CN105085679A - Fully human anti-RANKL antibody - Google Patents
Fully human anti-RANKL antibody Download PDFInfo
- Publication number
- CN105085679A CN105085679A CN201410168618.9A CN201410168618A CN105085679A CN 105085679 A CN105085679 A CN 105085679A CN 201410168618 A CN201410168618 A CN 201410168618A CN 105085679 A CN105085679 A CN 105085679A
- Authority
- CN
- China
- Prior art keywords
- antibody
- seqidno
- rankl
- fragment
- light chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The present invention relates to fully humanized antibodies, specifically to a fully human anti-RANKL antibody, a nucleic acid molecule encoding the antibody, and a composition containing the antibody. The present invention further relates to uses of the antibody in preparation of drugs for prevention and treatment of bone loss related diseases, especially osteoporosis, arthritis osteolysis damage or tumor bone metastasis and other diseases. According to the present invention, the antibody is specifically bound with human RANKL and blocks the activity of the human RANKL while the potential immunesuppression of the human RANKL on human body is reduced, the antibody has good stability, and the defect of the non-uniformity (heterogeneity) of the IgG2 antibody production on the batch quality identification is avoided.
Description
Technical field
The present invention relates to full humanized antibody, particularly, the present invention relates to the anti-RANKL antibody in total man source, encode this antibody nucleic acid molecule and comprise the composition of this antibody, the invention still further relates to described antibody for the preparation of the purposes of preventing and treating in the medicine of the diseases such as osteoporosis, sacroiliitis bone dissolved destruction or bone metastaes.
Background technology
Bone forming and bone remoulding have been coordinated by bone resorption osteoclast and bone forming scleroblast, in the growth and growth of people, be responsible for bone metabolism and skeletal reconstruction.Constantly absorb at the specific position bone of bone and formed, estimating that the bone total amount of in annual human body about 10% can be rebuild.Come from the osteoclast of Monocytes/Macrophages precursor, heavily can absorb bone after activation, final osteoclast generation apoptosis.Then, the newly-generated scleroblast deriving from preosteoblast/stroma cell forms sclerotin at absorption site.The development of osteoclast is controlled by scleroblast, closely cooperates to make bone resorption and forming process.Namely followed by one in each bone resorption circulation and take turns bone forming.The unbalance meeting of osteoclast and scleroblast activity causes increasing with bone loss (osteoporosis) or bone the skeletal abnormality that (osteopetrosis) is feature.(Khosla,Endocrinology,2001,142,5050-5055；Nakashimaetal.,Curr.Opin.Rheumatol.,2003,15,280-287).
Communication between scleroblast and osteoclast is realized by cytokine and iuntercellular effect.The cytokine played a crucial role in bone remoulding is receptor activator of nuclear factor κB ligand (receptoractivatorofNF-kappaBligand, RANKL).RANKL is the first TNF aglucon family member be found, be also referred to as tumour necrosis factor associated activation inducible factor (tumornecrosis-factor-relatedactivation-inducedcytokine, TRANCE), osteoprotegerin ligand (osteoprotegerinligand, OPGL), osteoclast differentiation factor (osteoclastdifferentiationfactor, ODF), tumour necrosis factor (aglucon) superfamily member 11 (tumornecrosisfactor (ligand) superfamilymember11, TNFSF11), RANKL was identified the cytokine of external evoked differentiation of osteoclast afterwards.(Andersonetal.,Nature,1997,390,175-179；Laceyetal.,Cell,1998,93,165-176；Wongetal.,J.Exp.Med.,1997,186,2075-2080；Yasudaetal.,Proc.Natl.Acad.Sci.USA,1998,95,3597-3602)。The gene of mankind RANKL is positioned at chromosome 13q14.RANKL expression level in bone, marrow and Lymphoid tissue the highest (Andersonetal., Nature, 1997,390,175-179; Laceyetal., Cell, 1998,93,165-176; Wongetal., J.Exp.Med., 1997,186,2075-2080; Yasudaetal., Proc.Natl.Acad.Sci.USA, 1998,95,3597-3602), also can detect in brain, the heart, kidney, skeletal muscle and skin (Kartsogiannisetal., Bone, 1999,25,525-534).
RANKL is assembled by three kinds of RANKL subunits and forms functional trimeric molecules.RANKL is anchored on cytolemma at first, at tumor necrosis factor alpha saccharase (metalloprotease-disintegrinTNF-alphaconvertase, TACE) under hydrolytic action, its extracellular partial protein (Lumetal. is discharged from cell surface, J.Biol.Chem., 1999,274,13613-13618).RANKL, to promotion differentiation of osteoclast, strengthens its vigor, and suppresses osteoclast apoptosis to be absolutely necessary (Fulleretal., J.Exp.Med., 1998,188,997-1001; Laceyetal., Cell, 1998,93,165-176; Lumetal., J.Biol.Chem., 1999,274,13613-13618; Yasudaetal., Proc.Natl.Acad.Sci.USA, 1998,95,3597-3602).RANK acceptor is at preosteoblast and marrow stromal cell surface expression.The expression of RANKL is just regulating or negative regulator by various hormone, cytokine, somatomedin and glucocorticosteroid, comprise Vitamin D3 500,000 I.U/GM, Rat parathyroid hormone 1-34, interleukin 1-β and TNF-α, these all can increase the expression (Kongetal. of RANKL, Immunol.Today, 2000,21,495-502).At bone resorption and osteoplastic beginning of cycle, RANKL is combined (Andersonetal., Nature, 1997,390,175-179 with the functional receptor RANK on osteoclast precursor cells surface; Laceyetal., Cell, 1998,93,165-176).The interaction of RANKL and RANK promotes the maturation of osteoclast, mature osteoclast is divided into multinuclear, has bone resorption function with expression specificity marker Tartrate resistant acid phosphatase (tartrate-resistantacidphosphatase, TRAP) be feature (Burgessetal., J.Cell.Biol., 1999,145,527-538; Hsuetal., Proc.Natl.Acad.Sci.U.S.A., 1999,96,3540-3545; Lumetal., J.Biol.Chem., 1999,274,13613-13618; Yasudaetal., Proc.Natl.Acad.Sci.USA, 1998,95,3597-3602).RANKL also can combine with water-soluble acceptor osteoprotegerin (OPG), and OPG expresses primarily of marrow stromal cell and the osteoclast cell maturation suppressing RANKL to mediate and activation (Laceyetal., Cell, 1998,93,165-176; Yasudaetal., Proc.Natl.Acad.Sci.USA, 1998,95,3597-3602).PTH is a kind of main regulatory factors of bone remoulding, expressing the expression simultaneously reducing OPG, promoting osteoclast activity (LeeandLorenzo, Endocrinology, 1999,140,3552-3561) by increasing RANKL.During current osteoblast differentiation, the mRNA level in-site of RANKL significantly declines, and OPGmRNA level significantly raises (Gorietal., Endocrinology, 2000,141,4768-4776).Dynamic movement between RANKL and OPG level makes the activity of osteoclast and then osteoblastic activity, completes bone resorption and osteoplastic circulation with this.
