CN101370511A - Activin-ActRlla antagonists and uses for promoting bone growth - Google Patents

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

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

Background technology

Skeletal diseases comprises from the osteoporosis to the fracture, has represented one group of pathological state, little can be effectively at the pharmaceutical preparation of these pathological states.The substitute is, the focus of treatment concentrates on and comprises in curing, motion and metatrophia physics and the behavior interference.For the various skeletal diseases of treatment, it will be useful that the healing potion that promotes bone growth and increase skeleton density is arranged.

Bone growth and mineralising depend on osteoblast and these two types of cell activity of osteoclast, although chondrocyte and vascular cell have also participated in the critical aspects of these processes.Development ground, skeleton form by two kinds of mechanism and take place, endochondral ossification and intramembranous ossification, and the former is responsible for the stringer bone formation, and the latter is responsible for the formation of topological flat bone such as skull skeleton.The endochondral ossification requirement form in proper order and growth plate in the degraded of cartilage structure, described growth plate is as the template of the formation of osteoblast, osteoclast, vascular cell and mineralising subsequently.In the intramembranous ossification process, skeleton directly forms in connective tissue.These two kinds of processes require osteoblastic infiltration and follow-up apposition.

The process that goes up similar osteogenetic growth order by a kind of surface at least of breaking fracture and other skeletal structure heals, and described osteogenetic growth comprises that cartilaginous tissue forms and mineralising subsequently.The process of union of fracture can take place in two ways.Direct or initial bone healing do not have a callus formation.Indirectly or the having the callus precursor stage of the bone healing of secondary.The initial stage healing of fracture relates to the transformation across the disruptive mechanical connection of closely arranging.Under appropriate condition, shown around disruptive skeleton absorptive cell that the tunnel absorbs and replied, and made up the path of puncture vessel and healing subsequently.The secondary healing of skeleton is carried out with the order of inflammatory reaction, the formation of cartilage crust, callus mineralising and callus reconstruct.In the inflammatory reaction stage, hematoma and hemorrhage formation come from the periosteum of damage position and breaking of perimyelis blood vessel.Inflammatory cell is invaded this zone.Form the stage in the cartilage crust, cell generates new blood vessel, fibroblast, intracellular organic matter and sustenticular cell, and the gap between the fracture fragmentation forms granulation tissue.Set up by fibrous or cartilaginous tissue (cartilage crust) across disruptive clinical healing.Osteoblast forms and regulates the mineralising of cartilage crust, and described then cartilage crust is replaced by lamellar bone and is subject to normal restructuring procedure.

Except fracture and other physics of skeletal structure broke, losing of skeleton content of mineral substances and bone density can be caused and may be caused the important medical problem by multiple different condition.The change of bone density betides individual whole life period in expected relatively mode.About time 30 years old, the skeleton of masculinity and femininity is by the linear growth of endochondral ossification growth dish and radially grow to the biggest quality.About 30 years old (for spongy bone, for example, flat bone such as vertebra and pelvis) and 40 years old (for cortical bone, for example, the long bone in limbs), bone loss slowly all takes place in masculinity and femininity.In the women, also exist the essence skeleton of final stage to lose, perhaps be owing to lack estrogen after the menopause.During this period, the women may additionally lose 10% quality on cortical bone, and additionally loses on the spongiosa interval and lose 25% quality.Whether progressive bone mass is lost and is caused pathological condition such as osteoporosis to depend critically upon individual initial bone mass and whether have the situation that increases the weight of.

Bone loss is a feature with the imbalance of normal bone restructuring procedure sometimes.Healthy skeleton often is subject to reconstruct.Reconstruct is absorbed beginning with skeleton by osteoclast.Absorbed then skeleton is replaced by new skeletal tissue, it is characterized in that osteoblast forms collagen and calcification subsequently.In the individuality of health, absorption and the speed that forms are equilibrated.Osteoporosis is a kind of chronic, progressive situation, to be labelling towards the transfer that absorbs, causes the comprehensive minimizing and the mineralization of skeleton of bone mass.The osteoporosis of human body is prior to clinical osteopenia (bone mineral density below the adult average of youth greater than a standard deviation but less than two standard deviations).In the world wide, roughly 700,000,000 5 thousand ten thousand people have osteoporotic risk.

Therefore, control osteoclast and the active method of osteoblast are to promoting that the fracture and the healing of other damage of skeleton and the treatment of disease (as osteoporosis, relevant with the loss and the mineralization of skeleton of bone mass) are effective.

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

Therefore, the purpose of this invention is to provide compositions and the method that promotes bone growth and mineralising.

Summary of the invention

Partly, the present invention has proved that the molecular energy with activin (activin) or activin IIA receptor (ActRIIa) antagonist activities (" activin antagonist " and " ActRIIa antagonist ") is enough in the increase skeleton density, promote bone growth, and/or increase bone strength.Especially, the present invention has proved the inhibitor of the soluble form of ActRIIa as the activin-ActRIIa signal, and promotes skeleton density, bone growth and the bone strength of increase in vivo.Yet, the pharmaceutical preparation that most promotion bone growths or inhibition are lost bone or as anti--catabolism preparation (also relating to usually) as " catabolism preparation " (as, biphosphonate) or as the anabolism preparation (as, parathyroid hormone, PTH, when dosage is suitable), the ActRIIa albumen of solubility shows double activity, has catabolism and anabolic effect.Therefore, the present invention establishes, and the antagonist of activin-ActRIIa signal path can be used to increase skeleton density and promote bone growth.Yet the ActRIIa of solubility may the mechanism by except the activin antagonism influence skeleton, and however, the present invention has proved that desirable therapeutic agent can select according to the activin-ActRIIa antagonist activities.Therefore, in some embodiments, the present invention the disease (as osteoporosis) that provides the method for using the activin-ActRIIa antagonist to treat to be associated or promote that this patient who needs the is arranged bone growth of (as in the patient of fracture) with low skeleton density or low bone strength, described activin-ActRIIa antagonist comprises that for example activin is 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 the nucleic acid that reduces 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 that comprises soluble, as to be bonded to activin activin-binding ActRIIa polypeptide.The ActRIIa polypeptide can be made into to comprise the pharmaceutical preparation of activin-binding ActRIIa and pharmaceutically acceptable carrier.Preferably, activin-binding ActRIIa polypeptide is with less than 1 micromole or less than the K of 100,10 or 1 nanomoles _DBe bonded to activin.According to circumstances, with respect to GDF11 and/or GDF8, activin-binding ActRIIa is optionally in conjunction with activin, preferably, and the K of activin _DAt least than the K of GDF11 and/or GDF8 _DHang down 10 times, 20 times or 50 times.Yet do not wish to be bound by specific mechanism of action, be contemplated that selectance that activin suppresses to be higher than GDF11/GDF8 has illustrated can not cause in the selection effect on the skeleton and continue measurable effect on muscle.In some embodiments, select to cause and be lower than 15%, be lower than 10% or be lower than ActRIIa that 5% muscle increases obtains desirable effect on skeleton dosage.Preferably, the purity of compositions is 95% at least, about other polypeptide composition, determines that by the chi exclusion chromatography more preferably, the purity of compositions is 98% at least.The activin-binding ActRIIa polypeptide that uses in such preparation can be any polypeptide disclosed in this invention, as have a polypeptide that is selected from SEQ ID Nos:2,3,7,12 or 13 aminoacid sequence, or be selected from the polypeptide that SEQID Nos:2,3,7 or 12 aminoacid sequence have at least 80%, 85%, 90%, 95%, 97% or 99% homogeneity.Activin-binding ActRIIa polypeptide can comprise the functional fragment of natural ActRIIa, as comprises at least 10,20 or 30 aminoacid (" tail ") of the sequence of the sequence that is selected from SEQ ID NOs:1-3 or SEQ ID Nos:2 (10 to 15 aminoacid that lack C-terminal).

With respect to naturally occurring ActRIIa polypeptide, solubility, activin-binding ActRIIa polypeptide can comprise the change (for example at ligand binding domains) of one or more aminoacid sequences.The example of altered ActRIIa polypeptide is provided at the 59-60 page or leaf of WO 2006/012627, incorporates this paper by reference at this.The change of aminoacid sequence can be for example, when the glycosylation that changes polypeptide when mammal, insecticide or other eukaryotic cell are produced, perhaps to change the proteolytic cleavage of polypeptide with respect to naturally occurring ActRIIa polypeptide.

Activin-binding ActRIIa polypeptide can be a fusion rotein, and it has a domain (for example ligand binding moiety of ActRIIa polypeptide) and one or more other domain that desirable characteristic (as the specific tissue of improved pharmacokinetics, easier purification, targeting etc.) is provided of ActRIIa polypeptide.For example, the domain of fusion rotein can strengthen the multimerization and/or in the purification one or more of formation, the fusion rotein of half-life, absorption/use, tissue positioned or distribution, protein complex in the body internal stability, body.Activin-binding ActRIIa fusion rotein can comprise that immunoglobulin Fc domain (wild type or mutant) or serum albumin or other provide the polypeptide portion of desirable characteristics (such as improved pharmacokinetics, improved solubility or improved stability).One preferred embodiment in, ActRIIa-Fc merges and to comprise relative destructuring connexon, it is between Fc domain and ActRIIa ectodomain.This non-structured row key word can be corresponding to roughly 15 aminoacid destructuring zones of the C-terminal end points (" tail ") of ActRIIa ectodomain, perhaps it can be 1,2,3,4 or 5 amino acid whose artificial sequence, or 5 relative length between aminoacid freely, or both mixture with 15,20,30,50 or more a plurality of secondary structure.Connexon can be rich in glycine and proline residue, and can, for example, contain the simple sequence of threonine/serine and glycine or repetitive sequence (for example, the TG of threonine/serine and glycine ₄Or SG ₄Singlet or repetitive sequence).Fusion rotein can comprise the subsequence of purification, merges as epi-position label, FLAG label, poly histidine sequence and GST.According to circumstances, the activin of solubility comprises one or more modified amino acid residue that are selected from down group in conjunction with IIA receptor polypeptide: aminoacid, the coupling of glycosylated aminoacid, polyethyleneglycol modified (PEGylated) aminoacid, the aminoacid of farnesylation (farnesylated), acetylizad aminoacid, biotinylated aminoacid, coupling connection liposome part are associated with the aminoacid of machine derivative formulations.Pharmaceutical preparation can comprise that also one or more additional compounds are such as the chemical compound that is used for the treatment of skeletal diseases.Preferably, pharmaceutical preparation is no pyrogen substantially.Usually, preferably ActRIIa albumen is expressed in mammal cell line, and described cell line is regulated the Natively glycosylated suitably probability with the adverse immune response of eliminating the patient of ActRIIa albumen.Human cell line and Chinese hamster ovary celI system are successfully used, and expect that other common mammalian expression systems also is useful.

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

In some aspects, the invention provides the nucleic acid of coding solubility activin-binding ActRIIa polypeptide.Isolating polynucleotide can comprise the coded sequence of all solubility activin-binding ActRIIa polypeptide as described above.For example, the ectodomain that isolating nucleic acid can comprise ActRIIa (for example, ligand binding domains) coded sequence, and the sequence of the endochylema domain of coded portion or whole membrane spaning domain and/or ActRIIa, but do not encode in membrane spaning domain or in the endochylema domain or the termination codon between ectodomain and membrane spaning domain or endochylema domain.For example, isolating polynucleotide can comprise total length ActRIIa polynucleotide sequence such as SEQ ID NO:4 or 5, or the sequence of part amputation, described polynucleotide further be included in 3 ' terminal before the translation of polynucleotide of making of at least 600 nucleotide or other cause ectodomain to be fused to the tanscription termination codon of 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 and is used for expression promoter, and the invention provides the cell that has transformed such recombination of polynucleotide.Preferably, described cell is a mammalian cell, such as Chinese hamster ovary celI.

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

In some aspects, activin-ActRIIa antagonist disclosed in this invention, such as the activin-binding ActRIIa polypeptide of solubility, can be used for a kind of is the method for purpose to promote bone growth or to increase skeleton density.In some embodiments, the invention provides the method that treatment is hanged down the skeleton density relevant disease or promote bone growth in the patient of this demand is arranged.This method can comprise the activin-ActRIIa antagonist of the object that this demand is arranged being used effective dose.In some aspects, the invention provides the medicine that uses activin-ActRIIa antagonist preparation treatment disease recited above or situation.

In some aspects, the invention provides the method that stimulates bone growth or increase the medicine of mineralization of skeleton of differentiating.Described method comprises: the medicine l to be measured that a) differentiates the ligand binding domains that is bonded to activin or ActRIIa polypeptide; B) effect of assessment medicine on bone growth or mineralising.

Description of drawings

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

Fig. 2 has shown that ActRIIa-hFc is bonded to activin and GDF11, by the BiaCore analyzing and testing.

Fig. 3 has shown the sketch map that the A-204 reporter gene is analyzed.The figure illustrates report carrier: pGL3 (CAGA) 12 (in EMBO 17:3091-3100, description being arranged, Dennler etc., 1998).The CAGA12 motif appears in the TGF-Beta reactive group (PAI-1 gene), so this carrier is generally used for the factor by Smad2 and 3 signal paths.

