 ° C and with 5% CO2. A cell bank can be generated to search for the existence of sclerostin neutralizing antibodies. A vial of frozen MC3T3-E1-BF cells was thawed by stirring in a 37 ° C water bath. Thawed cells are placed in 10 ml of expansion medium (Alpha-MEM / 10% FBS / PenStrepGlu) in a 50 ml tube and gently centrifuged for 5 minutes. The cells are resuspended in 4 ml of Alpha-MEM / 10% FBS / PenStrepGlu. After determining the number
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of cells using trypan blue and a hemacytometer, 1 x 106 cells are seeded in 50 ml of Alpha-MEM / 10% FBS / PenStrepGlu medium in a T175 flask.
When this passage is confluent (at approximately 7 days), the cells are trypsinized with trypsin / EDTA (0.05% trypsin; 0.53 mM EDTA), gently centrifuged for 5 minutes and then resuspended in 5 ml of Alpha-MEM / 10% FBS / PenStrepGlu. After determining the number of cells using trypan blue and a hemacytometer, 1 x 106 cells are seeded in 50 ml of 10% Alpha-MEM / FBS / PenStrepGlu medium for each T175 flask. The number of T175 flasks used for planting at this point depends on the total number of available cells and the desired number of flasks that should be taken to the next passage.
When this passage is confluent (approximately 3-4 days), the cells are trypsinized with trypsin / EDTA (0.05% trypsin; 0.53 mM EDTA), gently centrifuged for 5 minutes and then resuspended in 5 ml of Alpha-MEM / 10% FBS / PenStrepGlu. After determining the number of cells using trypan blue and a hemacytometer, 1 x 106 cells are seeded in 50 ml of 10% Alpha-MEM / FBS / PenStrepGlu medium per T175 flask. The number of T175 flasks used for planting at this point depends on the total number of available cells and the desired number of flasks that should be taken to the next passage.
When this passage is confluent (approximately 3-4 days), the cells are trypsinized with trypsin / EDTA (0.05% trypsin; 0.53 mM EDTA), gently centrifuged for 5 minutes and then resuspended in 5 ml of Alpha-MEM / 10% FBS / PenStrepGlu. After determining the number of cells using trypan blue and a hemacytometer, 1 x 106 cells are seeded in 50 ml of 10% Alpha-MEM / FBS / PenStrepGlu medium per T175 flask. The number of T175 flasks used for planting at this point depends on the total number of available cells and the desired number of flasks that were taken to the next passage. Extra cells are frozen at a rate of 1-2 x 106 living cells / ml in 90% FBS / 10% DMSO.
When this passage is confluent (approximately 3-4 days), the cells are trypsinized with trypsin / EDTA (0.05% trypsin; 0.53 mM EDTA), gently centrifuged for 5 minutes and then resuspended in 5 ml of Alpha-MEM / 10% FBS / PenStrepGlu. After determining the number of cells using trypan blue and a hemacytometer, the cells are frozen at a rate of 1-2 x 106 living cells / ml in 90% FBS / 10% DMSO. This "final passage" of frozen cells is the passage used for the screening test.
The cell culture for mineralization of MC3T3-E1-BF cells is performed as follows. The cell culture is carried out at 37 ° C and with 5% CO2. It is desirable to minimize the temperature and% of CO2 fluctuations during the cell culture mineralization procedure. An appropriate number of "final passage" vials prepared as described above are thawed by stirring in a 37 ° C water bath. Thawed cells are placed in 10 ml of expansion medium (Alpha-MEM / 10% FBS / PenStrepGlu) in a 50 ml tube and gently centrifuged for 5 minutes. The cells are resuspended in 4 ml of Alpha-MEM / 10% FBS / PenStrepGlu. After determining the number of cells using trypan blue and a hemacytometer, 2500 cells are seeded in 200 microliters of expansion medium per well in 96-well plates covered with collagen I (Becton Dickinson Labware, Cat. No. 354407).
