CN101370511B

CN101370511B - Activin-ActRlla antagonists and uses for promoting bone growth

Info

Publication number: CN101370511B
Application number: CN200680051538.9A
Authority: CN
Inventors: J·克诺普夫; J·西拉
Original assignee: Acceleron Pharma Inc
Current assignee: Acceleron Pharma Inc
Priority date: 2005-11-23
Filing date: 2006-11-22
Publication date: 2015-07-01
Anticipated expiration: 2026-11-22
Also published as: CN101370511A; UA98447C2; ZA200805408B; SI1973559T1; CN112457389A

Abstract

In certain aspects, the present invention provides compositions and methods for promoting bone growth and increasing bone density.

Description

The application of Activin-ActRIIa antagonist and promotion bone growth thereof

Related application

This application claims the provisional application number submitted on November 23rd, 2005 is 60/739,462, the provisional application number submitted on March 17th, 2006 is 60/783,322, in JIUYUE, 2006 No. 15 provisional application number submitted to are 60/844, the benefit of priority of 855, above-mentioned application is incorporated to the application by reference to entirety.

Background technology

Skeletal diseases, comprises from osteoporosis to fracture, represents one group of pathological state, rarely can effectively for the pharmaceutical preparation of these pathological states.The substitute is, the focus for the treatment of concentrates on and comprises on solidification, motion and metatrophia physics and behavioral intervention.For the various skeletal diseases for the treatment of, the healing potion promoting bone growth and increase skeleton density is had to be useful.

Bone growth and mineralising depend on the activity of osteoblast and this two types cell of osteoclast, although chondrocyte and vascular cell also take part in the critical aspects of these processes.Development ground, skeleton is formed and is occurred by two kinds of mechanism, and endochondral ossification and intramembranous ossification, the former is responsible for stringer bone formation, and the latter is responsible for the formation of topological flat bone as skull skeleton.Endochondral ossification requirement order is formed and the degraded of cartilage structure in growth plate, the formation of described growth plate as osteoblast, osteoclast, vascular cell and the template of mineralising subsequently.In intramembranous ossification process, skeleton is directly formed in connective tissue.This two kinds of osteoblastic infiltrations of schemes call and follow-up apposition.

Fracture and other skeletal structure are broken and to be healed by a kind of process of similar osteogenetic growth order at least on the surface, and described osteogenetic growth comprises cartilaginous tissue and formed and mineralising subsequently.The process of union of fracture can occur in two ways.Direct or initial bone healing there is no callus formation.Indirectly or the bone healing of secondary there is callus precursor phases.The initial stage of fracture heals the transformation related to across the mechanical connection broken of closely arranging.Under suitable conditions, show tunnel around the bone absorption cell broken and absorb response, and build the path of puncture vessel and healing subsequently.The order that the secondary healing of skeleton reconstructs with inflammatory reaction, soft callus formation, callus mineralising and callus is carried out.In the inflammatory reaction stage, hematoma and hemorrhage formation come from the periosteum of damage position and breaking of perimyelis blood vessel.Inflammatory cell invades this region.At cartilage crust formation stages, the blood vessel that Hemapoiesis is new, fibroblast, intracellular organic matter and sustenticular cell, the gap between fracture fragmentation forms granulation tissue.Set up by fibrous or cartilaginous tissue (cartilage crust) across the clinical healing broken.Osteoblast is formed and regulates the mineralising of cartilage crust, then described cartilage crust replace by lamellar bone and be limited to normal restructuring procedure.

Except fracture and skeletal structure other physical disruption except, the loss of bone mineral content and bone density can be caused by multiple different condition and may cause important medical problem.The change of bone density betides individual whole life period in relatively expected mode.Approximately to when 30 years old, the linear growth that the skeleton of masculinity and femininity is coiled by endochondral ossification growth and radial growth grow to the biggest quality.Such as, such as, about 30 years old (for spongy bone, flat bone such as vertebra and pelvis) and 40 years old (for cortical bone, the long bone in limbs), all there is bone loss slowly in masculinity and femininity.In women, the essence skeleton that also there is final stage is lost, and is perhaps because post menopausal lacks estrogen.During this period, women additionally may lose the quality of 10% on cortical bone, and the quality of extra loss mistake 25% on spongiosa interval.Whether progressive bone mass loss causes pathological condition such as osteoporosis depend critically upon individual initial bone quality and whether there is the situation increased the weight of.

Bone loss sometimes with the imbalance of normal bone restructuring procedure for feature.Healthy skeleton is often limited to reconstruct.Reconstruct and absorbed beginning with skeleton by osteoclast.Then absorbed skeleton replace by new skeletal tissue, it is characterized in that osteoblast forms collagen and calcification subsequently.In the individuality of health, absorption and the speed formed are balances.Osteoporosis is a kind of chronic, progressive situation, with the transfer towards absorption for labelling, causes comprehensive minimizing and the mineralization of skeleton of bone mass.The osteoporosis of human body is prior to clinical osteopenia (bone mineral density is greater than a standard deviation but is less than two standard deviations below the average of young adult).In world wide, roughly 75 thousand ten thousand people have osteoporotic risk.

Therefore, the method controlling osteoclast and osteoblast activity is effective to the treatment promoting healing of fracture and other damage of skeleton and disease (as osteoporosis, relevant to the loss of bone mass and mineralization of skeleton).

About osteoporosis, estrogen, calcitonin, Bone Gla protein and vitamin K, or the dietary calcium of heavy dose is all as therapeutic intervention.Other osteoporotic therapy approach comprises salt and the sodium fluoride of biphosphonate, parathyroid hormone, Sensipar (calcimimetic), Statins, lanthanum and strontium.But these Therapeutic Method are associated with adverse side effect usually.

Therefore, the object of this invention is to provide the compositions promoting bone growth and mineralising and method.

Summary of the invention

Partly, present invention demonstrates that the molecular energy with activin (activin) or activin IIA receptor (ActRIIa) antagonist activities (" activin antagonist " and " ActRIIa antagonist ") is enough in increase skeleton density, promote bone growth, and/or increase bone strength.Especially, present invention demonstrates that the inhibitor of the soluble form of ActRIIa as activin-ActRIIa signal, and the skeleton density promoting in vivo to increase, bone growth and bone strength.But, the pharmaceutical preparation that most promotion bone growths or suppression are lost bone or as anti-catabolism preparation (usually also relating to as " catabolism preparation ") (as, biphosphonate) or as anabolism preparation (as, parathyroid hormone, PTH, when appropriate dosage), the ActRIIa albumen display double activity of solubility, has catabolism and anabolic effect.Therefore, the present invention establishes, and the antagonist of activin-ActRIIa signal path may be used for increasing skeleton density and promoting bone growth.But the ActRIIa of solubility can affect skeleton by the mechanism except activin antagonism, however, present invention demonstrates that desirable therapeutic agent can be selected according to activin-ActRIIa antagonist activities.Therefore, in some embodiments, invention provides and use the method for activin-ActRIIa antagonist to treat the disease (as osteoporosis) be associated with low skeleton density or low bone strength or the bone growth promoting the patient's (as in the patient fractured) having this to need, described activin-ActRIIa antagonist comprises such as activin in conjunction with IIA receptor (activin-binding ActRIIa) polypeptide, anti-activin antibody, anti-ActRIIa antibody, the micromolecule of activin targeting or ActRIIa targeting and aptamer, and reduce the nucleic acid of activin and ActRIIa expression.In addition, the ActRIIa polypeptide of solubility promotes bone growth and does not cause lasting measurable increase of muscle quality.

In some aspects, the invention provides the polypeptide comprising activin-binding ActRIIa polypeptide that is solvable, that be bonded to activin.ActRIIa polypeptide can be made into the pharmaceutical preparation comprising activin-binding ActRIIa and pharmaceutically acceptable carrier.Preferably, activin-binding ActRIIa polypeptide is to be less than 1 micromole or to be less than the K of 100,10 or 1 nanomoles _dbe bonded to activin.Optionally, relative to GDF11 and/or GDF8, activin-binding ActRIIa optionally in conjunction with activin, preferably, the K of activin _dat least than the K of GDF 11 and/or GDF8 _dlow 10 times, 20 times or 50 times.But do not wish to be bound by specific mechanism of action, be contemplated that the Selection effect that activin suppresses the selectance higher than GDF11/GDF8 to describe on skeleton can not cause lasting measurable effect on muscle.In some embodiments, select to cause lower than 15%, lower than 10% or obtain the dosage of desirable effect lower than the ActRIIa that increases of muscle of 5% on skeleton.Preferably, the purity of compositions is at least 95%, about other polypeptide moiety, is determined by chi exclusion chromatography, and more preferably, the purity of compositions is at least 98%.The activin-binding ActRIIa polypeptide used in such preparation can be any polypeptide disclosed in this invention, be selected from as having SEQ ID Nos:2,3,7, the polypeptide of the aminoacid sequence of 12 or 13, or be selected from SEQID Nos:2,3, the aminoacid sequence of 7 or 12 has the polypeptide of at least 80%, 85%, 90%, 95%, 97% or 99% homogeneity.Activin-binding ActRIIa polypeptide can comprise the functional fragment of natural ActRIIa, is selected from least 10,20 or 30 aminoacid (" tail ") of the sequence of SEQ ID NOs:1-3 or the sequence of SEQ ID Nos:2 (lacking 10 to 15 aminoacid of C-terminal) as comprised.

Relative to naturally occurring ActRIIa polypeptide, solubility, activin-binding ActRIIa polypeptide can comprise the change (such as at ligand binding domains) of one or more aminoacid sequence.The example of altered ActRIIa polypeptide is provided in the 59-60 page of WO 2006/012627, is incorporated to by reference herein at this.The change of aminoacid sequence can be, such as, when changing the glycosylation of polypeptide when mammal, insecticide or other eukaryotic cell are produced, or changes the proteolytic cleavage of polypeptide relative to naturally occurring ActRIIa polypeptide.

Activin-binding ActRIIa polypeptide can be fusion rotein, and it has a domain (such as the ligand binding moiety of ActRIIa polypeptide) of ActRIIa polypeptide and one or more domain providing desirable characteristic (as the pharmacokinetics improved, easier purification, targeting are specifically organized) in addition.Such as, fusion rotein domain can reinforcement internal stability, Half-life in vivo, absorption/use, one or more in tissue positioned or distribution, the formation of protein complex, the multimerization of fusion rotein and/or purification.Activin-binding ActRIIa fusion rotein can comprise the polypeptide portion that immunoglobulin Fc domain (wild type or mutant) or serum albumin or other provide desirable characteristics (stability of the pharmacokinetics such as improved, the solubility of improvement or improvement).In one preferred embodiment, ActRIIa-Fc fusion comprises relative destructuring connexon, and it is between Fc domain and ActRIIa ectodomain.This non-structured row key word can correspond to roughly 15 aminoacid destructuring regions of the C-terminal end points (" tail ") of ActRIIa ectodomain, or it can be 1,2,3,4 or 5 amino acid whose artificial sequence, or 5 with 15,20,30,50 or more the relative length freely between aminoacid of secondary structure, or both mixture.Connexon can be rich in glycine and proline residue, and passable, such as, containing threonine/serine and the simple sequence of glycine or repetitive sequence (such as, the TG of threonine/serine and glycine ₄or SG ₄singlet or repetitive sequence).Fusion rotein can comprise the subsequence of purification, as epitope tag, FLAG label, polyhistidine sequence and GST merge.Optionally, the activin of solubility comprises one or more amino acid residue being selected from the modification of lower group in conjunction with IIA receptor polypeptide: glycosylated aminoacid, polyethyleneglycol modified (PEGylated) aminoacid, the aminoacid of farnesylation (farnesylated), acetylizad aminoacid, biotinylated aminoacid, be coupled the aminoacid of liposomal fraction, be coupled the aminoacid of organic derivative preparation.Pharmaceutical preparation also can comprise the compound that one or more additional compound is such as used for the treatment of skeletal diseases.Preferably, pharmaceutical preparation is without pyrogen substantially.Usually, preferably ActRIIa albumen is expressed in mammal cell line, and described cell line regulates ActRIIa albumen Natively glycosylated with the probability of the adverse immune response eliminating patient suitably.Human cell line and Chinese hamster ovary celI system are successfully applied, and expect that other common mammalian expression systems is also useful.

As described in the present invention, the ActRIIa-Fc that ActRIIa albumen is specified has desirable characteristic, comprises relative to GDF8 and/or GDF11 optionally in conjunction with activin, is greater than the serum half-life of two weeks in the high-affinity of ligand binding and animal model.In certain embodiments, the invention provides ActRIIa-Fc polypeptide and comprise the pharmaceutical preparation of such polypeptide and pharmaceutically acceptable excipient.

In some aspects, the invention provides the nucleic acid of encoding soluble activin-binding ActRIIa polypeptide.The polynucleotide be separated can comprise the coded sequence of all solubility activin-binding ActRIIa polypeptide as described above.Such as, the nucleic acid be separated can comprise the ectodomain of ActRIIa (such as, ligand binding domains) coded sequence, and the sequence of the cytoplasmic domain of coded portion or whole membrane spaning domains and/or ActRIIa, but in membrane spaning domain or in cytoplasmic domain or between ectodomain and membrane spaning domain or cytoplasmic domain termination codon of not encoding.Such as, the polynucleotide be separated can comprise total length ActRIIa polynucleotide sequence such as SEQ ID NO:4 or 5, or the sequence of partial cutaway, described polynucleotide be included in 3 ' end further before at least 600 nucleotide or other the translation of polynucleotide that makes cause ectodomain to be fused to the translation stop codon of the position of the total length ActRIIa of part truncate randomly.Preferred nucleotide sequence is SEQ IDNO:14.Nucleic acid disclosed in this invention is operably connected to the promoter for expressing, and the invention provides the cell having transformed such recombination of polynucleotide.Preferably, described cell is mammalian cell, such as Chinese hamster ovary celI.

In some aspects, the invention provides the method preparing solubility activin-binding ActRIIa polypeptide.Such method can be included in suitable cell (such as Chinese hamster ovary (CHO) cell) and express any nucleic acid disclosed in this invention (such as, SEQ ID NO:4,5 or 14).Such method can comprise: a) cultured cell is to express the ActRIIa polypeptide of solubility under appropriate conditions, and wherein said cell transformation has the expression constructs of solubility ActRIIa; And soluble ActRIIa polypeptide b) expressed by renaturation.Soluble ActRIIa polypeptide can as natural, partially purified or highly purified fragment renaturation.Purification can be completed by a series of purification step, comprise such as, one in described below, two or three or multiple, in any order: albumen affinity chromatograph (protein A chromatography), anion-exchange chromatography are (such as, Q agarose), hydrophobic chromatography (such as, phenyl sepharose), size exclusion chromatography and cation-exchange chromatography.

In some aspects, activin-ActRIIa antagonist disclosed in this invention, the activin-binding ActRIIa polypeptide of such as solubility, may be used for a kind of by the method promoting bone growth or increase for the purpose of skeleton density.In some embodiments, the invention provides the method for the treatment of low skeleton density relevant disease or promoting bone growth in the patient having this demand.The method can comprise the activin-ActRIIa antagonist to there being the object of this demand to use effective dose.In some aspects, the invention provides the medicine using activin-ActRIIa antagonist preparation treatment disease recited above or situation.