By discharging calcium from thesaurus in cell, RANKL induces the instantaneous rising of calcium contents (Komarovaetal., J.Biol.Chem., 2003,278,8286-8293) in osteoclast.After the homozygous mouse blocking RANKL gene is born three weeks, show serious growth retardation.Because scleroblast can not support osteoclast, RANKL deficient mice is owing to lacking the supporting function of scleroblast to osteoclast formation, show as serious osteopetrosis (sclerotin thickens), tooth eruption defect and osteoclast disappearance (Kongetal., Immunol.Today, 2000,21,495-502).
The function of RANKL is not only formation and the reconstruction of bone, RANKL deficient mice also shows T and bone-marrow-derived lymphocyte early differentiation defect and lymphoglandula and lacks, this shows that RANKL is the regulatory factor of lymphoid organ and lymphocyte development, (Kongetal., Immunol.Today, 2000,21,495-502).φt cell receptor stimulates the expression of induction RANKL gene, thus causes the activation (Wongetal., J.Biol.Chem., 1997,272,25190-25194) of c-JunN-terminal Kinase in T cell.RANKL also participates in function of immune system and regulates, by inhibited apoptosis as important survival factors (Lumetal., J.Biol.Chem., 1999,274, the 13613-13618 of marrow source dendritic cell; Wongetal., J.Exp.Med., 1997,186,2075-2080).In addition, during mouse pregnancy, alveolar breast structure grows the participation (Fataetal., Cell, 2000,103,41-50) also needing RANKL.
RANKL activates osteoclast inadequately can make bone resorption process unbalance, causes bone resorption to be greater than bone forming.In many osteoporosis, comprise the bone loss (Khosla all observing local or general in postmenopausal osteoporosis, the osteoporosis relevant to the age, periodontopathy, familial swelling property bone dissolving disease, osteitis deformans, Endocrinology, 2001,142,5050-5055).RANKLmRNA up-regulated has been reported in these diseases above-mentioned.
The feature of osteitis deformans has a large amount of abnormal osteoclast causing bone resorption to increase.RANKLmRNA in the bone marrow stromal cell system be separated in osteitis deformans patient and sufferer marrow expresses and raises.And the osteoclast precursor of osteitis deformans patient required RANKL concentration ratio normal marrow cell low (Menaaetal., J.Clin.Invest., 2000,105,1833-1838) in formation osteoclast process.
There is osteopenic other diseases, if rheumatoid arthritis, chronic viral infection, adult and leukemia of children are for feature (Kongetal., Immunol.Today, 2000,21,495-502) with t cell activation and osteoclasia.Rheumatoid arthritis is a kind of chronic inflammatory disease, and feature is the bone resorption of gradual Osteoclasts mediate.Containing the B cell that osteoclast precursor, the T cell expressing RANKL and OPG produce in rheumatoid arthritis synovial liquid.The scavenger cell of the cultivation in RA synovial fluid can rely on RANKL and be divided into osteoclast (Itonagaetal., J.Pathol., 2000,192,97-104).In the tentative rat model of arthritis that T cell relies on, show many Clinical symptoms of mankind RA, treated by OPG, suppress RANKL function thus osteoclasia (Kongetal., Nature, 1999,402,304-309) can be prevented.
The cancer that multiple myeloma is is prominent feature with osteoporosis and osteoclasia.Multiple myeloma cells promotes that RANKL expresses, and also suppresses the OPG of marrow stromal cell to express simultaneously, causes RANKL and OPG level exception that is unbalance and osteoclast to generate and activation (Pearseetal., Proc.Natl.Acad.Sci.USA, 2001,98,11581-11586).In addition, some cancer cells expression-secretion RANKL, causes malignant tumor patient hypercalcemia (Nagaietal., Biochem.Biophys.Res.Commun., 2000,269,532-536).After glucocorticoid treatment, in scleroblast, RANKL increases, and OPG expresses and reduces simultaneously, this can activate osteoclast formation, can produce severe osteoporosis (Hofbaueretal., Endocrinology with this whole body glucocorticoid treatment, 1999,140,4382-4389)..
Immunologic function is relevant to physiology of bone, and immunocyte expresses the molecule explanation that RANKL provides the bone density reduction that immune system disorder brings.Therefore by suppressing RANKL function, and then suppressing osteoclast activity, the osteoporosis (Kongetal., Immunol.Today, 2000,21,495-502) that immune inflammation causes can be improved.
Because RANKL participates in various diseases, the rise that RANKL expresses is relevant to polytype osteoporosis, therefore needs the inhibitor that effectively can suppress RANKL activity, with the disease that to treat with bone damage be feature.
Denosumab is the medicine that new approval is used for the treatment of Bone tumour disease.It is a kind of total man source anti-RANKLIgG2 type monoclonal antibody, is the inhibitor of a kind of effective suppression osteoclast formation and bone resorption.Be used for the treatment of Bone tumour and fracture that postmenopausal osteoporosis and solid tumor and multiple myeloma cause.
In the II phase clinical study that Lipton etc. shift about mammary cancer related bone, be divided into six groups at random, wherein five groups use denosumab, Primary Endpoint is that Bone turnover marker N-holds peptide to the ratio (uNTX/Cr) of uric creatinine, and assesses its security and skeletal related events (SREs)., less there is SRES (9%vs is to 16%) compared to Diphosphonate patient in the reduction of patient's most obviously display uNTX/Cr of injection denosumab.The placebo-controlled randomized trial directly comparing denosumab and Zoledronate one larger shows, denosumab is delaying or stoping the SRES performance in breast cancer patients Bone tumour excellent.For the inhibitor of RANKL, denosumab provides a kind of novel method of potential osteoclasia for the treatment of in the caused fracture of osteoporosis, the bone complications of malignant tumour and RA.But except being expressed by scleroblast, RANKL also produces by the synovial cell in activating T cell and RA, and the acceptor of RANKL, i.e. RANK, also by Monocytes/Macrophages and 1 expressed by dendritic cells.In mouse model, the disappearance of RANKL, RANK and OPG highlights the importance of this path in rodent immune system development and maturation, comprises the function of T cell and/or B cell.Pharmacology/toxicological study proposes more immunosuppressant problems, although determined the beneficial effect to bone density and incidence of fracture in clinical trial, must assess the risk of denosumab before the treatment.In clinical study, total incidence of the serious adverse reaction that denosumab group infects is higher than control group.The incidence of the infection that denosumab group is relevant to bacterium and no specific pathogen is higher than control group.Consider the risk of infection, carrying out immunosuppressant therapy or having in the patient of high infection risk, should avoid using denosumab.Except immunosuppressant excessive risk, denosumab is difficult to production control due to the complicacy of its IgG2 hypotype and free cysteine structure, and during this makes it produce in production technique exploitation development and GMP are produced, difficulty strengthens, and is difficult to carry out good quality control.
Summary of the invention
In order to reduce immunosuppression side effect and the infection risk of existing anti-RANKL antibody, and in order to more be conducive to scale operation and quality control, the invention provides the anti-RANKL antibody in new total man source.