The following is an example of a cell culture procedure. It is indicated that the day on which cells begin to be plated on plaques is a Wednesday. If a different day of the week is used as the starting day, that day will be the reference day of the daily schedule for the removal and addition of media throughout the process as indicated below. For example, if the cells are sown on a Tuesday, the medium should not be removed and added on the first Friday and Saturday, nor on the second Friday and Saturday. If you start on a Tuesday, the plates are prepared for the calcium test on the last Sunday. The cells are sown on Wednesday at the rate of 2500 cells in 200 pl of expansion medium. On Thursday the entire expansion medium is removed and 200 pl of differentiation medium are added. On Friday, 100 pl of medium are removed and 100 pl of freshly prepared differentiation medium are added. On Monday, 100 pl of medium are removed and 100 pl of freshly prepared differentiation medium are added. On Tuesday, 100 pl of medium are removed and 100 pl of freshly prepared differentiation medium are added. On Wednesday, 100 pl of medium are removed and 100 pl of freshly prepared differentiation medium are added. On Thursday, 100 pl of medium are removed and 100 pl of freshly prepared differentiation medium are added. On Friday, 100 pl of medium are removed and 100 pl of freshly prepared differentiation medium are added. The following Monday, plates for the calcium assay are prepared as follows: The plates are washed once with 10 mM Tris, HCl pH 7-8. 200 pl of 0.5 N HCl per well are added while working under an extraction hood. The plates are then frozen at -80 ° C. Just before measuring calcium, the plates are frozen-thawed twice, and then crushing with a multichannel pipette is used to disperse the contents of the plate. The contents of the plate are then allowed to settle at 4 ° C for 30 minutes, at which time an appropriate amount of supernatant is removed to measure calcium, using a commercialized calcium kit. An example and non-limiting kit is Calcium (CPC) Liquicolor, Cat. No. 0150-250, Stanbio Laboratory, Boerne, TX.
In this cell-based assay, sclerostin inhibits one or more of the sequence of events that lead to and include mineral deposition (i.e., sclerostin inhibits mineralization). Therefore, in experiments where sclerostin is included in the particular cell culture experiment, recombinant sclerostin is added to the medium starting on the first Thursday and thereafter every feeding day. In cases where
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an anti-sclerostin antibody is analyzed to see if it can neutralize sclerostin, that is, if it allows mineralization by neutralizing the ability of sclerostin to inhibit mineralization, the antibody is added to the medium starting on the first Thursday and thereafter every day of feeding. The antibody is previously incubated with the recombinant sclerostin in the differentiation medium for 45-60 minutes at 37 ° C and then this medium is used to feed the cells.
A 12-day mineralization protocol for MC3T3-E1-BF cells has been previously described. The mineralization of the original MC3T3-E1 cells is inhibited by recombinant sclerostin and this inhibition is blocked using an anti-sclerostin neutralizing antibody, for example, an anti-sclerostin antibody comprising the CDRs of SEQ ID NO: 245-247 and 78-80. The cell-based neutralization assay is further described in U.S. Patent Publication No. 7,592,429 in, for example, Example 8.
