CN105085679B - The anti-RANKL antibody of full source of people - Google Patents

Abstract

The present invention relates to full humanized antibodies, specifically, the present invention relates to the anti-RANKL antibody of full source of people, the nucleic acid molecules of the antibody and the composition comprising the antibody are encoded, the invention further relates to the antibody in preparation for preventing and treating and the purposes in the drug of the diseases such as bone lesion related disease especially osteoporosis, arthritis bone dissolved destruction or bone metastaes.Antibody of the invention is being kept and people RANKL specific bond, and while blocking its activity, it is reduced to the potential inhibitive ability of immunity of human body, while there is better stability, avoids the drawbacks of inhomogeneity (heterogeneity) of IgG2 antibody producing is brought to batch Quality Identification.

Description

The anti-RANKL antibody of full source of people

Technical field

The present invention relates to full humanized antibodies, in particular it relates to which the anti-RANKL antibody of full source of people, encodes the antibody Nucleic acid molecules and composition comprising the antibody, the invention further relates to the antibody preparation for prevent and treat osteoporosis, Purposes in the drug of the diseases such as arthritis bone dissolved destruction or bone metastaes.

Background technique

Bon e formation and bone remoulding are coordinated to complete by bone resorption osteoclast and bon e formation osteoblast, people development and at It is responsible for bone metabolism and skeletal reconstruction in length.It constantly absorbs and is formed in the specific position bone of bone, estimate in annual human body about 10% bone total amount can be rebuild.Derived from the osteoclast of Monocytes/Macrophages precursor, reabsorption can be carried out to bone after activation, most Apoptosis occurs for whole osteoclast.Then, from the newly-generated osteoblast of preosteoblast/stroma cell in absorption site Form sclerotin.The development of osteoclast is controlled by osteoblast, so that bone resorption and forming process are fitted close.I.e. in each bone Absorption cycle is followed by a wheel bon e formation.Osteoclast and osteoblast is movable unbalance will lead to bone loss (osteoporosis) Or bone increases the skeletal abnormality that (osteopetrosis) is characterized.(Khosla,Endocrinology,2001,142,5050-5055； Nakashima et al.,Curr.Opin.Rheumatol.,2003,15,280-287).

Communication between osteoblast and osteoclast is realized by cell factor and iuntercellular effect.Pass is played in bone remoulding One cell factor of key effect is receptor activator of nuclear factor κB ligand (receptor activator of NF- KappaB ligand, RANKL).RANKL is the first TNF aglucon family member being found, also referred to as tumor necrosis factor Associated activation inducible factor (tumor necrosis-factor-related activation-induced cytokine, TRANCE), osteoprotegerin ligand (osteoprotegerin ligand, OPGL), osteoclast differentiation factor (osteoclast Differentiation factor, ODF), 11 (tumor necrosis of tumor necrosis factor (aglucon) superfamily member Factor (ligand) superfamily member11, TNFSF11), RANKL was identified external evoked osteoclast later The cell factor of differentiation.(Anderson et al.,Nature,1997,390,175-179；Lacey et al.,Cell, 1998,93,165-176；Wong et al.,J.Exp.Med.,1997,186,2075-2080；Yasuda et al., Proc.Natl.Acad.Sci.USA,1998,95,3597-3602).The gene of mankind RANKL is located at chromosome 13q14. RANKL expression highest (Anderson et al., Nature, 1997,390,175- in bone, marrow and lymphoid tissue 179；Lacey et al.,Cell,1998,93,165-176；Wong et al.,J.Exp.Med.,1997,186,2075- 2080；Yasuda et al., Proc.Natl.Acad.Sci.USA, 1998,95,3597-3602), can also brain, the heart, kidney, (Kartsogiannis et al., Bone, 1999,25,525-534) is detected in skeletal muscle and skin.

RANKL is assembled by three kinds of RANKL subunits and is formed functional trimeric molecules.RANKL is initially anchored on cell membrane On, tumor necrosis factor α invertase (metalloprotease-disintegrin TNF-alpha convertase, TACE under hydrolysis), from cell surface discharge its extracellular partially protein (Lum et al., J.Biol.Chem., 1999,274,13613-13618).RANKL enhances its vigor, and inhibit osteoclast apoptosis to osteoclast differentiation is promoted It is essential (Fuller et al., J.Exp.Med., 1998,188,997-1001；Lacey et al.,Cell, 1998,93,165-176；Lum et al.,J.Biol.Chem.,1999,274,13613-13618；Yasuda et al., Proc.Natl.Acad.Sci.USA,1998,95,3597-3602).RANK receptor is in preosteoblast and marrow stromal cell Surface expression.The expression of RANKL is by various hormones, cell factor, growth factor and glucocorticoid positive regulator or negative regulator, packet Vitamine D3, parathyroid hormone, interleukin 1-β and TNF-α are included, these can all increase expression (the Kong et of RANKL al.,Immunol.Today,2000,21,495-502).In the beginning of cycle of bone resorption and bon e formation, RANKL and osteoclast The functional receptor RANK of precursor cell surface combines (Anderson et al., Nature, 1997,390,175-179； Lacey et al.,Cell,1998,93,165-176).The interaction of RANKL and RANK promotes the maturation of osteoclast, at Ripe osteoclast is divided into multicore, has the function of bone resorption with expression specificity marker Tartrate resistant acid phosphatase (tartrate-resistant acid phosphatase, TRAP) be characterized (Burgess et al., J.Cell.Biol., 1999,145,527-538；Hsu et al.,Proc.Natl.Acad.Sci.U.S.A.,1999,96,3540-3545；Lum et al.,J.Biol.Chem.,1999,274,13613-13618；Yasuda et al., Proc.Natl.Acad.Sci.USA,1998,95,3597-3602).RANKL can also be with water-soluble receptor osteoprotegerin (OPG) In conjunction with the osteoclast cell maturation and activation (Lacey et that OPG is mainly expressed by marrow stromal cell and RANKL is inhibited to mediate al.,Cell,1998,93,165-176；Yasuda et al.,Proc.Natl.Acad.Sci.USA,1998,95,3597- 3602).PTH is a kind of main regulatory factors of bone remoulding, and the expression of OPG is expressed while reduced by increasing RANKL, is promoted broken Bone cell activity (Lee and Lorenzo, Endocrinology, 1999,140,3552-3561).Current osteoblast differentiation When, the mRNA level in-site of RANKL is remarkably decreased, and the horizontal significantly raising of OPGmRNA (Gori et al., Endocrinology, 2000,141,4768-4776).Dynamic movement between RANKL and OPG level keeps the movable and then skeletonization of osteoclast thin The circulation of bone resorption and bon e formation is completed in the activity of born of the same parents with this.