Fig. 4 has shown ActRIIa-hFc (rhombus) and the effect of ActRIIa-mFc (square) on the GDF-8 signal in the analysis of A-204 reporter gene.Two kinds of albumen have showed that all it almost completely suppresses the signal that GDF-8 regulates in picomole concentration.

Fig. 5 has shown three kinds of different ActRIIa-hFc preparation effects on the GDF-11 signal in the analysis of A-204 reporter gene.

Fig. 6 shown BALB/c mouse that matched group and ActRIIa-mFc handle before the treatment phase in 12 weeks (above) and afterwards (below) the example of DEXA (dual intensity X line absorption mensuration) image.Light-colored part has shown the skeleton density that has increased.

Fig. 7 shown ActRIIa-mFc during BALB/c mouse 12 week treatment to the effect of bone mineral density quantitatively.Treatment is divided into the ActRIIa-mFc (square) of contrast (rhombus), 2mg/kg dosage, the ActRIIa-mFc (triangle) of 6mg/kg dosage and the ActRIIa-mFc (circle) of 10mg/kg dosage.

Fig. 8 shown ActRIIa-mFc during BALB/c mouse 12 week treatment to the effect of bone mineral content quantitatively.Treatment is divided into the ActRIIa-mFc (square) of contrast (rhombus), 2mg/kg dosage, the ActRIIa-mFc (triangle) of 6mg/kg dosage and the ActRIIa-mFc (circle) of 10mg/kg dosage.

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

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

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

Figure 12 has shown the skeleton density pQCT analysis result during 12 weeks of (OVX) mice of extracing ovary treat.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 has described the skeleton density pQCT analysis result during the treating in 12 weeks of mice 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 shown that the whole body DEXA of 12 week treatment after dates (A) analyze and femur between junctor inner analysis (B).The zone of high skeleton density has been described in the clear zone.

Figure 15 shown 12 weeks treatment after dates the femur stage casing between in the junctor pQCT analyze.Treatment is divided into contrast (PBS, dark bars) and ActRIIa-mFc (light bar).Four bars in left side show total skeleton density and four bars demonstration cortical bone density on right side.The first couple of per four bars represented the data that come from the mice of extracing ovary and second pair represented the data that come from the sham-operation mice.

Figure 16 shown 12 weeks treatment after dates the femur stage casing between in the junctor pQCT analyze and key content.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 the bars demonstrations in right side cortical bone content.The first couple of per four bars represented the data that come from the mice of extracing ovary and second pair represented the data that come from the sham-operation mice.

Figure 17 shown the femur stage casing between in the junctor pQCT analyze and the femur skin thickness.Treatment is divided into contrast (PBS, dark bars) or ActRIIa-mFc (light bar).Four bars in left side show the perimyelis girth and four bars hour periosteum girth on right side.The first couple of per four bars represented the data that come from the mice of extracing ovary and second pair represented the data that come from the sham-operation mice.

Figure 18 has described the femur mechanics testing result of 12 weeks treatment after date.Treatment is divided into contrast (PBS, dark bars) or ActRIIa-mFc (light bar).Two bars in left side have been represented the data that come from the mice of extracing ovary and two bars of back have been represented the data that come from the sham-operation mice.

Figure 19 has shown that ActRIIa-mFc is to the volumetrical influence of spongy bone.

Figure 20 has shown the influence of ActRIIa-mFc to the spongy bone structure of distal femur.

Figure 21 has shown the influence of ActRIIa-mFc to cortical bone.

Figure 22 has shown the influence of ActRIIa-mFc to skeletal mechanics intensity.

Figure 23 has shown that the ActRIIa-mFc of various dose is to the influence of skeleton characteristic under three various dose.

Figure 24 has shown that tectology measurement (histomorphometry) 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 the multiple identical total order that has The growth factor of column unit and structural motif. Known these albumen are to vertebrate and no vertebra Animal permitted eurypalynous cells play biological function. The member of superfamily is in embryonic development period Pattern formation and tissue differentiation in carry out critical function, and can affect multiple differentiation process, Comprise fatty generation, myogenesis, cartilage formation, heart generation, hemoposieis, neural giving birth to Become and the epithelial cell differentiation. This family be divided into two general branch: BMP/GDF and TGF-beta/Activin/BMP10 branch, its member have different, normally complementary effects. By handling TGF-beta family member's activity, usually can cause important life in the organism The of science variation. For example, Piedmont (Piedmontese) and Belgian Blue (Belgian Blue) Cattle breeds is with the intragenic disappearance function of GDF8 (also being called flesh chalone (myostatin)) Sudden change, it causes the phenomenal growth of muscle quality. Grobet etc., Nat Genet.1997,17 (1): 71-4. In addition, at human body, the increase of the allelic inactivation of GDF8 and muscle quality and (according to Title) anomaly intensity is relevant. Schuelke etc., N Engl J Med 2004,350:2682-8.

Activin (activin) is the dimer PGF that belongs to the TGF-beta superfamily. The activin that three kinds of citation forms (A, B and AB) arranged, it is two closely-related β Asias (the β of unit_A β _A、β _B β _BAnd β_A β _B) homology/heterodimer. Human genome also Coding activin C and activin E, it is expressed in liver at first. At the TGF-beta superfamily In, activins be can stimulate in the ovary and placenta cells in hormone produce, support neural thin Born of the same parents' survival, dependent cells type positivity or negativity ground affect cell cycle progression and exist at least Induce the only and multi-functional factor (DePaolo of mesoderm differentiation among the amphibian embryo Deng, 1991, Proc Soc Ep Biol Med.198:500-512; Dyson etc., 1997, Curr Biol.7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963). In addition, the erythroid differentiation factor (EDF) that separates the person monocytic cell leukaemia who certainly stimulated Be found identical with activin A (Murata etc., 1988, PNAS, 85:2434). Show activin A in ossis as erythropoietic natural positive regulating factor. One In a little tissues, the activin signal is by its relevant heterodimer, inhibin institute antagonism. For example, When hypophysis discharged follicular stimulating hormone (FSH), activin promoted the FSH secretion with synthetic, and Inhibin stops the FSH secretion with synthetic. Other regulated activin biologically active and/or combination To the albumen of activin comprise follicostatin (FS), follicostatin GAP-associated protein GAP (FSRP), α₂-macroglobulin, Cerberus and endothelium glycoprotein (endoglin).

The TGF-signal beta is by the different poly-complex of I type and II type serine/threonine kinase acceptor Regulate, the Smad albumen in its phosphorylation and activation downstream by the part activation (Massague, 2000, Nat.Rev.MoI.Cell Biol.1:169-178). These I types and II receptor are to stride film Albumen is by with the ligand binding ectodomain, the membrane spaning domain that are rich in the cysteine zone Form with the specific cytoplasm domain of serine/threonine with precognition. The I type is that signal is logical The road is necessary; The II type is that binding partner and expression I receptor are required. I type and II type activin Acceptor forms stable compound behind ligand binding, cause the I receptor by II receptor phosphorylation.

Two kinds of relevant II receptors, ActRIIa and ActRIIb have been accredited as the II of activin Receptor (Mathews and Vale, 1991, Cell 65:973-982; Attisano etc., 1992, Cell 68:97-108). Except activin, ActRIIa and ActRIIb can biochemical ground and other Several TGF-'beta ' family protein-interactings comprise BMP7, Noda1, 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 Be main activin I receptor, particularly for activin A, and ALK-7 also can With the acceptor as activins, particularly to activin B.

Prove such as the present invention, the ActRIIa polypeptide (sActRIIa) of solubility, it has shown TGF-beta family member with respect to other preferentially selects basically such as GDF8 or GDF11 Be bonded to activin A, can effectively promote in vivo bone growth and increase skeleton density. Yet do not wish to be subject to any specific mechanism, consider by in these research and adopt The combination that the activin that specific sActRIIa construction represents is very strong (picomole dissociation constant), The effect of expectation sActRIIa mainly is caused by activin antagonist effect. Do not consider machine System, the data here show that clearly the ActRIIa-activin antagonist is really at normal mouse And increased skeleton density in the osteoporotic mouse model. Please note that bone is dynamic organization, The increase of its growth or atrophy and density or minimizing depend on produces bone and stimulates mineralising The factor of (mainly being Gegenbaur's cell) and destruction and removal bone mineral matter (mainly are broken bone Cell) balance between the factor. The growth of bone and mineralising can by increase productivity because of Element or minimizing monkey wrench or both carry out simultaneously and increase. Term " promotion bone growth " " increase mineralization of skeleton " refers to the visible variation of physics of bone and is used for neutral ground Describe and change the mechanism that takes place in the bone.

The osteoporotic mouse model that adopts in the research described in the invention and bone growth/ It is that the Height Prediction tool is resultful that density is considered in human body, thereby, the invention provides and adopt In human body, promote bone with ActRIIa polypeptide and other activin-ActRIIa antagonist Growth and raising skeleton density. The Activin-ActRIIa antagonist comprises that for example, activin ties Close solubility ActRIIa polypeptide, be bonded to activin (particularly activinA or B subunit, Also refer to β A or β B) and interrupt the ActRIIa combination antibody, be bonded to ActRIIa also And interrupt the activin combination antibody, be selected from the non-antibody albumen of activin or ActRIIa combination (referring to for example, WO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989, US 2003/0133939, and this class among the US 2005/0238646 The method of the example of albumen and design and selection same protein), be selected from activin or ActRIIa In conjunction with the at random peptide that usually is conjugated to the Fc domain. Two kinds have activin or ActRIIa knot Close active different albumen (or other parts), particularly block respectively the I type and (for example, can Dissolubility I type activin acceptor) or II type (for example solubility II type activin acceptor) binding site The activin zygote, can couple together to produce the molecule of bi-functional combination. Nucleic acid is suitable The preparation of body, little molecule and other inhibition activin-ActRIIa signal shaft. Various eggs Have in vain the activin-ActRIIa antagonist activities, comprise inhibin (being inhibin α subunit) Although (inhibin not institute in a organized way in equal antagonism activin), follicostatin is (such as ovarian follicle Inhibin 288 and follicostatin 315), Cerberus, follicostatin GAP-associated protein GAP (FSRP), Endothelium glycoprotein (endoglin), activin C, α₂-macroglobulin and M108A are (at 108 Become alanine by methionine) sudden change activin A. Normally, the alternative form of activin, Particularly those are vicissitudinous at I receptor binding structural domain, can be bonded to the II receptor and Can not form active triple compounds, thereby as antagonist action. In addition, nucleic acid, all Such as antisense molecule, inhibition activinA, B, C or E or (especially) ActRIIa express SiRNA or ribozyme can be used as the activin-ActRIIa antagonist. Preferably, adopt The activin-ActRIIa antagonist will show that other member with respect to TGF-beta family is (relative In GDF8 and GDF11) inhibition activin institute conditioning signal selective. Solubility ActRIIb is bonded to activin, yet the albumen of wild type does not show with respect to GDF8/11 Be bonded to the significant selective of activin, and this can not providing is provided in preliminary experiment The desirable effect to bone, but still can cause the remarkable growth of muscle. Yet, have difference Binding characteristic ActRIIb change form identified (referring to for example, WO 2006/012627, the 55-59 page or leaf, mode is by reference incorporated the present invention into), and these eggs Desirable effect in vain can bone. Natural or the ActRIIb that changes can be by being coupled the Two activin selective binding compositions and additional specificity to activin is provided.

The used term of specification is applied specific at context of the present invention and each term Usually has it in the linguistic context in its ordinary meaning of this area. Some term is hereinafter or in explanation The other parts of book come into question, in order to describe the compositions and methods of the invention and how should Use and offer the guidance that the practitioner adds. The scope of any application of term or implication are in its institute Clearly in the special context of using.

Material object when " approximately " and " approaching " means the person's character of considering mensuration and precision usually The acceptable extent of error of measuring. Typically, exemplary extent of error given numerical value or Number range 20% in, preferably in 10%, more preferably in 5%.

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

Method of the present invention can comprise the step of mutual contrast sequence, comprises that the sequence of wild type and one or more mutant (sequence variants) compare.How the comparison that relatively generally includes the polymer sequence so for example, adopts sequence alignment program well known in the art/exclusive disjunction rule (for example, BLAST, FASTA and MEGALIGN can say).Those skilled in the art recognize easily, in such comparison, one comprise insert or the sudden change of deletion residue in, aligned sequences will produce " gap " (gap) (usually with dash or " A " expression) in the polymer sequence that does not comprise residue insertion or deletion.

" homologous ", its all grammatical form and spelling variant refer to and have the relation between two kinds of albumen of " the common origin of evolving ", comprise the albumen from the superfamily of organism of the same race, and from the homologous protein of organism not of the same race.Such albumen (and code nucleic acid) has homologous sequence, as sequence similarity reflection by them, and no matter existence from concordance percentage ratio aspect or by specific residue or motif and conservative site.

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

Yet in common usage and instant application, term " homology " when being modified such as " highly " such adverbial word, can refer to sequence similarity, and may maybe can not refer to and relate to common evolution origin.