First aspect present invention relates to the anti-RANKL antibody in total man source or its fragment, and it comprises the heavy chain as shown in SEQIDNO:1 or SEQIDNO:3 and the light chain as shown in SEQIDNO:5 or SEQIDNO:7; Or one or several that is selected from following (1)-(4):
(1) variable region sequences of wherein said heavy chain is: in the variable region sequences and SEQIDNO:9 sequence of described heavy chain through replacement, lack or add one or several amino acid and formed antibody or its fragment there is same or similar activity;
(2) variable region sequences of wherein said light chain is: in the variable region sequences and SEQIDNO:11 sequence of described light chain through replacement, lack or add one or several amino acid and formed antibody or its fragment there is same or similar activity;
(3) constant-region sequences of wherein said heavy chain is: in the constant-region sequences and SEQIDNO:13 sequence of described heavy chain through replacement, lack or add one or several amino acid and formed antibody or its fragment there is same or similar activity;
(4) constant-region sequences of wherein said light chain is: in the constant-region sequences and SEQIDNO:12 sequence of described light chain through replacement, lack or add one or several amino acid and formed antibody or its fragment there is same or similar activity.
In embodiments of the invention, the anti-RANKL antibody in described total man source or its fragment comprise the heavy chain as shown in SEQIDNO:1 and the light chain as shown in SEQIDNO:5.
In embodiments of the invention, the anti-RANKL antibody in described total man source or its fragment comprise the heavy chain as shown in SEQIDNO:3 and the light chain as shown in SEQIDNO:7.
Second aspect present invention relates to the nucleic acid molecule of separation, the anti-RANKL antibody in total man source of its any one of coding first aspect present invention or its fragment.
The nucleic acid molecule of any one according to a second aspect of the present invention, it comprises the heavy chain as shown in SEQIDNO:2 or SEQIDNO:4 and the light chain as shown in SEQIDNO:6 or SEQIDNO:8.
The nucleic acid molecule of any one according to a second aspect of the present invention, it comprises the heavy chain as shown in SEQIDNO:2 and the light chain as shown in SEQIDNO:6.
The nucleic acid molecule of any one according to a second aspect of the present invention, it comprises the heavy chain as shown in SEQIDNO:4 and the light chain as shown in SEQIDNO:8.
Third aspect present invention relates to recombinant vectors, and it contains the nucleic acid molecule of any one of second aspect present invention.
Fourth aspect present invention relates to reconstitution cell, and it contains the nucleic acid molecule of any one of second aspect present invention or the recombinant vectors of any one of the third aspect.
Fifth aspect present invention relates to composition, it contains the anti-RANKL antibody in total man source of any one of first aspect present invention or the reconstitution cell of its fragment, the nucleic acid molecule of any one of second aspect, the recombinant vectors of any one of the third aspect or any one of fourth aspect, and pharmaceutically acceptable carrier or vehicle.
The composition of any one according to a fifth aspect of the present invention, it also treats the therapeutical agent of inflammation or Immunological diseases containing at least one.
The composition of any one according to a fifth aspect of the present invention, wherein said therapeutical agent is selected from COX1 and COX2 inhibitor, prednisolone, methotrexate, chloroquine, S-Neoral.
Sixth aspect present invention also relates to the anti-RANKL antibody in total man source of any one of first aspect present invention or the purposes of the reconstitution cell of its fragment, the nucleic acid molecule of any one of second aspect, the recombinant vectors of any one of the third aspect or any one of fourth aspect in the medicine for the preparation of prevention or treatment and bone lesion relative disease.
The purposes of any one according to a sixth aspect of the present invention, wherein saidly includes but not limited to osteoporosis, sacroiliitis bone dissolved destruction, malignant tumour or malignant metastatic tumor of bone, paget's disease of bone (osteitis deformans), the inflammation (such as osteomyelitis) with osteoclasia, the autoimmune disorder with osteoclasia or rheumatoid arthritis, hypercalcemia, osteonecrosis, osteopenia with bone lesion relative disease.
The purposes of any one according to a sixth aspect of the present invention, wherein said malignant tumour includes but not limited to mammary cancer, prostate cancer, thyroid carcinoma, kidney, lung cancer, skin carcinoma, esophagus cancer, the rectum cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, multiple myeloma and lymphoma (Hokdkin disease).
The purposes of any one according to a sixth aspect of the present invention, wherein said osteoporosis includes but not limited to primary osteoporosis, internal secretion osteoporosis (includes but not limited to hyperthyroidism, hyperparathyroidism, hypercortisolism, acromegaly), osteoporotic heredity and apriori form (include but not limited to osteogenesis imperfecta, homocystinuria, menkes' syndrome, Lai-Dai syndrome), and due to the fixing osteoporosis of acra.
Seventh aspect present invention relates to the therapeutic agent that the anti-RANKL antibody in total man source of any one of first aspect present invention or the reconstitution cell of its fragment, the nucleic acid molecule of any one of second aspect, the recombinant vectors of any one of the third aspect or any one of fourth aspect and at least one treats inflammation or Immunological diseases and is used in for the preparation of preventing or treating the purposes in the medicine of bone lesion.
The purposes of any one according to a seventh aspect of the present invention, wherein said therapeutical agent is selected from COX1 and COX2 inhibitor, prednisolone, methotrexate, chloroquine, S-Neoral.
The purposes of any one according to a seventh aspect of the present invention, wherein saidly includes but not limited to osteoporosis, sacroiliitis bone dissolved destruction, malignant tumour or malignant metastatic tumor of bone, paget's disease of bone (osteitis deformans), the inflammation (such as osteomyelitis) with osteoclasia, the autoimmune disorder with osteoclasia or rheumatoid arthritis, hypercalcemia, osteonecrosis, osteopenia with bone lesion relative disease.
The purposes of any one according to a seventh aspect of the present invention, wherein said malignant tumour includes but not limited to mammary cancer, prostate cancer, thyroid carcinoma, kidney, lung cancer, skin carcinoma, esophagus cancer, the rectum cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, multiple myeloma and lymphoma (Hokdkin disease).
The purposes of any one according to a seventh aspect of the present invention, wherein said osteoporosis includes but not limited to primary osteoporosis, internal secretion osteoporosis (includes but not limited to hyperthyroidism, hyperparathyroidism, hypercortisolism, acromegaly), osteoporotic heredity and apriori form (include but not limited to osteogenesis imperfecta, homocystinuria, menkes' syndrome, Lai-Dai syndrome), and due to the fixing osteoporosis of acra.
The invention still further relates to the anti-RANKL antibody in total man source of any one of first aspect present invention or the reconstitution cell of its fragment, the nucleic acid molecule of any one of second aspect, the recombinant vectors of any one of the third aspect or any one of fourth aspect in preparation as the purposes in the medicine of RANKL inhibitor.
The invention still further relates to detection reagent or test kit, it contains antibody or its fragment of any one of first aspect present invention;
Preferably, described antibody or its antigen-binding portion thereof also can comprise detectable mark; Or
Preferably, described detection reagent or test kit also can comprise second antibody, antibody described in its specific recognition or its antigen-binding portion thereof; Or
Preferably, described second antibody also can comprise detectable mark.
The invention still further relates to antibody or the purposes of its fragment in preparation detection reagent or test kit of any one of first aspect present invention, wherein said detection reagent or test kit for detecting Nuclear factor kappa-B receptor activation factor ligand (RANKL), or for diagnosing Nuclear factor kappa-B receptor activation factor ligand relative disease.