By discharging calcium from intracellular repository, the calcium content in RANKL induction osteoclast is instantaneously increased (Komarova et al.,J.Biol.Chem.,2003,278,8286-8293).The homozygous mouse of RANKL gene is blocked to go out Life after three weeks, shows serious growth retardation.Since osteoblast cannot support osteoclast, RANKL deficient mice Due to lacking osteoblast to the supporting function of osteoclast formation, serious osteopetrosis (sclerotin thickens) is shown as, tooth is sprouted Defect and osteoclast missing (Kong et al., Immunol.Today, 2000,21,495-502) out.

The function of RANKL is not only in that the formation and reconstruction of bone, and RANKL deficient mice also shows T and B lymph Cell early differentiation defect and lymph node missing, this shows that RANKL is the regulatory factor of lymphoid organ and lymphocyte development, (Kong et al.,Immunol.Today,2000,21,495-502).The expression of T cell receptor stimulation induction RANKL gene, So as to cause c-Jun N- terminal Kinase in T cell activation (Wong et al., J.Biol.Chem., 1997,272, 25190-25194).RANKL also participates in function of immune system adjusting, by inhibiting Apoptosis as marrow source Dendritic Cells Important survival factors (Lum et al., J.Biol.Chem., 1999,274,13613-13618；Wong et al., J.Exp.Med.,1997,186,2075-2080).In addition, the development of alveolar breast structure is also required to RANKL's during mouse pregnancy It participates in (Fata et al., Cell, 2000,103,41-50).

RANKL, which inadequately activates osteoclast, can make bone resorption process unbalance, and bone resorption is caused to be greater than bon e formation.Permitted In more osteoporosis, including postmenopausal osteoporosis, age-related osteoporosis, periodontosis, familial Dilatancy bone dissolution disease, all observe in osteitis deformans topically or systemically bone loss (Khosla, Endocrinology, 2001,142,5050-5055).RANKL mRNA expression up-regulation is had reported in these above-mentioned diseases.

Osteitis deformans, which is characterized in having, leads to the increased a large amount of abnormal osteoclasts of bone resorption.Osteitis deformans patient RANKL mRNA expression in middle isolated bone marrow stromal cell system and sufferer marrow all increases.And osteitis deformans patient Osteoclast precursor required RANKL concentration during forming osteoclast it is lower than normal marrow cell (Menaa et al., J.Clin.Invest.,2000,105,1833-1838)。

With osteopenic other diseases, such as rheumatoid arthritis, chronic viral infection, adult and leukemia of children It is characterized by t cell activation and osteoclasia (Kong et al., Immunol.Today, 2000,21,495-502).Rheumatoid Property arthritis is a kind of chronic inflammatory disease, is characterized in the bone resorption of gradual Osteoclasts mediate.Rheumatoid arthritis is sliding The B cell generated in film liquid containing osteoclast precursor, the T cell of expression RANKL and OPG.Culture in RA synovial fluid it is huge Phagocyte can rely on RANKL and be divided into osteoclast (Itonaga et al., J.Pathol., 2000,192,97-104). In the tentative rat model of arthritis that T cell relies on, many Clinical symptoms of mankind RA are shown, are treated by OPG, pressed down RANKL function processed thus prevents osteoclasia (Kong et al., Nature, 1999,402,304-309).

Huppert's disease is using osteoporosis and osteoclasia as the cancer of prominent features.Multiple myeloma cells promote RANKL expression, while also inhibiting the OPG expression of marrow stromal cell, lead to the horizontal unbalance and osteoclast of RANKL and OPG Abnormal generate and activation (Pearse et al., Proc.Natl.Acad.Sci.USA, 2001,98,11581-11586).This Outside, some cancer cells express secretory RANKL, cause malignant tumor patient hypercalcinemia (Nagai et al., Biochem.Biophys.Res.Commun.,2000,269,532-536).After glucocorticoid treatment, in osteoblast RANKL increases, while OPG expression is reduced, this can activate osteoclast formation, can produce sternly with this whole body glucocorticoid treatment Weight osteoporosis (Hofbauer et al., Endocrinology, 1999,140,4382-4389)..

Immune function is related to physiology of bone, and it is close that immunocyte expression RANKL provides immune system disorder bring bone Reduced molecule is spent to explain.Therefore by inhibiting RANKL function, and then inhibit osteoclast activity, immune inflammation can be improved and led The osteoporosis (Kong et al., Immunol.Today, 2000,21,495-502) of cause.

Since RANKL participates in a variety of diseases, the up-regulation of RANKL expression is related to a plurality of types of osteoporosis, therefore It is required to effectively inhibit the active inhibitor of RANKL, to treat the disease characterized by bone damage.

Denosumab is the drug newly ratified for treating Bone tumour disease.It is a kind of anti-RANKL IgG2 type of full source of people Monoclonal antibody is a kind of effective inhibitor for inhibiting osteoclast formation and bone resorption.For treating postmenopausal women's bone Bone tumour caused by matter osteoporosis and solid tumor and Huppert's disease and fracture.

Lipton etc. is randomly divided into six groups about in the II phase clinical research of breast cancer correlation Bone tumour, wherein five groups Using denosumab, Primary Endpoint is ratio (uNTX/Cr) of the Bone turnover marker N- end peptide to urine creatinine, and to its safety Property and skeletal related events (SREs) are assessed.Inject drop of patient's majority evident from uNTX/Cr of denosumab It is low, less there is SRES compared to Diphosphonate patient (9%vs is to 16%).Directly relatively denosumab and Zoledronate One biggish placebo-controlled randomized trial shows that denosumab is delaying or preventing in breast cancer patients Bone tumour SRES is excellent in.For the inhibitor of RANKL, denosumab provides a kind of treat and fractures caused by osteoporosis, is pernicious The new method of potential osteoclasia in the bone complications and RA of tumour.However, RANKL can also in addition to being expressed by osteoblast It is generated by the synovial cell in activating T cell and RA, and the receptor of RANKL, i.e. RANK, also by Monocytes/Macrophages and tree Prominent shape cell expression.The missing of RANKL, RANK and OPG highlight this path in rodent immune system in mouse model Importance in system development and maturation, the function including T cell and/or B cell.Pharmacology/toxicological study proposes immunosupress Some problems, although having determined that the beneficial effect to bone density and incidence of fracture in clinical test, before the treatment The risk of denosumab must be assessed.The total incidence for the serious adverse reaction that denosumab group infects in clinical research is higher than Control group.The incidence of denosumab group infection relevant to bacterium and no specific pathogen is higher than control group.In view of infection Risk should be avoided in carrying out immunosuppressive therapy or patient with high infection risk using denosumab.In addition to exempting from The high risk that epidemic disease inhibits, denosumab are produced since the complexity of its IgG2 hypotype and free cysteine structure is difficult control, This increases its difficulty in production technology is developed in development and GMP production and produced, it is difficult to carry out good quality control.