In some aspects, the invention provides the method differentiating the medicine stimulating bone growth or increase mineralization of skeleton.Described method comprises: a) differentiate the medicine to be measured 1 being bonded to the ligand binding domains of activin or ActRIIa polypeptide; B) effect of medicine on bone growth or mineralising is assessed.

Accompanying drawing explanation

Fig. 1 shows the purification of ActRIIa-hFc expressed in Chinese hamster ovary celI.The albumen of purification is the unimodal of a good peak shape.

Fig. 2 shows ActRIIa-hFc and is bonded to activin and GDF11, is analyzed detect by BiaCore.

Fig. 3 shows the schematic diagram of A-204 reporter gene assays.The figure illustrates report carrier: pGL3 (CAGA) 12 (having description in EMBO 17:3091-3100, Dennler etc., 1998).CAGA12 motif appears in TGF-Beta reactive group (PAI-1 gene), and therefore this carrier is generally used for the factor by Smad 2 and 3 signal path.

Fig. 4 shows ActRIIa-hFc (rhombus) and ActRIIa-mFc (square) effect on GDF-8 signal in A-204 reporter gene assays.Two kinds of albumen all show its signal almost suppressing GDF-8 to regulate completely at picomolar concentrations.

Fig. 5 to show in A-204 reporter gene assays three kinds of different ActRIIa-hFc preparation effects on GDF-11 signal.

Fig. 6 show the BALB/c mouse of matched group and ActRIIa-mFc process before the treatment phase of 12 weeks (above) and rear (below) the example of DEXA (dual-energy X-ray absorption mensuration) image.Light-colored part shows the skeleton density added.

Fig. 7 shows ActRIIa-mFc quantitative to the effect of bone mineral density of BALB/c mouse 12 weeks treatments period.Treatment is divided into the ActRIIa-mFc (circle) of contrast (rhombus), the ActRIIa-mFc (square) of 2mg/kg dosage, the ActRIIa-mFc (triangle) of 6mg/kg dosage and 10mg/kg dosage.

Fig. 8 shows ActRIIa-mFc quantitative to the effect of bone mineral content of BALB/c mouse 12 weeks treatments period.Treatment is divided into the ActRIIa-mFc (circle) of contrast (rhombus), the ActRIIa-mFc (square) of 2mg/kg dosage, the ActRIIa-mFc (triangle) of 6mg/kg dosage and 10mg/kg dosage.

Fig. 9 shows ActRIIa-mFc to effect quantitative of C57BL6 mice Grafting Cancellous Bone Bolt mineral density of extracing ovary (OVX) or sham-operation more than 6 weeks.Treatment is divided into the ActRIIa-mFc (ActRIIa) of contrast (PBS) or 10mg/kg dosage.

Figure 10 shows ActRIIa-mFc quantitative to the effect of spongy bone of the 12 weeks treatments period of (OVX) C57BL6 mice extracing ovary.Treatment is divided into the ActRIIa-mFc (ActRIIa, dark bars) of contrast (PBS, light bar) or 10mg/kg dosage.

Figure 11 shows ActRIIa-mFc quantitative to the effect of spongy bone of the C57BL6 mice 6 weeks of sham-operation or 12 weeks treatment after dates.Treatment is divided into the ActRIIa-mFc (ActRIIa, dark bars) of contrast (PBS, light bar) or 10mg/kg dosage.

Figure 12 shows the skeleton density pQCT analysis result of 12 weeks treatments period of (OVX) mice of extracing ovary.Treatment is divided into the ActRIIa-mFc (ActRIIa, dark bars) of contrast (PBS, light bar) or 10mg/kg dosage.Y-axis: mg/ccm

Figure 13 describes the skeleton density pQCT analysis result of mice 12 weeks treatments period of sham-operation.Treatment is divided into the ActRIIa-mFc (ActRIIa, dark bars) of contrast (PBS, light bar) or 10mg/kg dosage.Y-axis: mg/ccm

Figure 14 A and Figure 14 B shows the whole body DEXA treating after date (A) for 12 weeks and analyzes and the indirect In vivo analysis of femur (B).Clear zone describes the region of high skeleton density.

Between the femur stage casing that Figure 15 shows 12 weeks treatment after dates, pQCT analyzes in junctor.Treatment is divided into contrast (PBS, dark bars) and ActRIIa-mFc (light bar).Four bars in left side show four bar display cortical bone density on total skeleton density and right side.The first couple of every four bars represents the data that come from the mice of extracing ovary and second to representing the data coming from sham-operation mice.

Between the femur stage casing that Figure 16 shows 12 weeks treatment after dates, pQCT analyzes and key content in junctor.Treatment is divided into vehicle Control (PBS, dark bars) or ActRIIa-mFc (light bar).Four bars in left side show total skeleton content and four, right side bar shows cortical bone content.The first couple of every four bars represents the data that come from the mice of extracing ovary and second to representing the data coming from sham-operation mice.

Figure 17 to show between femur stage casing pQCT in junctor and analyzes and femoral cortex thickness.Treatment is divided into contrast (PBS, dark bars) or ActRIIa-mFc (light bar).Four bars in left side show four bars hour periosteum girth on perimyelis girths and right side.The first couple of every four bars represents the data that come from the mice of extracing ovary and second to representing the data coming from sham-operation mice.

Figure 18 describes the femur mechanics testing result of 12 weeks treatment after dates.Treatment is divided into contrast (PBS, dark bars) or ActRIIa-mFc (light bar).Two bars in left side represent the data that come from the mice of extracing ovary and two bars below represent the data coming from sham-operation mice.

Figure 19 shows the impact of ActRIIa-mFc on spongy bone volume.

Figure 20 shows the impact of ActRIIa-mFc on the cancellous bone structure of distal femur.

Figure 21 shows the impact of ActRIIa-mFc on cortical bone.

Figure 22 shows the impact of ActRIIa-mFc on skeletal mechanics intensity.

Figure 23 shows under three various dose, and the ActRIIa-mFc of various dose is on the impact of skeleton characteristic.

Figure 24 shows tectology measurement (histomorphometry) and shows that ActRIIa-mFc has dual anabolism and anti-absorbing activity.

Detailed Description Of The Invention

1. summarize

Transforming growth factor beta (TGF-beta) superfamily comprises and multiplely has identical consensus sequence unit and the somatomedin of structural motif.These albumen known are permitted eurypalynous cells play biological function to vertebrates and invertebrates.The member of superfamily performs critical function in the pattern formation and histo-differentiation of embryonic development period, and can multiple differentiation process be affected, comprise lipogenesis, muscle generation, Subchondral drilling, heart generation, hemoposieis, neural generation and epithelial cell differentiation.This family is divided into two general branches: BMP/GDF and TGF-beta/Activin/BMP10 branch, and its member has different, normally complementary effects.By handling the activity of TGF-beta family member, physiology's change important in organism usually can be caused.Such as, Piedmont (Piedmontese) and Belgian Blue (Belgian Blue) cattle breeds are with the sudden change of GDF8 (being also called flesh chalone (myostatin)) intragenic disappearance function, and it causes the remarkable growth of muscle quality.Grobet etc., Nat Genet.1997,17 (1): 71-4.In addition, at human body, increase and (it is said) anomaly intensity of the allelic inactivation of GDF8 and muscle quality are relevant.Schuelke etc., N Engl J Med 2004,350:2682-8.

Activin (activin) is the dimer polypeptide growth factor belonging to TGF-beta superfamily.Have the activin of three kinds of primitive forms (A, B and AB), it is two closely-related β subunit (β _aβ _a, β _bβ _band β _aβ _b) homology/heterodimer.Human genome is also encoded activin C and activin E, and it expresses at first in liver.In TGF-beta superfamily, activins can stimulate in ovary and in placenta cells, hormone produces, supports Neuronal Survival, dependent cells type positivity or negatively affect cell cycle progression and in Amphibian embryo, at least induce the only of mesodermal differentiation and the multi-functional factor (DePaolo etc., 1991, Proc Soc Ep Biol Med.198:500-512; Dyson etc., 1997, CurrBiol.7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963).In addition, the erythroid differentiation factor (EDF) being separated the human monocytic leukaemia's cell certainly stimulated is found identical with activin A (Murata etc., 1988, PNAS, 85:2434).Show activin A in medullary cavity as erythropoietic natural positive regulating factor.In some tissues, activin signal is by its relevant heterodimer, inhibin institute antagonism.Such as, when hypophysis release follicule-stimulating hormone (FSH) (FSH), activin promotes FSH secretion and synthesis, and inhibin stops FSH secretion and synthesis.Other scalable activin biological activity and/or the albumen being bonded to activin comprise follicostatin (FS), follicostatin associated protein (FSRP), alpha2-macroglobulin, Cerberus and Endoglin (endoglin).

TGF-signal beta is regulated by the different poly-complex of I type and II type serine/threonine kinase receptor, Smad albumen (the Massague in its phosphorylation and activation downstream by ligand activation, 2000, Nat.Rev.MoI.Cell Biol.1:169-178).These I types and II receptor are transmembrane proteins, form by with being rich in the ligand binding ectodomain of cysteine regions, membrane spaning domain and the specific cytoplasmic domain of serine/threonine with precognition.I type is that signal path is necessary; II type is binding partner and expresses needed for I receptor.I type and II type activin receptor form stable complex after ligand binding, cause I receptor by II receptor phosphorylation.

Two kinds of relevant II receptors, ActRIIa and ActRIIb, has been accredited as II receptor (Mathews and Vale, 1991, the Cell 65:973-982 of activin; Attisano etc., 1992, Cell 68:97-108).Except activin, ActRIIa and ActRIIb can biochemically with other several TGF-'beta ' family protein-interacting, comprise BMP7, Nodal, GDF8 and GDF11 (Yamashita etc., 1995, J.Cell Biol.130:217-226; Lee and McPherron, 2001, Proc.Natl.Acad.Sci.98:9306-9311; Yeo and Whitman, 2001, MoI.Cell 7:949-957; Oh etc., 2002, Genes Dev.16:2749-54).ALK4 is main activin I receptor, and particularly for activin A, and ALK-7 also can as the receptor of activins, particularly to activin B.

As the present invention prove, the ActRIIa polypeptide (sActRIIa) of solubility, which show relative to other TGF-beta family member such as GDF8 or GDF11 substantially prioritizing selection be bonded to activin A, can effectively promote in vivo bone growth and increase skeleton density.But do not wish to be limited to any specific mechanism, consider the combination (picomolar dissociation constants) that the activin that represented by the specific sActRIIa construction that adopts in these research is very strong, expect that the effect of sActRIIa is mainly caused by activin antagonist effect.Do not consider mechanism, data here clearly show, ActRIIa-activin antagonist adds skeleton density really in normal mouse and osteoporotic mouse model.Please note that skeleton is dynamic organization, the increase of its growth or atrophy and density or minimizing depend on the balance between the factor and destruction of producing skeleton and stimulation mineralising (mainly osteoblast) and the factor removing bone mineral (mainly osteoclast).The growth of skeleton and mineralising can be carried out simultaneously by increasing productivity factor or minimizing monkey wrench or both and increase.Term " promotion bone growth " and " increase mineralization of skeleton " refer to the visible change of physics of skeleton and are used for describing in skeleton the mechanism changing generation neutrally.

The osteoporotic mouse model adopted in research described in the invention and bone growth/density are considered to be that high predicted has effect in human body, thus, the invention provides and adopt ActRIIa polypeptide and other activin-ActRIIa antagonist in human body, promote bone growth and raising skeleton density.Activin-ActRIIa antagonist comprises, such as, activin is in conjunction with soluble ActRIIa polypeptide, be bonded to activin (particularly activinA or B subunit, also refer to β A or β B) and interrupt ActRIIa combine antibody, be bonded to ActRIIa and interrupt activin combine antibody, be selected from non-antibody protein that activin or ActRIIa combine (see such as, WO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989, US 2003/0133939, and the example of this albuminoid in US 2005/0238646 and the method for design and selection same protein), be selected from the random peptide being usually conjugated to Fc domain that activin or ActRIIa combines.Two kinds of different albumen (or other parts) with activin or ActRIIa binding activities, particularly block I type (such as respectively, soluble Type I activin receptor) or the activin zygote of II type (such as solubility II type activin receptor) binding site, can couple together to produce the molecule that bi-functional combines.The preparation of aptamer, micromolecule and other suppression activin-ActRIIa signal shaft.Various albumen has activin-ActRIIa antagonist activities, comprise inhibin (i.e. inhibin α subunit) (although inhibin not in a organized way in equal antagonism activin), follicostatin (as follicostatin 288 and follicostatin 315), Cerberus, follicostatin associated protein (FSRP), Endoglin (endoglin), activin C, α ₂-macroglobulin and M108A (becoming alanine from methionine at 108) suddenly change activin A.Normally, the alternative form of activin, particularly those are vicissitudinous at I receptor binding structural domain, can be bonded to II receptor and can not form active triple complex, thus as antagonist action.In addition, nucleic acid, such as antisense molecule, the siRNA suppressing activinA, B, C or E or (especially) ActRIIa to express or ribozyme, can be used as activin-ActRIIa antagonist.Preferably, the activin-ActRIIa antagonist adopted is by the selectivity of display relative to the suppression activin institute conditioning signal of other member (relative to GDF8 and GDF 11) of TGF-beta family.The ActRIIb of solubility is bonded to activin, but the albumen of wild type does not show the significant selectivity being bonded to activin relative to GDF8/11, and preliminary experiment display, thisly can not provide the desirable effect to skeleton, but still the remarkable growth of muscle can be caused.But, there is changing form of the ActRIIb of different binding characteristics identified (see such as, WO2006/012627,55-59 page, is incorporated to the present invention by way of reference), and these albumen can desirable effect on skeleton.ActRIIb that is natural or that change can provide the additional specificity to activin by being coupled the 2nd activin selective binding composition.

Usually there is in the special context that description term used is applied at context of the present invention and each term its its ordinary meaning in this area.Some term comes into question hereinafter or in the other parts of description, in order to describe the compositions and methods of the invention and how to apply the guidance being supplied to practitioner and adding.The scope of any application of term or implication are clearly in its special context applied.

" approximately " and " close " means the acceptable extent of error of quantity measure when considering the person's character of measurement method and precision usually.Typically, exemplary extent of error within 20% of given numerical value or numerical range, preferably within 10%, more preferably within 5%.

Or specifically in biology system, term " approximately " and " close " may mean the value in some levels, preferably within given numerical value 5 times, more preferably within 2 times.Except as otherwise noted, the given numerical value of the present invention is close, and mean when not expressly providing, term " approximately " or " close " can be inferred.

Method of the present invention can comprise the step of mutual compared sequences, and the sequence comprising wild type compares with one or more mutant (sequence variants).Such comparison generally includes the comparison of polymer sequences, such as, how adopt alignment programs well known in the art/or algorithm (such as, BLAST, FASTA and MEGALIGN, can say).Those skilled in the art easily recognize, in such comparison, one comprise insert or delete residue sudden change in, aligned sequences by do not comprise insertion or delete residue polymer sequences in produce " gap " (gap) (usually representing with dash or " A ").

" homology ", its all grammatical form and spelling variant, refer to the relation between two kinds of albumen with " common evolutionary origin ", comprise the albumen of the superfamily from homologous organism, and from the homologous protein of organism not of the same race.Such albumen (and code nucleic acid) has the sequence of homology, as what reflected by their sequence similarity, no matter from concordance percentage ratio aspect or the existence by specific residue or motif and conserved positions.