In the present invention, term " antibody " refers to the immunoglobulin molecules usually formed identical polypeptide chain (often pair has " gently " (L) chain and " weight " (H) chain) by two.Light chain of antibody can be categorized as κ and lambda light chain.Heavy chain can be categorized as μ, δ, γ, α or ε, and respectively the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE.In light chain and heavy chain, variable region and constant region are connected by about 12 or more amino acid whose " J " district, and heavy chain also comprises about 3 or more individual amino acid whose " D " districts.Each heavy chain is by variable region of heavy chain (V
h) and CH (C
h) composition.CH is by 3 structural domain (C
h1, C
h2 and C
h3) form.Each light chain is by variable region of light chain (V
l) and constant region of light chain (C
l) composition.Constant region of light chain is by a domain C
lcomposition.The constant region of antibody can mediated immunity sphaeroprotein and host tissue or the factor, comprises the combination of first component (C1q) of immune various cell (such as, effector cell) and classical complement system.V
hand V
ldistrict also can be subdivided into has denatured region (being called complementary determining region (CDR)), is scattered with the comparatively conservative region being called framework region (FR) therebetween.Each V
hand V
lby in the following order: 3 CDR and 4 FR that FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arrange from N-terminal to C-terminal form.Variable region (the V that each heavy chain/light chain is right
hand V
l) form antibody binding site respectively.Amino acid follows KabatSequencesofProteinsofImmunologicalInterest (NationalInstitutesofHealth to the distribution of each region or structural domain, Bethesda, (1987and1991)), or Chothia & Lesk (1987) J.Mol.Biol.196:901-917 Md.; The definition of the people such as Chothia (1989) Nature342:878-883.Term " antibody " does not limit by the method for any specific generation antibody.Such as, it comprises, especially, and recombinant antibodies, monoclonal antibody and polyclonal antibody.Antibody can be the antibody of different isotype, such as, and IgG (such as, IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.
In the present invention, " fragment " of term antibody refers to one or more parts of full length antibody, and the ability of the same antigen (such as, RANKL) that described part keeps binding antibody to combine, competes the specific binding to antigen with complete antibody.Usually see, (Paul, W., ed., the 2nd edition, RavenPress, N.Y. (1989), it integrates with in full herein, for all objects with it by reference for FundamentalImmunology, Ch.7.By recombinant DNA technology or enzymatic or the chemical disruption generation antigen-binding portion thereof of passing through complete antibody.In some cases, antigen-binding portion thereof comprises Fab, Fab', F (ab')
2, Fd, Fv, dAb and complementary determining region (CDR) fragment, single-chain antibody (such as, scFv), chimeric antibody, double antibody (diabody) and such polypeptide, it comprises the antibody that is enough to give polypeptid specificity antigen binding capacity at least partially.
In the present invention, term " Fd fragment " means by V
hand C
hthe antibody fragment of 1 structural domain composition; Term " Fv fragment " means by the V of the single armed of antibody
land V
hthe antibody fragment of structural domain composition; Term " dAb fragment " means by V
hantibody fragment people such as (, Nature341:544-546 (1989)) Ward of structural domain composition; Term " Fab fragment " means by V
l, V
h, C
land C
hthe antibody fragment of 1 structural domain composition; Term " F (ab')
2fragment " mean the antibody fragment of two the Fab fragments comprised by the disulfide bridge connects on hinge area.
In some cases, the fragment of antibody is single-chain antibody (such as, scFv), wherein V
land V
hstructural domain by can be produced as single polypeptide chain linker pairing formed monovalent molecule (see, such as, the people such as Bird, the people such as Science242:423-426 (1988) and Huston, Proc.Natl.Acad.Sci.USA85:5879-5883 (1988)).This type of scFv molecule can have general structure: NH
2-V
l-joint-V
h-COOH or NH
2-V
h-joint-V
l-COOH.Suitable prior art joint is made up of the GGGGS aminoacid sequence repeated or its variant.Such as, can use there is aminoacid sequence (GGGGS)
4joint, but also can use its variant (people (1993) such as Holliger, Proc.Natl.Acad.Sci.USA90:6444-6448).Other joints used in the present invention are by the people such as Alfthan (1995), ProteinEng.8:725-731, the people such as Choi (2001), Eur.J.Immunol.31:94-106, the people such as Hu (1996), the people such as CancerRes.56:3055-3061, Kipriyanov (1999), the people such as J.Mol.Biol.293:41-56 and Roovers (2001), CancerImmunol. describes.
In some cases, antibody is double antibody, that is, bivalent antibody, wherein V
hand V
lstructural domain is expressed on single polypeptide chain, but use too short linker so that do not allow to match between two structural domains of same chain, thus force the complementary domain of structural domain and another chain to match and produce two antigen-binding sites (see, such as, HolligerP. people is waited, Proc.Natl.Acad.Sci.USA90:6444-6448 (1993), and the people such as PoljakR.J., Structure2:1121-1123 (1994)).
Routine techniques well known by persons skilled in the art can be used (such as, recombinant DNA technology or enzymatic or chemical disruption method) the above-mentioned fragment of antibody is obtained from given antibody (such as monoclonal antibody BA05-1, BA05-2), and with the fragment of the mode identical with the mode for complete antibody with regard to specificity screening antibody.
In the present invention, described carrier can be cloning vector or expression vector.Described expression vector is such as prokaryotic expression carrier, carrier for expression of eukaryon, phage vector or virus vector, is the carrier that this area is conventional.Wherein said prokaryotic expression carrier is such as PET28a, pGEMEx1, pGEM7ZF (+) or pBAD24 etc., described carrier for expression of eukaryon is such as pBudCE4.1, pcDNA3.1, pCMV-Tag2B or pGL3basic etc., in embodiments of the invention, described carrier for expression of eukaryon is pcDNA3.1, and described virus vector is such as retrovirus, slow virus, adenovirus and adeno-associated virus.
In the present invention, described cell can be prokaryotic cell prokaryocyte or eukaryotic cell.Described eukaryotic cell is such as mammalian cell.Described cell can pass through to the above-mentioned nucleic acid molecule of introducing/transfection in prokaryotic cell prokaryocyte or eukaryotic cell or carrier and obtain.
In the present invention, described prokaryotic cell prokaryocyte can be such as intestinal bacteria, described eukaryotic cell can be such as yeast cell, insect cell (as Sf21), mammalian cell, and described Mammals can be such as NSO cell, Chinese hamster ovary celI, 293 cells.In one embodiment of the invention, described cell is eukaryotic cell.In one embodiment of the invention, described eukaryotic cell is 293F cell.
In the present invention, the transfection method of any kind known in the art acquisition transfection can be utilized to have the host cell of specific nucleic acid or carrier.Such as, by electroporation or microinjection, nucleic acid is introduced in cell.Or, lipofectin reagent can be used as FuGENE6, X-tremeGENE and LipofectAmine.Or, by nucleic acid being introduced in cell based on the suitable virus vector of retrovirus, slow virus, adenovirus and adeno-associated virus.
In the present invention, described osteoporosis includes but not limited to primary osteoporosis, internal secretion osteoporosis (includes but not limited to hyperthyroidism, hyperparathyroidism, hypercortisolism, acromegaly), osteoporotic heredity and apriori form (include but not limited to osteogenesis imperfecta, homocystinuria, menkes' syndrome, Lai-Dai syndrome), and due to the fixing osteoporosis of acra.
In the present invention, described malignant tumour includes but not limited to mammary cancer, prostate cancer, thyroid carcinoma, kidney, lung cancer, esophagus cancer, the rectum cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, multiple myeloma and lymphoma (Hokdkin disease).
The invention provides the new anti-RANKL antibody that structure is different from denosumab.