Summary of the invention

In order to reduce the immunosupress side effect for having anti-RANKL antibody and infection risk, and it is big in order to be more advantageous to Large-scale production and quality control, the present invention provides the anti-RANKL antibody of new full source of people.

First aspect present invention is related to the anti-RANKL antibody of full source of people or its segment, and it includes such as SEQ ID NO:1 or SEQ Heavy chain shown in ID NO:3 and the light chain as shown in SEQ ID NO:5 or SEQ ID NO:7；Or it is selected from following (1)-(4) In one or several:

(1) wherein variable region sequences of the heavy chain are as follows: in variable region sequences, that is, SEQ ID NO:9 sequence of the heavy chain The middle antibody by replacing, missing or adding one or several amino acid and being formed or its segment are with same or similar activity；

(2) wherein variable region sequences of the light chain are as follows: in variable region sequences, that is, SEQ ID NO:11 sequence of the light chain By the antibody for replacing, missing or adding one or several amino acid and being formed or its segment with same or similar work in column Property；

(3) wherein constant-region sequences of the heavy chain are as follows: in constant-region sequences, that is, SEQ ID NO:13 sequence of the heavy chain By the antibody for replacing, missing or adding one or several amino acid and being formed or its segment with same or similar work in column Property；

(4) wherein constant-region sequences of the light chain are as follows: in constant-region sequences, that is, SEQ ID NO:12 sequence of the light chain By the antibody for replacing, missing or adding one or several amino acid and being formed or its segment with same or similar work in column Property.

In embodiments of the invention, the anti-RANKL antibody of the full source of people or its segment include such as SEQ ID NO:1 institute The heavy chain and the light chain as shown in SEQ ID NO:5 shown.

In embodiments of the invention, the anti-RANKL antibody of the full source of people or its segment include such as SEQ ID NO:3 institute The heavy chain and the light chain as shown in SEQ ID NO:7 shown.

Second aspect of the present invention is related to isolated nucleic acid molecules, and the full source of people of any one of coding first aspect present invention is anti- RANKL antibody or its segment.

The nucleic acid molecules of any one according to a second aspect of the present invention, it includes such as SEQ ID NO:2 or SEQ ID NO:4 institutes The heavy chain and the light chain as shown in SEQ ID NO:6 or SEQ ID NO:8 shown.

Nucleic acid molecules any one of according to a second aspect of the present invention, it includes the heavy chain as shown in SEQ ID NO:2 and The light chain as shown in SEQ ID NO:6.

Nucleic acid molecules any one of according to a second aspect of the present invention, it includes the heavy chain as shown in SEQ ID NO:4 and The light chain as shown in SEQ ID NO:8.

Third aspect present invention is related to recombinant vector, the nucleic acid molecules containing any one of second aspect of the present invention.

Fourth aspect present invention is related to recombinant cell, the nucleic acid molecules containing any one of second aspect of the present invention or The recombinant vector of any one of three aspects.

Fifth aspect present invention is related to composition, and the anti-RANKL of full source of people containing any one of first aspect present invention is anti- Any one of the recombinant vector of any one of the nucleic acid molecules of any one of body or its segment, second aspect, the third aspect or fourth aspect Recombinant cell and pharmaceutically acceptable carrier or excipient.

Composition any one of according to a fifth aspect of the present invention also contains at least one treatment inflammation or immunological diseases Therapeutic agent.

Composition any one of according to a fifth aspect of the present invention, wherein the therapeutic agent be selected from COX1 and COX2 inhibitor, Prednisolone, methotrexate (MTX), chloroquine, cyclosporin.

Sixth aspect present invention further relate to any one of first aspect present invention the anti-RANKL antibody of full source of people or its segment, The recombinant vector of any one of the nucleic acid molecules of any one of second aspect, the third aspect or the recombinant cell of any one of fourth aspect exist Preparation is for preventing or treating and the purposes in the drug of bone lesion related disease.

The purposes of any one according to a sixth aspect of the present invention, wherein described and bone lesion related disease includes but is not limited to bone Matter is loose, arthritis bone dissolved destruction, malignant tumour or malignant metastatic tumor of bone, paget's disease of bone (osteitis deformans), adjoint The inflammation (such as osteomyelitis) of osteoclasia, with osteoclasia autoimmune disease or rheumatoid arthritis, hypercalcinemia, Osteonecrosis, osteopenia.

The purposes of any one according to a sixth aspect of the present invention, wherein the malignant tumour includes but is not limited to breast cancer, it is preceding Column gland cancer, thyroid cancer, kidney, lung cancer, cutaneum carcinoma, cancer of the esophagus, the carcinoma of the rectum, bladder cancer, cervix cancer, oophoroma, liver cancer, stomach Bowel cancer, Huppert's disease and lymthoma (Hodgkin's disease).

The purposes of any one according to a sixth aspect of the present invention, wherein the osteoporosis includes but is not limited to primary sclerotin It is loose, endocrine osteoporosis (including but not limited to hyperthyroidism, hyperparathyroidism, Cushing syndrome, limb Hold loose disease), (including but not limited to osteogenesis imperfecta, homocystinuria, Men Kesi are comprehensive with apriori form for the heredity of osteoporosis Simulator sickness, Lai-wear syndrome), and the osteoporosis fixed due to acra.

Seventh aspect present invention is related to the anti-RANKL antibody of full source of people or its segment, of any one of first aspect present invention The recombinant cell of the nucleic acid molecules of any one of two aspects, the recombinant vector of any one of third aspect or any one of fourth aspect with extremely It is few a kind of to treat inflammation or the therapeutic agent of immunological diseases uses in preparing the drug for preventing or treating bone lesion Purposes.

The purposes of any one according to a seventh aspect of the present invention, wherein the therapeutic agent is selected from COX1 and COX2 inhibitor, sprinkles Ni Songlong, methotrexate (MTX), chloroquine, cyclosporin.