Bone specific alkaline phosphatase assay - An example of a bone specific alkaline phosphatase assay 10 is described in International Patent Publication No. WO 2008/115732 and in US Patent 7,744,874. The following is an example protocol. C2C12 cells (ATCC, CRL 1772) are plated at 3000-5000 cells / well in a 96-well tissue culture plate in MEM medium supplemented with 5% bovine fetal serum. The plate is incubated at 37 ° C in 5% CO2 overnight. The antibody is diluted in 0.5X Wnt3a conditioned medium (prepared as described in WO 2008/115732) until several final concentrations are achieved. The medium is removed from the seeded cells and a solution of premixed anti-sclerostin-BMP4 antibody (human or mono-cynomolgous) (150 pl) is added, which provides a final antibody concentration of 30 pg / ml at 0.5 pg / ml, a final BMP-4 concentration of 25 ng / ml, a final sclerostin protein concentration of 1.0 pg / ml and the conditioned medium is at a 0.5X concentration. The plate is then incubated at 37 ° C for 72 hours in 5% CO2. The medium is removed from the cells, which are washed once with PBS, and frozen and thawed three times alternating between -80 ° C and 37 ° C. Alkaline phosphatase activity is measured by adding alkaline phosphatase substrate (PNPP 1 stage, Pierce No. 37621) (150 pl / well). The cell plate is incubated for 60 minutes at room temperature, at which time the optical density (OD) is measured at 405 nm to determine the activity of the alkaline phosphatase. The IC50 calculations can be performed using, for example, the SigmaPlot Regression Wizard with an adjustment equation of 4 sigmoid parameters.
BMP2-induced MC3T3 cell mineralization test - An example of BMP2-induced mineralization test on MC3T3 cells is described in International Patent Publication No. WO 2009/047356. Briefly, MC3T31b cells are seeded in 96-well plates (for example, 6 x 103 cells / well or 2 x 103 cells / well) in 100 pl of test culture medium (maintenance culture medium without G418) and are incubate for three days to reach confluence. The test culture medium is changed and the compounds to be analyzed with 10 mM b-glycerophosphate and 50 pM ascorbic acid are added. Before addition to the cells, sclerostin and a candidate antibody are previously incubated in a separate plate for two hours at room temperature. 2.1 or 2.8 nM BMP-2 (R&D Systems, Cat. No. 355-BM-010) is applied to the 96-well plates of the assay before applying the sclerostin-antibody mixture. The cells are incubated for 14 days. At the end of the incubation, the cells are washed twice with 200 μl of PBS / well, 50 μl of 0.5 M HCl are added to each well, and the plates are frozen at -20 ° C for a minimum of 24 hours . The plates are thawed at room temperature for 2 hours for analysis. Ten 10 pl of each well is transferred to a new plate and exposed to a working calcium solution (1: 5) (200 pl). The optical density is measured after an incubation period of 5-30 minutes at 595 nm in a microplate reader. The absorbance is translated into micrograms of calcium according to a standard curve, which allows the determination of the degree of mineralization induced by BMP-2.
Cell-based wnt signaling assay - An example of a cell-based signaling assay using the super top flash indicator protein (STF) is described in International Patent Publication No. WO 2009/047356. HEK293 cells are transfected with pcDNA3 + (480 ng); SuperTopFlash (STF) (20 ng); and phRL-CMV (0.5 ng) for control wells and pcDNA-wntl (20 ng); pcDNA3 + (460 ng); SuperTopFlash (STF) (20 ng); and phRL-CMV (0.5 ng) for Wntl treatment wells. The plasmids are mixed 10 with 1.6 pl of lipofectamine 2000 diluted in 50 pl of OptiMEM® and incubated for 30 minutes at room temperature before application to the cells. Once applied, the cells are incubated at 37 ° C for five hours in 5% CO2.