Term " sequence similarity ", its all grammatical form, refers to conforming degree or maybe may can not have the correspondence between the nucleic acid of common evolutionary origin or aminoacid sequence.

But in Common usage and instant application, term " homology ", when the adverbs modify such by such as " highly ", can refer to sequence similarity, and maybe may can not refer to and relate to common evolutionary origin.

2.ActRIIa polypeptide

In some aspects, the present invention relates to ActRIIa polypeptide.As the present invention apply, term " ActRIIa " refers to from any kind and comes from by sudden change or other activin IIa receptor family of ActRIIa protein variant of modifying.ActRIIa of the present invention can be understood as any existing form that authenticated.The transmembrane protein when member of ActRIIa family is usual, it is by having the ligand binding ectodomain, the membrane spaning domain that are rich in cysteine regions and having the cytoplasmic domain of activity of serine/threonine kinases of prediction.

Term " ActRIIa polypeptide " comprises any variant (comprising mutant, fragment, fusant and plan peptide form) including any naturally occurring ActRIIa family member and remain with useful activity.Such as, ActRIIa polypeptide comprises and derives from any polypeptide at least with ActRIIa polypeptide with the sequence of about 80% conforming known ActRIIa, preferably at least 85%, 90%, 95%, 97%, 99% or higher concordance.Such as, ActRIIa polypeptide of the present invention can be bonded to ActRIIa albumen and/or activin and suppress its function.Preferably, ActRIIa polypeptide promotes bone growth and bone mineralising.The example of ActRIIa polypeptide comprises the people ActRIIa polypeptide (such as, SEQ ID NOs:2,3,7 and 12) of people ActRIIa Precursor Peptide (SEQ ID NO:1) and solubility.

People ActRIIa precursor polypeptide sequence is as follows:

MGAA′AKLAFAVFLISCSSGAILGRSETQECLFFNANWEKDRT

QTGVEPCYGDKDKRRHCFATWK ISGSIEIVKQGCWLDDINCY

DRTDCVEKKD SPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNP

VTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVL

VPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEY

VAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTS

VDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGL

AYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLA

LKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDM

YAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQE

VVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARL

SAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL(SEQ ID NO：1)

Signal peptide indicates with single underscore; Ectodomain indicates with runic, and potential N-connection glycosylation site indicates with double underline.

(born of the same parents are outer) of people ActRIIa solubility, the peptide sequence of processing are as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSPVTPKPP(SEQ ID NO：2)

The C-end " tail " of ectodomain indicates with underscore.The sequence deleting " tail " (Δ 15 sequence) is as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM (SEQ ID NO：3)

The nucleotide sequence following (the 164-1705 position nucleotide of the Genebank number of logining _ 001616) of encoding human ActRIIa Precursor Peptide albumen:

ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTCTT

CAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGC

TAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGT

GACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTT

CCATTGAAATAGTGA′A′ACAAGGTTGTTGGCTGGATGAT′ATCAACTGCTATGA

CAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGC

TGTGAGGGCAATATGTGTAATGAAA′AGTTTTCTTATTTTCCAGAGATGGAAG

TCACACAGCCCACTTCAAATCCAGTTAC′ACCTA′AGCCACCCT′ATTACAACAT

CCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGT

GCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTC

CAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACC

ACTGCA′GTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAA

GCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACA

AACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCA

TGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGAT

GTGGATCTTTGGCTGATCAC′AGCATTTCATGAAA′AGGGTTC′ACTATCAGACT

TTCTTAAGGCTA′ATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAAC

CATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGAT

GGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAATGTGCTGT

TGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATT

TGAGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGG

TACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCAT

TTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTC

TCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAG

GAGG′AAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGC

ATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAAT

GGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGA′AGCC

AGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAA

CAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAA

TGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA (SEQ IDNO：4)

The nucleotide sequence of encoding human ActRIIa solubility (born of the same parents are outer) polypeptide is as follows:

ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGG

AAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGA

TAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAA

ATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTG

ATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGG

CAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAG CCCACTTCAAATCCAGTTACACcTAAGCCACCC(SEQ ID NO：5)

In specific embodiment, the present invention relates to the ActRIIa polypeptide of solubility.As described in the present invention, term " soluble ActRIIa polypeptide " is often referred to the polypeptide that generation comprises the ectodomain of ActRIIa albumen.Terminology used here " soluble ActRIIa polypeptide " comprises the naturally occurring ectodomain of any ActRIIa albumen and any variant (comprising mutant, fragment and plan peptide form) thereof.Activin-is retain the polypeptide being bonded to the ability of activin, particularly activinAA, AB or BB in conjunction with ActRIIa polypeptide.Preferably, activin-is bonded to activinAA in conjunction with ActRIIa polypeptide with the dissociation constant of 1nM or lower.Below the aminoacid sequence of people ActRIIa precursor protein is provided in.ActRIIa albumen be bonded to activin and normally solvable, and be thus called that the activin-of solubility is in conjunction with ActRIIa polypeptide.The activin-of solubility comprises the soluble polypeptide illustrated by SEQ ID NOs:2,3,7,12 and 13 in conjunction with the example of ActRIIa polypeptide.SEQ ID NO:7 refers to ActRIIa-hFc, and further describes in an embodiment.The activin-of other solubility comprises in conjunction with ActRIIa polypeptide add a signal sequence except the ectodomain of ActRIIa albumen, such as, honey bee melittin targeting sequencing (SEQ ID NO:8), tissue plasminogen activator (TPA) leading (SEQ ID NO:9) or natural ActRIIa leading (SEQ ID NO:10).The ActRIIa-hFc polypeptide illustrated in SEQ ID NO:13 is as the leading application of TPA.

The polypeptide that the functional activity fragment of ActRIIa polypeptide can be produced by the corresponding nucleic fragment of coding ActRIIa polypeptide by screening and obtaining.In addition, fragment can carry out chemosynthesis by using the known Merrifield solid phase f-Moc such as traditional in technology or t-Boc chemistry.Fragment can be produced (restructuring ground or chemosynthesis) and for test differentiate those can exercise ActRIIa protein antagonist (inhibitor) function or by the peptide acyl fragment of activin conditioning signal.

The library of the recombinant polypeptide of the modification that the variant of the functional activity of ActRIIa polypeptide can be produced by the corresponding mutagenic treatment nucleic acid of screening coding ActRIIa polypeptide and obtaining.Variant can be produced and for test to differentiate those can exercise ActRIIa protein antagonist function or by activin conditioning signal.In some embodiments, the functional variant thereof of ActRIIa polypeptide comprises the aminoacid sequence with the aminoacid sequence being selected from SEQ ID NOs:2 or 3 with at least 75% homogeneity.In some example, functional variant thereof has the aminoacid sequence with the aminoacid sequence being selected from SEQ ID NOs:2 or 3 with at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity.

Functional variant can by order to such as strengthen therapeutic efficiency or stability (such as, between the ability of anti-protein degradation in junctor in shelf-life and body) object modify the structure of ActRIIa polypeptide and generate, the ActRIIa polypeptide of such modified when retained by selection activin in conjunction with time, be considered to the functionally equivalent of naturally occurring ActRIIa polypeptide.The ActRIIa polypeptide modified also can be produced, such as, by amino acid replacement, deletion or interpolation.Such as, reasonably can expect and substitute separately leucine with isoleucine or valine, substitute separately aspartic acid with glutamic acid, substitute separately threonine with serine or large impact can not be brought on the biologic activity of produced molecule with another aminoacid of amino acid replacement (such as, conservative variants) that structure is relevant.Conservative replacement is the replacement occurred between those side chains in family aminoacid of being correlated with.Whether the result of the change of ActRIIa polypeptid acid sequence is that the ability that function homology easily produces response by assessment ActRIIa polypeptide variants in the mode of the ActRIIa polypeptide being similar to wild type in cell is determined.

In some embodiments, present invention encompasses the specific sudden change of ActRIIa polypeptide to change the glycosylation of polypeptide.Such sudden change can be selected to introduce or to eliminate one or more glycosylation site, and O-connects or N-connects glycosylation site.The glycosylation recognition site of agedoite-connection generally includes tripeptide sequence, asparagine-X-threonine (or asparagine-X-serine) (X here can be any aminoacid), it is identified specifically by suitable cellular glycosylation enzymes.Transformation also can be added or substitute the ActRIIa polypeptide (glycosylation site for O-connects) to wild type and carry out by one or more serine or threonine residues.Glycosylation recognition site first or the 3rd amino acid sites one of or both each seed amino acids simultaneously replace or delete that (and/or deleting at second site amino acids) cause at the tripeptide sequence modified is non-glycosylated.Another method increasing carbohydrate number on ActRIIa polypeptide can be coupled glucosides to ActRIIa polypeptide by chemical Coupling or enzyme.Depend on adopted CGCM, sugar can be affixed to (a) arginine and histidine; (b) free carboxyl group; The free sulfhydryl group of (c) free sulfhydryl group such as cysteine; The free hydroxyl of (d) free hydroxyl such as serine, threonine or hydroxyproline; The aromatic residues of (e) aromatic residues such as phenylalanine, tyrosine or tryptophan; Or the amide group of (f) glutamine.These methods, at WO 87/05330 (within 1987, JIUYUE discloses on the 11st) and Aplin and Wriston (1981) CRC Crit.Rev.Biochem., have description in 259-306 page, and are incorporated to the present invention by reference.The one or more carbohydrates removed on ActRIIa polypeptide can be completed by the method for chemistry and/or zymetology.Chemical deglycosylation can relate to, and such as, ActRIIa polypeptide is exposed to compound trifluoromethanesulfonic acid or equivalent compound.This process causes major part except the sugar (N-acetyl group glucose or GalNAc) connected or the excision of all sugar, and keeps the complete of aminoacid sequence simultaneously.Chemical deglycosylation is at Hakimuddin etc. (1987) Arch.Biochem.Biophys.259:52 and Edge etc. there is further description in (1981) Anal.Biochem.118:131.The zymetology excision of the carbohydrate on ActRIIa polypeptide can by using by Thotakura etc. and the various interior glycosidase described by (1987) Meth.Enzymol.138:350 or outer glycosidase realize.The sequence of ActRIIa polypeptide can suitably regulate, and it depends on the type of adopted expression system, as mammal, yeast, insecticide and plant cell all can introduce different glycosylation patterns, its can affect by the aminoacid sequence of peptide.Under normal circumstances, ActRIIa polypeptide for the mankind can be expressed can provide in correct glycosylated mammal cell line, as HEK293 cell or Chinese hamster ovary celI system, also may be used for expressing although it is expected to other mammalian expression cell system, the yeast cells system with the glycosylase of engineering and insect cell line.

The present invention covers the complete set mutant of method, particularly ActRIIa polypeptide and the method for truncation mutant that generate muton further; The storehouse of combination mutant is particularly useful for the variant sequence thereof of identification function.The object of screening such combinatorial library is to produce, and such as, or as the variant of agonist or the ActRIIa polypeptide as antagonist action, or alternatively, has the mutant of all new activity.Various screening technique provides below, and such method can be used for assessing variant.Such as, the variant of ActRIIa polypeptide can be prevented by being bonded to ActRIIa part ActRIIa ligand binding to ActRIIa polypeptide or disturb the ability of the signal caused by ActRIIa part to screen.

The activity of ActRIIa polypeptide or its variant also can be tested on cell base or in vivo.Such as, ActRIIa polypeptide variants can be assessed in the effect expressing the gene relating to skeleton production or bone destruction.If needed, this can at the ActRIIa ligandin of one or more restructuring (such as, complete during existing activin), and cell can be transfected to produce ActRIIa polypeptide and/or its variant and (optionally) ActRIIa part.Similarly, ActRIIa polypeptide can be administered to mice or other animal, and assesses one or more skeleton characteristics such as density or volume.The cure rate of fracture also can be evaluated.Dual-energy x-ray absorption measurement (DEXA) is the quantitative technique of the bone density in a kind of maturation, non-invasive assessment animal.The maincenter DEXA system of people may be used for the bone density assessing vertebra and pelvis.These are the best predictor of total skeleton density.Periphery DEXA system may be used for the density assessing periphery skeleton, comprises such as, the skeleton of hands, wrist, ankle and foot.Traditional x-ray PS, comprises cat scan, may be used for assessment bone growth and union of fracture.The mechanical strength of skeleton also can be evaluated.

The variant in combination source can generate, and it has optionally relative to naturally occurring ActRIIa polypeptide or the potential of common growth.Similarly, sudden change variant can be caused to have be different from dramatically corresponding wild type ActRIIa polypeptide born of the same parents in the half-life.Such as, the albumen of change is for Proteolytic enzyme or other cellular processes causing it destroyed or the inactivation of natural A ctRIIa polypeptide or more stable or more unstable on the contrary.Such variant and their gene of encoding, can utilize the level by regulating the half-life of ActRIIa polypeptide to change ActRIIa polypeptide.Such as, short-half-life can cause more instantaneous biological effect and can allow the level of the restructuring ActRIIa polypeptide controlled more closely in patient body.In Fc fusion rotein, sudden change can be in connexon (if there is) and/or Fc part to change the half-life of albumen.

Restructuring library can be produced by the mode of the degeneracy gene library in coded polypeptide library, and described each polypeptide includes ActRIIa peptide sequence potential at least partially.Such as, the oligonucleotide mixture of synthesis can connect into zymetology gene order and make the degeneracy form of potential ActRIIa polypeptide nucleotide sequence can be used as independent expression of polypeptides, or alternatively, the form (such as, phage display) as larger fusion rotein is expressed.

There is the method that much, the library of potential congener produces from degenerate oligonucleotide sequence.The chemosynthesis of degeneracy gene order can be carried out in automatic dna synthesizer, and the gene of synthesis is connected into the carrier of suitable expression subsequently.The synthesis of degenerate oligonucleotide is well known in the art (see such as, Narang, SA (1983) Tetrahedron 39:3; Itakura etc., (1981) recombinant DNA, Proc.3rd Cleveland Sympos.Macromolecules, editor .AG Walton, Amsterdam:Elsevier 273-289 page; Itakura etc., (1984) Annu.Rev.Biochem.53:323; Itakura etc., (1984) Science 198:1056; Ike etc., (1983) Nucleic Acid Res.11:477).These technology be applied to other albumen orthogenesis (see such as, Scott etc., (1990) Science 249:386-390; Roberts etc., (1992) PNAS USA 89:2429-2433; Devlin etc., (1990) Science249:404-406; Cwirla etc., (1990) PNAS USA 87:6378-6382; And United States Patent (USP) 5,223,409,5,198,346 and 5,096,815)

Or the sudden change of other form can be used for producing combinatorial library.Such as, the variant of ActRIIa polypeptide can produce and suddenly change and similar (Ruf etc., (1994) Biochemistry 33:1565-1572 by adopting such as alanine to screen; Wang etc., (1994) J.Biol.Chem.269:3095-3099; Balint etc., (1993) Gene 137:109-118; Grodberg etc., (1993) Eur.J.Biochem.218:597-601; Nagashima etc., (1993) J.Biol.Chem.268:2888-2892; Lowman etc., (1991) Biochemistry 30:10832-10838; With Cunningham etc., (1989) Science 244:1081-1085), by linker-scanning mutant (Gustin etc., (1993) Virology 193:653-660; Brown etc., (1992) MoI.CellBiol.12:2644-2652; McKnight etc., (1982) Science 232:316), by saturation mutation (Meyers etc., (1986) Science 232:613), by PCR suddenly change (Leung etc., (1989) Method Cell MoI Biol 1:11-19) or pass through random mutation, comprise (the Miller et a1. such as chemical mutation, (1992) A Short Course in Bacterial Genetics, CSHLPress, Cold Spring Harbor, NY; And Greener etc., (1994) Strategies in MoIBiol 7:32-34) from library, screening and separating is out.Linker-scanning mutant, in particular in combination setting, is a kind of method of ActRIIa polypeptide of attractive discriminating truncate (biological activity) form.