The purposes of any one according to a seventh aspect of the present invention, wherein described and bone lesion related disease includes but is not limited to bone Matter is loose, arthritis bone dissolved destruction, malignant tumour or malignant metastatic tumor of bone, paget's disease of bone (osteitis deformans), adjoint The inflammation (such as osteomyelitis) of osteoclasia, with osteoclasia autoimmune disease or rheumatoid arthritis, hypercalcinemia, Osteonecrosis, osteopenia.

The purposes of any one according to a seventh aspect of the present invention, wherein the malignant tumour includes but is not limited to breast cancer, it is preceding Column gland cancer, thyroid cancer, kidney, lung cancer, cutaneum carcinoma, cancer of the esophagus, the carcinoma of the rectum, bladder cancer, cervix cancer, oophoroma, liver cancer, stomach Bowel cancer, Huppert's disease and lymthoma (Hodgkin's disease).

The purposes of any one according to a seventh aspect of the present invention, wherein the osteoporosis includes but is not limited to primary sclerotin It is loose, endocrine osteoporosis (including but not limited to hyperthyroidism, hyperparathyroidism, Cushing syndrome, limb Hold loose disease), (including but not limited to osteogenesis imperfecta, homocystinuria, Men Kesi are comprehensive with apriori form for the heredity of osteoporosis Simulator sickness, Lai-wear syndrome), and the osteoporosis fixed due to acra.

The invention further relates to the anti-RANKL antibody of the full source of people of any one of first aspect present invention or its segments, second aspect The recombinant vector of any one of the nucleic acid molecules of any one, the third aspect or the recombinant cell of any one of fourth aspect are in preparation conduct Purposes in the drug of RANKL inhibitor.

The invention further relates to detection reagent or kits, contain the antibody of any one of first aspect present invention or its piece Section；

Preferably, the antibody or its antigen-binding portion thereof also may include detectable label；Or

Preferably, the detection reagent or kit also may include secondary antibody, antibody described in specific recognition or its Antigen-binding portion thereof；Or

Preferably, the secondary antibody also may include detectable label.

The invention further relates to the antibody of any one of first aspect present invention or its segment in preparation detection reagent or kit In purposes, wherein the detection reagent or kit be for detecting Nuclear factor kappa-B receptor activation factor ligand (RANKL), or Person is for diagnosing Nuclear factor kappa-B receptor activation factor ligand related disease.

In the present invention, term " antibody " refers to usually (each pair of to have " light " (L) chain by two pairs of identical polypeptide chains With " weight " (H) chain) composition immunoglobulin molecules.Antibody light chain can be classified as κ and lambda light chain.Heavy chain can be classified as μ, δ, γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.It, can in light chain and heavy chain Become area to connect with constant region by area " J " of about 12 or more amino acid, heavy chain also includes about 3 or more amino Area " D " of acid.Each heavy chain is by heavy chain variable region (V_H) and heavy chain constant region (C_H) composition.Heavy chain constant region is by 3 structural domains (C_H1、C_H2 and C_H3) it forms.Each light chain is by light chain variable region (V_L) and constant region of light chain (C_L) composition.Constant region of light chain is by one Domain C_LComposition.The constant region of antibody can mediated immunity globulin and host tissue or the factor, including the various of immune system The combination of the first component (C1q) of cell (for example, effector cell) and classical complement system.V_HAnd V_LArea can also be subdivided into tool There is denatured region (referred to as complementary determining region (CDR)), is interspersed with the more conservative region for being known as framework region (FR).Respectively V_HAnd V_LBy in the following order: arranged from amino terminal to carboxyl terminal 3 of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 CDR and 4 FR composition.Variable region (the V of each heavy chain/light chain pair_HAnd V_L) it is respectively formed paratope.Amino acid is to each area The distribution of domain or structural domain follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and1991)) or Chothia&Lesk (1987)J.Mol.Biol.196:901-917；The definition of Chothia et al. (1989) Nature342:878-883.Term is " anti- Body " is not limited by any specific method for generating antibody.Such as comprising, particularly, recombinant antibodies, monoclonal antibody and Polyclonal antibody.Antibody can be the antibody of different isotypes, for example, IgG is (for example, IgG1, IgG2, IgG3 or IgG4 are sub- Type), IgA1, IgA2, IgD, IgE or IgM antibody.

In the present invention, " segment " of term antibody refers to that one or more parts of full length antibody, the part are kept The ability for the same antigen (for example, RANKL) that binding antibody is combined competes the specific binding to antigen with complete antibody. Usually referring to Fundamental Immunology, Ch.7 (Paul, W., ed., second edition, Raven Press, N.Y. (1989), it is incorporation by reference in its entirety, for all purposes.It can be by recombinant DNA technology or by complete The enzymatic or chemical disruption of antibody generate antigen-binding portion thereof.In some cases, antigen-binding portion thereof includes Fab, Fab', F (ab')₂, Fd, Fv, dAb and complementary determining region (CDR) segment, single-chain antibody (for example, scFv), chimeric antibody, double antibody (diabody) and such polypeptide, it includes at least part for the antibody for being enough to assign polypeptid specificity antigen binding capacity.

In the present invention, term " Fd segment " means by V_HAnd C_HThe antibody fragment of 1 structural domain composition；Term " Fv segment " Mean by the V of the single armed of antibody_LAnd V_HThe antibody fragment of structural domain composition；Term " dAb segment " means by V_HStructural domain composition Antibody fragment (Ward et al., Nature341:544-546 (1989))；Term " Fab segment " means by V_L、V_H、C_LAnd C_H1 knot The antibody fragment of structure domain composition；Term " F (ab')₂Segment " means two Fab comprising connecting by the disulphide bridges on hinge area The antibody fragment of segment.

In some cases, the segment of antibody is single-chain antibody (for example, scFv), wherein V_LAnd V_HStructural domain is by making it The connector that can be produced as single polypeptide chain match to be formed monovalent molecule (see, e.g., Bird et al., Science242: 423-426 (1988) and Huston et al., Proc.Natl.Acad.Sci.USA85:5879-5883 (1988)).Such scFv Molecule can have general structure: NH₂-V_LConnector-V_H- COOH or NH₂-V_HConnector-V_L-COOH.Suitable prior art connector It is made of duplicate GGGGS amino acid sequence or its variant.For example, can be used has amino acid sequence (GGGGS)₄Connector, But its variant (Holliger et al. (1993), Proc.Natl.Acad.Sci.USA90:6444-6448) can also be used.It can use In other connectors of the invention by Alfthan et al. (1995), Protein Eng.8:725-731, Choi et al. (2001), Eur.J.Immunol.31:94-106, Hu et al. (1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), (2001) J.Mol.Biol.293:41-56 and Roovers et al., Cancer Immunol. description.