The antibodies are previously mixed with SOST to generate a series of dilutions. One ml of medium is prepared for each dilution, and 450 pl are added to each well after removing the transfection mixture. The cells are incubated with the antibody-SOST mixtures for 18-20 hours. At the end of the incubation, the medium is removed and 300 µl of 1X passive lysis buffer (Promega, Cat No. E194A) is added to lyse the cells. Next, luciferase activity is measured using the Dual-Glo luciferase system (Promega, No. Cat E2940) with 30 pl of lysates in duplicates. Normally 30 pl of Dual-Glo luciferase substrates (firefly luciferase; for STF) and 30 pl of Dual-Glo Stop and Glo (Renilla luciferase) substrates are used for the control of transfection efficiency. The luminescence signals are measured with the Mithras LB940 instrument (Berthold Technologies). The ratio of firefly and Renilla luciferases is calculated. The final results are expressed by setting the Wntl value without SOST as 1. Additional details of the test are provided in International Patent Publication No. WO 2009/047356.
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Example 2
This Example illustrates the ability of a sclerostin inhibitor, namely, a monoclonal anti-sclerostin antibody (Scl-Ab), to treat a bone space defect in a primate subject. This example also illustrates that treatment with a sclerostin inhibitor, specifically an anti-sclerostin monoclonal antibody (Scl-Ab), increased the area and cortical thickness for a period of 28 weeks without inducing adverse effects, such as an increase in cortical porosity in the bones of primate subjects.
Segmental defects (0.5 cm space size) were created in the middle of the left ulna in 26 cynomolgus monkeys (males, aged 4-4.5 years). After surgery, the left arm is immobilized with fiberglass throughout the period of the experiment. The animals were separated into two treatment groups: Group A (n = 10) and Group B (n = 16). The monkeys were injected subcutaneously weekly with a vehicle (Group A) or with Scl-Ab (Group B), at a dose of 30 mg / kg, starting immediately after surgery and continued for 28 weeks after surgery .
The animal's body weight was measured at week 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. X-rays of the left arm were taken immediately after surgery and at week 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 28. Tetracycline (a bone marker), calcein (fluorescent green ink staining the calcification tissues) and alizarin (ink fluorescent red staining the calcification tissues) at weeks 6-7.5, 18-19.5 and 26-27.5 respectively. Histomorphometric analysis of the femoral shaft without fracture after 28 weeks of treatment was analyzed.
At week 28, only 1 in 10 (10%) of the A (control) monkeys had completely bridged the defect. On the contrary, only 6 of 16 (38%) of the monkeys of Group B (Scl-Ab treatment group) had completely bypassed the defect. These results show that the inhibition of sclerostin by Scl-mAb can fill a gap or space between bone segments to successfully treat a bone space defect in humans.
The results indicated that treatment with Scl-Ab increased the area d
and cortical thickness in the femoral shaft at week 28 in the treatment animals. See Figures 2A and 2B. The monkeys who had received the treatment with Scl-mAb also showed a higher rate of endocortical and periodic bone formation. See Figures 3A and 3B. It should be noted that treatment with Scl-mAb over a period of 28 weeks did not significantly increase cortical porosity in the femoral shaft compared to control monkeys. See Figure 4.
The combined results described in this Example demonstrate that treatment with Scl-Ab is not only useful for the treatment of skeletal defects, such as bone space defects, but also that the administration of Scl-Ab for a period greater than 6 months increases bone formation and bone mass without producing adverse effects, such as an increase in cortical porosity. The increase in cortical porosity is associated with an increased risk of fractures and a decrease in bone strength.