Well known in the art for screening the far-ranging technology of the gene prod in the combinatorial library that obtained by point mutation and truncate, and, thus, screening is had to the cDNA library of the gene prod of certain characteristic.Such technology is adapted to the gene library that rapid screening is generated by the combinatorial mutagenesis of ActRIIa polypeptide usually.The technology being used most widely for screening large gene library generally includes clone gene library and enters reproducible expression vector, transforms suitable cell with the vector library obtained and express combination gene under the condition that the carrier of the gene detecting the product that desirable activity makes separation coding detect is relatively easy.Preferred method comprises the cell signal analysis of activin binding analysis and activin adjustment.

In some embodiments, any natural ActRIIa polypeptide that is present in of modification after ActRIIa polypeptide of the present invention may further include translation.Such modification includes but not limited to, acetylation, carboxylated, glycosylation, phosphorylation, lipidization and acyl group.As a result, the ActRIIa polypeptide after modifying can comprise non-amino acid element, such as Polyethylene Glycol, lipid, polysaccharide or monosaccharide and phosphate.Such non-amino acid element can be tested for other ActRIIa polypeptide variants as described in the present invention like that in the effect functionally of ActRIIa polypeptide.When ActRIIa polypeptide generates in cell by shearing the nascent form of ActRIIa polypeptide, after translation, process is also important to the correct folding of protein and/or function.Different cell line (such as CHO, Hela, MDCK, 293, WI38, NIH-3T3 or HEK293) for behavior after such translation, there is special cellular machineries and distinctive mechanism, and selectedly can be used for guaranteeing correct modification and the process of ActRIIa polypeptide.

In some aspects, the functional variant thereof of ActRIIa polypeptide or modified forms comprise the fusion rotein with ActRIIa polypeptide and one or more Fusion domain at least partially.The example of known such Fusion domain includes but not limited to polyhistidine, Glu-Glu, glutathione transferase (GST), thioredoxin, A albumen, G-protein, immunoglobulin heavy chain constant region (Fc), maltose-binding protein (MBP) or human serum albumin.Fusion domain can be selected to give desirable characteristic.Such as, some Fusion domains are specifically for passing through affinity protein purification fusion rotein.For reaching the object of affinity purification, adopt the substrate resin that such as glutathion, amylase and nickel are puted together or cobalt is puted together that affinity chromatograph is correlated with.Much such substrate can obtain with the form of " test kit ", such as Pharmacia GST purification system and QIAexpress ^tMsystem (Qiagen) and useful (HISg) fusion partner.As other example, Fusion domain can be selected to contribute to detecting ActRIIa polypeptide.The example in such detection architecture territory comprises various fluorescin (such as GFP) and " Epitope tag ", the short peptide sequence of its normally obtainable specific antibody.The known Epitope tag for specific monoclonal antibody easily obtains, and comprises FLAG, influenza virus hemagglutinin (HA) and c-myc labelling.In some instances, Fusion domain has the protease cutting site such as Xa factor or thrombin, and it allows relevant protease part ground digestion fusion rotein and thus therefrom discharges recombiant protein.Then the albumen of release be separated from Fusion domain by chromatography subsequently.Some preferred embodiment in, ActRIIa peptide fusion has the domain (" regulator " domain) stablizing ActRIIa polypeptide in vivo.Meaned any material that can increase serum half-life by " stablizing ", do not consider that whether it be because reduce destructions, the reduction of kidney removing or other medicines kinetic effect.The fusion of the known Fc part with immunoglobulin can give far-ranging albumen with desirable pharmacokinetic properties.In addition, merge human serum albumin and can give desirable characteristic.The Fusion domain of other type selectable comprises poly domain (such as dimerization, four is gathered) and Functional domains, and (it gives other biological function, such as stimulate bone growth or muscle growth further, as desired).

As specific example, the invention provides the fusion rotein comprising and merging to the soluble extracellular domains of the ActRIIa of Fc domain (such as SEQID NO:6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV D(A)VSHEDPEVKF

NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC K(A)VSNKAL

PVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN

GQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALH N(A)HYT QKSLSLSPGK ^*

Selectively, Fc domain has one or more sudden change in residue (such as aspartic acid 265, lysine 322 and agedoite 434).In some example, the mutant Fc domain with one or more sudden change (such as aspartic acid 265 suddenlys change) like this has the ability being bonded to Fc γ receptor of reduction relative to the Fc domain of wild type.In other example, the MHC I class that is bonded to that the mutant Fc domain with one or more sudden change (such as winter amide 434 suddenlys change) like this has increase relative to the Fc domain of wild type is correlated with the ability of Fc receptor (FcRN).

Certainly, the different elements of fusion rotein can arrange in any and that conceivable function is consistent mode.Such as, ActRIIa polypeptide can be placed in the C-terminal in hetero-structures territory, or alternatively, hetero-structures territory can be placed in the C-terminal of ActRIIa polypeptide.ActRIIa polypeptide domain and hetero-structures territory do not need to be close in fusion rotein, and additional domain or aminoacid sequence can comprise one of C-terminal or N-terminal to two domain or be between two domains.

In some embodiments, ActRIIa polypeptide of the present invention contains one or more modification can stablizing ActRIIa polypeptide.Such as, such modification strengthens the ActRIIa polypeptide half-life in vivo, strengthens the circulating half-life of ActRIIa polypeptide or reduces the protein degradation of ActRIIa polypeptide.Stable modification like this includes but not limited to that fusion rotein (comprises, such as, comprise the fusion rotein in ActRIIa polypeptide and stabilizer structure territory), the modification of glycosylation site (comprises, such as, additional glycosylation sites is to ActRIIa polypeptide) and the modification of carbohydrate (comprise, such as, from ActRIIa polypeptide, carbohydrate is removed).With regard to fusion rotein, ActRIIa peptide fusion is to the stabilizer structure territory (such as Fc domain) of such as IgG molecule.Apply as the present invention, term " stabilizer structure territory " not only refers to the Fusion domain (such as Fc) in fusion rotein, and the nonprotein comprising such as carbohydrate is modified or the nonproteinaceous polymers of such as Polyethylene Glycol.

In some embodiments, the invention enables the ActRIIa polypeptide that can obtain unpack format and/or purified form, it is separated from or substantially dissociates from other albumen on the contrary.ActRIIa polypeptide produces by expressing from recombinant nucleic acid usually.

3. the nucleic acid of coding ActRIIa polypeptide

In some aspects, the invention provides separation and/or restructuring coding any ActRIIa polypeptide (such as soluble ActRIIa polypeptide) comprises the nucleic acid of fragment, functional variant thereof and fusion rotein disclosed in this invention.Such as, SEQ ID NO:4 encodes naturally occurring people ActRIIa Precursor Peptide, and SEQ ID NO:5 encodes the ectodomain of processing of ActRIIa.Target nucleic acid can be strand or double-strand.Such nucleic acid can be DNA molecular or RNA molecule.These nucleic acid may be used for, and such as, manufacture the method for ActRIIa polypeptide or direct therapeutic agent (such as, in gene therapy method).

In some aspects, the target nucleic acid should understanding coding ActRIIa polypeptide further comprises the nucleic acid of the variant of SEQID NO:4 or 5.Variant nucleotide sequences comprises the different sequence that one or more nucleotide replaces, adds or delete, such as allele variant.

In some embodiments, the invention provides to have with SEQ ID NO:4 or 5 at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity be separated or the nucleotide sequence of recombinating.Those of ordinary skill in the art should understand complementary with SEQ ID NO:4 or 5 and can comprise within the scope of the invention with the nucleotide sequence of the variant complementation of SEQ ID NO:4 or 5.In further embodiment, nucleotide sequence of the present invention can be separated, restructuring and/or merge with heterologous nucleotide sequence or in DNA library.

In other embodiments, nucleic acid of the present invention is hybridized under can also being included in high stringency conditions specify nucleotide sequence, the complementary series of SEQ ID NO:4 or 5 or the nucleotide sequence of its fragment to SEQ ID NO:4 or 5.As discussed above, those of ordinary skill in the art should easily understand, and promote that the suitable high stringency conditions of DNA hybridization can change.Such as, can hybridize at about 45 DEG C in 6.0 × sodium chloride/sodium citrate (SSC), wash with 2.0 × SSC at 50 DEG C subsequently.Such as, the salinity of washing step can be selected from 2.0 × SSC, low high stringency conditions to the 0.2 × SSC of 50 DEG C, the high high stringency conditions of 50 DEG C.In addition, the temperature of washing step can from the high high stringency conditions of the low high stringency conditions to about 65 DEG C of the room temperature of about 22 DEG C.Temperature and salt can change, or temperature or salinity can to change in another contingent condition be keep constant.Gather at some embodiment, the invention provides and hybridize under the low high stringency conditions of 6 × SSC, room temperature, subsequently at room temperature with the nucleic acid of 2 × SSC washing.

The nucleic acid molecules that be separated different from the nucleic acid in SEQ ID NO:4 or 5 due to the degeneracy of genetic codon is also included within scope of the present invention.Such as, a large amount of aminoacid is specified by the codeword triplet of more than.Point to identical amino acid whose codon or synonymous codon (such as, CAU and CAC is the synonymous codon of histidine) can cause " silence " to suddenly change, it can not affect the aminoacid sequence of protein.But we expect and really cause the polymorphism of the DNA sequence of the change of the aminoacid sequence of target protein to be present in mammalian cell.The variation on one or more nucleotide (at the most the nucleotide of 3-5%) that those skilled in the art should understand the nucleic acid of encode specific protein may reside in the individual party of given kind due to natural allelic variation.The variation of any or whole such nucleotide and the amino acid polymorphism that causes are all within the scope of the invention.

In some embodiments, the exercisable one or more adjustment nucleotide sequences being connected to expression constructs of recombinant nucleic acid of the present invention.Regulate what connection calculate sequence be usually suitable for express apply appeal to cell.Very eurypalynous suitable expression vector for various host cell known in this field and suitable adjustment sequence.Normally, described one or more adjustment nucleotide sequences can include but not limited to promoter sequence, leading or signal sequence, ribosome binding site, transcription initiation and terminator sequence, translation initiation and terminator sequence and enhancer or activate subsequence.Structure well known in the art or induction promoter contain by the present invention.Promoter or naturally occurring promoter, or the hybrid promoter of more than one promoter composition element.Expression constructs can be present on intracellular episome, such as plasmid, or expression constructs is inserted in chromosome.In one preferred embodiment, expression vector comprises selectable marker gene to allow to select to transform host.Selectable marker gene is well known in the art and changes with the host cell adopted.

Of the present invention in some, target nucleic acid is provided in an expression vector comprising the nucleotide sequence of coding ActRIIa polypeptide, and may be operably coupled at least one and regulate sequence.Regulate sequence to be art-recognized and be selected to guide the expression of ActRIIa polypeptide.Correspondingly, term regulates sequence to comprise promoter, enhancer and other expression control element.Exemplary adjustment sequence description is in Goeddel; Gene expression technique; Enzymology method, Academic Press, San Diego, CA (1990).Such as, controlling arbitrarily the expression control sequenc that DNA sequence is expressed on a large scale, when being operably connected, can be used for the expression of the DNA sequence expressing coding ActRIIa polypeptide.Useful expression control sequenc like this comprises, such as, early stage or the late promoter of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoter, lac operon system, tryptophan system, TAC or TRC system, the T7 promoter expressed is guided by t7 rna polymerase, the main operation of bacteriophage lambda and starting region, the control area of fd goat albumen, the promoter of glycerol 3-phosphate acid kinase or other glycolytic ferment, the promoter such as Pho5 of acid phosphatase, the promoter of yeast α-Hybridization Factor, the sequence of the polyhedron promoter of rhabdovirus system and other known control prokaryotic cell or eukaryotic cell or their viral gene and the expression of various recombinant thereof.Should understand, the design of expression vector depends on host cell such as to be transformed and/or wants the factor of selection of the protein types of expressing.In addition, also should consider the quantity of vector copies, control the ability of the expression of number of copies and other albumen any such as antibiotic marker coded by carrier.

Recombinant nucleic acid of the present invention can by connecting the gene of clone or its fragment enters to be applicable to or in prokaryotic cell or in eukaryotic cell (yeast, birds, insecticide or mammal) or the carrier of expressing in both and producing.The production expression vector of restructuring ActRIIa polypeptide comprises plasmid and other carrier.Such as, suitable carrier comprises the plasmid of following classes: the plasmid in the plasmid that the pBR322-for expressing in such as colibacillary prokaryotic cell originates, pEMBL-source, the plasmid in pEX-source, the plasmid in pBTac-source and the plasmid in pUC-source.

Some mammalian expression vectors include simultaneously and help protokaryon sequence that carrier breeds in antibacterial and be one or morely expressed in eukaryotic eukaryotic transcription unit.The carrier in PcDNAI/amp, pcDNAI/neo, pRC/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg source is the example of the mammalian expression vector of applicable eukaryotic cell transfection.The sequence that a part in these carriers is originated by bacterial plasmid modify, such as pBR322, with contribute in prokaryotic cell and eukaryotic cell copying and drug resistance select.Another is chosen as, and the derivant such as bovine papilloma virus (BPV-1) or eb virus (pHEBo, pREP source and p205) of virus are used in the transient expression of albumen in eukaryotic cell.The example of other virus (comprising retrovirus) expression system can find in the description of gene therapy import system below.It is well known in the art for preparing in the process of plasmid and transformed host organism the various methods adopted.Protokaryon and eukaryotic expression system and common regrouping process is applicable to while other, see Molecular Cloning A Laboratory Manual, the third edition, by Sambrook, Fritsch and Maniatis edits (Cold Spring Harbor Laboratory Press, 2001).In some example, it is suitable for adopting bovine papilloma virus expression system to express recombinant polypeptide.The example of such bovine papilloma virus expression system comprises the carrier (such as comprising the beta galactosidase of pBlueBacIII) in the carrier (such as pVL1392, pVL1393 and pVL941) in pVL-source, the carrier (such as pAcUW1) of pAcUW-Lianyuan and pBlueBac-source.

In a preferred embodiment, carrier is designed to productive target ActRIIa polypeptide in Chinese hamster ovary celI, such as Pcmv-Script carrier (Stratagene, La Jolla, Calif), pcDNA4 carrier (Invitrogen, Carlsbad, Calif) and pCI-neo carrier (Promega, Madison, Wisc).Apparently, target gene construction can be used for the expression in the cell causing target ActRIIa polypeptide to breed in the medium, such as, to produce albumen, comprises fusion rotein or mutant protein, for purification.

The invention still further relates to the host cell of transfection recombination, described host gene comprises coded sequence (such as, the SEQ ID NO of one or more target ActRIIa polypeptide of encoding; 4 or 5).Host cell can be any protokaryon or eukaryotic cell.Such as, ActRIIa polypeptide of the present invention gram is expressed in antibacterial, such as escherichia coli, insect cell (such as adopting bovine papilloma virus expression system), yeast or mammalian cell.Other suitable host cell is known in those skilled in the art.