In some cases, antibody is double antibody, that is, bivalent antibody, wherein V_HAnd V_LStructural domain table in single polypeptide chain Reach, but using too short connector so that do not allow to match between two structural domains of same chain, thus force structural domain with The complementary domain of another chain match and generate two antigen-binding sites (see, e.g., Holliger P. et al., Proc.Natl.Acad.Sci.USA90:6444-6448 (1993) and Poljak R.J. et al., Structure2:1121- 1123(1994))。

It can be used routine techniques well known by persons skilled in the art (for example, recombinant DNA technology or enzymatic or chemical disruption Method) obtain the above-mentioned segment of antibody from given antibody (such as monoclonal antibody BA05-1, BA05-2), and with for complete Segment of the identical mode of the mode of whole antibody with regard to specificity screening antibody.

In the present invention, the carrier can be cloning vector or expression vector.The expression vector is, for example, protokaryon table It is carrier commonly used in the art up to carrier, carrier for expression of eukaryon, phage vector or viral vectors.The wherein protokaryon table It is, for example, PET28a, pGEM Ex1, pGEM7ZF (+) or pBAD24 etc. up to carrier, the carrier for expression of eukaryon is, for example, PBudCE4.1, pcDNA3.1, pCMV-Tag2B or pGL3basic etc., in embodiments of the invention, the eukaryotic expression Carrier is pcDNA3.1, and the viral vectors is, for example, retrovirus, slow virus, adenovirus and adeno-associated virus.

In the present invention, the cell can be prokaryotic cell or eukaryocyte.The eukaryocyte is, for example, that lactation is dynamic Object cell.The cell can be and introducing/transfection into prokaryotic cell or eukaryocyte above-mentioned nucleic acid molecules or carrier It obtains.

In the present invention, the prokaryotic cell can be for example Escherichia coli, and the eukaryocyte for example can be yeast Cell, insect cell (such as Sf21), mammalian cell, the mammal for example can be NSO cell, Chinese hamster ovary celI, 293 Cell.In one embodiment of the invention, the cell is eukaryocyte.In one embodiment of the invention, institute Stating eukaryocyte is 293F cell.

In the present invention, can use any kind of transfection method known in the art obtain transfection have specific nucleic acid or The host cell of carrier.For example, nucleic acid can be introduced into cell by electroporation or microinjection.Alternatively, fat transfection can be used Reagent such as FuGENE6, X-tremeGENE and LipofectAmine.Alternatively, can be by being based on retrovirus, slow virus, gland Nucleic acid is introduced into cell by the appropriate viral vectors of virus and adeno-associated virus.

In the present invention, the osteoporosis includes but is not limited to primary osteoporosis, endocrine osteoporosis (including But it is not limited to hyperthyroidism, hyperparathyroidism, Cushing syndrome, acromegalia), the heredity of osteoporosis With apriori form (including but not limited to osteogenesis imperfecta, homocystinuria, menkes' syndrome, Lai-wear syndrome), and due to The fixed osteoporosis of acra.

In the present invention, the malignant tumour includes but is not limited to breast cancer, prostate cancer, thyroid cancer, kidney, lung Cancer, cancer of the esophagus, the carcinoma of the rectum, bladder cancer, cervix cancer, oophoroma, liver cancer, gastrointestinal cancer, Huppert's disease and lymthoma (what Outstanding king's evil).

The present invention provides structures different from the new anti-RANKL antibody of denosumab.

In some embodiments of the present invention, the present invention construct from denosumab mutant library and by its Expression uses RANKL as target on yeast surface display system, carries out two-wheeled screening in vitro, and selection has lower The anti-RANKL antibody mutants of Kd and longer dissociation half-life period, the variant of selection is after stability test and signature analysis Whole antibody is converted into suitable IgG4 skeleton.

In some embodiments of the present invention, anti-RANKL antibody of the invention and its segment are broken up as osteoclast With mature inhibitor, there is very strong affinity with RANKL, can block RANKL in conjunction with RANKL receptor.

In some embodiments of the present invention, anti-RANKL antibody of the invention and its segment are to T cell and B cell function It can influence smaller, therefore have lesser immunosuppressive action and better security window, it is contemplated that in malignant tumour or pernicious swollen On tumor Bone tumour, there is better potential applicability in clinical practice.

In some embodiments of the present invention, antibody of the invention and its segment have better homogeneity and stabilization Property avoids the drawbacks of inhomogeneity (heterogeneity) of IgG2 antibody producing is brought to batch Quality Identification.

Detailed description of the invention

The combination of Fig. 1 antibody BA05-1 and BA05-2 and RANKL

Fig. 2 antibody BA05-1 and BA05-2 inhibit ability of the RANKL in conjunction with its homoreceptor RANK

Inhibiting effect of the anti-RANKL antibody of Fig. 3 to the RANKL osteoclast differentiation induced

The effect of TNF α and the IL-6 release of Fig. 4 antibody BA05-1 and BA05-2 Cytokines in Peripheral Blood Mononuclear (PBMC)

Effect of Fig. 5 antibody BA05-1 and BA05-2 to RANKL positive T cell in PBMC culture

Internal inhibiting effect of Fig. 6 single dose BA05-1 and BA05-2 antibody to machin CTX-1

Internal inhibiting effect of Fig. 7 single dose BA05-1 and BA05-2 antibody to machin TRACP-5B

The serum stability of Fig. 8 antibody BA05-1 and BA05-2 (wherein ordinate indicates the complete, antibody without degradation)

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.

The present invention constructs the peptide library of the CDR region random mutation sequence containing Denosumab, and is shown thin in yeast Cellular surface, storage capacity are about 1x10⁷, use RANKL as target, carry out two-wheeled screening in vitro using flow cytometry, select Select the anti-RANKL antibody mutants with lower Kd and longer dissociation half-life period.In following embodiment i.e. using the library into Row screening.

Target RANKL therein is the ECD structural domain of RANKL, is purchased from R/D Systems (article No. 390-TN-010).