2.ActRIIa polypeptide

In some aspects, the present invention relates to the ActRIIa polypeptide.Applied as the present invention, term " ActRIIa " refers to from any kind and comes from activin IIa receptor family by the sudden change or the ActRIIa protein variant of other modification.ActRIIa of the present invention can be understood as any existing form of having differentiated.The transmembrane protein when member of ActRIIa family is common, it is by having the part that is rich in the cysteine zone in conjunction with ectodomain, membrane spaning domain with have the Cytoplasm domain of the activity of serine/threonine kinases of prediction.

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

People ActRIIa precursor peptide sequence is as follows:

MGAA′AKLAFAVFLISCSSGAILGRSETQECLFFNANWEKDRT

QTGVEPCYGDKDKRRHCFATWK ISGSIEIVKQGCWLDDINCY

DRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNP

VTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVL

VPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEY

VAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTS

VDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGL

A?YLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLA

LKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRID

M

YAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQ

E

VVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARL

SAG?CVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL

(SEQ?ID?NO：1)

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

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

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFAT

WKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCE

GNMCNEKFSYFPEM EVTQPTSNPVTPKPP(SEQ?ID?NO：2)

The C-end " tail " of ectodomain indicates with underscore.The sequence of having deleted " tail " (△ 15 sequences) is as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFAT

WKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCE

GNMCNEKFSYFPEM(SEQ?ID?NO：3)

The nucleotide sequence following (the 164-1705 position nucleotide of the Genebank number of logining _ 001616) of coding people ActRIIa precursor polypeptide protein:

ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCT

CCTGTTCTT

CAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTT

CTTTAATGC

TAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAAC

CGTGTTATGGT

GACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAAT

ATTTCTGGTT

CCATTGAAATAGTGA′A′ACAAGGTTGTTGGCTGGATGAT′ATCA

ACTGCTATGA

CAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATA

TTTTTGTTGC

TGTGAGGGCAATATGTGTAATGAAA′AGTTTTCTTATTTTCCAG

AGATGGAAG

TCACACAGCCCACTTCAAATCCAGTTAC′ACCTA′AGCCACCCT

′ATTACAACAT

CCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTG

TCATTTGT

GCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTG

TACTTGTTC

CAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAG

GGTTGAAACC

ACTGC′AGTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTG

TGTCTGGAAA

GCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAA

TACAGGACA

AACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTG

GAATGAAGCA

TGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCAC

CAGTGTTGAT

GTGGATCTTTGGCTGATCAC′AGCATTTCATGAAA′AGGGTTC′A

CTATCAGACT

TTCTTAAGGCTA′ATGTGGTCTCTTGGAATGAACTGTGTCATAT

TGCAGAAAC

CATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGC

CTAAAAGAT

GGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAA

AATGTGCTGT

TGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGG

CCTTAAAATT

TGAGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGG

TACCCGGAGG

TACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAA

GGGATGCAT

TTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGA

ACTGGCTTC

TCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTG

CCATTTGAG

GAGG′AAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAA

GTTGTTGTGC

ATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAAC

ATGCTGGAAT

GGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGA

CGCAGA′AGCC

AGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATG

CAGAGACTAA

CAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAAT

GGTGACAAA

TGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA(SEQ?IDNO：4)

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

ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATG

CTAATTGGG

AAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATG

GTGACAAAGA

TAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGT

TCCATTGAA

ATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATG

ACAGGACTG

ATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTG

CTGTGAGGG

CAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAA

GTCACACAG?CCCACTTCAAATCCAGTTACACcTAAGCCACCC

(SEQ?ID?NO：5)

In specific embodiment, the present invention relates to the ActRIIa polypeptide of solubility.As described in the invention, term " solubility ActRIIa polypeptide " is often referred to the polypeptide that generation comprises the proteic ectodomain of ActRIIa.Terminology used here " solubility ActRIIa polypeptide " comprises the proteic naturally occurring ectodomain of any ActRIIa and any variant (comprising mutant, fragment and plan peptide form) thereof.Activin-is the polypeptide that keeps the ability that is bonded to activin, particularly activinAA, AB or BB in conjunction with the ActRIIa polypeptide.Preferably, activin-is bonded to activinAA in conjunction with the ActRIIa polypeptide with 1nM or lower dissociation constant.Below the aminoacid sequence of people ActRIIa precursor protein is provided at.The proteic activin and normally soluble that is bonded to of ActRIIa, and thereby the activin-that is called solubility in conjunction with the ActRIIa polypeptide.The activin-of solubility comprises as SEQ ID NOs:2,3,7,12 and 13 illustrated soluble polypeptides 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 the ActRIIa polypeptide add a signal sequence except that the proteic ectodomain of ActRIIa, for example, bee variety phallotoxins 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 that illustrates in SEQ ID NO:13 is as the leading application of TPA.

The functional activity fragment of ActRIIa polypeptide can be obtained by the polypeptide that the corresponding nucleic fragment of coding ActRIIa polypeptide is produced by screening.In addition, fragment can knownly be come chemosynthesis in technology such as traditional Merrifield solid phase f-Moc or t-Boc chemistry by using.Fragment can be produced (reorganization ground or chemosynthesis) and be used 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 obtains.Variant can be produced and be used to test differentiate those can exercise ActRIIa protein antagonist function or by the activin conditioning signal.In some embodiments, the functional variant of ActRIIa polypeptide comprises the aminoacid sequence that has at least 75% homogeneity with the aminoacid sequence that is selected from SEQ ID NOs:2 or 3.In some example, functional variant has the aminoacid sequence that has at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity with the aminoacid sequence that is selected from SEQ ID NOs:2 or 3.

Functional variant can be by for such as (for example strengthening therapeutic efficiency or stability, between the ability of shelf-life and intravital anti-protein degradation in the junctor) purpose modify the structure of ActRIIa polypeptide and generate, the ActRIIa polypeptide of such modified when selected reservation activin in conjunction with the time, be considered to the functionally equivalent of naturally occurring ActRIIa polypeptide.The ActRIIa polypeptide of modifying also can be produced, for example, and by amino acid replacement, deletion or interpolation.For example, can reasonably expect and substitute leucine separately, substitute aspartic acid separately, substitute threonine separately or can not bring big influence the biologic activity of the molecule that produced with another aminoacid of amino acid replacement (for example, conservative sudden change) of structurally associated with serine with glutamic acid with isoleucine or valine.Conservative replacement is the replacement that takes place between the relevant aminoacid of those side chains in family.Whether the result of the change of ActRIIa polypeptid acid sequence is that the function homology produces the ability of replying by assessment ActRIIa polypeptide variants in the mode of the ActRIIa polypeptide that is similar to wild type easily and determines in cell.

In some embodiments, the present invention has been contained the specific sudden change of ActRIIa polypeptide so that change the glycosylation of polypeptide.Such sudden change can be selected so that introduce or eliminate one or more glycosylation sites, and O-connects or N-connects glycosylation site.The glycosylation recognition site of agedoite-connection generally includes tripeptide sequence, agedoite-X-threonine (or agedoite-X-serine) (the X here can be any aminoacid), and it is discerned specifically by suitable cell glycosylase.Transform also and can be undertaken by one or more serines or threonine residues interpolation or the ActRIIa polypeptide (for the glycosylation site of O-connection) that substitutes to wild type.First or the 3rd amino acid sites of glycosylation recognition site one of or both each seed amino acids simultaneously replace or deletion (and/or second site aminoacid deletion) causes non-glycosylated at the tripeptide sequence of modifying.Another method that increases the carbohydrate number on the ActRIIa polypeptide can be coupled glucosides to the ActRIIa polypeptide by chemical Coupling or enzyme.Depend on the CGCM that is adopted, sugar can be affixed to (a) arginine and histidine; (b) free carboxyl group; (c) free sulfhydryl group of free sulfhydryl group such as cysteine; (d) free hydroxyl of free hydroxyl such as serine, threonine or hydroxyproline; (e) aromatic residues of aromatic residues such as phenylalanine, tyrosine or tryptophan; Or (f) amide group of glutamine.These methods have description in the 259-306 page or leaf, and incorporate the present invention into by reference at WO 87/05330 (JIUYUE disclosed on the 11st in 1987) and Aplin and Wriston (1981) CRC Crit.Rev.Biochem..Can finish the one or more carbohydrates that remove on the ActRIIa polypeptide by the method for chemistry and/or zymetology.The chemistry deglycosylation can relate to, and for example, the ActRIIa polypeptide is exposed to chemical compound trifluoromethanesulfonic acid or equivalent chemical compound.This processing causes the excision of the most of or all sugar except that the sugar (N-acetyl group glucose or N-acetyl group galactosamine) that connects, and keeps the complete of aminoacid sequence simultaneously.The chemistry deglycosylation is at Hakimuddin etc. (1987) Arch.Biochem.Biophys.259:52 and Edge etc. among (1981) Anal.Biochem.118:131 further description is arranged.The excision of the zymetology of the carbohydrate on the ActRIIa polypeptide can be by using by Thotakura etc. and described various interior glycosidase of (1987) Meth.Enzymol.138:350 or outer glycosidase are realized.The sequence of ActRIIa polypeptide can suitably be regulated, and it depends on the type of the expression system that is adopted, and all can introduce different glycosylation patterns as mammal, yeast, insecticide and plant cell, and it can be influenced by the aminoacid sequence of peptide.Generally, being used for human ActRIIa polypeptide can provide correct glycosylated mammal cell line to express, as HEK293 cell or Chinese hamster ovary celI system, although can expect that other mammal express cell system, yeast cells system and the insect cell line with glycosylase of engineering also can be used for expressing.

The complete combination mutant of the method, particularly ActRIIa polypeptide that generate muton and the method for truncate mutation body have been contained in the present invention further; The storehouse of combination mutant is particularly useful for the variant sequence of identification function.The purpose of screening such combinatorial library is to produce, for example, or as agonist or as the ActRIIa variant polypeptides of antagonist action, or alternatively, have all new active mutants.Various screening techniques provide below, and such method can be used for assessing variant.For example, the ActRIIa variant polypeptides can prevent that the ActRIIa part is bonded to the ActRIIa polypeptide or disturbs the ability of the signal that causes by the ActRIIa part to screen by being bonded to the ActRIIa part.

The activity of ActRIIa polypeptide or its variant also can be tested on cell base or in vivo.For example, can assess the ActRIIa polypeptide variants relates to skeleton production or the destructive gene of skeleton in expression effect.If desired, this can (for example, finish during existing activin), and cell can be transfected to produce ActRIIa polypeptide and/or its variant and (according to circumstances) ActRIIa part at the ActRIIa of one or more reorganization ligandin.Similarly, the ActRIIa polypeptide can be administered to mice or other animal, and assesses one or more skeleton characteristic such as density or volumes.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 sophisticated, non-invasive assessment animal.People's maincenter DEXA system can be used to assess the bone density of vertebra and pelvis.These are the best predictor of total skeleton density.Periphery DEXA system can be used to assess the density of periphery skeleton, for example comprises the skeleton of hands, wrist, ankle and foot.Traditional x ray image system comprises cat scan, can be used to assess 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 with respect to naturally occurring ActRIIa polypeptide or the potential of common growth.Similarly, sudden change can cause variant to have being different from dramatically the half-life in the born of the same parents of corresponding wild type ActRIIa polypeptide.For example, the albumen of change causes its ruined cell process or the inactivation of natural A ctRIIa polypeptide or more stable or more unstable on the contrary for Proteolytic enzyme or other.Such variant and their gene of encoding can be used to change by the half-life of regulating the ActRIIa polypeptide level of ActRIIa polypeptide.For example, the short-half-life level that can cause more instantaneous biological effect and can allow more closely to control the intravital reorganization of patient ActRIIa polypeptide.In the Fc fusion rotein, sudden change can be in connexon (if there is) and/or Fc part to change the proteic half-life.

The reorganization library can be by the coded polypeptide library the mode of degeneracy gene library produce, described each polypeptide includes the potential ActRIIa peptide sequence of at least a portion.For example, synthetic oligonucleotide mixture can connect into to zymetology gene order makes the degeneracy form of potential ActRIIa polypeptide nucleotide sequence can be used as independent expression of polypeptides, or alternatively, express as the form (for example, phage display) of bigger fusion rotein.

The method that the library of potential congener produces from degenerate oligonucleotide sequence in having a lot.The chemosynthesis of degeneracy gene order can be carried out in automatic dna synthesizer, and synthetic gene is connected into the carrier of suitable expression usefulness subsequently.Degenerate oligonucleotide synthesize be known in the art (referring to for example, Narang, SA (1983) Tetrahedron 39:3; Itakura etc., (1981) recombinant DNA, Proc.3rd Cleveland Sympos.Macromolecules, editor .AG Walton, Amsterdam:Elsevier 273-289 page or leaf; 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 proteic orthogenesis (referring to for example, 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)

Perhaps, the sudden change of other form can be used for producing combinatorial library.For example, the ActRIIa variant polypeptides can produce and alanine screens sudden change and similar (Ruf etc., (1994) Biochemistry 33:1565-1572 by for example adopting; 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 mutagenesis sudden change (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 sudden change (Leung etc., Method Cell MoI Biol 1:11-19) or pass through random mutation (1989), comprise (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHLPress such as chemical mutation, Cold Spring Harbor, NY; And Greener etc., (1994) Strategies in MoIBiol 7:32-34) screening and separating is come out from the library.The linker-scanning mutagenesis sudden change in combination is set, is the method for the ActRIIa polypeptide of a kind of attractive discriminating truncate (biological activity) form in particular.