Correspondingly, the present invention relates to the method for productive target ActRIIa polypeptide further.Such as, the expression vector having transformed coding ActRIIa polypeptide can be cultivated to allow ActRIIa expression of polypeptides under appropriate conditions.ActRIIa polypeptide can from cell and comprise ActRIIa polypeptide substrate mixture secrete and separate.Selection is in addition, ActRIIa polypeptide can to stay in Cytoplasm or as membrane component and harvesting, dissolving protein isolate.Cell culture comprises host cell, substrate and other side-product.Suitable cell culture substrate is well known in the art.Target ActRIIa polypeptide from cell culture substrate, host cell or can be separated simultaneously from both, adopt technology purifying protein well known in the art, comprise ion-exchange chromatography, affinity purification (such as, A albumen post can be used for purification ActRIIa-Fc fusion rotein) that immunoaffinity purification that gel permeation chromatography, ultrafiltration, electrophoresis, employing are specific to the antibody of the defined epitope of ActRIIa polypeptide and employing are bonded to the preparation of the domain being fused to ActRIIa polypeptide.In one preferred embodiment, ActRIIa polypeptide is the fusion rotein including the domain helping purification.In one preferred embodiment, purification is reached by a series of column chromatography steps, comprise, such as, three or more the following steps with any order: A protein chromatographic, Q agarose chromatography, phenyl sepharose chromatography, size exclusion chromatography and cation-exchange chromatography.Purification can be exchanged by virus filtration and buffering and be completed.As the present invention prove, ActRIIa-hFc protein purification to determine to be greater than with size exclusion chromatography 98% and determine to be greater than with SDS PAGE 95% purity.Such purity is enough for obtaining on the skeleton of mice the effect wanted and the acceptable drug safety in mice, rat and non-human primates.

In another embodiment, the fusion gene of coding purification leader sequence, poly-(histidine)/enterokinase cleavage site point sequence of ActRIIa polypeptide desirable part N-terminal position of such as recombinating, can allow to adopt Ni ²⁺metal-resin is by the warm albumen expressed by affinitive layer purification.Purification leader sequence removed by enterokinase process subsequently to provide purification ActRIIa polypeptide (such as, see Hochuli etc., (1987) J Chromatography 411:177; And Janknecht etc., PNAS USA 88:8972).

The technology preparing fusion rotein is well known in the art.In essence, the connection of various DNA fragmentations of different peptide sequences of encoding is completed by corresponding conventional art, adopt that blunt-ended or stagger-ended end connects, Restriction Enzyme digestion to provide suitable end, suitable when fill up sticky end, alkaline phosphatase treatment to avoid undesired connection, and enzyme connects.In another embodiment, fusion gene can comprise automatic dna synthesizer to synthesize by conventional art.Or, the pcr amplification of genetic fragment can adopt adapter-primer to perform, described adapter-primer make two continuous print can with after annealing with pendency complementary between the genetic fragment producing chimeric gene sequence (see such as, Current Protocols inMolecular Biology, the editors such as Ausubel, John Wiley & Sons:1992).

4. optionally Activin and ActRIIa antagonist

The antagonist of digital proof provided by the present invention activin-ActRIIa signal can be used for promoting bone growth and bone mineralising.Although the ActRIIa polypeptide of solubility, particularly ActRIIa-Fc, it is preferred antagonist, and the antagonist of even now affects skeleton (such as by the mechanism of more than activin antagonism, activin suppresses to have the indicator suppressing spectroscopic molecular activity trend, comprise, possible, other member of TGF-beta superfamily, and such colony suppresses the effect that may cause on desired skeleton), the activin-ActRIIa antagonist that it is expected to other type is effective, comprise anti-activin (such as A, B, C or E) antibody, anti-ActRIIa antibody, antisense thing, the RNAi suppressing ActRIIa to produce or the inhibitor of ribozymal nucleic acid and other activin or ActRIIa, particularly those destroy the inhibitor that activin-ActRIIa combines.

The antibody reacted with ActRIIa polypeptide (such as, the ActRIIa polypeptide of solubility) specifically and or competitive binding and coordinate with ActRIIa polypeptide or the material of the signal that suppresses ActRIIa to regulate on the contrary can be used as the antagonist of ActRIIa polypeptide active.In addition, the antibody reacted with activinA polypeptide specifically and destroy the antibody that ActRIIa is combined and also can be used as antagonist.

Adopt the immunogen from ActRIIa polypeptide or activin polypeptide, by standard method (see, such as, A Laboratory Manual is edited (Cold SpringHarbor Press:1988) by Harlow and Lane) prepare anti-albumen/anti-polypeptide antiserum or monoclonal antibody.Mammal, such as mice, hamster or rabbit can with the immunogen form of ActRIIa polypeptide, the anti-genic fragment that can cause antibody response or fusion protein immunizations.The technology of giving albumen or immunogenicity of polypeptides comprises conjugate vectors or other technology well known in the art.The immunogenic portion of ActRIIa or activin polypeptide can be deposited at adjuvant and use in case.Immunologic process detects by detecting antibody titre in plasma or serum.Standard ELISA or other immunization method can use to assess antibody horizontal together with the immunogen as antigen.

After with the antigen formulations immune animal of ActRIIa polypeptide, obtain antiserum, if needed, polyclonal antibody can be separated from serum.For producing monoclonal antibody, can antibody produced cell (lymphocyte) be collected from the animal of immunity and merge to produce hybridoma by the Somatic Fusion step of standard and the immortalized cells of such as myeloma cell.Such technology is well known in the art, comprise, such as, hybridoma technology (starts to be developed by Kohler and Milstein most, (1975) Nature, 256:495-497), human B cell hybridoma's technology (Kozbar etc., (1983) Immunology Today, 4:72) and EBV-hybridoma technology produce human monoclonal antibodies (Cole etc., (1985) monoclonal antibody and treatment of cancer, Alan R.Liss, Inc.77-96 page).Hybridoma can screen to produce the antibody reacted with ActRIIa polypeptid specificity by immunochemistry, and monoclonal antibody is separated from the culture medium comprising such hybridoma.

Term used herein " antibody " be intended to comprise those also can with the fragment of target polypeptides specific reaction.Antibody can adopt traditional technology fragmentation, and screens fragment by the same method for whole antibody described above.Such as, F (ab) ₂fragment can by generating with pepsin.The F (ab) produced ₂fragment can process with less disulfide bridge bond to produce Fab fragment.The total length human molecular of ActRIIa or activin polypeptide affinity that antibody of the present invention is intended to comprise two special, strand, chimeric, humanization further and is given by least one antibody CDR region.Antibody can comprise the additional label (such as, label can be radiosiotope, fluorescent chemicals, enzyme or cofactors) that also can be detected in addition further.

In some embodiments, antibody is recombinant antibodies, its term includes arbitrary partly with the antibody that Protocols in Molecular Biology produces, comprise antibody that CDR transplants or chimeric antibody, people's or the assembling antibody of antibody domain, single-chain antibody and single domain antibody (such as, the people V that select from library _halbumen or camelid V _hHalbumen).In some embodiments, antibody of the present invention is monoclonal antibody, and in some embodiments, the invention enables the method for producing new antibodies to become possibility.Such as, a kind of production is bonded to the method for the monoclonal antibody of ActRIIa polypeptide or activin polypeptide specifically, comprise the immunogenic composition of the detectable immunoreactive antigen polypeptide of the stimulation including effective amount using doses to mice, antibody producing cells is obtained (such as from mice, cell in spleen) and by antibody producing cells and myeloma cell fusion to obtain antibody producing hybridoma, detect antibody producing hybridoma to differentiate to produce the monoclonal hybridoma of specific binding antigen.Once obtain, from cell culture medium, breed hybridoma, under the condition of culture of the cell of the production specific binding antigen optionally in hybridoma source.Monoclonal antibody can from cell culture medium purification.

Adjective " reacts with it specifically " and herein means for antibody, be meant to, as usually understood in this area, have between the interested antigen of antibody in particular organisms sample (such as, ActRIIa polypeptide) and other non-interested antigen fully optionally.Some adopt antibody method in, in such as treatment use, in conjunction with high degree of specificity be necessary.Monoclonal antibody usually has larger trend (compared with polyclonal antibody) and effectively distinguishes between the polypeptide of desirable antigen and cross reaction.Affect antibody: the antibody specific feature that interacts is the affinity of antibody to antigen.Although desired specificity can reach the scope of different affinity, preferred antibody has about 10 usually ^-6, 10 ^-7, 10 ^-8, 10 ^-9or less affinity (dissociation constant).Combining especially closely between given activin and ActRIIa, we expect that the anti-activin antibody of neutrality or anti-ActRIIa antibody have 10 usually ^-10or less dissociation constant.

In addition, for screening antibodies to identify that the technology of desirable antibody can affect the characteristic of the antibody of acquisition.Such as, if antibody is used for conjugated antigen in the solution, it may be suitable that test solution combines.Various different technology can be used for interaction between test antibody and antigen to differentiate the antibody of certain desired.Such technology comprises ELISAs, surface plasma body resonant vibration binding analysis (such as, the Biacore ^tMbinding assay, Biacore AB, Upps ala, Sweden), sandwich assay (such as, the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Maryland), western immunoblotting, immunoprecipitation and SABC.

The example of the nucleic acid compound kind of Activin or ActRIIa antagonist comprises antisensenucleic acids, RNAi construction and catalytic nucleic acid construction.Nucleic acid compound can be strand or double-strand.Double-strandednucleic acid compound can also comprise cantilever or non-complementary region, and one of them or another one fragment are strands.Single chain compound can comprise the region of self-complementary, means that compound is formed and is called " hair clip " or " stem ring " structure, have the region of a double-stranded helical structure.Nucleic acid compound can comprise being complementary to and comprises being no more than 1000, being no more than 500, being no more than 250, being no more than 100 or be no more than the nucleotide sequence in region of 50,35,30,25,22,20 or 18 nucleotide of total length ActRIIa nucleotide sequence or activin β A or activin β B nucleotide sequence.Preferred complementary region is at least 8 nucleotide, optionally at least 10 or at least 15 nucleotide, and optionally between 15 to 25 nucleotide.Complementary region can belong to intron, the coded sequence of target transcripton or non-coding sequence such as coding sequence portion.Normally, nucleic acid compound has the length of 8 to about 500 nucleotide or base pair, and optionally length can be about 14 to about 50 nucleotide.Nucleic acid can be DNA (specifically using as antisensenucleic acids), RNA or RNA:DNA heterocomplex.Any chain can comprise the mixture of DNA and RNA, and is not easy the form of the modification being divided into DNA or RNA.In addition, double chain compound can be DNA:DNA, DNA:RNA or RNA:RNA, and any chain can comprise the mixture of DNA and RNA, and is not easy the form of the modification being divided into DNA or RNA.Nucleic acid compound can comprise arbitrary various modification, comprises one or more modifications of skeleton (sugar-phosphate portion of natural acid, comprises internucleoside linkage) or base portion (purine of natural acid or pyrimidinium moiety).It is long that antisense nucleic acid compound preferably has about 15 to about 30 nucleotide, and usually comprise one or more modification to improve stability in such as serum, in cell or in the characteristic of the possible administering position of compound, described administering position is such as stomach (when oral administration) and lungs (for suction compound).When RNAi construction, the complementary chain to target transcripton is RNA or its modification normally.Other chain can be RNA, DNA or other variant any.The two-phase part of double-strand or strand " hair clip " RNAi construction is preferably 18 to 40 length of nucleotides, and be optionally about 21 to 23 length of nucleotides, as long as it can be used as Dicer, substrate works.Catalysis or enzymatic nucleic acid can be ribozyme or DNA enzymatic, and also can comprise the form of modification.Nucleic acid compound in physiological conditions, with nonsense or there is justice to control the concentration that substantially do not produce or do not tell on, contacts with cell, can suppress the expression of the target of about 50%, 75%, 90% or more.The concentration of the effect of preferred detection nucleic acid compound is 1,5 and 10 micromole.Nucleic acid compound can be used for test case as the effect on bone growth and bone mineralising.

5. screening technique

In some aspects, the present invention relates to adopt ActRIIa polypeptide (such as, soluble ActRIIa polypeptide) and activin polypeptide to identify can the compound (medicine) of exciting or antagonism activin-ActRIIa signal path.Can test to assess the ability that they regulate bone growth and bone mineralising in vivo by this Screening and Identification medicament, optionally, these compounds can test to assess the ability of their regulate tissue growth in vivo further in animal.

Have and much more very determine activin and ActRIIa polypeptide with the approach of the therapeutic agent of regulate tissue growth for screening by target.In some embodiments, the high flux screening of compound can be used for the medicament differentiating the regulating effect of interference activin or ActRIIa on skeleton.In some embodiments, the method is used for the suppression of screening and identification specificity or reduces the compound that ActRIIa polypeptide is bonded to activin.Or the method is bonded to the compound of activin for the identification of strengthening ActRIIa polypeptide.In another embodiment, compound is identified by itself and the interactional ability of activin or ActRIIa polypeptide.

The version of various method is enough and in scope disclosed by the invention, but can being understood by those of ordinary skill in the art of clearly not describing of those the present invention.As described in the invention, detection compound of the present invention (medicament) is by any combinational chemistry manufacture.Or target compound can be the biomolecule of in vivo naturally occurring or external synthesis.Its compound as tissue growth regulator ability (medicament) to be measured by such as antibacterial, yeast, plant or other organism (such as, natural prodcuts) produce, or chemical production (such as, micromolecule, comprise and intend peptide), or restructuring ground produces.The detection compound that the present invention is contained comprises non-peptide organic molecule, peptide, polypeptide, plan peptide, sugar, hormone and nucleic acid molecules.In specific embodiment, test agents is that molecular weight is less than about 2000 daltonian little organic molecules.

Detection compound of the present invention can be single, discrete entity, or has the library of larger complexity, such as with storehouse prepared by combinatorial chemistry.These storehouses can comprise, such as, and the organic compound of ethanol, alkyl halide, amine, amide, ester, aldehyde, ether and other classification.Detection compound existence in the detection system can be independent form or with the form of the mixture of compound, particularly in initial screening step.Optionally, compound optionally can contribute to compound separation with other compound deriving and Derived Populations.The example of nonrestrictive derivant colony comprise biotin, fluorescein, digoxin, green fluorescent protein, isotope, polyhistidine, magnetic bead, glutathione transferase (GST), optical activation cross-linking agent (photoactivatible crosslinkers) or its combine arbitrarily.

In the drug screening programs of many detection compound storehouses and natural extract, high-throughout method in order to maximize required for detection compound quantity within preset time.This method under cell-free environment, such as can purification or half purifying protein everywhere, and usually preferably as " main " screening technique, it allows fast-developing and relatively easily detects the change of target molecule of detected compound adjustment.In addition, the cytotoxicity of detection compound or bioavailability can be ignored in system in vitro, the method then mainly concentrate on the effect of medicine on target molecule, and it can show in the change of the binding affinity of ActRIIa polypeptide and activin.