In some embodiments of the present invention, the present invention constructs the mutant library that derives from denosumab and is expressed on yeast surface display system, use RANKL as target, carry out two-wheeled screening in vitro, select the anti-RANKL antibody mutants with lower Kd and longer transformation period of dissociating, the variant of selection is converted into whole antibody with suitable IgG4 skeleton after stability test and signature analysis.
In some embodiments of the present invention, anti-RANKL antibody of the present invention and fragment thereof, as differentiation of osteoclast and ripe inhibitor, have very strong avidity with RANKL, can block RANKL and RANKL receptors bind.
In some embodiments of the present invention, anti-RANKL antibody of the present invention and fragment thereof to T cell and B cell function effect less, therefore there are less immunosuppressive action and better security window, it is expected to, in malignant tumour or malignant metastatic tumor of bone, have better potential applicability in clinical practice.
In some embodiments of the present invention, antibody of the present invention and fragment thereof have better homogeneity and stability, the drawback that the unhomogeneity (heterogeneity) avoiding IgG2 antibody producing is brought to batch quality evalution.
Accompanying drawing explanation
The combination of Fig. 1 antibody BA05-1 and BA05-2 and RANKL
The ability that Fig. 2 antibody BA05-1 and BA05-2 suppresses RANKL to be combined with its homoreceptor RANK
The anti-RANKL antibody of Fig. 3 is to the restraining effect of the differentiation of osteoclast that RANKL induces
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)
Fig. 5 antibody BA05-1 and BA05-2 is to the effect of RANKL positive T cell in PBMC culture
Fig. 6 single dose BA05-1 and BA05-2 antibody are to restraining effect in the body of cynomolgus monkey CTX-1
Fig. 7 single dose BA05-1 and BA05-2 antibody are to restraining effect in the body of cynomolgus monkey TRACP-5B
The serum stability (wherein ordinate zou represents complete, without the antibody of degrading) of Fig. 8 antibody BA05-1 and BA05-2
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
The present invention constructs the peptide storehouse of the CDR district random mutation sequence containing Denosumab, and is illustrated in yeast cell surface, and storage capacity is about 1x10
7, use RANKL as target, utilize flow cytometry to carry out two-wheeled screening in vitro, select the anti-RANKL antibody mutants with lower Kd and longer transformation period of dissociating.Namely this storehouse is adopted to screen in following examples.
Target RANKL is wherein the ECD structural domain of RANKL, purchased from R/DSystems (article No. 390-TN-010).
The design of embodiment 1:BA05-1 and BA05-2 antibody sequence and expression
Be below the sequence of the variable region of heavy chain of AMG162:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPGTTVIMSWFDPWGQGTLVTVSS(SEQIDNO：9)
Be below the light chain constant region sequence of AMG162:
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：10)
Its original heavy chain IgG2 constant-region sequences is changed into IgG4 constant-region sequences, forms BA05-1 sequence of heavy chain:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPGTTVIMSWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNo：1)
Constant-region sequences is wherein:
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：13)
The CDR region that frame is combined with antigen of not optimizing of its original variable region of heavy chain is optimized to attempt to increase and RANKL affinity through yeastdisplay, then is combined with heavy chain IgG4 constant-region sequences, formation BA05-2 sequence of heavy chain:
QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWIRQAPGKGLEWVSGITGSGSSTYYADSAKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDPGTTVIMSWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNo：3)
Be below the sequence of the variable region of light chain of AMG162:
EIVLTQSPGTLSLSPGERATLSCRASQSVRGRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGSSPRTFGQGTKVEIK(SEQIDNo：11)
Be below constant region of light chain ck sequence:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQIDNo：12)
Above-mentioned variable region of light chain is combined with constant region of light chain CL (k) sequence, namely forms BA05-1 sequence of light chain:
EIVLTQSPGTLSLSPGERATLSCRASQSVRGRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGSSPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQIDNo：5)
The non-Optimization Framework sequence of its original variable region of light chain is optimized to attempt to increase and RANKL affinity through yeastdisplay, it is combined with constant region of light chain CL (k) sequence, namely forms the sequence of light chain of BA05-2:
EIVMTQSPATLSLSPGERATLSCRASQSLRGRYLAWYQQKPGKAPKLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQYASSPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQIDNo：7)
Embodiment 2:BA05-1 and the preparation of BA05-2 antibody expression
The heavy chain full length DNA sequence of BA05-1 is:
GAGGTGCAGCTCCTGGAGAGCGGCGGAGGCCTGGTGCAGCCCGGAGGAAGCCTGCGGCTCTCCTGCGCCGCTAGCGGATTCACATTCTCCAGCTACGCTATGAGCTGGGTCAGGCAGGCTCCTGGCAAGGGACTCGAGTGGGTGAGCGGCATCACCGGATCCGGCGGATCCACATACTATGCCGATTCCGTCAAGGGAAGGTTCACAATCTCCCGGGACAACAGCAAGAACACCCTCTACCTCCAGATGAACAGCCTGCGGGCCGAGGACACAGCCGTCTACTATTGCGCCAAAGACCCCGGAACCACCGTGATCATGAGCTGGTTCGATCCCTGGGGACAGGGAACCCTCGTGACAGTGTCCAGCGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCCAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA(SEQIDNO：2)。
The light chain full length DNA sequence of BA05-1 is:
GAGATCGTCCTGACACAGAGCCCCGGAACCCTCTCCCTCTCCCCCGGCGAAAGGGCTACCCTCTCCTGCAGGGCTTCCCAATCCGTGAGGGGACGGTACCTCGCTTGGTACCAGCAAAAGCCCGGACAAGCTCCTCGGCTGCTCATTTACGGCGCCAGCAGCAGAGCCACAGGCATTCCCGACCGGTTCAGCGGCAGCGGCAGCGGCACAGACTTCACACTGACAATCTCCCGGCTGGAACCCGAAGACTTCGCTGTGTTCTACTGCCAACAGTACGGATCCAGCCCCAGGACCTTCGGCCAAGGCACAAAGGTCGAGATTAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG(SEQIDNO：6)。
The heavy chain full length DNA sequence of BA05-2 is:
CAGGTCCAGCTCGTGGAATCCGGAGGCGGACTGGTCAAGCCTGGCGGATCCCTCAGGCTCTCCTGTGCCGCTTCCGGCTTCACATTCAGCTCTTACGGAATGAGCTGGATTAGGCAAGCCCCTGGCAAAGGCCTCGAGTGGGTCTCCGGAATCACCGGCAGCGGCTCCAGCACCTATTACGCTGACAGCGCCAAAGGCAGATTTACAATCAGCAGGGATAACGCTAAGAATTCCCTCTACCTCCAGATGAATTCCCTCAGGGCTGAGGATACCGCTGTGTATTACTGTGCCAGGGACCCTGGCACAACCGTCATCATGAGCTGGTTTGACCCTTGGGGACAGGGAACCCTCGTGACAGTGAGCTCCGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCCAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA(SEQIDNO：4)。
The light chain full length DNA sequence of BA05-2 is:
GAGATTGTGATGACCCAATCCCCTGCCACACTGAGCCTGAGCCCCGGAGAGCGGGCCACACTGAGCTGCCGGGCCAGCCAGAGCCTGCGGGGCCGGTACCTCGCCTGGTACCAACAGAAACCCGGAAAGGCTCCCAAACTGCTCATCTATGGCGCTTCCACAAGGGCTACCGGAATCCCTGCCCGGTTCAGCGGCAGCGGATCCGGAACAGACTTCACACTGACAATCAGCTCCCTCCAGCCTGAGGATTTCGCTGTGTATTACTGTCAGCAATACGCTTCCAGCCCCCGGACCTTTGGACAGGGAACCAAAGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG(SEQIDNO：8)。
The coding CH (HC) of BA05-1 and BA05-2 and the DNA of constant region of light chain (LC) are synthesized by IDT (USA).In order to express anti-RANKL antibody, VK fragment is cloned into HindIII and the NheI site of the pcDNA3.1 carrier (Invitrogen) containing people CK fragment, VH fragment is cloned into HindIII and the BsiWI site of the pcDNA3.1 carrier (Invitrogen) containing human IgG 4CH fragment.PcDNA3.1 carrier wherein containing people CK fragment and the pcDNA3.1 carrier containing human IgG 4CH fragment build (CK fragment wherein and the sequence of CH fragment are the part in aforementioned DNA sequence dna) by the present inventor.