The far-ranging technology that is used for screening the gene prod of the combinatorial library that obtains by point mutation and truncate is well known in the art, and, thus, screening is had the cDNA library of the gene prod of certain specific character.Such technology is adapted to the gene library of rapid screening by the combinatorial mutagenesis generation of ActRIIa polypeptide usually.The technology that is used most widely for screening big gene library generally includes the clone gene library and goes into reproducible expression vector, transforms suitable cell and make and express combination gene under the relatively easy condition of the carrier of the gene that separates the product that coding detected detecting desirable activity with the vector library that obtains.Preferable methods comprises the cell signal analysis that activin binding analysis and activin regulate.

In some embodiments, ActRIIa polypeptide of the present invention may further include any natural ActRIIa of the being present in polypeptide of modification after the translation.Such modification includes but not limited to, acetylation, carboxylated, glycosylation, phosphorylation, lipidization and acyl groupization.The result is that the ActRIIa polypeptide after the modification can comprise non-aminoacid element, such as Polyethylene Glycol, lipid, polysaccharide or monosaccharide and phosphate.The effect of non-aminoacid element on the function of ActRIIa polypeptide like this can be tested for other ActRIIa polypeptide variants as described in the present invention like that.When the ActRIIa polypeptide generated in cell by the nascent form of shearing the ActRIIa polypeptide, translation back process also was important to proteinic correct folding and/or function.Different cell line (such as CHO, Hela, MDCK, 293, WI38, NIH-3T3 or HEK293) has special cell machine and distinctive mechanism for behavior after such translation, and can selectedly be used for guaranteeing the process of correct modification and ActRIIa polypeptide.

In some aspects, the functional variant of ActRIIa polypeptide or modified forms comprise the fusion rotein with at least a portion ActRIIa polypeptide and one or more fusion structures territory.The example in known such fusion structure territory includes but not limited to poly histidine, Glu-Glu, glutathione transferase (GST), thioredoxin, A albumen, G albumen, immunoglobulin heavy chain constant region (Fc), maltose-binding protein (MBP) or human serum albumin.Can select the fusion structure territory to give desirable characteristic.For example, some fusion structure territories specifically are used for dividing isolated fusion protein by affinity chromatograph.For reaching the purpose of affinity purification, adopt the relevant resin that substrate such as glutathion, amylase and nickel are puted together or cobalt is puted together of affinity chromatograph.Many 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, can select the fusion structure territory to help to detect the ActRIIa polypeptide.The example in such detection architecture territory comprises various fluorescins (for example GFP) and " epi-position labelling ", and it is the short peptide sequence of obtainable specific antibody normally.Known epi-position labelling for specific monoclonal antibody obtains easily, comprises FLAG, influenza virus hemagglutinin (HA) and c-myc labelling.In some instances, the fusion structure territory has such as the protease cutting site for Xa factor or thrombin, and it allows relevant protease partly to digest fusion rotein and thereby therefrom discharges recombiant protein.The albumen that discharges is the chromatography by subsequently and separating from the fusion structure territory then.Some preferred embodiment in, the ActRIIa polypeptide merges the domain (" regulator " domain) of stablizing the ActRIIa polypeptide in vivo.Mean any material that can increase serum half-life by " stablizing ", do not consider whether it is because reduced reduction or other medicines kinetic effect that destruction, kidney are removed.Known Fc fusion partly with immunoglobulin can be given far-ranging albumen with desirable pharmacokinetic properties.In addition, merge the human serum albumin and can give desirable characteristic.The fusion structure territory of selectable other type comprises that (it gives other biological function, such as further stimulation bone growth or muscle growth, as desired) for poly domain (for example dimerization, four is gathered) and functional structure territory.

As specific example, the invention provides and comprise the fusion rotein that merges to the solubility ectodomain of the ActRIIa of Fc domain (for example SEQID NO:6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV D(A)VSH

EDPEVKF

NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

KC K(A)VSNKAL

PVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD

IAVEWESN

GQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALH N(A)HYT?QKSLSLSPGK ^*

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

Certainly, the different elements of fusion rotein can be arranged in the corresponding to mode of any and conceivable function.For example, the ActRIIa polypeptide can place the C-terminal of allos domain, or alternatively, the allos domain can place the C-terminal of ActRIIa polypeptide.ActRIIa polypeptide structure territory and allos domain need not 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 the one or more modifications that can stablize the ActRIIa polypeptide.For example, such modification strengthens the ActRIIa polypeptide half-life in vivo, strengthens the protein degradation of the circulating half-life or the minimizing ActRIIa polypeptide of ActRIIa polypeptide.Stable modification like this includes but not limited to that fusion rotein (comprises, for example, the fusion rotein that comprises ActRIIa polypeptide and stabilizer structure territory), the modification of glycosylation site (comprises, for example, additional glycosylation site is to the ActRIIa polypeptide) and the modification of carbohydrate (comprise, for example, from the ActRIIa polypeptide, remove carbohydrate).With regard to fusion rotein, the ActRIIa polypeptide merges to the stabilizer structure territory (for example Fc domain) such as the IgG molecule.Applied as the present invention, term " stabilizer structure territory " not only refers to the fusion structure territory (for example Fc) in the fusion rotein, and comprises such as the nonprotein of carbohydrate and modifying or such as the nonprotein polymer of Polyethylene Glycol.

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

3. the nucleic acid of coding ActRIIa polypeptide

In some aspects, the invention provides the nucleic acid that any ActRIIa polypeptide of coding (for example solubility ActRIIa polypeptide) isolating and/or reorganization comprises fragment, functional variant and fusion rotein disclosed in this invention.For example, the SEQ ID NO:4 naturally occurring people ActRIIa precursor polypeptide of encoding, and the ectodomain of the processing of SEQ ID NO:5 coding ActRIIa.Target nucleic acid can be strand or double-stranded.Such nucleic acid can be dna molecular or RNA molecule.These nucleic acid can be used for, and for example, make the method or the direct therapeutic agent (for example, in gene therapy method) of ActRIIa polypeptide.

In some aspects, the target nucleic acid that should further understand coding ActRIIa polypeptide comprises the nucleic acid of the variant of SEQID NO:4 or 5.The variant nucleotide sequence comprises the different sequence that one or more nucleotide replace, add or delete, such as allele variant.

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

In other embodiment, nucleic acid of the present invention can also be included in hybridizes complementary series or its segmental nucleotide sequence of specifying nucleotide sequence, SEQ ID NO:4 or 5 to SEQ ID NO:4 or 5 under the rigorous condition.As discussed above, those of ordinary skills should understand easily, promote the suitable rigorous condition of DNA hybridization to change.For example, can hybridize at about 45 ℃, wash with 2.0 x SSC at 50 ℃ subsequently in 6.0 x sodium chloride/sodium citrate (SSC).For example, the salinity of washing step can be selected from 2.0 x SSC, 50 ℃ low rigorous condition to 0.2 x SSC, 50 ℃ high rigorous condition.In addition, the temperature of washing step can be from the low rigorous condition of about 22 ℃ room temperature to about 65 ℃ high rigorous condition.Temperature and salt can change, and perhaps temperature or salinity can be to keep constant in another contingent condition change.Gather the nucleic acid that the invention provides hybridization under the low rigorous condition of 6 x SSC, room temperature, at room temperature washs subsequently with 2 x SSC at some embodiment.

The isolated nucleic acid molecule different with the nucleic acid in SEQ ID NO:4 or 5 is also included within the scope of the present invention owing to the degeneracy of genetic codon.For example, a large amount of aminoacid are specified by more than one codeword triplet.Point to identical amino acid whose codon or synonymous codon (for example, CAU and CAC are the synonymous codon of histidine) and can cause " silence " sudden change, it can not influence proteinic aminoacid sequence.Yet we expect that the polymorphism of DNA sequence of variation of the aminoacid sequence that causes target protein really is present in the mammalian cell.The variation on one or more nucleotide (nucleotide of 3-5% at the most) that those skilled in the art should understand the nucleic acid of encode specific protein may reside in the individual party of given kind owing 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 adjusting nucleotide sequences that are connected to expression constructs of recombinant nucleic acid of the present invention.Regulate that what connection and calculate sequence and be suitable for expressing the applied cell of appealing to usually.Suitable expression vector and the proper regulation sequence that is used for a lot of types of various host cells known in this field.Normally, described one or more adjusting nucleotide sequence 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 activation subsequence.Promoter structure well known in the art or inductive is contained 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 the intracellular episome, and such as plasmid, or expression constructs is inserted in the chromosome.One preferred embodiment in, expression vector comprises selectable marker gene allow to select to transform the host.Selectable marker gene is well known in the art and changes with the host cell that adopts.

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

Recombinant nucleic acid of the present invention can by connect cloned genes its fragment goes into to be fit to or in prokaryotic cell or in eukaryotic cell (yeast, birds, insecticide or mammal) or the carrier of in both, expressing produce.The production of reorganization ActRIIa polypeptide comprises plasmid and other carrier with expression vector.For example, suitable carriers comprises the plasmid of following type: the plasmid in the plasmid in the plasmid that the pBR322-that is used for expressing such as colibacillary prokaryotic cell originates, pEMBL-source, the plasmid in pEX-source, pBTac-source and the plasmid in pUC-source.

Some mammalian expression vectors include simultaneously help protokaryon sequence and the one or more eukaryotic eukaryotic transcription unit that is expressed in that carrier is bred in antibacterials.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 that is fit to the mammalian expression vector of eukaryotic cell transfection.The sequence that a part in these carriers is originated by bacterial plasmid is modified, such as pBR322, to help duplicating and the drug resistance selection in prokaryotic cell and eukaryotic cell.Another is chosen as, and derivant such as the bovine papilloma virus (BPV-1) of virus or eb virus (pHEBo, pREP source and p205) are used in proteic transient expression in the eukaryotic cell.The example of other virus (comprising retrovirus) expression system finds in the description of gene therapy import system below.The whole bag of tricks that is adopted in the process of preparation plasmid and conversion host organisms is well known in the art.Other the time be fit to protokaryon and eukaryotic expression system and common regrouping process, referring to Molecular Cloning A Laboratory Manual, the third edition, by Sambrook, Fritsch and Maniatis edit (Cold Spring Harbor Laboratory Press, 2001).In some example, it is suitable adopting the bovine papilloma virus expression system to come the express recombinant polypeptide.The example of such bovine papilloma virus expression system comprises the carrier (such as pVL1392, pVL1393 and pVL941) in pVL-source, the carrier (such as pAcUW1) of pAcUW-Lianyuan and the carrier (such as the beta galactosidase that comprises pBlueBacIII) in pBlueBac-source.

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

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

Correspondingly, the present invention relates to the method for productive target ActRIIa polypeptide further.For example, having transformed coding ActRIIa polypeptide expression carrier can cultivate under suitable condition to allow the ActRIIa expression of polypeptides.The ActRIIa polypeptide can and comprise the mixture of substrate of ActRIIa polypeptide secretion and separates from cell.Selection in addition is, the ActRIIa polypeptide can be stayed in the Cytoplasm or as membrane component and harvesting, dissolving and 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 can or separate from both simultaneously from cell culture substrate, host cell, adopt technology purifying protein well known in the art, comprise that ion-exchange chromatography, gel permeation chromatography, ultrafiltration, electrophoresis, employing are specific to the immunoaffinity purification of antibody of defined epitope of ActRIIa polypeptide and the affinity purification (for example, A albumen post can be used for purification ActRIIa-Fc fusion rotein) that employing is bonded to the preparation of the domain that is fused to the ActRIIa polypeptide.One preferred embodiment in, the ActRIIa polypeptide is the fusion rotein that includes the domain that helps purification.One preferred embodiment in, purification reaches by a series of column chromatography step, comprise, for example, with three or more following steps of any order: A protein chromatographic, Q agarose chromatography, phenyl sepharose chromatography, size exclusion chromatography and cation-exchange chromatography.Purification can be finished by virus filtration and buffering exchange.Prove that as the present invention the ActRIIa-hFc protein purification is to determine greater than 98% to determine purity greater than 95% with SDS PAGE with size resistance row chromatography.Such purity is enough for effect of wanting in acquisition on the skeleton of mice and the acceptable drug safety in mice, rat and non-human primates.

In another embodiment, the fusion gene of coding purification targeting sequencing, poly-(the histidine)/enterokinase cleavage site sequence such as the desirable part N-terminal of reorganization ActRIIa polypeptide position can allow to adopt Ni ²⁺Metal-resin is by the expressed warm albumen of affinitive layer purification.The purification targeting sequencing subsequently by enterokinase handle remove with ActRIIa polypeptide that purification is provided (for example, referring to Hochuli etc., (1987) J Chromatography 411:177; And Janknecht etc., PNAS USA 88:8972).