Only illustrate, in exemplary screening technique of the present invention, interested compound contacts with the ActRIIa polypeptide that usually can be bonded to activin being separated also purification.The compositions comprising ActRIIa part is added subsequently in the mixture of compound and ActRIIa polypeptide.The detection of ActRIIa/activin complex and quantitatively provide a kind of deterministic compound and suppress (or reinforcement) complex between ActRIIa polypeptide and activin to form the method for effect.The dose-effect curve that the effect of compound generates by the data adopting the various concentration of test compounds and obtain and assessing.In addition, check analysis can be used for providing the benchmark compared.Such as, in check analysis, the activin being separated also purification is added in the compositions comprising ActRIIa polypeptide, the quantitative ActRIIa/activin complex when not having test compounds.Should understand, common, the order of reactant mixing can change, and can mix simultaneously.In addition, the cell extract of purifying protein and solute is replaced to can be used for providing suitable cell-free assay system.

The formation of the complex of ActRIIa polypeptide and activin can by various technology for detection.Such as, complex formed adjustment can adopt the albumen of such as detectable label such as radiolabeled (such as, ³²p, ³⁵s, ¹⁴c or ³h), fluorescein-labelled (such as FITC) or the ActRIIa polypeptide of enzyme labelling or activin, detect quantitatively by immunoassay or chromatography.

In some embodiments, present invention encompasses fluorescence polarization method and the application of FRET (fluorescence resonance energy transfer) (FRET) in the interaction protein-bonded with it of direct or indirect measurement ActRIIa polypeptide.Further, other detecting pattern, such as based on (PCTPublication WO 96/26432 and the United States Patent (USP) 5 of fiber waveguide, 677,196), surface plasma body resonant vibration (SPR), surface charge sensing and surperficial stress sensing, all consistent with many embodiments of the present invention.

In addition, present invention encompasses the application of interaction trap method (being also called " two-hybrid method "), with identify destroy or strengthen ActRIIa polypeptide with it protein-bonded interactional medicament.See such as, United States Patent (USP) 5,283,317; Zervos etc. (1993) Cell 72:223-232; Madura etc. (1993) J Biol Chem 268:12046-12054; Bartel etc. (1993) Biotechniques 14:920-924; And (1993) the Oncogene 8:1693-1696 such as Iwabuchi.In specific embodiment, present invention encompasses application reverse two hybrid system with identify separation ActRIIa polypeptide with it protein-bonded interactional compound (such as, micromolecule or peptide).See such as, Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; And United States Patent (USP) 5,525,490; 5,955,280; With 5,965,368.

In some embodiments, target compound is identified by itself and the interactional ability of ActRIIa or activin polypeptide of the present invention.Interaction between compound and ActRIIa or activin polypeptide is covalency or non-covalent.Such as, this interaction can adopt biochemical method in body to identify on protein level,

Described biochemical method comprises photo-crosslinking, isotope-labeled ligand binding and affinity chromatograph (Jakoby WB et al., 1974, Methods in Enzymology 46:1).In some example, compound screens by the method being attached to the compound of activin or ActRIIa polypeptide that such as detects based on some mechanism.This can comprise solid phase or liquid phase combines.Or, the gene of coding activin or ActRIIa polypeptide can with reporting system (such as, beta galactosidase, luciferase or green fluorescent protein) to be together transfected in cell and preferably with high flux screening to screen library, or with the together transfection of the individuality in library.Other mechanism based on associated methods can be used for, and such as, detects the binding analysis of Gibbs free.Binding analysis can be fixed to hole, pearl or chip or the antibody capture be immobilized or the target that decomposes by capillary electrophoresis together carry out.The compound be locked can adopt colorimetry or fluorescence or surface plasma body resonant vibration to measure usually.

In some aspects, the invention provides method and medicament that adjustment (stimulate or suppress) skeleton formed and increased bone mass.Therefore, the compound of any qualification can in full cell or tissue, in vitro or body build-in test, to determine its ability regulating bone growth or bone mineralising.Various method well known in the art can be used for this object.

Such as, ActRIIa or activin polypeptide or the effect of detection compound on skeleton or cartilage-derived growth are divided into osteoblast to determine (see such as by the induction or osteoblast of measuring Ms × 2 on cell base, (Daluiski etc., Nat Genet.2001,27 (1): 84-8; Hino etc., Front Biosci.2004,9:1520-9).The example that other cell base is analyzed comprise evaluating objects ActRIIa or activin polypeptide osteogenic activity and in mesenchymal progenitor cells and osteoblast test compounds.Illustrate, the recombinant adenovirus of expressing activin or ActRIIa polypeptide can build for infecting multipotency mesenchymal progenitor cells C3H10T 1/2, osteoclast precursor C2C12 and osteoblast TE-85.Osteogenic activity is determined by measuring the induction of alkali phosphatase, Bone Gla protein and Matrix Mineralization subsequently (see, such as, Cheng etc., Jbone JointSurg Am.2003,85-A (8): 1544-52).

The present invention also covers the method detecting skeleton or cartilage-derived growth in body.Such as, Namkung-Matthai etc., Bone, 28:80-86 (2001) disclose a kind of osteoporosis model, its bone repair early stage after have studied fracture.Kubo etc., SteroidBiochemistry & Molecular Biology, 68:197-202 (1999) also discloses a kind of osteoporosis model, and it have studied the bone repair in fracture rear late period.Andersson etc., J.Endocrinol.170:529-537 disclose the osteoporosis model of mice, and mice removes ovary, it causes mice to lose essence bone mineral content and bone mineral density, wherein the Cancellous bone loss bone mineral density of general 50%.The skeleton density removing the mice of ovary can be improved by the factor using some such as parathyroid hormones.In some aspects, the present invention application of fracture healing method well known in the art.These methods comprise Fracture Technique, histologic analysis and biomechanical analysis, it is at such as United States Patent (USP) 6,521, there is description in 750, integrally the degree of the foundation disclosed in it and measurement fracture is incorporated to the present invention with the experimental technique of the process of reparation with the form quoted.

6. exemplary therapeutic application

In some embodiments, activin-ActRIIa antagonist of the present invention (such as, ActRIIa polypeptide) treatment can be used to or prevent and treat the disease relevant to skeletal injury or situation, no matter described damage is such as, by fracture, loss or demineralization.In some embodiments, the invention provides the method for the skeletal injury by the individuality for the treatment of to the activin-ActRIIa antagonist (particularly ActRIIa polypeptide) of individual administering therapeutic effective dose or prevent this to need.In some embodiments, the invention provides by promoting the bone growth of the individuality having this to need or the method for mineralising to the activin-ActRIIa antagonist (particularly ActRIIa polypeptide) of individual administering therapeutic effective dose.These methods are preferably conceived to treatment and the prophylactic treatment of animal, more preferably, are conceived to people.In some embodiments, the invention provides the application of activin-ActRIIa antagonist (particularly the ActRIIa polypeptide of solubility and the neutrality antibody of targeting activin or ActRIIa) in the disease that treatment is relevant to the bone strength of low skeleton density or reduction.

As the present invention apply, the therapeutic agent of " prevention " disease or situation refers in a statistics sample, relative to not treating sample, the disease for the treatment of sample or the compound of situation generation can be reduced, or relative to not treating sample, postponing outbreak or palliating a disease or the compound of the order of severity of one or more symptoms of situation.Term used herein " treatment " comprises prevents the situation of specifying or determines improve or eliminate this situation once this situation.In both in any one situation, the therapeutic agent of prevention or treatment and expection is used result and is considered by the doctor doing to diagnose.

The invention provides comprise skeleton and/or Subchondral drilling, prevention skeleton is lost, increase the method for bone mineralising or the demineralization of prevention skeleton.Such as, target activin-ActRIIa antagonist has been applied to the cartilage defect in the healing and human or animal for the treatment of osteoporosis and fracture.ActRIIa or activin polypeptide is effective to the patient being diagnosed as subclinical low skeleton density, and it is as the protective measure developed osteoporosis.

In a special embodiment, method and composition of the present invention has the medical effect in the union of fracture and cartilage defect of people and other animal.Goal approach and compositions also have preventative application in closed and the open reduction of the fracture and artificial joint improvement is fixing.The De novo skeleton of skeletonization Chemicals induction is formed with the reparation helping that geneogenous, wound causes or that tumor resection causes craniofacial deformity, is also effective in medical and beauty treatment.In some example, target activin-ActRIIa antagonist can provide a kind of environment attracting skeleton to form cell, stimulates skeleton to form the growth of cell or the differentiation of induction skeleton formation precursor.Activin-ActRIIa antagonist of the present invention is also effective in osteoporotic treatment.

Method and composition of the present invention can be applied to the situation losing bone for or cause to lose bone as feature, such as osteoporosis (comprising secondary osteoporosis), hyperparathyroidism, hypercortisolism (Cushing ' s disease), cypress Zhe Shi disease (Paget ' s disease), hyperthyroidism, chronic dysentery or malabsorption, renal tubular acidosis or anorexia nervosa.

Osteoporosis can be caused by various factors or associated.Concerning women, particularly postclimacteric women, body weight is low, and the sitting behavior caused is all the risk factor of osteoporosis (lose bone mineral density, cause risk of bone fracture).The people with arbitrary following features can be used as with the candidate of ActRIIa antagonism treatment: the postclimacteric and women of do not ingest estrogen or other Hormone Replacement Therapy; The postclimacteric women of high (more than 5 feet 7 inches) or thin (being less than 125 pounds); There is the male of relevant clinical condition of losing bone; Apply the known people causing the medicine of losing bone, comprise such as prednisone (Prednisone ^tM) glucocorticoid, various anti-tic medicines such as phenytoin Sodium (Dilantin ^tM) and some Barbiturate, or the thyroid alternative medicine of high dose; There is type i diabetes, hepatic disease, kidney disease or there is the people of osteoporosis family history; There is the people of high turnover (such as, in urine sample, collagen is too much); There is thyroid status such as hyperthyroid people; The people fractured after slight wound; There is the people of the x-ray evidence of spinal fracture or other phenomenon osteoporotic.

As mentioned above, osteoporosis also can be and another kind of disease association or the situation relevant with the use of certain medicine.The osteoporosis that drug induced or other medical conditions causes is called as secondary osteoporosis.In the situation of one hypercortisolism (Cushing ' s disease), the too much hydrocortisone that health produces causes osteoporosis and fracture.The most common medicine relevant to secondary osteoporosis is glucocorticoid, the medicine that a class acts on as hydrocortisone, a kind of natural hormone produced by adrenal gland.Although the proper level of thyroxin (it is produced by thyroid) is that skeleton development is necessary, excessive thyroxin reduces bone amount sustainably.The people particularly dialysed when kidney is problematic take in high dose comprise the antacid of aluminum time can cause bone loss.The medicament of other caused secondary osteoporosis comprises phenytoin (phenytoin Sodium (Dilantin)) for preventing epilepsy and Barbiturate; Methotrexate (Rheumatrex, Immunex, Folex PFS), the medicine of a kind of arthritis for some types, cancer or immunological diseases; Ring spore toxin (Sandimmune, Neoral), is a kind ofly used for the treatment of some autoimmune diseasees and the medicine of Immunosuppression system in the patient body of organ transplantation; Luteinising hormone-releasing hormo agonist (Lupron, Zoladex), is used for the treatment of carcinoma of prostate and endometriosis; Heparin (Calciparine, Liquaemin), anticoagulation medicine; And cholestyramine (Questran) and colestipol (Colestid), be used for the treatment of hypercholesterolemia.The bone loss that treatment of cancer causes is familiar with widely and is known as the bone loss (CTIBL) for the treatment of of cancer induction.Bone tumour can cause hole on skeleton, and it is by with activin-ActRIIa antagonist for treating and correction.

In one preferred embodiment, the ActRIIa of activin-ActRIIa antagonist, particularly solubility disclosed in this invention, can apply and cancer patient.There is the patient of certain tumor (such as, prostatic, mammary gland, multiple myeloma or anyly cause hyperthyroid tumor), there is due to the bone loss of tumor inducing and the reason of Bone tumour and therapeutic agent the excessive risk of bone loss.Or even when lacking the evidence of bone loss or Bone tumour, such patient can with activin-ActRIIa antagonist for treating.The bone loss of patient or the evidence of Bone tumour can also be monitored, when indicator for displaying risk rises with activin-ActRIIa antagonist for treating.Normally, adopt the change of DEXA (research of dual-energy x-ray bone density measurement) scanning assessment bone density, and the indicator of skeleton reconstruct can be used for the probability assessing Bone tumour.Serum marker can be monitored.The special alkali phosphatase (BSAP) of skeleton is a kind of enzyme existed in osteoblast.Bone tumour and other BSAP blood level causing skeleton to reconstruct the patient of the situation increased rise.Bone Gla protein and procollagen peptide are formed relevant with Bone tumour to skeleton equally.In the patient with the Bone tumour that carcinoma of prostate causes, the rising of BSAP detected, and in the Bone tumour that causes of existence in various degree and breast carcinoma.High-caliber skeletal form generation albumen 7 (BMP-7) be present in Bone tumour in the carcinoma of prostate of skeleton, but be not present in the Bone tumour that bladder, skin, liver or lungs cancer cause.I type carboxyl terminal peptide (ICTP) is the cross-linking agent found in a kind of collagen formed during bone absorption.Due to skeleton destroyed and transformation constantly, ICTP can find at whole body.But, Bone tumour occur position, its level by significant higher than the region at normal bone.The high level of ICTP is found in the Bone tumour that prostate, lungs and breast carcinoma causes.Another kind of collagen crosslinking agent, I type amino terminal peptide (NTx), when bone is changed and ICTP together produce.Comprise in the Bone tumour that lungs, prostate and breast carcinoma causes in a variety of various cancers, the amount of NTx rises.Further, the rising of NTx level is along with the development process of Bone tumour.Therefore, this labelling can be used for the degree detecting Bone tumour and measurement disease.Other absorption labelling comprises pyridine ether and deoxidation pyridine ether.Any growth absorbing labelling and Bone tumour labelling indicates the treatment of needs of patients activin-ActRIIa antagonist.