Again through DH5 ɑ Bacterial Transformation, positive colony is determined in plasmid extraction and order-checking.Sequencing result is consistent with the encoding sequence of the antibody of record.For expressing restructuring BA05-1 and BA05-2, respectively by BA05-1 and BA05-2 light chain and heavy chain plasmid cotransfection in 293-F cell (293fectin, purchased from Invitrogen company), adopt FreeStyle
tM293Expression substratum (purchased from Invitrogen company), cell after 100ml culturing bottle cultivation transfection 5 days, centrifugal, culture supernatant is collected after 0.22um membrane filtration, adopt ProteinA post (5mlMabSelect prepacked column, purchased from GEBiosciences company) purifying, with 10 column volume level pad (20mM phosphoric acid buffers, 150mMNaCl, pH7.0) after, the flow velocity of 2ml/ minute will filter rear supernatant upper prop, after rinsing 10 column volumes, with elution buffer (10mM Trisodium Citrate, pH3.5) wash-out 5 column volumes, the antibody of wash-out is adopted 1MTrisHCl, with pH to 6.5 in pH8.0, then the antibody after dialysis purifying is in PBS, antibody concentration is measured with ultraviolet method (280nm wavelength), measure antibody purity.
The engineered method that the present invention adopts those of ordinary skill in the art to know prepares object human antibody, only describe in the present embodiment and adopt by 293-F cell transient expression, the method obtaining analysis calibrating antibody samples, but, those skilled in the art also can be come by bacterium, yeast, virus and other eukaryotic cell expression system, as Chinese ovary hamster cell (CHO) stable cell lines, prepare, produce human antibody of the present invention.
Embodiment 3: flow cytomery BA05-1andBA05-2 antibody is to the combination of RANKL
By the combination of Flow cytometry anti-RANKL antibody BA05-1andBA05-2 and RANKL (total length RANKL) rotaring redyeing 293 cell (Invitrogen) surperficial RANKL.Anti-RANKL antibody BA05-1, BA05-2, AMG162 of 293-RANKL transfectional cell and different concns hatch 1h at 4 DEG C.Use PBS washed cell, and add anti-human igg (H+L)--PE (1:200)/anti-human igg-R-PE (1:1000) antibody 4 DEG C hatches 30min.After PBS washing, cell is resuspended in 1mlPBS, then uses flow cytometer (BDFACScan) to analyze.As shown in Figure 1, the binding ability of BA05-1 and RANKL is suitable with AMG162, and the binding ability of BA05-2 and RANKL is slightly better than AMG162.
Embodiment 4:BA05-1 and BA05-2 blocks the combination of RANKL and its homoreceptor
As shown in Figure 2, the ability of three kinds of anti-RANKL antibody blocking RANKL and its receptors bind is detected.Particularly, before experiment, people RANK (2 μ g/ml are purchased from R & D, article No. 683-RK-100) is coated on 96 orifice plates by 16h.Then the 0.5 μ g/ml biotin labeling RANKL (eBioscience, article No. 13-6619-82) with different concns recombinant human RANKL antibody BA05-1, BA05-2, AMG162 preincubate is added, 37 DEG C, 30min.Add the avidin (Pierce, article No. N100) of the HRP mark of 50ul1:5000 dilution, hatch 1h for detecting and wrapping the RANKL be combined by RANK for 37 DEG C.The ability that anti-RANKLBA05-1 and BA05-2 antibodies specific blocks RANKL and RANK combination is suitable with AMG162.
Embodiment 5: anti-RANKL antibody BA05-1 and BA05-2 is to the restraining effect of the differentiation of osteoclast that RANKL induces
Detect the ability (see Fig. 3) that anti-RANKL antibody BA05-1 and BA05-2 suppresses the RAW differentiation of osteoclast of RANKL induction.RAW264.7 cell hatches 4 days from anti-RANKL antibody BA05-1, BA05-2, AMG162 of a certain amount of people RANKL (40ng/ml) and different amount altogether in cell culture medium.At the end of 4 days, utilize infiltration and acidifying to carry out Tartrate resistant acid phosphatase (TRAP) active coloring to cell, then use p-nitrophenyl phosphate (pNPP) to process 5min.Add 50 μ l0.5MNaOH solution termination reactions.Utilizing TRAP activity that p-nitrophenyl phosphate is converted into p-NP, being undertaken quantitatively by measuring 405nm place optical density(OD).Result shows, and the differentiation of osteoclast that BA05-1 and BA05-2 induces RANKL is inhibited, and has similar activity to AMG162.
Embodiment 6: antibody BA05-1 and BA05-2 is to the suppression of RANKL immune function
Utilize Ficoll density gradient centrifugation separating peripheral blood mononuclear cells (PBMC) from the whole blood of heparin process.Hatch 4h altogether with anti-RANKL antibody and PBMC and carry out pre-treatment, pretreated PBMC and fixing RANK-Fc fusion rotein (purchased from R/DSystems, article No. 683-RK-100) at 37 DEG C, 5%CO
2cultivate 48h under condition, make RANKL form polymer .VEGF-Fc fusion rotein (purchased from R/DSystems, article No. 357-KD-050) as Isotype control.TNFa and IL-6 level in culture supernatant utilizes the ELISA method of OptEIA (BDBiosciences, SanDiego, CA) to measure to specifications.Utilize flow cytometer to pass through two dyeing T cell tagged molecule (PE-coupling AntiCD3 McAb) and detect RANKL positive T cell, and detect RANK-Fc fusion rotein by hatching with the goat anti-human igg two anti-(Sigma) of FITC coupling.
Before inducing RANKL signal by fixing RANK-Fc, with anti-RANKL antibody pre-treatment PBMC, reduce the release of TNF α and IL-6.But compared with AMG162, the restraining effect less (Fig. 4) of BA05-1 and BA05-2 antibody on cell factor release.This less may cause (Fig. 5) due to the consumption of RANKL positive T cell.