The technology of preparation fusion rotein is well known in the art.In essence, the connection of various dna fragmentations of different peptide sequences of encoding is finished by corresponding conventional art, adopt blunt-ended or the stagger-ended end connects, Restriction Enzyme digestion is filled up sticky end, alkaline phosphatase treatment avoiding undesired connection to provide under suitable end, the suitable situation, and enzyme connects.In another embodiment, fusion gene can comprise that automatic dna synthesizer synthesizes by conventional art.Perhaps, the pcr amplification of genetic fragment can adopt the joint primer to carry out, described joint primer makes two successive can dangling (referring to for example with complementation between the genetic fragment that produces chimeric gene sequence with after annealing, Current Protocols inMolecular Biology, editors such as Ausubel, John Wiley ﹠amp; Sons:1992).

4. optionally Activin and ActRIIa antagonist

Digital proof provided by the present invention the antagonist of activin-ActRIIa signal can be used for promoting bone growth and bone mineralising.Although the ActRIIa polypeptide of solubility, ActRIIa-Fc particularly, it is preferred antagonist, and the antagonist of even now can (for example influence skeleton by the mechanism of more than activin antagonism, activin suppresses to have the indicator that suppresses the active trend of spectroscopic molecular, comprise, possible, other member of TGF-beta superfamily, and the inhibition of such colony may cause the effect on the desirable skeleton), can expect that the activin-ActRIIa antagonist of other type is effective, comprise anti--activin (A for example, B, C or E) antibody, anti-ActRIIa antibody, the antisense thing, suppress RNAi or the inhibitor of ribozymal nucleic acid and other activin or ActRIIa, the particularly bonded inhibitor of those destruction activin-ActRIIa that ActRIIa produces.

Specifically with the antibody of ActRIIa polypeptide (for example, the ActRIIa polypeptide of solubility) reaction and or competitive in conjunction with and the material that cooperates with the ActRIIa polypeptide or suppress the signal of ActRIIa adjusting on the contrary can be used as the antagonist of ActRIIa polypeptide active.In addition, specifically with the antibody of activinA polypeptide reaction and destroy the bonded antibody of ActRIIa and also can be used as antagonist.

Employing is from the immunogen of ActRIIa polypeptide or activin polypeptide, can by standard method (referring to, for example, A Laboratory Manual edits (Cold SpringHarbor Press:1988) by Harlow and Lane) preparation is anti--albumen/anti--polypeptide antiserum or monoclonal antibody.Mammal, such as mice, hamster or rabbit can be with the immunogen form of ActRIIa polypeptide, can cause the antigenicity fragment or the fusion protein immunization of antibody response.The technology of giving albumen or immunogenicity of polypeptides comprises carrier or other technology well known in the art of puting together.The immunogenicity part of ActRIIa or activin polypeptide can be used under the situation that adjuvant exists.Immunologic process can detect by detecting the titre of antibody in blood plasma or serum.Standard ELISA or other immunization method can be with using as antigenic immunogen with the assessment antibody horizontal.

After antigenicity preparation immune animal with the ActRIIa polypeptide, obtain antiserum, if desired, can from serum, separate polyclonal antibody.Be to produce monoclonal antibody, can collect antibody produced cell (lymphocyte) and merge to produce hybridoma from the animal of immunity by the somatic cell fusion steps of standard and immortalized cells such as the myeloma cell.Such technology is well known in the art, comprise, for example, hybridoma technology (begins by Kohler and Milstein exploitation, (1975) Nature most, 256:495-497), human B cell hybridoma technology (Kozbar etc., (1983) Immunology Today, 4:72) and the EBV-hybridoma technology produce human monoclonal antibodies (Cole etc., (1985) monoclonal antibody and treatment of cancer, Alan R.Liss, the Inc.77-96 page or leaf).Hybridoma can screen the antibody that reacts with the ActRIIa polypeptid specificity to produce by immunochemistry, and monoclonal antibody is separated from the culture medium that comprises 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 with the same method that is used for whole antibody described above.For example, F (ab) ₂Fragment can be by generating with pepsin.The F (ab) that produces ₂Fragment can be handled with less disulfide bridge bond and produce the Fab fragment.Antibody of the present invention further is intended to comprise two special, strand, chimeric, humanizations and the ActRIIa that given by at least one antibody CDR zone or the total length human molecular of activin polypeptide affinity.Antibody can further comprise label (for example, label can be radiosiotope, fluorescent chemicals, enzyme or cofactors) additional in addition and can be detected.

In some embodiments, antibody is recombinant antibodies, its term has comprised the antibody that partly produces with Protocols in Molecular Biology arbitrarily, comprise antibody that CDR transplants or chimeric antibody, the people's or the antibody structure territory selected from the library of assembling antibody, single-chain antibody and single domain antibody (for example, people V _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.For example, a kind of production is bonded to the monoclonal antibody method of ActRIIa polypeptide or activin polypeptide specifically, comprise the immunogenic composition of using the detectable immunoreactive antigen polypeptide of stimulation that comprises effective dose of doses to mice, (for example from mice, obtain antibody producing cells, cell in the spleen) and with antibody producing cells and myeloma cell merge to obtain the antibody producing hybridoma, detect the antibody producing hybridoma to differentiate the monoclonal hybridoma that to produce the specificity conjugated antigen.In case obtain, from cell culture medium, breed hybridoma, according to circumstances under the condition of culture of the cell of the production specificity conjugated antigen in hybridoma source.Monoclonal antibody can be from cell culture medium purification.

Adjective " reaction with it specifically " refers to antibody at this, the meaning is, as the common sense of this area, antibody in the particular organisms sample interested antigen (for example, ActRIIa polypeptide) and other non-interested antigen between have fully optionally.Adopt in the method for antibody at some, in using such as treatment, bonded high degree of specificity is necessary.Monoclonal antibody has bigger trend (comparing with polyclonal antibody) usually and distinguishes between the polypeptide of desirable antigen and cross reaction effectively.Influence antibody: the antibody specific feature that interacts is that antibody is to antigenic affinity.Although desirable specificity can reach the scope of different affinitys, usually preferred antibody has about 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9Or affinity still less (dissociation constant).Combination especially closely between given activin and the ActRIIa, we expect that the anti-activin antibody of neutrality or anti-ActRIIa antibody have 10 usually ^-10Or dissociation constant still less.

In addition, be used to screen antibody can influence the antibody of acquisition with the technology of identifying desirable antibody characteristic.For example, if antibody is used at the solution conjugated antigen, the test solution combination may be suitable.Various technology can be used for interaction between test antibody and the antigen to differentiate the antibody of certain desired.Such technology comprises ELISAs, surface plasma body resonant vibration binding analysis (for example, the Biacore ^TMBinding assay, Biacore AB, Uppsala, Sweden), sandwich assay (for example, 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-stranded.The double-strandednucleic acid chemical compound can also comprise cantilever or non-complementary zone, and one of them or another one fragment are strands.Single chain compound can comprise self complementary zone, means that compound formation is called " hair clip " or " stem ring " structure, has the zone 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 the zone 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 8 nucleotide at least, optionally at least 10 or at least 15 nucleotide, and optionally between 15 to 25 nucleotide.Complementary region can belong to the coded sequence of intron, target transcripton or non-coding sequence such as the coded sequence part.Normally, nucleic acid compound has 8 length to about 500 nucleotide or base pair, and length can be about 14 to about 50 nucleotide according to circumstances.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 the form that is not easy to be divided into the modification of 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 the form that is not easy to be divided into the modification of DNA or RNA.Nucleic acid compound can comprise various arbitrarily modifications, comprises one or more modifications of skeleton (sugar of natural acid-phosphoric acid part comprises bonding between nucleoside) or base portion (purine of natural acid or pyrimidine part).Antisense nucleic acid compound preferably have about 15 to about 30 nucleotide long, and comprise one or more modifications usually to improve such as the stability in the serum, in cell or in the characteristic of the possible administration position of chemical compound, described administration position be such as stomach (under case of oral administration) and lungs (for the suction chemical compound).Under the situation of RNAi construction, complementary normally RNA or its modification of chain to the target transcripton.Other chain can be RNA, DNA or any other variant.The two-phase of two strands or strand " hair clip " RNAi construction partly is preferably 18 to 40 length of nucleotides, is about 21 to 23 length of nucleotides according to circumstances, as long as it can be used as the Dicer substrate and works.Catalytic or enzymatic nucleic acid can be ribozyme or DNA enzyme, and also can comprise the form of modification.Nucleic acid compound with nonsense or the concentration that has justice control not produce or do not tell on substantially, contacts with cell under physiological condition, can suppress about 50%, 75%, 90% or the expression of more target.The preferred concentration that detects the effect of 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 (for example, solubility ActRIIa polypeptide) and activin polypeptide to identify can excitement or the chemical compound (medicine) of antagonism activin-ActRIIa signal path.Can test to assess them by this Screening and Identification medicament and to regulate the ability of bone growth and bone mineralising in vivo, according to circumstances, these chemical compounds can be further in animal test to assess their abilities of regulate tissue growth in vivo.

There is much more very being used to screen and decide activin and ActRIIa polypeptide approach with the therapeutic agent of regulate tissue growth by target.In some embodiments, the high flux screening of chemical compound can be used for differentiating the medicament that disturbs activin or the regulating effect of ActRIIa on skeleton.In some embodiments, this method is used to screen and identifies the specificity inhibition or reduce the chemical compound that the ActRIIa polypeptide is bonded to activin.Perhaps, this method is used to identify that enhancing ActRIIa polypeptide is bonded to the chemical compound of activin.In another embodiment, chemical compound can be identified by itself and activin or the interactional ability of ActRIIa polypeptide.

The version of the whole bag of tricks is enough and in scope disclosed by the invention, however can being understanded of clearly not describing of those the present invention by those of ordinary skills.As described in the invention, detection compound of the present invention (medicament) can be by any combinational chemistry manufacturing.Perhaps, target compound can be in vivo naturally occurring or external synthetic biomolecule.Its chemical compound as tissue growth regulator ability to be measured (medicament) can be produced by for example antibacterial, yeast, plant or other organism (for example, natural prodcuts), or chemical production (for example, micromolecule comprises and intends peptide), or reorganization 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 one, discrete entity, or the library with bigger complexity, such as the storehouse with the combinatorial chemistry preparation.These storehouses can comprise, for example, and the organic compound of ethanol, alkyl halide, amine, amide, ester, aldehyde, ether and other classification.The existence of detection compound in detection system can be independent form or with the form of the mixture of chemical compound, particularly in the initial screening step.According to circumstances, chemical compound can help compound separation with other compound deriving and the colony of deriving according to circumstances.The example of nonrestrictive derivant colony comprises that biotin, fluorescein, digoxin, green fluorescent protein, isotope, poly histidine, magnetic bead, glutathione transferase (GST), optical activation cross-linking agent (photoactivatible crosslinkers) or its make up arbitrarily.

In the drug screening program of many detection compound storehouse and natural extract, high-throughout method is needed in order to maximize detection compound quantity in preset time.This method under acellular environment, preferred usually as " main " screening technique such as purification or half purifying protein everywhere, it allows change fast-developing and that relatively easily detect the target molecule that detected chemical compound regulates.In addition, the cytotoxicity of detection compound or bioavailability can be ignored in vitro system, this method then mainly concentrate on the effect of medicine on target molecule, and it can show in the change of binding affinity of ActRIIa polypeptide and activin.

Only illustrate, in exemplary screening technique of the present invention, the ActRIIa polypeptide that can be bonded to activin usually of purification contacts compound of interest with separating also.Add the compositions that comprises the ActRIIa part in the mixture of chemical compound and ActRIIa polypeptide subsequently.The detection of ActRIIa/activin complex and the complex that quantitatively provides a kind of definite chemical compound to suppress between (or reinforcement) ActRIIa polypeptide and the activin form the method for rendeing a service.The dose-effect curve that the data that the effectiveness of chemical compound can obtain by the various concentration that adopt test compounds generate is assessed.In addition, check analysis can be used for providing the benchmark of comparison.For example, in check analysis, the activin of separation and purification is added in the compositions that comprises the ActRIIa polypeptide, is not having quantitative ActRIIa/activin complex under the situation of test compounds.Should understand, common, the blended order of reactant can change, and can mix simultaneously.In addition, replace the cell extract of purifying protein and solute to can be used for the acellular analytical system that provides suitable.

The formation of the complex of ActRIIa polypeptide and activin can be by various technology for detection.For example, the adjusting that forms of the complex albumen that can adopt detectable label for example such as radiolabeled (for example, ³²P, ³⁵S, ¹⁴C or ³H), ActRIIa polypeptide or activin fluorescein-labelled (for example FITC) or enzyme labelling, detect quantitatively by immunoassay or chromatography.

In some embodiments, 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 have been contained in the present invention.Further, other detecting pattern is such as (PCTPublication WO 96/26432 and the United States Patent (USP) 5,677 based on fiber waveguide, 196), surface plasma body resonant vibration (SPR), surface charge sensing and surperficial stressed sensing, all consistent with many embodiments of the present invention.