The design and expression of embodiment 1:BA05-1 and BA05-2 antibody sequence

The following are the sequences of the heavy chain variable region of AMG162:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGSGGSTYYADSVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYCAKDPGTTVIMSWFDPWGQGTLVTVSS (SEQ ID NO:9)

The following are the heavy chain constant region sequences of AMG162:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFG TQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK (SEQ ID NO:10)

Its original heavy chain IgG2 constant-region sequences is changed into IgG4 constant-region sequences, BA05-1 sequence of heavy chain is formed:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGSGGSTYYADSVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYCAKDPGTTVIMSWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID No:1)

Constant-region sequences therein are as follows:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK (SEQ ID NO:13)

The CDR region domain for being not optimised frame and antigen binding of its original heavy chain variable region is excellent by yeast display Change to attempt increase and RANKL compatibility, then in conjunction with heavy chain IgG4 constant-region sequences, form BA05-2 sequence of heavy chain:

QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWIRQAPGKGLEWVSGITGSGSSTYYADSAKGRFTISRDNAKN SLYLQMNSLRAEDTAVYYCARDPGTTVIMSWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID No:3)

The following are the sequences of the light chain variable region of AMG162:

EIVLTQSPGTLSLSPGERATLSCRASQSVRGRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVFYCQQYGSSPRTFGQGTKVEIK (SEQ ID No:11)

The following are constant region of light chain ck sequences:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID No:12)

By above-mentioned light chain variable region in conjunction with constant region of light chain CL (k) sequence, that is, form BA05-1 sequence of light chain:

EIVLTQSPGTLSLSPGERATLSCRASQSVRGRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVFYCQQYGSSPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID No:5)

By its original light chain variable region be not optimised Frame sequence by yeast display optimize with attempt increase with RANKL compatibility forms the sequence of light chain of BA05-2 by it in conjunction with constant region of light chain CL (k) sequence:

EIVMTQSPATLSLSPGERATLSCRASQSLRGRYLAWYQQKPGKAPKLLIYGASTRATGIPARFSGSGSGTDFTLTIS SLQPEDFAVYYCQQYASSPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID No:7)

The preparation of embodiment 2:BA05-1 and BA05-2 antibody expression

The heavy chain full length DNA sequence of BA05-1 are as follows:

GAGGTGCAGCTCCTGGAGAGCGGCGGAGGCCTGGTGCAGCCCGGAGGAAGCCTGCGGCTCTCCTGCGCCGCTAGCGG ATTCACATTCTCCAGCTACGCTATGAGCTGGGTCAGGCAGGCTCCTGGCAAGGGACTCGAGTGGGTGAGCGGCATCA CCGGATCCGGCGGATCCACATACTATGCCGATTCCGTCAAGGGAAGGTTCACAATCTCCCGGGACAACAGCAAGAAC ACCCTCTACCTCCAGATGAACAGCCTGCGGGCCGAGGACACAGCCGTCTACTATTGCGCCAAAGACCCCGGAACCAC CGTGATCATGAGCTGGTTCGATCCCTGGGGACAGGGAACCCTCGTGACAGTGTCCAGCGCTAGCACCAAGGGCCCAT CCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTAC TTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCA ACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGC CCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCCAAACCCAAGGACACTCTCATGATCTCCCG GACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCAT CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGA TGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAG GCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA (SEQ ID NO:2).

The light chain full length DNA sequence of BA05-1 are as follows:

GAGATCGTCCTGACACAGAGCCCCGGAACCCTCTCCCTCTCCCCCGGCGAAAGGGCTACCCTCTCCTGCAGGGCTTC CCAATCCGTGAGGGGACGGTACCTCGCTTGGTACCAGCAAAAGCCCGGACAAGCTCCTCGGCTGCTCATTTACGGCG CCAGCAGCAGAGCCACAGGCATTCCCGACCGGTTCAGCGGCAGCGGCAGCGGCACAGACTTCACACTGACAATCTCC CGGCTGGAACCCGAAGACTTCGCTGTGTTCTACTGCCAACAGTACGGATCCAGCCCCAGGACCTTCGGCCAAGGCAC AAAGGTCGAGATTAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO:6).

The heavy chain full length DNA sequence of BA05-2 are as follows:

CAGGTCCAGCTCGTGGAATCCGGAGGCGGACTGGTCAAGCCTGGCGGATCCCTCAGGCTCTCCTGTGCCGCTTCCGG CTTCACATTCAGCTCTTACGGAATGAGCTGGATTAGGCAAGCCCCTGGCAAAGGCCTCGAGTGGGTCTCCGGAATCA CCGGCAGCGGCTCCAGCACCTATTACGCTGACAGCGCCAAAGGCAGATTTACAATCAGCAGGGATAACGCTAAGAAT TCCCTCTACCTCCAGATGAATTCCCTCAGGGCTGAGGATACCGCTGTGTATTACTGTGCCAGGGACCCTGGCACAAC CGTCATCATGAGCTGGTTTGACCCTTGGGGACAGGGAACCCTCGTGACAGTGAGCTCCGCTAGCACCAAGGGCCCAT CCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTAC TTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCA ACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGC CCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCCAAACCCAAGGACACTCTCATGATCTCCCG GACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCAT CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGA TGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAG GCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA (SEQ ID NO:4).

The light chain full length DNA sequence of BA05-2 are as follows:

GAGATTGTGATGACCCAATCCCCTGCCACACTGAGCCTGAGCCCCGGAGAGCGGGCCACACTGAGCTGCCGGGCCAG CCAGAGCCTGCGGGGCCGGTACCTCGCCTGGTACCAACAGAAACCCGGAAAGGCTCCCAAACTGCTCATCTATGGCG CTTCCACAAGGGCTACCGGAATCCCTGCCCGGTTCAGCGGCAGCGGATCCGGAACAGACTTCACACTGACAATCAGC TCCCTCCAGCCTGAGGATTTCGCTGTGTATTACTGTCAGCAATACGCTTCCAGCCCCCGGACCTTTGGACAGGGAAC CAAAGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO:8).

The DNA of the heavy chain constant region (HC) and constant region of light chain (LC) that encode BA05-1 and BA05-2 is closed by IDT (USA) At.In order to express anti-RANKL antibody, VK segment is cloned into the pcDNA3.1 carrier (Invitrogen) of the segment of CK containing someone VH segment is cloned into the pcDNA3.1 carrier (Invitrogen) containing human IgG 4CH segment by Hind III and Nhe I site Hind III and BsiW I site.Wherein the pcDNA3.1 carrier of the segment of CK containing someone and contain human IgG 4CH segment By the present inventor's building, (sequence of CK segment and CH segment therein is one in aforementioned DNA sequence dna to pcDNA3.1 carrier Point).