Activin-ActRIIa antagonist can be used with other medicines agents.Co-administered by single compound prescription, by use or interval time has used simultaneously.If Activin-ActRIIa antagonist is together used with other skeleton activator, have special advantage.Patient can from activin-ActRIIa antagonist and picked-up calsium supplement, vitamin D, suitable exercise and/or (in some cases) other medication combined be benefited using.The example of other medicine comprises, diphosphonate (Alendronate sodium, ibandronate and risedronate sodium), calcitonin, estrogen, parathyroid hormone and raloxifene.Diphosphonate (Alendronate sodium, ibandronate and risedronate sodium), calcitonin, estrogen, parathyroid hormone and raloxifene affect the skeleton reconstruct cycle and are classified as anti-absorbing the drug.Skeleton reconstructs by two different stages; Bone absorption and skeleton are formed.The anti-bone absorption part but the skeleton forming section not slowing down the cycle that slow down or stagnate the skeleton reconstruct cycle of absorbing the drug.As a result, new synthesis speed continues to be greater than absorption rate, skeleton density increases always.Fragment of parathyroid hormone, a kind of form of parathyroid hormone, it increases the skeleton synthesis speed in skeleton reconstruction cycle.Alendronate sodium has been recognized for postclimacteric osteoporotic prevention (5 milligrams of every days or weekly 35 milligrams) and treatment (10 milligrams of every days or weekly 70 milligrams).Alendronate sodium reduces bone loss, increases bone density and reduces the risk of bone fracture of spinal column, wrist and hip.Alendronate sodium be also recognized for the glucocorticoid inducible caused as these medicines of prolonged application (i.e. prednisone and cortisone) in treatment masculinity and femininity osteoporosis and for the osteoporotic treatment in male.Alendronate sodium adds that vitamin D is recognized for the postclimacteric osteoporosis (weekly 70 milligrams, add vitamin D) for the treatment of women, and is used for the treatment of to improve the bone amount suffering from osteoporotic male.Ibandronate is recognized for the postclimacteric osteoporosis of prevention and therapy.Monthly take once (150 milligrams), ibandronate must taking monthly on the same day.Ibandronate reduces bone loss, increases bone density and reduces the risk of spinal fracture.Risedronate sodium is recognized for the postclimacteric osteoporosis of prevention and therapy.Every day takes (dosage of 5 milligrams) or take weekly (35 milligrams of dosage or 35 milligrams of dosage add calcium), and risedronate sodium slows down bone loss, increases skeleton density and reduces the risk of spinal fracture and non-spinal fracture.Risedronate sodium is also recognized for masculinity and femininity with the osteoporosis of the glucocorticoid inducible preventing and/or treating these medicines of chronic administration (i.e. prednisone and cortisone) and cause.Calcitonin is that a kind of natural existence relates to calcium adjustment and the metabolic hormone of skeleton.Women after 5 years menopause, calcitonin slows down bone loss, increases spinal bone density and may alleviate the relevant pain of fracture.Calcitonin reduces the risk of spinal fracture.Calcitonin can be used as injection (50-100IU every day) or nose sprays into (200IU every day) use.Estrin treatment (ET)/hormone therapy (HT) is recognized for prevention of osteoporosis.ET reduces bone loss as shown, increases the skeleton density of spinal column and hip, and reduces the hip of postclimacteric women and the risk of spinal fracture.It is form with pill or skin patch that ET uses the most common, and the low dosage of about 0.3 milligram of every day of its release or the standard dose of about 0.625 milligram of every day, even and if just start also still effective after 70 years old.When taking in separately estrogen, it can increase the risk that women develops cervix uteri wall cancer (carcinoma of endometrium).For eliminating this risk, the prescription that health care provider is outputed is that progesterone hormone and estrogen combine the women for those with intact uteri.ET/HT alleviates menopausal symptom and display has beneficial effect to bone health.Side effect comprises colporrhagia, breast tenderness, emotion disturbance and gallbladder disease.Raloxifene, 60 milligrams of every days, is recognized for the postclimacteric osteoporosis of prevention and therapy.It derives from a series of medicine being called selective estrogen receptor modulators (SERMs), and it is developed into provides estrogenic beneficial effect and do not have potential defect.Raloxifene increases appraisal and reduces the risk of spinal fracture.Do not obtain data yet to prove that raloxifene can reduce the risk of hip and other non-spinal non-fracture.The formation of the new skeleton of this medicine irritation and increase bone mineral density significantly.In postclimacteric women, the reduction of the fracture of spinal column, hip, foot, rib and wrist is significant.In male, the reduction of the fracture in spinal column is significant, but does not have enough data to assess the reduction of the fracture of other position.Fragment of parathyroid hormone from using, as on the same day 24 hours injection.

7. pharmaceutical composition

In some embodiments, activin-ActRIIa antagonist of the present invention (such as, ActRIIa polypeptide) and pharmaceutically acceptable carrier compound.Such as, ActRIIa polypeptide can be used separately or as the composition of pharmaceutical formulation (therapeutic combination).The approach formula that target compound anyly can be suitable for being applied to the mankind or veterinary is used.

In some embodiments, Therapeutic Method of the present invention comprises and systemically uses compositions or as implantation body or equipment local application.When using, be naturally acceptable form on pyrogen-free, physiology for therapeutic combination of the present invention.Treatment effective agent except ActRIIa antagonist can optionally include compositions described above, in the method for the invention its can with target compound (such as, ActRIIa polypeptide) simultaneously or one after the other use.

Normally, ActRIIa antagonist is in parenteral administration.Being applicable to the pharmaceutical composition of parenteral, to comprise one or more ActRIIa polypeptide and one or more pharmaceutically acceptable sterile isotonic aqueous solutions or nonaqueous solvent, dispersant, suspension or Emulsion, maybe can the recombinate sterilized powder of sterile injectable solution used or dispersion above combined, and it can comprise the medicine of antioxidant, buffer agent, antibacterial, solute that imparting formula and the blood etc. of receptor are in the future opened or suspension or thickening.The example of the suitable water carrier or nonaqueous carrier that are applied to pharmaceutical composition of the present invention comprises water, ethanol, polyol (such as glycerol, propylene glycol, Polyethylene Glycol, etc.) and suitable mixture, vegetable oil (such as olive oil), and injectable organic ester (such as ethyl oleate).Suitable mobility can be kept, such as, by application encrusting substance matter (such as lecithin), in the case of a dispersion by the granular size required by maintenance, and by application surface activating agent.

In addition, compositions can be loaded into capsule or the form of injection for being released into target tissue site (such as, skeleton).In some embodiments, compositions of the present invention comprise can discharge one or more therapeutic compound (such as ActRIIa polypeptide) to target tissue site (such as, skeleton) substrate, there is provided a kind of structure for developmental tissue and, most desirably, can be absorbed in body.Such as, substrate can provide the slow releasing of ActRIIa polypeptide.Such substrate can be formed for other material implanting medical applications by existing.

The selection of stroma ground substance is based on biological adaptation degree, biodegradability, mechanical property, outward appearance and interfacial characteristics.The application-specific of objective composition will determine the formula be suitable for.The potential substrate for compositions is biodegradable and chemically clearly calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and condensing model.Other potential material is biodegradable and is biologically perfectly clear, such as skeleton or dermal collagen.Other substrate comprises pure protein or extracellular matrix components.Other potential substrate right and wrong are biodegradable and chemically clearly, the hydroxyapatite such as sintered, bio-vitric, aluminate or other pottery.Substrate can be made up of the combination of the material of any type mentioned above (such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate).Bioceramic can change in the composition, such as in calcium-aluminum-phosphoric acid and in the process changing aperture, granular size, grain shape and biodegradability.

In some embodiments, method of the present invention can be oral administration, such as, with capsule, cathets, pill, tablet, buccal tablet (adopts flavoured base, normally sucrose and Radix Acaciae senegalis or tragacanth), powder, granule or as the solution in water or nonaqueous solvent or suspension, or as Water-In-Oil or as oil-in-water liquid emulsion, or as elixir or syrup, or (adopt inert base as lozenge, such as gel and glycerol, or sucrose and Radix Acaciae senegalis) and/or the form of collutory etc., often kind of medicament as active component all comprising predetermined volume.The form that medicament also can be used as pill, electuary or paste is used.

In the solid-state dosage form (capsule, tablet, pill, dragee, powder, granule etc.) of oral administration, one or more therapeutic compound of the present invention can mix with one or more pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate), and/or any below material: (1) filler or diluent, such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binding agent, such as, such as, methylcellulose, alginate, gel, polyvinylpyrrolidone, sucrose and/or Radix Acaciae senegalis; (3) wetting agent, such as glycerol; (4) disintegrating agent, such as aga agar, calcium carbonate, potato starch or tapioca, alginic acid, some silicate and sodium carbonate; (5) solution slow releasing agent, such as paraffin; (6) absorption enhancer, such as quaternary ammonium compound; (7) wetting agent, such as, such as, hexadecanol and glyceryl monostearate; (8) absorbent, such as Kaolin and bentonite; (9) lubricant, such as Talcum, calcium stearate, answers antacid magnesium and solid polyethylene glycol, lauryl sulfate base sodium and composition thereof; (10) coloring agent.When capsule, tablet and pill, pharmaceutical composition can comprise buffering medicament.The solid-state composition of similar type also can be used for using the soft filling as adjuvant of such as lactose or toffee and high molecular weight polyethylene glycol etc. and the filler of hard-filled gelatin capsule.

Liquid forms for oral administration comprises pharmaceutically acceptable emulsion, microemulsion, solution, syrup and elixir.In addition to the active ingredient (s, liquid forms comprises the conventional inert diluent (such as water or other solvent) in this area, cosolvent and emulsifying agent (such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, phenylpropanol, benzyl benzoate, propylene glycol, 1, fatty acid ester of 3-butanediol, oil (especially, Semen Gossypii, Semen arachidis hypogaeae, Semen Maydis, plumule, Fructus Canarii albi, Semen Ricini and Oleum sesami), glycerol, tetrahydrofurfuryl carbinol, Polyethylene Glycol and sorbitan and composition thereof.Besides inert diluents, Orally administered composition also comprises the auxiliary agent of such as wetting agent, emulsifying and suspending agent, sweeting agent, spice, coloring agent, perfuming and antiseptic.

Suspending agent, except active ingredient beyond the region of objective existence, comprise the suspending agent of such as ethoxylated isostearyl alcohol (ethoxylatedisostearyl alcohols), polyoxyethylene sorbitol and sorbitan, microcrystalline Cellulose, partially aluminium hydroxide (aluminum metahydroxide), bentonite, aga agar and tragacanth and composition thereof.

The present composition also can comprise auxiliary agent, such as antiseptic, wetting agent, emulsifying agent and dispersant.By comprising various antimicrobial drug and preventing of microbial activities guaranteed by antifungal agent (such as, para, methaform, phenol etc.).Also isotonic agent is comprised in compositions, such as sugar, sodium chloride etc. time necessary.In addition, the prolongation of injectable drug absorbs the medicament such as aluminum monostearate and being mingled with of gel by postponing absorption and brings.

Certainly, dosage regimen is made after attending doctor considers the factor that various change target compound of the present invention (such as, ActRIIa polypeptide) acts on.Described various factors includes but not limited to, needs the skeleton weight formed, the degree that bone density is lost, the position of bone injury, the situation of damage skeleton, age of patient, sex and diet, the order of severity of the disease of any promotion bone loss, time of application and other clinical factor.Optionally, dosage can change according to the type of compounds in the matrix type used in reconstruction and compositions.Other known somatomedin be added in the middle of final compositions also can affect dosage.Process is monitored by the periodical evaluation (such as, X-ray (comprising DEXA), histomorphometricall and tetracycline marker) of bone growth and/or reparation.

Mouse experiment demonstrate when each doses at intervals of compound and dosage enough reach 0.2 microgram/kilogram or higher blood drug level time, the effect of ActRIIa-Fc on skeleton is detectable,, and serum level be 1 microgram/kilogram or 2 micrograms/kilogram or higher be that to obtain the remarkable result of skeleton density and intensity necessary.Although there is no the ActRIIa-Fc of more high dose due to side effect can be caused and undesirable sign, therapeutic scheme be designed to reach 0.2 and 15 micrograms/kilogram between blood drug level, be optionally 1 to 5 micrograms/kilogram between.In the mankind, 0.2 microgram/kilogram serum level reach by 0.1 mg/kg or higher single dose, 1 microgram/kilogram serum level reach by 0.3 mg/kg or higher single dose.The serum half-life of the molecule observed about 20 days by 30 days between, be longer than most Fc fusion rotein substantially, and therefore obtain lasting effective serum level, such as, by the dosage of the 0.2-0.4 mg/kg weekly or on two all bases, or interval longer between higher dosage and dosage.Such as, the dosage of 1-3 mg/kg can be used for monthly or on bimonthly basis, and effect on skeleton is enough lasting, such dosage for be only every 3,4,5,6,9,12 or more months once necessary.

In some embodiments, present invention also offers the gene therapy producing ActRIIa polypeptide in body.Such therapy reaches its therapeutic effect by introducing ActRIIa polynucleotide sequence in the cell or tissue suffering from disease listed above.Sending by using recombinant expression carrier such as embedded virus or colloidal dispersion system of ActRIIa polynucleotide sequence.What be preferred for ActRIIa polynucleotide sequence therapeutic delivery is the use of target liposomes.

What the present invention instructed variously can be used for utilizing the viral vector of gene therapy to comprise adenovirus, herpesvirus, cowpox, or preferably, RNA viruses is retrovirus such as.Preferably, retroviral vector is the derivant of mice or avian retrovirus.The retroviral example that can insert single exogenous gene includes but not limited to: moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), MuMTV (MuMTV) and rous sarcoma virus (RSV).Other retrovirus can merge multiple gene in a large number.All these carriers transferable or merge gene with selected marker make transduce cell can identified and generate.Retroviral vector makes target-specific by adhering to such as sugar, glycolipid or protein.Preferred targeting reaches by adopting antibody.Those skilled in the art will appreciate that special polynucleotide sequence can be inserted into reverse transcription virus gene group or adhere to virus envelope protein to send with allowing to comprise the retroviral vector target-specific of ActRIIa polynucleotide.In one preferred embodiment, carrier targeting skeleton or cartilage.

Or, organizational structure cell by the calcium phosphate transfection of routine directly with the plasmid transfection of encoding retrovirus structural gene gag, pol and env.Then these cells are to comprise the vector plasmid transfection of interested gene.The cell release retroviral vector obtained is in culture medium.

Another ActRIIa polynucleotide Targeting delivery system is dispersion system of colloid.Dispersion system of colloid comprises macromolecular complex, Nano capsule, microsphere, pearl and liposome system and comprises water in oil emulsion, micelle, mixed micelle and liposome.The preferred dispersion system of colloid of the present invention is liposome.Liposome is artificial membrane vesicle, and it can be effective as the dispersible carrier in external or body.RNA, DNA and complete virion together can encapsulate with aqueous solution inside, and with biologically active form dispersate cell (see, such as, Fraley, etc., Trends Biochem.Sci., 6:77,1981).The method adopting liposome vectors high-efficiency transfection gene is well known in the art, see such as, and Mannino, etc., Biotechniques, 6:682,1988.The combination of liposome composition normally phospholipid, usually and steroid particularly cholesterol combination.Also other phosphate ester or other lipid can be used.The physical characteristic of liposome depends on the existence of pH, ionic strength and bivalent cation.

The example that can be used for the lipid producing liposome comprises phosphatidyl compound, such as phosphatidyl glycerol, lecithin, Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, sphingomyelins, cerebroside and ganglioside.The example of exemplary phospholipid comprises Ovum Gallus domesticus Flavus lecithin, dipalmitoyl phosphatidyl choline and distearyl phospholipid ester choline.The targeting of liposome also may be based on, such as, organ specificity, cell-specific and organelle-specificity and known in the art other.

Embodiment

The present invention is here general description, will be easier to understand by referring to following embodiment, and included embodiment, only for explaining some embodiment and some embodiment of the present invention, is not intended for use to limit the present invention.

Embodiment 1:ActRIIa-Fc fusion rotein

Applicant constructs the solubility ActRIIa fusion rotein with the people ActRIIa ectodomain merging the pure man or mice Fc domain, has minimal connexon between the two.Construction refers to ActRIIa-hFc and ActRIIa-mFc respectively.