In addition, the application of interaction trap method (also being called " double cross method ") has been contained in the present invention, with identify to destroy or strengthen the ActRIIa polypeptide with it protein-bonded interactional medicament.Referring to for example, 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) Oncogene 8:1693-1696 such as Iwabuchi.In specific embodiment, the present invention contained use reverse two-hybrid system with identify separate the ActRIIa polypeptide with it protein-bonded interactional chemical compound (for example, micromolecule or peptide).Referring to for example, 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 ActRIIa of the present invention or the interactional ability of activin polypeptide.Interaction between chemical compound and ActRIIa or the activin polypeptide is covalency or non-covalent.For example, this interaction can adopt the interior biochemical method of body to identify on protein level,

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

In some aspects, the invention provides method and the medicament that adjusting (stimulating or inhibition) skeleton forms and increases bone mass.Therefore, any compounds identified can be regulated the ability of bone growth or bone mineralising to determine it in full cell or tissue, at external or body build-in test.Various methods well known in the art can be used for this purpose.

For example, ActRIIa or activin polypeptide or the effect of detection compound on skeleton or cartilage-derived growth can by on cell base, measure Msx2 induce or osteoblast is divided into osteoblast and determines (referring to for example, (Daluiski etc., Nat Genet.2001,27 (1): 84-8; Hino etc., Front Biosci.2004,9:1520-9).The example of analyzing on other cell base comprises the osteogenic activity of evaluating objects ActRIIa or activin polypeptide and test compounds in a mesenchymal progenitor cell and osteoblast.Illustrate, the recombinant adenovirus of expressing activin or ActRIIa polypeptide can make up and be used to infect mesenchymal progenitor cell C3H10T1/2 between multipotency, osteoblast precursor C2C12 and osteoblast TE-85.Osteogenic activity subsequently by measure alkali phosphatase, Bone Gla protein and substrate mineralising induce determine (referring to, for example, Cheng etc., Jbone JointSurg Am.2003,85-A (8): 1544-52).

The method that detects skeleton or cartilage-derived growth in the body has also been contained in the present invention.For example, Namkung-Matthai etc., Bone, 28:80-86 (2001) discloses a kind of osteoporosis rat model, and it has studied the early stage bone repair in fracture back.Kubo etc., SteroidBiochemistry ﹠amp; Molecular Biology, 68:197-202 (1999) also discloses a kind of osteoporosis rat model, and it has studied the bone repair in fracture back late period.Andersson etc., J.Endocrinol.170:529-537 disclose the osteoporosis model of mice, and mice is to remove ovary, and it causes mice to lose essence bone mineral content and bone mineral density, and wherein spongy bone has been lost general 50% bone mineral density.The skeleton density of removing the mice of ovary can improve by using some factors such as parathyroid hormone.In some aspects, the present invention has used fracture healing method well known in the art.These methods comprise fracture technology, histologic analysis and biomechanical analysis, it is at for example United States Patent (USP) 6, description is arranged in 521,750, integrally incorporate the degree of its disclosed foundation and measurement fracture and the experimental technique of the process of reparation into the present invention with the form of quoting.

6. exemplary treatment is used

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

Applied as the present invention, the therapeutic agent of " prevention " disease or situation refers in a statistics sample, with respect to not treating sample, can reduce the disease of treatment sample or the chemical compound that situation takes place, perhaps with respect to not treating sample, postpone outbreak or palliate a disease or the chemical compound of the order of severity of one or more symptoms of situation.Term used herein " treatment " comprise the specified situation of prevention or in case this situation improve when determining or eliminate this situation.Under any situation, the therapeutic agent of prevention or treatment and expection is used the result and is considered by the doctor who does diagnosis among both.

The invention provides and comprise that skeleton and/or cartilage form, the bone mineralising is lost, increased to the prevention skeleton or the method for prevention skeleton demineralization.For example, target activin-ActRIIa antagonist has been applied to treat the healing of osteoporosis and fracture and the cartilage defect among the human or animal.ActRIIa or activin polypeptide are effectively to the patient who is diagnosed as subclinical low skeleton density, and it is as the protective measure to the osteoporosis development.

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

Method and composition of the present invention can be applied to lose bone for or to cause losing bone be the situation of 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 or associated by various factors.Concerning the women, particularly postclimacteric women, body weight is low, and the sitting behavior that causes all is the risk factor of osteoporosis (lose bone mineral density, cause risk of bone fracture).People with arbitrary following feature can be used as the candidate with the treatment of ActRIIa antagonism: the women of postclimacteric and do not ingest estrogen or other Hormone Replacement Therapy; The postclimacteric women of high (above 5 feet 7 inches) or thin (being less than 125 pounds); Male with the relevant clinical condition of losing bone; Use the known people who causes the medicine of losing bone, comprise such as prednisone (Prednisone ^TM) glucocorticoid, various anti-tic medicine such as phenytoin Sodium (Dilantin ^TM) and some Barbiturate, or the thyroid alternative medicine of high dose; The people who has type i diabetes, hepatic disease, kidney disease or have the osteoporosis family history; People with high turnover (for example, collagen is too much in the urine sample); Has the thyroid situation such as hyperthyroid people; The people who fractures after the slight wound; People with x ray evidence of spinal fracture or osteoporotic other phenomenon.

Mention as top, osteoporosis also can be and another kind of disease association or the situation relevant with the use of certain medicine.Osteoporosis drug induced or that other medical conditions causes is called as secondary osteoporosis.In a kind of situation of being hypercortisolism (Cushing ' s disease), the too much hydrocortisone that health produced causes osteoporosis and fracture.The most common medicine relevant with secondary osteoporosis is glucocorticoid, and a class is as the medicine of hydrocortisone effect, a kind of natural hormone that is produced by the adrenal gland.Although the proper level of thyroxin (it is produced by thyroid) is that skeleton development is necessary, excessive thyroxin reduces the bone amount sustainably.When taking in the aluminiferous antacid of bag of high dose, the problematic particularly people of dialysis of kidney can cause bone loss.The medicament of other caused secondary osteoporosis comprises phenytoin (phenytoin Sodium (Dilantin)) and the Barbiturate that is used to prevent epilepsy; Methotrexate (Rheumatrex, Immunex, Folex PFS), a kind of medicine that is used for arthritis, cancer or the immunological diseases of some types; (Sandimmune Neoral), a kind ofly is used for the treatment of some autoimmune diseasees and suppresses immune medicine in patient's body of organ transplantation ring spore toxin; (Lupron Zoladex), is used for the treatment of carcinoma of prostate and endometriosis to the luteinising hormone-releasing hormo agonist; 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 by understanding and quilt are become the inductive bone loss of treatment of cancer (CTIBL) widely.Bone shifts can cause the hole on skeleton, it can pass through with activin-ActRIIa antagonist for treating and correction.

One preferred embodiment in, the ActRIIa of activin-ActRIIa antagonist, particularly solubility disclosed in this invention can use and the cancer patient.Patient's (for example, prostatic, mammary gland, multiple myeloma or anyly cause hyperthyroid tumor) with certain tumor is because the bone loss of tumor inducing and bone shift and therapeutic agent former thereby have the excessive risk of bone loss.Or even under the situation of the evidence that lacks the transfer of bone loss or bone, such patient can be with the activin-ActRIIa antagonist for treating.Can also monitor the evidence that patient's bone loss or bone shift, when the indicator for displaying risk rises with the 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 assessing the probability that bone shifts.Can monitor serum marker.The special alkali phosphatase (BSAP) of skeleton is a kind of enzyme that exists in osteoblast.Bone shifts and other causes the patient's of the situation that skeleton reconstruct increases BSAP blood levels to rise.Bone Gla protein and procollagen peptide form with skeleton equally and bone shifts relevant.In having the patient that bone that carcinoma of prostate causes shifts, detect the rising of BSAP, and in the bone transfer that causes of existence in various degree and breast carcinoma.High-caliber skeletal form generation albumen 7 (BMP-7) is present in that bone is transferred in the carcinoma of prostate of skeleton, but is not present in during bone that bladder, skin, liver or lungs cancer cause shifts.I type carboxyl terminal peptide (ICTP) is the cross-linking agent of finding in a kind of collagen that forms during skeleton absorbs.Because skeleton is destroyed constantly and transformation, ICTP can both find at whole body.Yet, shifting the position that takes place at bone, its level will be higher than the zone at normal bone significantly.The high level of ICTP is found in during bone that prostate, lungs and breast carcinoma causes shifts.Another kind of collagen crosslinking agent, I type amino terminal peptide (NTx), when bone is changed and ICTP together produce.In a variety of various cancers comprised that bone that lungs, prostate and breast carcinoma causes shifts, the amount of NTx rose.And the rising of NTx level is accompanied by the development process that bone shifts.Therefore, this labelling can be used for detecting the degree that bone shifts and weighs disease.Other absorption labelling comprises pyridine ether and deoxidation pyridine ether.Any growth that absorbs labelling and bone metastatic marker has shown the treatment of needs of patients activin-ActRIIa antagonist.

The Activin-ActRIIa antagonist can be co-administered with the other medicines preparation.Co-administered can be by the unitary composite prescription, by using simultaneously or using blanking time and finish.If the Activin-ActRIIa antagonist is together used with other skeleton activator, have special advantage.The patient can be benefited from other medication combined using of activin-ActRIIa antagonist and picked-up calsium supplement, vitamin D, suitable exercise and/or (under some situation).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 influence the skeleton reconstruct cycle and are classified as anti-absorbing the drug.Skeleton reconstruct is by two different stages: skeleton absorbs and skeleton forms.Anti-absorbing the drug slowed down or stagnated the skeleton absorption portion in skeleton reconstruct cycle but do not slow down the skeleton formation part in cycle.The result is, new formation speed continues greater than absorption rate, and skeleton density increases always.The parathyroid hormone fragment, a kind of form of parathyroid hormone, its skeleton that increases in the skeleton reconstruction cycle forms speed.Alendronate sodium is used for postclimacteric prevention of osteoporosis (5 milligrams every day or weekly 35 milligrams) and treatment (10 milligrams every day or weekly 70 milligrams) by approval.Alendronate sodium reduces bone loss, bone density improving and the risk of bone fracture that reduces spinal column, wrist and hip.Alendronate sodium also is used for the treatment of the osteoporosis of the glucocorticoid inducible that is caused as these medicines of prolonged application (being prednisone and cortisone) in the masculinity and femininity and the osteoporotic treatment that is used for the male by approval.Alendronate sodium adds that vitamin D is used for the treatment of women's postclimacteric osteoporosis (weekly 70 milligrams add vitamin D) by approval, and is used for the treatment of the bone amount of suffering from osteoporotic male with improvement.Ibandronate is used for prevention and treats postclimacteric osteoporosis by approval.Took once (150 milligrams) in every month, ibandronate must be every month take on the same day.Ibandronate reduces bone loss, bone density improving and the risk that reduces spinal fracture.Risedronate sodium is used for prevention and treats postclimacteric osteoporosis by approval.Take (5 milligrams dosage) every day or take (35 milligrams of dosage or 35 milligrams of dosage add calcium) weekly, risedronate sodium slows down bone loss, the risk that increases skeleton density and reduce spinal fracture and non-spinal fracture.Risedronate sodium also is used for the osteoporosis of masculinity and femininity with the glucocorticoid inducible that prevents and/or treats these medicines of chronic administration (being prednisone and cortisone) and caused by approval.Calcitonin is that a kind of natural existence relates to calcium adjusting and the metabolic hormone of skeleton.In later women in 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 approved to be used for prevention of osteoporosis.ET reduces bone loss as shown, increases the skeleton density of spinal column and hip, and reduces postclimacteric women's the hip and the risk of spinal fracture.It is form with pill or skin patch that ET uses the most common, and it discharges low dosage or the standard dose of about 0.625 milligram of every day of about 0.3 milligram of every day, even and just begin also still effective after 70 years old.When independent absorption estrogen, it can increase the risk that the women develops cervix uteri wall cancer (carcinoma of endometrium).For eliminating this risk, the prescription that health care provider is left is that progesterone hormone and estrogen combination are used for the women that those have complete uterus.ET/HT alleviates menopausal symptom and demonstration has beneficial effect to bone health.Side effect comprises colporrhagia, breast tenderness, emotion disturbance and gallbladder disease.Raloxifene, is approved to be used for prevention and to treat postclimacteric osteoporosis 60 milligrams of every days.It derives from a series of medicine that is called selective estrogen receptor modulators (SERMs), and it is developed into and estrogenic beneficial effect is provided and does not have potential defective.Raloxifene increases the risk of estimating and reducing spinal fracture.Do not obtain data can reduce hip and non-other non-spinal fracture with the proof raloxifene risk yet.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.Among the male, the reduction of the fracture in the spinal column is significant, but does not have enough data to assess the reduction of the fracture of other position.The parathyroid hormone fragment is from using, as injecting in 24 hours on the same day.

7. pharmaceutical composition

In some embodiments, activin-ActRIIa antagonist of the present invention (for example, ActRIIa polypeptide) and pharmaceutically acceptable carrier compound.For example, the ActRIIa polypeptide can be used separately or as the composition of pharmaceutical formulation (therapeutic combination).Target compound can anyly be suitable for being applied to the mankind or veterinary's approach prescription and use.