The conversion of DH5 ɑ bacterium is again passed by, plasmid, which is extracted and is sequenced, determines positive colony.The antibody of sequencing result and record Coded sequence it is consistent.BA05-1 and BA05-2 is recombinated for expression, is respectively total to BA05-1 and BA05-2 light chain and heavy chain plasmid Transfection is into 293-F cell (293fectin is purchased from Invitrogen company), using FreeStyle^TM293Expression training It cell 5 days after supporting base (being purchased from Invitrogen company), 100ml culture bottle culture transfection, is received after centrifugation, the filtering of 0.22um film Collect culture supernatant, using Protein A column (5ml MabSelect prepacked column is purchased from GE Biosciences company) purifying, uses After 10 column volume equilibration buffers (20mM phosphoric acid buffer, 150mMNaCl, pH7.0), 2ml/ minutes flow velocitys will filter after on Clear upper prop after rinsing 10 column volumes, elutes 5 column volumes with elution buffer (10mM sodium citrate, pH3.5), will elute Antibody use 1M TrisHCl, in pH8.0 and pH to 6.5, then the antibody after dialysis purification uses ultraviolet method into PBS (280nm wavelength) measures antibody concentration, measures antibody purity.

The present invention prepares purpose human antibody using the method for genetic engineering well known within the skill of those ordinarily skilled, this Only description is used through 293-F cell transient expression, the method for obtaining analysis calibrating antibody samples in embodiment, however, this Field technical staff can also be by bacterium, yeast, virus and other eukaryotic cell expression systems come such as Chinese ovary hamster is thin Born of the same parents (CHO) stable cell lines, to prepare, produce human antibody of the invention.

Embodiment 3: combination of the flow cytomery BA05-1and BA05-2 antibody to RANKL

It is transfected by the anti-RANKL antibody BA05-1and BA05-2 of Flow cytometry and RANKL (overall length RANKL) The combination of 293 cells (Invitrogen) surface RANKL.The anti-RANKL antibody of 293-RANKL transfection cell and various concentration BA05-1, BA05-2, AMG162 are in 4 DEG C of incubation 1h.Cell is washed with PBS, and anti-human igg (H+L) is added -- PE (1: 200)/anti-human igg-R-PE (1:1000) 4 DEG C of incubation 30min of antibody.After being washed with PBS, cell is resuspended in 1mlPBS, so It is analyzed afterwards with flow cytometer (BD FACScan).As shown in Figure 1, the binding ability and AMG162 of BA05-1 and RANKL Quite, and the binding ability of BA05-2 and RANKL is slightly better than AMG162.

The combination of embodiment 4:BA05-1 and BA05-2 blocking RANKL and its homoreceptor

As shown in Fig. 2, ability of the three kinds of anti-RANKL antibody blocking RANKL of detection in conjunction with its receptor.Specifically, it tests People RANK (2 μ g/ml, be purchased from R&D, article No. 683-RK-100) are coated on 96 orifice plates by preceding 16h.It is subsequently added into and various concentration 0.5 μ g/ml biotin labeling RANKL of recombined human RANKL antibody BA05-1, BA05-2, AMG162 preincubate (eBioscience, article No. 13-6619-82), 37 DEG C, 30min.The Avidin of the diluted HRP label of 50ul1:5000 is added (Pierce, article No. N100), 37 DEG C are incubated for the RANKL that 1h is used to detect be coated in conjunction with RANK.Anti- RANKL BA05-1 and BA05-2 antibody specificity blocks the ability in conjunction with RANKL and RANK suitable with AMG162.

Embodiment 5: inhibiting effect of the anti-RANKL antibody BA05-1 and BA05-2 to the RANKL osteoclast differentiation induced

Detect anti-RANKL antibody BA05-1 and BA05-2 inhibit RANKL induction the differentiation of RAW osteoclast ability (see Fig. 3).RAW264.7 cell and a certain amount of people RANKL (40ng/ml) and different amounts of anti-RANKL antibody BA05-1, BA05- 2, AMG162 is incubated for 4 days altogether in cell culture medium.At the end of 4 days, anti-tartaric acid acid is carried out to cell using infiltration and acidification Then acid phosphatase (TRAP) vital staining handles 5min with p-nitrophenyl phosphate (pNPP).It is molten that 50 μ l0.5MNaOH are added Liquid terminates reaction.P-nitrophenol is converted by p-nitrophenyl phosphate using TRAP activity, passes through optical density at measurement 405nm It is quantified.The results show that the osteoclast differentiation that BA05-1 and BA05-2 induces RANKL is inhibited, and with AMG162 has similar activity.

Embodiment 6: inhibition of the antibody BA05-1 and BA05-2 to RANKL immune function

Using Ficoll density gradient centrifugation from heparin processed whole blood separating peripheral blood mononuclear cells (PBMC). It is incubated for 4h altogether with PBMC with anti-RANKL antibody to be pre-processed, pretreated PBMC and fixed RANK-Fc fusion protein (be purchased from R/D Systems, article No. 683-RK-100) at 37 DEG C, 5%CO₂Under the conditions of cultivate 48h so that RANKL formed poly Body .VEGF-Fc fusion protein (being purchased from R/D Systems, article No. 357-KD-050) is used as Isotype control.In culture supernatant TNFa and IL-6 level using OptEIA (BD Biosciences, San Diego, CA) ELISA method to specifications into Row measurement.RANKL positive T cells are detected by double dyeing T cell mark molecules (PE- is coupled AntiCD3 McAb) using flow cytometer, And goat anti-human igg's secondary antibody (Sigma) by being coupled with FITC is incubated for detection RANK-Fc fusion protein.

Before inducing RANKL signal by fixed RANK-Fc, PBMC is pre-processed with anti-RANKL antibody, reduces TNF α With the release of IL-6.But compared with AMG162, the inhibiting effect of BA05-1 and BA05-2 antibody on cell factor release is smaller (Fig. 4).This may be as caused by the consumption less of RANKL positive T cell (Fig. 5).