Purification is as follows from the ActRIIa-hFc (SEQ ID NO:7) of Chinese hamster ovary celI system:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSI

EIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEV

TQPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVL

HODWLNGKEYKCKVSNKALPVPIEKTISKAKGOPREPOVYTLPPSREEMTK

NOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPGK

ActRIIa-hFc and ActRIIa-mFc protein expression is in Chinese hamster ovary celI system.Consider three kinds of different targeting sequencings:

(i) honey bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ IDNO:8)

(ii) tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO:9)

(iii) natural: MGAAAKL AFAVFLISCS SGA (SEQ ID NO:10)

Selected form adopts TPA leading and has following not processed aminoacid sequence:

MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQT

GVEPCYGDKDKRRHCFATWKNIS GSIEIVKQGCWLDDINCYDRTDCVEKKD

SPEVYFCCCEGNMCNEKF SYFPEMEVTQPT SNPVTPKPPTGGGTHTCPPCPA

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEQYNSTYIVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIE

KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK(SEQ ID NO：13)

This peptide is coded by nucleotide sequence below:

ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGT GGAG

CAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAG

GAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAAC

TGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTG

CTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTT

GGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAA

AGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATG

AAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAAT

CCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCAC

CGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC

CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT

GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG

GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA

GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG

CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA

AAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA

GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGG AGATG

ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA

GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT

ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAT

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT

CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGTAAATGAGAATTC(SEQ ID NO：14)

ActRIIa-hFc and ActRIIa-mFc complies with recombinant expressed significance.As shown in Figure 1, protein purification is single good albumen peak shape.N-terminal order-checking discloses the unique sequence of ILGRSETQE (SEQ ID NO:11).Purification reaches by a series of column chromatography steps, comprises, such as, and three or more following step, in any order: A protein chromatographic, Q agarose chromatography, phenyl sepharose chromatography, size exclusion chromatography and cation-exchange chromatography.Purification exchanges by virus filtration and buffering and completes.ActRIIa-hFc protein purification to determine to be greater than with size exclusion chromatography 98% and determine to be greater than with SDS PAGE 95% purity.

ActRIIa-hFc and ActRIIa-mFc shows the high-affinity with part particularly activinA.GDF-11 or ActivinA (" ACA ") adopts the aminoacid of standard to be coupled program and is fixed on Biacore CM5 chip.ActRIIa-hFc and ActRIIa-mFc albumen is loaded on system, and measures its combination.ActRIIa-hFc is with 5 × 10 ^-12dissociation constant (K _d) be bonded to activin, and protein is with 9.96 × 10 ^-9k _dbe bonded to GDF11.See Fig. 2.The situation of ActRIIa-mFc is similar.

A-204 reporter gene assays assesses the effect of ActRIIa-hFc albumen on signal transmission by GDF-11 and ActivinA.Cell line: human rhabdomyosarcoma's (deriving from muscle).Report carrier: pGL3 (CAGA) 12 (Described in Dennler etc., 1998, EMBO 17:3091-3100.) is see Fig. 3.CAGA12 motif is present in TGF-beta reactive group (PAI-1 gene), and therefore this carrier is generally used for the signal factor by Smad2 and 3.

First day: be separated A-204 cell and enter 48 orifice plates.

Second day: A-204 cell and 10 μ g pGL3 (CAGA) 12 or pGL3 (CAGA) 12 (10 μ g)+pRLCMV (1 μ g) and Fugene together transfection.

3rd day: add the factor (diluting into 0.1%BSA substrate).Inhibitor need before adding to cell with factor preincubate 1 hour.6 as a child, and cell washs with PBS, and dissolved cell.

Follow by luciferase assay.Usually in the method, without any the existence of inhibitor, ActivinA demonstrates the reporter gene expression of stimulation about 10 times and ED50 ~ 2 nanograms/milliliter.GDF-11:16 doubly stimulates, ED50: ~ 1.5 nanograms/milliliter.GDF-8 shows the effect of similar GDF-11.

As shown in Fig. 4, the signal that ActRIIa-hFc and ActRIIa-mFc suppresses GDF-8 to regulate under picomolar concentrations.As shown in Figure 5, three kinds of different ActRIIa-hFc preparations suppress GDF-11 signal together with the IC50 close to 200pM.

ActRIIa-hFc is highly stable in pharmacokinetic analysis.The ActRIIa-hFc polypeptide of rat administration 1mg/kg, 3mg/kg or 10mg/kg, and the Plasma Drug Level of albumen was measured at 24,48,72,144 and 168 hours.In an independent research, the dosage of rat is 1mg/kg, 10mg/kg or 30mg/kg.In rats, ActRIIa-hFc there is the serum half-life of 11-14 days and the cyclical level of medicine after 2 weeks very high (11 μ g/ml, 110 μ g/ml or 304 μ g/ml, respectively for the initial dose of 1mg/kg, 10mg/kg or 30mg/kg).In machin, plasma half-life is substantially greater than 14 days and the cyclical level of medicine is 25 μ g/ml, 304 μ g/ml or 1440 μ g/ml (corresponding respectively to the initial concentration of 1mg/kg, 10mg/kg or 30mg/kg).PRELIMINARY RESULTS display in the mankind, serum drug drug half-life is between about 20 to 30 days.

Embodiment 2:ActRIIa-hFc promotes bone growth in vivo

Normal female mice (BALB/c) gives the ActRIIa-mFc of 1mg/kg/ agent, 3mg/kg/ agent or 10mg/kg/ agent level, and Per-Hop behavior 2 times, bone mineral density and bone mineral content are determined by DEXA, see Fig. 6.

In female BAl BIc/c, DEXA scanning shows the therapeutic outcome as ActRIIa-mFc, the remarkable increase (> 20%) of bone mineral density and content.See Fig. 7 and 8.

Therefore, the antagonist of ActRIIa causes the bone density of normal female mice and the increase of content.As subsequent step, have detected the effect of ActRIIa-mFc on the mouse model removing ovary.

Andersson etc. (2001), determine that the mice of removing ovary stands suitable bone loss (probably lost the spongy bone of 50% in 6 weeks after surgery), and the bone loss in these mices is by candidate therapeutic agent such as parathyroid hormone extract for treating.

Applicant adopts (OVX) female C57BL6 mice of removal ovary or the sham-operation female mice in 4-5 age in week.Postoperative 8 weeks, start to treat with ActRIIa-mFc (10mg/kg, twice weekly) or contrast (PBS).Bone density is weighed by CT scan.

As Fig. 9 display, after 6 weeks, relative to the mice of sham-operation, mice that is untreated, that remove ovary shows suitable cancellous bone density loss.ActRIIa-mFc processed group remains the bone density of sham operated rats level.When the treatment of 6 weeks and 12 weeks, ActRIIa-mFc causes the growth that OVX mice spongy bone is suitable.See Figure 10.After the treatment of 6 weeks, relative to PBS matched group, bone density adds 24%.After 12 weeks, increase to 27%.

In sham-operation mice, ActRIIa-mFc also result in the suitable increase of spongy bone.See Figure 11.After 6 weeks and 12 weeks, relative to contrast, treatment creates the growth of 35%.

In other one group of experiment, remove ovary (OVX) mice or sham-operation mice as above and process more than 12 weeks with ActRIIa-mFc (10mg/kg, twice weekly) or contrast (PBS).Be similar to the result of above-described ActRIIa-mFc, the OVX mice accepting ActRIIa-mFc shows that cancellous bone density adds 15% as far back as process 4 weeks time, and after the process of 12 weeks, add 15% (Figure 12).Accept ActRIIa-mFc sham-operation mice show cancellous bone density equally add 25% as far back as process 4 weeks time, and after the process of 12 weeks, add 32% (Figure 13).

With ActRIIa-mFc process after 12 weeks, in whole body and a junctor, femur DEXA analyzes display, processes in removal ovary mice and sham-operation mice (Figure 14 A and Figure 14 B, the growth all induction of skeleton density respectively).These results also by pQCT in junctor between femur stage casing analyze support, which demonstrate with ActRIIa-mFc process after 12 weeks, total skeleton density and cortical bone density all significantly rise.The contrast of vehicle treated removes the skeleton density of ovary mice displaying compared with the contrast sham-operation mice of vehicle treated (Figure 15).Except skeleton density, bone amount also increases with ActRIIa-mFc process.Between femur stage casing, in junctor, pQCT analytical proof is all significantly rising with ActRIIa-mFc process 12 weeks rear total bone amount and cortex bone amount, and the mice of removing the mice of ovary and the vehicle Control process of sham-operation all illustrates comparable bone amount (Figure 16).Between femur stage casing, in junctor, pQCT analysis also shows, the mice of ActRIIa-mFc process does not show the change of periosteum girth, and ActRIIa-mFc process causes the reduction of perimyelis girth to show, and the increase of cortical thickness is (Figure 17) of being caused by the growth of femur inner surface.The mechanical test of femur determines that ActRIIa-mFc can increase the external characteristic (maximum load, rigidity and energy to failure) of skeleton, and it contributes to the remarkable increase of skeleton intrinsic characteristic (ultimate strength).With the mice of the removal ovary of ActRIIa-mFc process illustrate higher than sham-operation, the bone strength of the increase of the control level of vehicle treated, show that osteoporosis is phenotypic and reverse (Figure 18) completely.

These digital proofs activin-ActRIIa antagonist can increase bone density in female mice, and, in addition, in osteoporotic mouse model, correct the defect of bone density, bone content and maximum bone strength.

In another group experiment, mice removed ovary or sham-operation at the 4th week, accepts placebo or ActRIIa-mFc (2 times weekly, 10mg/kg) (RAP-11 as in Figure 19-24) at first, then carry out 12 weeks at the 12nd week.Assess various bone parameters.As shown in figure 19, ActRIIa-mFc increases OVX and the vertebra spongy bone volume of sham operated rats and the ratio (BV/TV) of total measurement (volume).ActRIIa-mFc also improves the structure (Figure 20) of spongy bone, increases periosteum thickness (Figure 21) and improves bone strength (Figure 22).As shown in figure 23, ActRIIa-mFc produces gratifying effect in the dosage range from 1mg/kg to 10mg/kg.

The tissue morphology measurement of skeleton carries out at the 2 time-of-week points of sham-operation mice.These data presented in fig. 24, prove that ActRIIa-mFc has a dual effect, and suppress bone absorption and promote bone growth.Therefore, ActRIIa-mFc promotes bone growth (anabolism effect) and suppresses bone absorption (Anticatabolism effect).

Embodiment 4: the ActRIIa-Fc albumen substituted

A kind of alternative construction can delete 15 aminoacid of ActRIIa ectodomain C-terminal tail (last).The sequence of such construction is as follows: (Fc part is following to be lined out) (SEQ ID NO:12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSI

EIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM TG

GGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV

KFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCK

VSNKALPVPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLVKGFYPS

DIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCS VMHEALHNHYTOKSLSLSPGK

The integration of list of references

Be incorporated to the present invention with the form of list of references, as each publication or patent especially and indicate individually and be incorporated to list of references all publications that the present invention mentions and entirety.

Although discuss the specific embodiment of goal object, description is above exemplary and is nonrestrictive.By reading description above and following claim, many variations are obvious to those skilled in the art.The whole protection domain of the present invention should be determined with reference to the claim protection domain suitable with it and description and such variation.

Claims

1. activin-is in conjunction with an ActRIIa polypeptide, and it comprises the aminoacid sequence be made up of the aminoacid sequence of SEQ ID NO:7.

2. activin-according to claim 1 is in conjunction with ActRIIa polypeptide, considers protein impurities, and the purity of wherein said polypeptide is at least 95%, and it is determined by size exclusion chromatography.

3. the activin-described in claim 1 or 2 in conjunction with ActRIIa polypeptide, wherein said polypeptide relative to GDF-11 to the selectivity dissociation constant of activin showing at least 10 times.

4. a pharmaceutical preparation, it comprises activin-any one of claim 1-3 in conjunction with ActRIIa polypeptide and the pharmaceutically acceptable adjuvant of at least one.

5. pharmaceutical preparation according to claim 4, wherein said preparation is apyrogeneity material substantially.

6. the polynucleotide be separated, it comprises the coded sequence of the activin-any one of claim 1-3 in conjunction with ActRIIa polypeptide.

7. the polynucleotide of separation according to claim 6, the polynucleotide of wherein said separation comprise the sequence of SEQ ID NO:14.

8. a recombination of polynucleotide, it comprises promoter sequence and is operably connected to polynucleotide described in claim 6 or 7.

9. a cell, it transforms with the recombination of polynucleotide any one of claim 6-8, and wherein said cell is not the plant cell of the plant variety that can develop into whole plant.

10. cell according to claim 9, wherein said cell is mammalian cell.

11. cells according to claim 10, wherein said cell is Chinese hamster ovary celI or human cell.

12. 1 kinds manufacture the method for activin-in conjunction with ActRIIa polypeptide, and the method comprises:

A) cultured cell under the condition of applicable expression ActRIIa polypeptide, wherein said cell transforms with the recombination of polynucleotide any one of claim 6-8; And

B) activin-expressed by renaturation is in conjunction with ActRIIa polypeptide.

13. 1 kinds of pharmaceutical preparatioies, it comprises:

A the activin-any one of () claims 1 to 3 is in conjunction with ActRIIa polypeptide; And

B () second bone-active agent, wherein said second bone-active agent is selected from: bisphosphonate, estrogen, selective estrogen receptor modulators, parathyroid hormone, calcitonin, calsium supplement and Vitamin D supplements.

Activin-any one of 14. claim 1-3 is in conjunction with ActRIIa polypeptide, or the pharmaceutical preparation any one of claim 4-5, and wherein said polypeptide is glycosylated.

15. 1 peptide species, it comprises SEQ ID NO:7.

16. 1 kinds of soluble polypeptides according to claim 15.

17. 1 kinds of polypeptide according to claim 16, it is bonded to activin.

18. 1 kinds of polypeptide according to claim 17, it is bonded to activin to be less than 1 micromolar dissociation constant.

19. 1 kinds of polypeptide according to claim 18, it is bonded to activin with the dissociation constant being less than 100 nanomoles.

20. 1 kinds of polypeptide according to claim 19, it is bonded to activin with the dissociation constant being less than 10 nanomoles.

21. 1 kinds of polypeptide according to claim 20, it is bonded to activin with the dissociation constant being less than 1 nanomole.

22. 1 kinds of polypeptide according to claim 15, its purity is at least 95%.

23. 1 kinds of polypeptide according to claim 22, its purity is at least 98%.

24. 1 kinds of polypeptide according to claim 15, it is acetylizad.

25. 1 kinds of polypeptide according to claim 15, it is carboxylated.

26. 1 kinds of polypeptide according to claim 15, it is glycosylated.

27. 1 kinds of polypeptide according to claim 15, it is phosphorylation.

28. 1 kinds of polypeptide according to claim 15, it is lipidization.

29. 1 kinds of polypeptide according to claim 15, it is acidylate.

30. 1 kinds of polypeptide according to claim 15, it comprises Polyethylene Glycol.

31. 1 kinds of polypeptide according to claim 15, it comprises lipid.

32. 1 kinds of polypeptide according to claim 15, it comprises phosphoric acid.

33. 1 kinds of polypeptide according to claim 15, it comprises polysaccharide.

34. 1 kinds of polypeptide according to claim 15, it comprises monosaccharide.

35. 1 kinds of polypeptide according to claim 15, it is Pegylation.

36. 1 kinds of polypeptide according to claim 15, it is farnesylation.

37. 1 kinds of polypeptide according to claim 15, it is biotinylated.

38. 1 kinds of polypeptide according to claim 15, it is coupled to organic derivative.

39. activin-according to claim 1 are in conjunction with ActRIIa polypeptide, and wherein said polypeptide is bonded to activin.

The method of 40. claim 12, wherein said activin-is soluble polypeptide in conjunction with ActRIIa polypeptide.