权利要求:
Claims (15)
[1]
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1. An effective amount of an anti-sclerostin antibody for use in a method of treating a bone space defect in a mammalian subject, wherein said antibody is a sclerostin inhibitor, said method comprising administering the antibody to the subject, in wherein the anti-sclerostin antibody is administered during a treatment period lasting at least 20 weeks, and in which the bone space defect comprises a space between two bone segments of at least 5 mm.
[2]
2. The anti-sclerostin antibody for use in the method of claim 1, wherein the anti-sclerostin antibody is administered at a weekly dose of 1 mg / kg to 50 mg / kg per week.
[3]
3. The anti-sclerostin antibody for use in the method of claim 1, wherein the treatment period lasts 28 weeks.
[4]
4. The anti-sclerostin antibody for use in the method of claim 1, wherein the bone space defect is selected from the group consisting of a comminuted fracture, an unconsolidated fracture, a segmental skeletal defect, created bone defects surgically, surgically treated bone defects and bone defects created from a traumatic bone injury or disease (including arthritis, developmental deformity, tumor removal (resection) or infection removal).
[5]
5. The anti-sclerostin antibody for use in the method of claim 4, wherein the bone space defect is caused by the removal of infected bone sections or the removal of bone cancer.
[6]
6. The anti-sclerostin antibody for use in the method of claim 1, wherein the method further comprises administering a second therapeutic bone strengthening agent selected from the group consisting of parathyroid hormone, a bisphosphonate, an anti-RANKL antibody and an anti-DKK-1 antibody.
[7]
7. The anti-sclerostin antibody for use in the method of any one of claims 1-6, wherein the anti-sclerostin antibody is administered:
(a) in an amount of 30 mg / kg per week; I
(b) once a week for the duration of the treatment period.
[8]
8. The anti-sclerostin antibody for use in the method of any one of claims 1-7, wherein treatment with the anti-sclerostin antibody does not result in a substantial increase in cortical porosity in the subject's bone. .
[9]
9. The anti-sclerostin antibody for use in the method of any one of claims 1-8, wherein the anti-sclerostin antibody is administered subcutaneously.
[10]
10. The anti-sclerostin antibody for use in the method of any one of claims 1-9, wherein the anti-sclerostin antibody is an immunoglobulin comprising a heavy chain and a light chain.
[11]
11. The anti-sclerostin antibody for use in the method of any one of claims 1-9, wherein the anti-sclerostin antibody is an antibody or fragment thereof that demonstrates a sclerostin binding affinity of SEQ ID. NO: 1 less than or equal to 1 x 10-7 M.
[12]
12. The anti-sclerostin antibody for use in the method of any one of claims 1-11, wherein the anti-sclerostin antibody binds to a sclerostin polypeptide comprising the amino acid sequence indicated in SEQ ID NO : 1, in which:
(a) said anti-sclerostin antibody binds to the sequence of SEQ ID NO: 6;
(b) said anti-sclerostin antibody binds to the sequence of at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or
(c) said anti-sclerostin antibody binds to the sequence of at least one of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72 or SEQ ID NO: 73.
[13]
13. The anti-sclerostin antibody for use in the method of any one of claims 1-11, wherein the anti-sclerostin antibody cross-blocks the binding of at least one of the Ab-A, Ab-B antibodies, AbC, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23 and Ab- 24 to sclerostin and / or is cross-blocked at its binding to sclerostin by at least one of the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab -3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab- 13, Ab-14, Ab-15 , Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24, in which antibodies Ab-A to Ab-D and Ab-1 through Ab-24 have the heavy and light chain sequences identified in Figure 1.
[14]
14. The anti-sclerostin antibody for use in the method of any one of claims 1-11, wherein the anti-sclerostin antibody comprises a CDR-H1 of SEQ ID NO: 245, a CDR-H2 of SEQ ID NO: 246, a CDR-H3 of SEQ ID NO: 247, a CDR-L1 of SEQ ID NO: 78, a CDR-L2 of SEQ ID NO: 79 and a CDR-L3 of SEQ ID NO: 80.
5
[15]
15. The anti-sclerostin antibody for use in the method of claim 14, wherein the anti-sclerostin antibody:
(a) comprises heavy chains comprising SEQ ID NO: 378 and light chains comprising the
10 SEQ ID NO 376; or
(b) it has heavy chains of SEQ ID NO: 145 or SEQ ID NO: 392 and light chains of SEQ ID NO: 141.

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NO2739311T3|2018-07-21|

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PL2739311T3|2018-08-31|

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AU2012290083A1|2014-03-13|

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EP2739311A1|2014-06-11|

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US10538584B2|2020-01-21|

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JP2017214374A|2017-12-07|

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US20140271654A1|2014-09-18|

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PT2739311T|2018-03-26|

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JP2014521692A|2014-08-28|

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US20200115444A1|2020-04-16|

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EP2739311B9|2018-09-19|

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JP2021183610A|2021-12-02|

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WO2013019954A1|2013-02-07|

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JP2019167348A|2019-10-03|

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CA2842432A1|2013-02-07|

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JP6301832B2|2018-03-28|

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DK2739311T3|2018-04-23|

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EP2739311B1|2018-02-21|

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SI2739311T1|2018-07-31|

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LT2739311T|2018-06-11|

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ES2667554T3|2018-05-11|

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MX2014001416A|2014-10-17|

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HRP20180736T1|2018-06-29|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201161515191P| true| 2011-08-04|2011-08-04|

---

US201161515191P|2011-08-04|

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PCT/US2012/049331|WO2013019954A1|2011-08-04|2012-08-02|Method for treating bone gap defects|

---