Embodiment 7: the internal validity of anti-RANKL antibody BA05-1 and BA05-2

PBS (n=2), the BA05- of 10mg/kg of six difference notch grafts in Healthy female machin (4.5 years old) by single dose The BA05-2 (n=2) of 1 (n=2) or 10mg/kg.Type I collagen egg is crosslinked by monitoring the end serum bone biomarker C- The blood plasma level of white (CTX-1) assesses validity.Foundation level (first 1 week of experiment), the 1st week and the 2nd week blood serum sample are collected, And it freezes in -70 DEG C.Change of serum C TX-1 is measured using commercially available ELISA kit (Mybiosource.com, MBS737402) Level, the kit are specific to the amino acid sequence of Type I collagen PROTEIN C end end peptide, generation when detecting osteoclastic property bone resorption Type I collagen protein fragments.Color density (O.D.) value of according to standard sample concentration formulates standard curve.Each sample CTX-1 concentration obtains (Fig. 6) from this standard curve.

Measurement blood serum substituting marker CTX-1 level shows that BA05-1 and BA05-2 is acute and bone is greatly reduced The level of CTX-1 in absorption shows that bone remoulding is reduced.Similarly, the BA05-1 and BA05-2 of single dose significantly reduce TRAP Activity shows to significantly suppress osteoclastic activity (Fig. 7).TRAP activity reduction monkey Tartrate resistant acid phosphatase 5B (TRACP- 5B) ELISA kit (Mybiosource.com, MBS025470) measures.

Embodiment 8: the serum stability of anti-RANKL antibody BA05-1 and BA05-2

It is incubated for altogether two weeks under the conditions of anti-RANKL antibody BA05-1, BA05-2 and AMG162 and 37 DEG C of 50% human serum.Size Exclude the purity and homogeneity of chromatography (Size Exclusion Chromatography, SEC) HPLC monitoring antibody purification.Sample Product pass through analyticalSuperdex20010/30Tricorn column (GE in PBS (pH7.4) with the flow velocity of 1ml/min Healthcare).UV detection is carried out at 214 and 280nm.IgG size is determined using gel filtration standards (Bio-Rad).Fig. 8 It has been shown that, under the conditions of being incubated for two weeks for 37 DEG C, BA05-1 and BA05-2 ratio AMG162 is more stable in human serum.

Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (26)

1. the complete anti-RANKL antibody of source of people or its segment, it includes the heavy chain as shown in SEQ ID NO:1 and such as SEQ ID NO: Light chain shown in 5.

2. the complete anti-RANKL antibody of source of people or its segment, it includes the heavy chain as shown in SEQ ID NO:3 and such as SEQ ID NO: Light chain shown in 7.

3. isolated nucleic acid molecules encode the anti-RANKL antibody of full source of people or its segment of claims 1 or 2.

4. the nucleic acid molecules of claim 3, it includes the heavy chain as shown in SEQ ID NO:2 and as shown in SEQ ID NO:6 Light chain.

5. the nucleic acid molecules of claim 3, it includes the heavy chain as shown in SEQ ID NO:4 and as shown in SEQ ID NO:8 Light chain.

6. recombinant vector, the nucleic acid molecules containing any one of claim 3-5.

7. recombinant cell, the recombinant vector of nucleic acid molecules or claim 6 containing any one of claim 3-5.

8. composition, any one of the anti-RANKL antibody of the full source of people containing claims 1 or 2 or its segment, claim 3-5 Nucleic acid molecules, the recombinant vector of claim 6 or the recombinant cell of claim 7 and pharmaceutically acceptable carrier or Excipient.

9. the composition of claim 8 also contains the therapeutic agent of at least one treatment inflammation or immunological diseases.

10. the composition of claim 9, wherein the therapeutic agent is selected from COX1 and COX2 inhibitor, prednisolone, first ammonia butterfly Purine, chloroquine, cyclosporin.

11. the nucleic acid molecules of any one of the anti-RANKL antibody of the full source of people of claims 1 or 2 or its segment, claim 3-5, power Benefit requires 6 recombinant vector or the recombinant cell of claim 7 preparing for preventing or treating and bone lesion related disease Purposes in drug.

12. the purposes of claim 11, wherein described broken selected from osteoporosis, the dissolution of arthritis bone with bone lesion related disease Bad, malignant tumour or malignant metastatic tumor of bone, paget's disease of bone (osteitis deformans) are broken with the inflammation of osteoclasia, with bone Bad autoimmune disease, hypercalcinemia, osteonecrosis and osteopenia.

13. the purposes of claim 12, wherein the autoimmune disease of the adjoint osteoclasia is rheumatoid arthritis.

14. the purposes of claim 12, wherein the inflammation of the adjoint osteoclasia is osteomyelitis.

15. the purposes of claim 12, wherein the malignant tumour is selected from breast cancer, prostate cancer, thyroid cancer, kidney, lung Cancer, cutaneum carcinoma, cancer of the esophagus, bladder cancer, cervix cancer, oophoroma, liver cancer, gastrointestinal cancer, Huppert's disease and lymthoma.

16. the purposes of claim 15, wherein the gastrointestinal cancer is the carcinoma of the rectum.

17. the purposes of claim 15, wherein the lymthoma is Hodgkin's disease.

18. the purposes of claim 12, wherein the osteoporosis is selected from primary osteoporosis, endocrine osteoporosis, bone The loose heredity of matter and apriori form and the osteoporosis fixed due to acra.

19. the purposes of claim 18, wherein the endocrine osteoporosis is selected from hyperthyroidism, parathyroid function Hyperfunction, Cushing syndrome and acromegalia.

20. the purposes of claim 18, wherein the heredity of the osteoporosis and apriori form are selected from osteogenesis imperfecta, homocystine Urine disease, menkes' syndrome and Lai-wear syndrome.

21. the nucleic acid molecules of any one of the anti-RANKL antibody of the full source of people of claims 1 or 2 or its segment, claim 3-5, power Benefit requires 6 recombinant vector or the recombinant cell of claim 7 preparing the purposes in the drug as RANKL inhibitor.

22. detection reagent or kit contain the antibody of claims 1 or 2 or its segment.

23. detection reagent described in claim 22 or kit, wherein the antibody or its antigen-binding portion thereof also may include Detectable label.

24. detection reagent described in claim 22 or kit, wherein the detection reagent or kit also include second anti- Body, antibody or its antigen-binding portion thereof described in specific recognition.

25. detection reagent described in claim 24 or kit, wherein the secondary antibody also may include detectable mark Note.

26. the purposes of the antibody of claims 1 or 2 or its segment in preparation detection reagent or kit, wherein the detection Reagent or kit for detecting Nuclear factor kappa-B receptor activation factor ligand, or for diagnose Nuclear factor kappa-B receptor activation because Sub- ligand-associated disease.