In some embodiments, Therapeutic Method of the present invention is used compositions or as implantation body or equipment local application with comprising general.When using, being used for therapeutic combination of the present invention naturally is pyrogen-free, the last acceptable form of physiology.Treatment effective agent except that the ActRIIa antagonist can optionally include compositions described above, and it can or one after the other be used with target compound (for example, the ActRIIa polypeptide) while in the method for the invention.

Normally, the ActRIIa antagonist is in parenteral administration.The pharmaceutical composition that is fit to parenteral comprises 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 used sterile injectable solution in front or dispersion is combined, and it can comprise antioxidant, buffer agent, antibacterial, give prescription and receptor's in the future the solute of blood etc. or the medicine of suspension or thickening.Suitable applications comprises that in the water carrier of pharmaceutical composition of the present invention or the example of nonaqueous carrier water, ethanol, polyol are (such as glycerol, propylene glycol, Polyethylene Glycol, or the like) and suitable mixture, vegetable oil (such as olive oil), and injectable organic ester (such as ethyl oleate).Can keep suitable flowability, for example,, under the situation of dispersion, pass through to keep desired granular size by using encrusting substance matter (such as lecithin), and by the application surface activating agent.

In addition, compositions can be loaded into capsule or inject to being released into the form of target tissue site (for example, skeleton).In some embodiments, compositions of the present invention comprise can discharge one or more treatment chemical compounds (for example ActRIIa polypeptide) to target tissue site () substrate for example, skeleton, for developmental tissue provide a kind of structure and, most desirably, can be absorbed in the body.For example, substrate can provide the slow release of ActRIIa polypeptide.Such substrate can be formed by existing other material of implanting medical applications that is used for.

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 be determined the prescription that suits.The potential substrate that is used for compositions is biodegradable and chemically clearly calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and poly-anhydride.Other potential material is biodegradable and biologically is 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, such as agglomerating hydroxyapatite, bio-vitric, aluminate or other pottery.Substrate can be made up of the combination (such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate) of the material of the top any kind of mentioning.Bioceramic can change in compositions, such as in calcium-aluminum-phosphoric acid and in the processing that changes aperture, granular size, grain shape and biodegradability.

In some embodiments; method of the present invention can be an oral administration; for example; with capsule; cathets; pill; tablet; buccal tablet (adopts flavoured base; normally sucrose and Radix Acaciae senegalis or tragacanth); powder; granule or as solution in water or the nonaqueous solvent or suspension; or as Water-In-Oil or as oil-in-water liquid emulsion; or as elixir or syrup; or as lozenge (employing inert base; such as gel and glycerol; or sucrose and Radix Acaciae senegalis) and/or the form of collutory or the like, every kind of medicament that all comprises the amount of pre-determining as active component.Medicament also can be used as the form of pill, electuary or paste and uses.

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

The liquid dosage form that is used for oral administration comprises pharmaceutically acceptable emulsion, microemulsion, solution, syrup and elixir.Except that active component, liquid dosage form comprises this area inert diluent (such as water or other solvent), cosolvent and emulsifying agent commonly used (such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, phenylpropanol, benzyl benzoate, propylene glycol, 1, fatty acid ester of 3-butanediol, oil (Semen Gossypii, Semen arachidis hypogaeae, corn, plumule, Fructus Canarii albi, Semen Ricini and Oleum sesami especially), glycerol, tetrahydrofurfuryl carbinol, Polyethylene Glycol and sorbitan and composition thereof.Except that inert diluent, Orally administered composition also comprises the auxiliary agent such as wetting agent, emulsifying and suspending agent, sweeting agent, spice, coloring agent, perfuming and antiseptic.

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

The present composition also can comprise auxiliary agent, such as antiseptic, wetting agent, emulsifying agent and dispersant.Can guarantee preventing of microbial activities by comprising various antimicrobial drugs and antifungal agent (for example, para, methaform, phenol or the like).Also comprise isotonic agent in the compositions in the time of necessary, such as sugar, sodium chloride or the like.In addition, the prolongation of injectable drug absorption can be by bringing medicament such as aluminum monostearate and being mingled with of gel of postponing to absorb.

Certainly, dosage regimen is considered through the attending doctor to make after the factor of various changes target compound of the present invention (for example, ActRIIa polypeptide) effect.Described various factors includes but not limited to, needs the skeleton weight of formation, the degree that bone density is lost, the position of bone injury, the situation of damage skeleton, patient's age, sex and diet, the order of severity of the disease of any promotion bone loss, time of application and other clinical factor.According to circumstances, dosage can change according to matrix type that uses in rebuilding and the type of compounds in the compositions.Other known somatomedin that is added in the middle of the final compositions also can influence dosage.Process can be monitored by the periodical evaluation (for example, X ray (comprising DEXA), histomorphometricall and tetracycline marker) of bone growth and/or reparation.

Mouse experiment has proved when the each administration at interval of chemical compound and dosage enough reach 0.2 microgram/kilogram or higher blood drug level, the effect of ActRIIa-Fc on skeleton is detectable,, and the serum drug level be 1 microgram/kilogram or 2 microgram/kilograms or higher be that to obtain the remarkable result of skeleton density and intensity necessary.Although do not have the ActRIIa-Fc of high dose more owing to can cause side effect and undesirable sign, therapeutic scheme is designed to reach the blood drug level between 0.2 and 15 microgram/kilograms, is between 1 to 5 microgram/kilogram according to circumstances.In the mankind, the serum drug level of 0.2 microgram/kilogram can reach by 0.1 mg/kg or higher single dose, and the serum drug level of 1 microgram/kilogram can reach by 0.3 mg/kg or higher single dose.The serum half-life of observed molecule is between about 20 days to 30 days, be longer than most Fc fusion rotein substantially, therefore and obtain lasting effectively serum drug level, for example, by weekly or the dosage of the 0.2-0.4 mg/kg on two all bases, or longer interval between higher dosage and dosage.For example, the dosage of 1-3 mg/kg can be used for every month or bimonthly basis on, and the effect on the skeleton is enough lasting, such dosage for only be per 3,4,5,6,9,12 or more a plurality of months once necessary.

In some embodiments, the present invention also provides the gene therapy that produces the ActRIIa polypeptide in the body.Such therapy can reach its therapeutic effect by introducing the ActRIIa polynucleotide sequence in the cell or tissue of disease listed on suffer from.Sending of ActRIIa polynucleotide sequence can be finished by using recombinant expression carrier such as embedded virus or colloidal dispersion system.What be preferred for that ActRIIa polynucleotide sequence therapeutic sends is the use of target liposomes.

The present invention instructed, and the various viral vector that can be used for being used to gene therapy comprise adenovirus, herpesvirus, cowpox, or preferably, RNA viruses is such as retrovirus.Preferably, retroviral vector is the retroviral derivant of mice or birds.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 a plurality of genes in a large number.All these carriers are transferable or merge gene and make that with selected marker the cell of transduction can be identified and generate.Retroviral vector can be made target-specific by adhering to for example 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 the reverse transcription virus gene group or adhere to virus envelope protein with the retroviral vector target-specific that allows to comprise the ActRIIa polynucleotide send.One preferred embodiment in, carrier targeting skeleton or cartilage.

Perhaps, the organizational structure cell can be by conventional calcium phosphate transfection directly with the plasmid transfection of encoding hiv reverse transcriptase structural gene gag, pol and env.These cells are to comprise the vector plasmid transfection of interested gene then.The cell that obtains discharges retroviral vector to culture medium.

Another ActRIIa polynucleotide targeting delivery system is a dispersion system of colloid.Dispersion system of colloid comprises that macromolecular complex, Nano capsule, microsphere, pearl and liposome system comprise water in oil emulsion, micelle, mixed micelle and liposome.The preferred dispersion system of colloid of the present invention is a liposome.Liposome is the synthetic membrane vesicle, and it can be effective as external or intravital dispersible carrier.RNA, DNA and complete virion can together encapsulate with internal water solution, and with biologically active form dispersate cell (referring to, for example, Fraley, etc., Trends Biochem.Sci., 6:77,1981).It is well known in the art adopting the method for the efficient rotaring redyeing gene of liposome vectors, referring to for example, and Mannino, etc., Biotechniques, 6:682,1988.Liposome composition is the combination of phospholipid normally, common and particularly cholesterol combination of steroid.Also can use other phosphate ester or other lipid.The physical characteristic of liposome depends on the existence of pH, ionic strength and bivalent cation.

The example that can be used for producing the lipid of liposome comprises the phosphatidyl chemical 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 based on, for example, organ specificity, cell-specific and organelle specificity and known in the art other.

Embodiment

The present invention here is general description, and by being more readily understood with reference to following embodiment, included embodiment only is used to explain 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

The applicant has made up the solubility ActRIIa fusion rotein with the people ActRIIa ectodomain that merges the pure man or mice Fc domain, and minimal connexon is arranged 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:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKN

ISGSI

EIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYF

PEMEV

TQPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEV

TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVS

VLTVL

HODWLNGKEYKCKVSNKALPVPIEKTISKAKGOPREPOVYTLPPSR

EEMTK

NOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLY

SKLTV?DKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPGK

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

(i) bee variety phallotoxins (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 have a following not processed aminoacid sequence:

MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDR

TNQT

GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDC

VEKKD

SPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTC

PPCPA

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

VDGVE

VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

PVPIE

KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE

WESNG

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNH?YTQKSLSLSPGK(SEQ?ID?NO：13)

This peptide is coded by following nucleotide sequence:

ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGT

GGAG

CAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAAC

TCAG

GAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATC

AAAC

TGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTG

TTTTG

CTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGT

TGTT

GGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAA

AAAA

AGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGT

AATG

AAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTT

CAAAT

CCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGC

CCAC

CGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCT

TCCCC

CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC

ACAT

GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC

AACTG

GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC

GGGA

GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGT

CCTG

CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC

CAACA

AAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAG

GGCA

GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGG

AGATG

ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT

CCCA

GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC

AACT

ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC

TCTAT

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG

TCTTCT

CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA

AGAG?CCTCTCCCTGTCTCCGGGTAAATGAGAATTC(SEQ?ID?NO：

14)

ActRIIa-hFc and ActRIIa-mFc comply with recombinant expressed significance.As shown in Figure 1, protein purification is single good albumen peak shape.The N-terminal order-checking has disclosed the unique sequence of ILGRSTQE (SEQ ID NO:11).Purification can reach by a series of column chromatography step, comprises, for example, the step that three or more is following, in any order: A protein chromatographic, Q agarose chromatography, phenyl sepharose chromatography, size resistance row's chromatography and cation-exchange chromatography.Purification can be finished by virus filtration and buffering exchange.The ActRIIa-hFc protein purification is to determine greater than 98% to determine purity greater than 95% with SDS PAGE with size resistance row chromatography.

ActRIIa-hFc and ActRIIa-mFc have shown and the part high-affinity of activinA particularly.GDF-11 or ActivinA (" ACA ") adopt standard amino acid coupling connection program to be fixed on the Biacore CM5 chip.ActRIIa-hFc and ActRIIa-mFc albumen are loaded on system, and measure 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.Referring to Fig. 2.The situation of ActRIIa-mFc is similar.

The analysis of A-204 reporter gene is assessed the effect of ActRIIa-hFc albumen on signal transmits 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 referring to Fig. 3.The CAGA12 motif is present in TGF-beta reactive group (PAI-1 gene), so this carrier is generally used for the signal factor by Smad2 and 3.

First day: separate the A-204 cell and go into 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 together transfection of Fugene.

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

Follow by the luciferase analysis.Usually in the method, without any the existence of inhibitor, ActivinA demonstrates stimulates about 10 times reporter gene expression 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 showing among Fig. 4, ActRIIa-hFc and ActRIIa-mFc suppress the signal that GDF-8 regulates under picomole concentration.As shown in Figure 5, three kinds of different ActRIIa-hFc preparations suppress the GDF-11 signal with the IC50 near 200pM.

ActRIIa-hFc is highly stable in pharmacokinetic analysis.The ActRIIa-hFc polypeptide of rat administration 1mg/kg, 3mg/kg or 10mg/kg, and proteic drug plasma level 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 rat, the cyclical level that ActRIIa-hFc has 11-14 days serum half-life and medicine very high after 2 weeks (11 μ g/ml, 110 μ g/ml or 304 μ g/ml are respectively for the initial dose of 1mg/kg, 10mg/kg or 30mg/kg).In machin, plasma half-life 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) greater than the cyclical level of 14 days and medicine basically.PRELIMINARY RESULTS among the mankind shows that the 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 administration is 2 times weekly, and bone mineral density and bone mineral content are determined by DEXA, referring to Fig. 6.

In female BALB/c, DEXA scanning shows the therapeutic outcome as ActRIIa-mFc, the remarkable increase (〉 20% of bone mineral density and content).Referring to 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, detected the effect of ActRIIa-mFc on the mouse model of removing ovary.

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

The applicant adopts (OVX) the female C57BL6 mice or the sham-operation female mice of the removal ovary in age in 4-5 week.In 8 weeks of postoperative, begin to treat with ActRIIa-mFc (10mg/kg, twice weekly) or contrast (PBS).Weigh bone density by CT scan.