Embodiment 7: validity in the body of anti-RANKL antibody BA05-1 and BA05-2
The notch graft of six Healthy female cynomolgus monkey (4.5 years old) difference is by PBS (n=2), the BA05-1 (n=2) of 10mg/kg or the BA05-2 (n=2) of 10mg/kg of single dose.The blood plasma level being cross-linked NTx albumen (CTX-1) by monitoring serum bone biomarker C-end assesses validity.Collect basal level (testing first 1 week), the 1st week and the 2nd week serum sample, and frozen in-70 DEG C.Utilize commercially available ELISA kit (Mybiosource.com, MBS737402) change of serum C TX-1 level is measured, this test kit is specific to the aminoacid sequence of NTx PROTEIN C end end peptide, the NTx protein fragments produced during to detect brokenly bone bone resorption.Color density (O.D.) value of according to standard sample concentration formulates typical curve.The CTX-1 concentration of each sample draws (Fig. 6) from this typical curve.
Measure blood serum substituting mark CTX-1 level to show, BA05-1 and BA05-2 is acute and reduce the level of CTX-1 in bone resorption significantly, shows that bone remoulding reduces.Similarly, BA05-1 and BA05-2 of single dose significantly reduces TRAP activity, shows to significantly suppress brokenly bone active (Fig. 7).TRAP activity is measured with reducing monkey Tartrate resistant acid phosphatase 5B (TRACP-5B) ELISA kit (Mybiosource.com, MBS025470).
Embodiment 8: the serum stability of anti-RANKL antibody BA05-1 and BA05-2
Two weeks are hatched altogether under anti-RANKL antibody BA05-1, BA05-2 and AMG162 and 50% human serum, 37 DEG C of conditions.Size-exclusion chromatography (SizeExclusionChromatography, SEC) HPLC monitors purity and the homogeneity of antibody purification.Sample passes through analyticalSuperdex20010/30Tricorn post (GEHealthcare) with the flow velocity of 1ml/min in PBS (pH7.4).214 and 280nm place carry out UV detection.Gel filtration standards (Bio-Rad) is utilized to determine IgG size.Fig. 8 shows, and under hatching two weeks conditions at 37 DEG C, BA05-1 and BA05-2 is more stable in human serum than AMG162.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (19)
1. the anti-RANKL antibody in total man source or its fragment, it comprises the heavy chain as shown in SEQIDNO:1 or SEQIDNO:3 and the light chain as shown in SEQIDNO:5 or SEQIDNO:7; Or one or several that is selected from following (1)-(4):
(1) variable region sequences of wherein said heavy chain is: in the variable region sequences and SEQIDNO:9 sequence of described heavy chain through replacement, lack or add one or several amino acid and formed antibody or its fragment there is same or similar activity;
(2) variable region sequences of wherein said light chain is: in the variable region sequences and SEQIDNO:11 sequence of described light chain through replacement, lack or add one or several amino acid and formed antibody or its fragment there is same or similar activity;
(3) constant-region sequences of wherein said heavy chain is: in the constant-region sequences and SEQIDNO:13 sequence of described heavy chain through replacement, lack or add one or several amino acid and formed antibody or its fragment there is same or similar activity;
(4) constant-region sequences of wherein said light chain is: in the constant-region sequences and SEQIDNO:12 sequence of described light chain through replacement, lack or add one or several amino acid and formed antibody or its fragment there is same or similar activity.
2. the anti-RANKL antibody in the total man source of claim 1 or its fragment, it comprises the heavy chain as shown in SEQIDNO:1 and the light chain as shown in SEQIDNO:5.
3. the anti-RANKL antibody in the total man source of claim 1 or its fragment, it comprises the heavy chain as shown in SEQIDNO:3 and the light chain as shown in SEQIDNO:7.
4. the nucleic acid molecule be separated, the anti-RANKL antibody in total man source of any one of its coding claim 1-3 or its fragment.
5. the nucleic acid molecule of claim 4, it comprises the heavy chain as shown in SEQIDNO:2 or SEQIDNO:4 and the light chain as shown in SEQIDNO:6 or SEQIDNO:8.
6. the nucleic acid molecule of claim 5, it comprises the heavy chain as shown in SEQIDNO:2 and the light chain as shown in SEQIDNO:6.
7. the nucleic acid molecule of claim 5, it comprises the heavy chain as shown in SEQIDNO:4 and the light chain as shown in SEQIDNO:8.
8. recombinant vectors, it contains the nucleic acid molecule of any one of claim 4-7.
9. reconstitution cell, it contains the nucleic acid molecule of any one of claim 4-7 or the recombinant vectors of claim 8.
10. composition, it contains the anti-RANKL antibody in total man source of any one of claim 1-3 or the reconstitution cell of its fragment, the nucleic acid molecule of any one of claim 4-7, the recombinant vectors of claim 8 or claim 9, and pharmaceutically acceptable carrier or vehicle.
The composition of 11. claims 10, it also treats the therapeutical agent of inflammation or Immunological diseases containing at least one.
The composition of 12. claims 11, wherein said therapeutical agent is selected from COX1 and COX2 inhibitor, prednisolone, methotrexate, chloroquine, S-Neoral.
The anti-RANKL antibody in total man source of 13. any one of claim 1-3 or the purposes of the reconstitution cell of its fragment, the nucleic acid molecule of any one of claim 4-7, the recombinant vectors of claim 8 or claim 9 in the medicine for the preparation of prevention or treatment and bone lesion relative disease.
The purposes of 14. claims 13, wherein saidly comprises with bone lesion relative disease: osteoporosis, sacroiliitis bone dissolved destruction, malignant tumour or malignant metastatic tumor of bone, paget's disease of bone (osteitis deformans), the inflammation (such as osteomyelitis) with osteoclasia, the autoimmune disorder with osteoclasia or rheumatoid arthritis, hypercalcemia, osteonecrosis, osteopenia.
The purposes of 15. claims 14, wherein said malignant tumour comprises: mammary cancer, prostate cancer, thyroid carcinoma, kidney, lung cancer, skin carcinoma, esophagus cancer, the rectum cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, multiple myeloma and lymphoma (Hokdkin disease).
The purposes of 16. claims 14, wherein said osteoporosis comprises primary osteoporosis, internal secretion osteoporosis (includes but not limited to hyperthyroidism, hyperparathyroidism, hypercortisolism, acromegaly), osteoporotic heredity and apriori form (include but not limited to osteogenesis imperfecta, homocystinuria, menkes' syndrome, Lai-Dai syndrome), and due to the fixing osteoporosis of acra.
The anti-RANKL antibody in total man source of 17. any one of claim 1-3 or the reconstitution cell of its fragment, the nucleic acid molecule of any one of claim 4-7, the recombinant vectors of claim 8 or claim 9 are being prepared as the purposes in the medicine of RANKL inhibitor.
18. detection reagent or test kit, it contains antibody or its fragment of any one of claim 1-3;
Preferably, described antibody or its antigen-binding portion thereof also can comprise detectable mark; Or
Preferably, described detection reagent or test kit also can comprise second antibody, antibody described in its specific recognition or its antigen-binding portion thereof; Or
Preferably, described second antibody also can comprise detectable mark.