Show that as Fig. 9 after 6 weeks, with respect to the mice of sham-operation, mice untreated, that remove ovary shows suitable spongy bone density loss.The ActRIIa-mFc processed group has kept the bone density of sham operated rats level.When the treatment in 6 weeks and 12 weeks, ActRIIa-mFc causes the suitable growth of OVX mice spongy bone.Referring to Figure 10.After the treatment in 6 weeks, with respect to the PBS matched group, bone density has increased by 24%.After 12 weeks, increase to 27%.

In the sham-operation mice, ActRIIa-mFc has also caused the suitable increase of spongy bone.Referring to Figure 11.After 6 weeks and 12 weeks, with respect to contrast, treatment has produced 35% growth.

In other one group of experiment, aforesaid removal ovary (OVX) mice or sham-operation mice are handled more than 12 weeks with ActRIIa-mFc (10mg/kg, twice weekly) or contrast (PBS).Be similar to the result of above-described ActRIIa-mFc, accept the OVX mice of ActRIIa-mFc and show that spongy bone density has increased by 15% when handling for 4 weeks, and after the processing in 12 weeks, increased by 15% (Figure 12).Accept ActRIIa-mFc the sham-operation mice show that equally spongy bone density has increased by 25% when handling for 4 weeks, and after the processing in 12 weeks, increased by 32% (Figure 13).

After handling for 12 weeks with ActRIIa-mFc, femur DEXA analyzes and shows in a whole body and the junctor, handles that (Figure 14 A and Figure 14 B have all induced the growth of skeleton density in respectively) removing ovary mice and sham-operation mice.These results also by pQCT in the junctor between the femur stage casing analyze support that it has proved that after handling for 12 weeks with ActRIIa-mFc total skeleton density and cortical bone density all significantly rise.Skeleton density that the ovary mice shows compare with the contrast sham-operation mice of vehicle treated (Figure 15) is removed in the contrast of vehicle treated.Except that skeleton density, the bone amount is also handled with ActRIIa-mFc to be increased.Between the femur stage casing in the junctor pQCT analytical proof all significantly rise handling 12 total bone amounts in week back and cortical bone amount with ActRIIa-mFc, all showed comparable bone amount (Figure 16) and remove the mice that the vehicle Control of the mice of ovary and sham-operation handles.Also demonstration of pQCT analysis in the junctor between the femur stage casing, the mice that ActRIIa-mFc handles does not show the variation of periosteum girth, and ActRIIa-mFc to handle the increase that reduces to show cortical thickness that causes the perimyelis girth be (Figure 17) that the growth by the femur inner surface causes.The mechanical test of femur determines that ActRIIa-mFc can increase the external characteristic of skeleton (maximum load, rigidity and energy to failure), and it helps the remarkable increase of skeleton intrinsic characteristic (ultimate strength).The mice of the removal ovary of handling with ActRIIa-mFc has been showed the bone strength of the increase that is higher than control level sham-operation, vehicle treated, shows phenotypic reverse fully (Figure 18) of osteoporosis.

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

In another group experiment, mice is removed ovary or sham-operation in the 4th week, and (2 times weekly, and 10mg/kg) (as the RAP-11 among Figure 19-24) carried out for 12 weeks again accepting placebo or ActRIIa-mFc the 12nd week at first.Assess various skeleton 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-weeks point of sham-operation mice.These data that present in Figure 24 prove that ActRIIa-mFc has a dual effect, and suppress the skeleton absorption and promote bone growth.Therefore, ActRIIa-mFc promotes bone growth (anabolism effect) and suppresses skeleton to absorb (anti-catabolism effect).

Embodiment 4: alternate ActRIIa-Fc albumen

A kind of alternate construction can be deleted 15 aminoacid of ActRIIa ectodomain C-terminal tail (last).The sequence of such construction is as follows: (lining out below the Fc part) (SEQ ID NO:12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKN

ISGSI

EIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYF

PEM TG

GGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

EDPEV

KFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGK

EYKCK

VSNKALPVPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLV

KGFYPS

DIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGN

VFSCS?VMHEALHNHYTOKSLSLSPGK

The integration of list of references

All publications and patent that the present invention mentioned are integrally incorporated the present invention into the form of list of references, indicate with list of references especially and individually as each publication or patent and incorporate into.

Although the specific embodiment of goal object is discussed, top description is exemplary and is nonrestrictive.By reading top description and following claim, many changes are tangible 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 change.

Claims (54)

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

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

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

4. pharmaceutical preparation, the activin-that comprises among the claim 1-3 each is in conjunction with ActRIIa polypeptide and acceptable accessories.

5. the described pharmaceutical preparation of claim 4, wherein said preparation are the apyrogeneity material substantially.

6. isolating polynucleotide comprise among the claim 1-3 coded sequence of each activin-in conjunction with the ActRIIa polypeptide.

7. described isolating polynucleotide of claim 6, wherein said isolating polynucleotide comprise the sequence of SEQ ID NO:14.

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

9. one kind with each recombination of polynucleotide cell transformed among the claim 6-8.

10. the described cell of claim 9, wherein said cell is a mammalian cell.

11. the described cell of claim 10, wherein said cell are Chinese hamster ovary celI or human cell.

12. a labelling activin-comprises in conjunction with the method for ActRIIa polypeptide:

A) cultured cell under the condition that is fit to expression solubility ActRIIa polypeptide, wherein said cell transforms with each recombination of polynucleotide among the claim 6-8; And

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

13. a method that promotes bone growth, increases skeleton density or bone strength, this method comprise target is used the polypeptide of organizing under being selected from of effective dose:

A) comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with SEQ ID NO:2;

B) comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with SEQ ID NO:3; And

C) comprise the polypeptide of at least 50 continuous amino acids that are selected from SEQ ID NO:2.

14. the described method of claim 13, wherein said polypeptide have one or more following characteristics:

I) with at least 10 ^-7The K of M _DBe bonded to the ActRIIa part; And

Ii) in cell, suppress the ActRIIa signal.

15. the method for claim 13 or 14, wherein said polypeptide is a fusion rotein, comprise, one or more in the one or more polypeptide portions except that the domain of ActRIIa polypeptide, described polypeptide portion enhanced stability, body in the formation of half-life, picked-up/use, tissue positioned or distribution, protein complex and/or the purification.

16. the method for claim 15, wherein said fusion rotein comprise the polypeptide portion that is selected from down group: immunoglobulin Fc domain and serum albumin.

17. the method for each of claim 13-16, wherein said polypeptide comprise one or more modified amino acid residue of organizing below that are selected from: the aminoacid of glycosylated aminoacid, Pegylation, the aminoacid of farnesylation, acetylizad aminoacid, biotinylated aminoacid, the aminoacid that is coupled to the aminoacid of lipid part and is coupled to organic derivative.

18. a method for the treatment of the skeleton relevant disease, this method comprise, give the activin or the ActRIIa antagonist that have this object that needs to use effective dose.

19. the method for claim 18, wherein said activin or ActRIIa antagonist are activin or ActRIIa antagonist polypeptide.

20. the method for claim 19, wherein said activin or ActRIIa antagonist are selected from following group:

A) comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with SEQ ID NO:2;

B) comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with SEQ ID NO:3; And

C) comprise the polypeptide of at least 50 continuous amino acids that are selected from SEQ ID NO:2.

21. the method for claim 19 or 20, wherein said activin or ActRIIa antagonist polypeptide have one or more following characteristics:

I) with at least 10 ^-7The K of M _DBe bonded to the ActRIIa part; And

Ii) in cell, suppress the ActRIIa signal.

22. each method of claim 19-21, wherein said activin or ActRIIa antagonist polypeptide are warm albumen, comprise, one or more in the one or more polypeptide portions except that the domain of ActRIIa polypeptide, described polypeptide portion enhanced stability, body in the formation of half-life, picked-up/use, tissue positioned or distribution, protein complex and/or the purification.

23. the method for claim 22, wherein said fusion rotein comprise the polypeptide portion that is selected from down group: immunoglobulin Fc domain and serum albumin.

24. each method of claim 19-23, wherein said activin or ActRIIa antagonist polypeptide comprise one or more modified amino acid residue of organizing below that are selected from: the aminoacid of glycosylated aminoacid, PEGization, the aminoacid of farnesylation, acetylizad aminoacid, biotinylated aminoacid, the aminoacid that is coupled to the aminoacid of lipid part and is coupled to organic derivative.

25. each method of claim 18-24, the disease that wherein said skeleton is relevant is selected from following group: primary osteoporosis and secondary osteoporosis.

26. each method of claim 18-24, the disease that wherein said skeleton is relevant is selected from following group: the osteoporosis that postclimacteric osteoporosis, gonad function are low, the osteoporosis that tumor causes, osteoporosis, metastatic tumor of bone, multiple myeloma and the cypress Zhe Shi disease that treatment of cancer causes.

27. each method of claim 18-26, wherein said method also comprises uses second kind of skeleton activator.

28. the method for claim 27, wherein said skeleton activator is selected from following group: bisphosphonate, estrogen, selective estrogen receptor modulators, parathyroid hormone, calcitonin, calsium supplement and vitamin D supplement.

29. a pharmaceutical preparation comprises:

(a) activin or ActRIIa antagonist; And

(b) the second skeleton activator.

30. a method of identifying the preparation that promotes bone growth or bone density improving, this method comprises:

A) evaluation and activin or ActRIIa antagonist polypeptide competitiveness are bonded to the test agents of the ligand binding domains of ActRIIa polypeptide; And

B) effect of assessment medicament on tissue growth.

31.Activin or ActRIIa antagonist polypeptide is used for the treatment of application in the medicine of skeleton relevant disease in preparation.

32. a method of preventing the skeleton relevant disease, this method comprise activin or the ActRIIa antagonist of the object that these needs are arranged being used effective dose.

33. the method for claim 32, wherein said object has the cancer relevant with metastatic tumor of bone.

34. the method for claim 32-33, wherein said object to bone density lose, skeleton absorbs or the indicator of metastatic tumor of bone is male.

35. each method of claim 32-34, wherein said to as if the receptor of the modality of cancer treatment relevant with bone loss.

36. each method of claim 32-34, wherein said object has the cancer relevant with bone loss.

37. each activin-of claim 1-3 is in conjunction with the ActRIIa polypeptide, claim 4-5 or 29 each pharmaceutical preparatioies, or each method of claim 13-28, and wherein said polypeptide is glycosylated.

38. one kind promotes bone growth and suppresses the method that skeleton absorbs in the patient, described method comprises the ActRIIa-Fc fusion rotein of the patient being used effective dose, and wherein said ActRIIa-Fc fusion rotein comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO:3 has at least 90% homogeneity.

39. the method for claim 38, wherein said ActRIIa-Fc fusion rotein comprise the aminoacid sequence that the aminoacid sequence with SEQ ID NO:3 has at least 95% homogeneity.

40. the method for claim 38 or 39, wherein said ActRIIa-Fc fusion rotein comprises the aminoacid sequence of SEQ ID NO:3.

41. each method of claim 38-40, wherein said ActRIIa-Fc fusion rotein comprises the aminoacid sequence of SEQ ID NO:2.

42. each method of claim 38-41, wherein said method cause patient's skeletal muscle mass less than 10% growth.

43. each method of claim 38-41, wherein said ActRIIa-Fc fusion rotein is used and is made the intravital blood drug level of patient reach 0.2 μ g/kg at least.

44. each method of claim 38-43, wherein said ActRIIa-Fc fusion rotein has the aminoacid sequence of SEQ ID NO:7.

45. each method of claim 38-44, wherein said ActRIIa-Fc fusion rotein has 15 to 30 days serum half-life.

46. each method of claim 38-45, wherein said ActRIIa-Fc fusion rotein is administered to the patient and is no more than once in a week.

47. each method of claim 38-46, wherein said ActRIIa-Fc fusion rotein is administered to the patient and is no more than every month once.

48. be selected from down the application of polypeptide in the medicine of preparation promotion bone growth, increase skeleton density or increase bone strength of group: (a) comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with SEQ ID NO:2; (b) comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with SEQ ID NO:3; And the polypeptide that (c) comprises at least 50 continuous amino acids that are selected from SEQ ID NO:2.

49.Activin or ActRIIa antagonist polypeptide is used for preventing the application of the medicine of skeleton relevant disease in preparation.

50. comprise the application of ActRIIa-Fc fusion rotein in the medicine of preparation promotion bone growth and the absorption of inhibition skeleton that has the aminoacid sequence of at least 90% homogeneity with the aminoacid sequence of SEQ ID NO:3.

51. be selected from the polypeptide of following group, be used to promote bone growth, bone density improving or increase bone strength: (a) comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with SEQ ID NO:2; (b) comprise the polypeptide that has the aminoacid sequence of at least 90% homogeneity with SEQ ID NO:3; And the polypeptide that (c) comprises at least 50 continuous amino acids that are selected from SEQ ID NO:2.

52.Activin or ActRIIa antagonist polypeptide is used for the treatment of skeleton relevant disease.

53.Activin or ActRIIa antagonist polypeptide is used to prevent the skeleton relevant disease.

54. comprising the ActRIIa-Fc fusion rotein that aminoacid sequence with SEQ ID NO:3 has the aminoacid sequence of at least 90% homogeneity is used to promote bone growth and suppresses skeleton absorb.

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