The antibody of 19. any one of claim 1-3 or the purposes of its fragment in preparation detection reagent or test kit, wherein said detection reagent or test kit for detecting Nuclear factor kappa-B receptor activation factor ligand, or for diagnosing Nuclear factor kappa-B receptor activation factor ligand relative disease.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410168618.9A CN105085679B (en) | 2014-04-25 | 2014-04-25 | The anti-RANKL antibody of full source of people |
| HK16100757.5A HK1212719A1 (en) | 2014-04-25 | 2016-01-22 | Fully human anti-rankl antibody rankl |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410168618.9A CN105085679B (en) | 2014-04-25 | 2014-04-25 | The anti-RANKL antibody of full source of people |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105085679A true CN105085679A (en) | 2015-11-25 |
| CN105085679B CN105085679B (en) | 2019-01-11 |
Family
ID=54567075
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410168618.9A Active CN105085679B (en) | 2014-04-25 | 2014-04-25 | The anti-RANKL antibody of full source of people |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN105085679B (en) |
| HK (1) | HK1212719A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109890845A (en) * | 2016-10-28 | 2019-06-14 | 伊莱利利公司 | Anti- RANKL antibody and application thereof |
| CN111793135A (en) * | 2020-05-11 | 2020-10-20 | 廊坊天光生物技术有限公司 | Antibody pair for detecting RANKL content in serum and application thereof |
| CN113456811A (en) * | 2021-05-27 | 2021-10-01 | 华中科技大学同济医学院附属协和医院 | Application of IgG in preparing medicine for preventing and treating bone diseases |
| CN117264071A (en) * | 2023-11-22 | 2023-12-22 | 江苏迈威康新药研发有限公司 | Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003086289A2 (en) * | 2002-04-05 | 2003-10-23 | Amgen Inc. | Human anti-opgl neutralizing antibodies as selective opgl pathway inhibitors |
| CN101796072A (en) * | 2007-05-24 | 2010-08-04 | 埃博灵克斯股份有限公司 | Amino acid sequences directed against RANK-l and polypeptides comprising the same for the treatment of bone diseases and disorders |
| CN102741286A (en) * | 2010-03-26 | 2012-10-17 | 刘庆法 | Anti human RANKL monoclonal antibodies developed by PAE technology and uses thereof |
| US8414890B2 (en) * | 2008-08-19 | 2013-04-09 | Regeneron Pharmaceuticals, Inc. | Human antibodies to human RANKL, encoding nucleic acids and methods of treatment |
-
2014
- 2014-04-25 CN CN201410168618.9A patent/CN105085679B/en active Active
-
2016
- 2016-01-22 HK HK16100757.5A patent/HK1212719A1/en unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003086289A2 (en) * | 2002-04-05 | 2003-10-23 | Amgen Inc. | Human anti-opgl neutralizing antibodies as selective opgl pathway inhibitors |
| CN101796072A (en) * | 2007-05-24 | 2010-08-04 | 埃博灵克斯股份有限公司 | Amino acid sequences directed against RANK-l and polypeptides comprising the same for the treatment of bone diseases and disorders |
| US8414890B2 (en) * | 2008-08-19 | 2013-04-09 | Regeneron Pharmaceuticals, Inc. | Human antibodies to human RANKL, encoding nucleic acids and methods of treatment |
| CN102741286A (en) * | 2010-03-26 | 2012-10-17 | 刘庆法 | Anti human RANKL monoclonal antibodies developed by PAE technology and uses thereof |
Non-Patent Citations (6)
| Title |
|---|
| ALAIN BECK等: "Strategies and challenges for the next generation of therapeutic antibodies", 《NATURE REVIEWS IMMUNOLOGY》 * |
| IVAN CORREIA: "Stability of IgG isotypes in serum", 《MABS》 * |
| JANICE M. REICHERT等: "Development trends for monoclonal antibody cancer therapeutics", 《NATURE REVIEWS DRUG DISCOVERY》 * |
| JOCHEN G SALFELD: "Isotype selection in antibody engineering", 《NATURE BIOTECHNOLOGY》 * |
| ROB C. AALBERSE等: "IgG4 breaking the rules", 《IMMUNOLOGY》 * |
| TAKAHIKO ITO等: "Effects of subclass change on the structural stability of chimeric，humanized, and human antibodies under thermal stress", 《PROTEIN SCIENCE》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109890845A (en) * | 2016-10-28 | 2019-06-14 | 伊莱利利公司 | Anti- RANKL antibody and application thereof |
| CN109890845B (en) * | 2016-10-28 | 2022-07-01 | 伊莱利利公司 | anti-RANKL antibodies and uses thereof |
| CN111793135A (en) * | 2020-05-11 | 2020-10-20 | 廊坊天光生物技术有限公司 | Antibody pair for detecting RANKL content in serum and application thereof |
| CN113456811A (en) * | 2021-05-27 | 2021-10-01 | 华中科技大学同济医学院附属协和医院 | Application of IgG in preparing medicine for preventing and treating bone diseases |
| CN117264071A (en) * | 2023-11-22 | 2023-12-22 | 江苏迈威康新药研发有限公司 | Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof |
| CN117264071B (en) * | 2023-11-22 | 2024-03-22 | 江苏迈威康新药研发有限公司 | Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| HK1212719A1 (en) | 2016-06-17 |
| CN105085679B (en) | 2019-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111971062B (en) | Monoclonal antibody preparation against human PD-1, combined medicament and application thereof | |
| CN110582511B (en) | anti-N3 pGlu amyloid beta peptide antibodies and uses thereof | |
| US20240025983A1 (en) | Monoclonal antibody against nerve growth factor, and encoding gene and use thereof | |
| CN110366560B (en) | anti-B7-H4 antibody, antigen binding fragment thereof and medical application thereof | |
| BR112020017676A2 (en) | CD73 ANTAGONIZING ANTIBODY | |
| US20220185901A1 (en) | Bispecific antibodies binding ALK-1 and BMPR-2 | |
| CN103772504B (en) | Humanization anti-epidermal growth factor receptor antibody and application thereof | |
| CN106661103A (en) | Improved a protofibril binding antibodies | |
| CN112243443B (en) | anti-TROP-2 antibodies, antigen-binding fragments thereof, and medical uses thereof | |
| JP2022547850A (en) | Anti-TIGIT immune inhibitor and application | |
| JP6952888B2 (en) | Anti-PACAP antibody | |
| EP3024849A2 (en) | Novel antibodies for the diagnosis and treatment of rheumatoid arthritis | |
| CN109988240B (en) | anti-GPC-3 antibodies and uses thereof | |
| CN111196849B (en) | Anti-sclerostin antibodies, antigen-binding fragments thereof, and medical uses thereof | |
| CN105085679A (en) | Fully human anti-RANKL antibody | |
| CN101233155B (en) | Human anti-epidermal growth factor receptor antibody | |
| WO2020172597A1 (en) | Combination therapy involving anti-cd39 antibodies and anti-pd-1 or anti-pd-l1 antibodies | |
| JP2021533770A (en) | Anti-IL-1β antibody and pharmaceutical compositions thereof and their use | |
| BR112020015961A2 (en) | GREMLIN-1 INHIBITOR FOR TREATING A BONE FRACTURE OR BONE DEFECT | |
| TWI454481B (en) | Antibodies that bind tgf-alpha and epiregulin | |
| CN107840885A (en) | GM CSF antibody and application thereof | |
| JP2022514693A (en) | MUC18-specific antibody | |
| CN114437227A (en) | Bispecific antibodies and uses thereof | |
| EP4159859A1 (en) | Anti-human nr1 antibody derivative | |
| WO2023030311A1 (en) | Antigen-binding protein targeting siglec15 and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1212719 Country of ref document: HK |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |