WO2015101241A1 - 一种抗人rankl抗体、其人源化抗体及它们的药物组合物和用途 - Google Patents

一种抗人rankl抗体、其人源化抗体及它们的药物组合物和用途 Download PDF

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WO2015101241A1
WO2015101241A1 PCT/CN2014/095236 CN2014095236W WO2015101241A1 WO 2015101241 A1 WO2015101241 A1 WO 2015101241A1 CN 2014095236 W CN2014095236 W CN 2014095236W WO 2015101241 A1 WO2015101241 A1 WO 2015101241A1
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seq
amino acid
acid sequence
variable region
heavy chain
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PCT/CN2014/095236
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French (fr)
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周清
冯晓
粘伟红
李玲云
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嘉和生物药业有限公司
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Priority to CN201480017990.8A priority Critical patent/CN105102482B/zh
Priority to EP14876325.3A priority patent/EP3091030B1/en
Priority to ES14876325T priority patent/ES2824101T3/es
Publication of WO2015101241A1 publication Critical patent/WO2015101241A1/zh
Priority to CN201510901094.4A priority patent/CN105732812B/zh
Priority to US14/757,970 priority patent/US9896510B2/en
Priority to EP15202649.8A priority patent/EP3085709B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the differentiation signal of osteoclasts is transmitted by osteogenesis/mesenchymal stem cells.
  • Various factors that stimulate bone resorption transmit signals of osteoclast formation to osteogenic/mesenchymal stem cells, and induce the expression of Osteoclast differentiation factor-1 (ODF-1), also known as nucleus.
  • Osteoclast differentiation factor-1 also known as nucleus.
  • Factor- ⁇ B receptor activating factor ligand Receptor activator of nuclear factor ⁇ B ligand, RANKL
  • RANKL can directly bind to osteoclast differentiation and activation receptor (ODAR) on the cell membrane of osteoclast precursor, also known as Receptor activator of nuclear factor ⁇ B (RANK).
  • the signal is introduced into the osteoclast precursor cells, causing a cascade reaction that stimulates osteoclast formation, differentiation and maturation.
  • OPG/RANKL/RANK system is a key signaling pathway that plays an important regulatory role. It has been reported in the literature that some systemic metabolic diseases such as osteoporosis, rheumatoid diseases, tumors and fracture healing exist. The enhancement of bone remodeling activity is closely related to the RANK/RANKL/OPG system.
  • osteoclasts Many diseases cause bone loss by increasing the number of osteoclasts and/or enhancing the activity of osteoclasts, such as postmenopausal and senile osteoporosis, malignant tumors complicated with humoral hypercalcemia, tumor metastasis, Paget 's bone disease, rheumatoid arthritis, hyperparathyroidism, prosthetic bone autolysis and so on.
  • Postmenopausal estrogen levels decrease, IL21, IL26, TNF- ⁇ gene expression increases, promotes osteoclast proliferation, differentiation, fusion, inhibits apoptosis, increases bone resorption, and unbalances bone metabolism coupling, resulting in bone Loose and loose.
  • the treatment of osteoporosis mainly starts from two aspects: (1) promoting bone formation of osteoblasts; and (2) inhibiting bone resorption of osteoclasts.
  • Blocking RANKL/RANK signaling can inhibit osteoclast formation and activity, block bone resorption, and treat osteoporosis, which has proven to be a viable pathway.
  • Prolia produced by Amgen, USA, is a fully human-derived human RANKL (human RANKL, or hRANKL) monoclonal antibody obtained by immunizing human IgG transgenic mouse XenoMouse, which blocks RANKL/RANK signaling and effectively inhibits osteoclastogenesis. And activity, blocking bone resorption and bone destruction.
  • Monoclonal antibody drugs have the advantages of high specificity, small side effects and remarkable curative effect, and have become an important tool for fighting cancer, infectious diseases and autoimmune diseases.
  • cell fusion and hybridoma technology are still the most reliable methods for preparing monoclonal antibodies.
  • the commonly used animals are mice, rats, sheep, rabbits and other animals.
  • the most commonly used are rodents including rats and Mouse.
  • the monoclonal antibody obtained by the method is of animal origin, and the development of animal-derived monoclonal antibody into antibody is applied to human body, and must be humanized to reduce human anti-animal antibody reaction caused by heterologous antibody (Human) Anti-Animal Antibodies (HAAA) can also activate the human immune system more effectively, reduce the clearance rate of antibody drugs, and prolong the half-life.
  • Complementary Determining Regions are regions that interact with epitope amino acids on an antigen molecule with the support of antibody framework regions (FRs).
  • the precise docking of the CDR regions with antigenic epitopes is the molecular basis for antibody affinity and specificity, while the CDRs region conformation of native parental antibodies represents the highest affinity and best antigen binding specificity.
  • changes in the amino acid of the FRs region cause changes in the conformation of the CDRs region to decrease the affinity.
  • Studies have shown that in all amino acids in the FRs region, most of the amino acid substitutions have only a slight effect on the conformation of the CDRs region, and will not have a serious impact on antibody affinity, but there are also a few key amino acids. Once these key amino acids are replaced by the corresponding amino acids in an adult, a large change in the conformation of the CDRs is caused, which in turn causes a severe decrease in antibody affinity.
  • the technical problem to be solved by the present invention is to provide an anti-human RANKL antibody, a humanized antibody thereof, and a pharmaceutical composition thereof, and use thereof.
  • the invention provides an anti-human RANKL antibody which specifically binds to the amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-human RANKL antibody comprises a heavy chain and a light chain, wherein: a) the heavy chain comprises a variable region of one of the amino acid sequences of SEQ ID NOs: 2-9; and b) The light chain comprises the variable region shown as one of the amino acid sequences of SEQ ID NOS: 10-17; and the antibody binds to RANKL, blocking RANK interaction with RANKL.
  • the anti-human RANKL antibody is a single chain antibody or a humanized antibody, or a murine monoclonal antibody obtained by hybridoma technology.
  • the invention also relates to nucleic acids encoding anti-human RANKL antibody molecules and host cells containing the nucleic acids.
  • the invention also relates to methods of making or producing the anti-human RANKL antibody molecules, nucleic acids, host cells, products, and compositions described herein.
  • the invention provides a humanized antibody against an anti-human RANKL antibody, the humanized antibody specifically binding to human RANKL, and the heavy chain variable region of the humanized antibody is selected from the group consisting of SEQ ID NO
  • the amino acid sequence shown in 6, 23, 25, 27 or 29, the light chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 14, 31, 33, 35, 37 or 39.
  • the heavy chain constant region of the humanized antibody is from human IgG2, the light chain constant region is from human Kappa; or the heavy chain constant region of the humanized antibody is selected from the group consisting of SEQ ID NO:41
  • the amino acid sequence, the light chain constant region is selected from the amino acid sequence set forth in SEQ ID NO:43.
  • the humanized antibody is an antigen-binding fragment selected from the group consisting of scFv, (scFv) 2 , Fab, Fab' or F(ab') 2 .
  • the entire heavy chain of the humanized antibody is selected from the amino acid sequence set forth in one of SEQ ID NOs: 46-50, and/or the entire light chain is selected from SEQ ID NOs: 51-56 One of the amino acid sequences shown.
  • the present invention provides a pharmaceutical composition, wherein the active ingredient is the anti-human RANKL antibody or the humanized antibody.
  • the present invention provides the use of the anti-human RANKL antibody or the humanized antibody for the preparation of a medicament for treating a bone loss disease.
  • the present invention provides a method of improving a condition of a patient suffering from a bone loss disease or treating a patient suffering from a bone loss disease, the method comprising administering to the patient a therapeutically effective amount of the anti-human RANKL antibody Or the humanized antibody or the pharmaceutical composition administered to the patient.
  • RANKL The natural human RANKL protein exists in three forms in vivo, a full-length transmembrane protein, a membrane-bound protein that does not contain intracellular regions, and a soluble protein (starting at 136 Gly to 317 Asp with a total of 182 amino acids). Studies have shown that under physiological conditions, RANKL mainly exists in the transmembrane form on the surface of osteoblasts, but under pathological conditions, matrix metalloproteinase 14 (MMP14) and metalloproteinase 10 (ADAM10) cleave transmembrane morphology of RANKL from the cell surface.
  • MMP14 matrix metalloproteinase 14
  • ADAM10 metalloproteinase 10
  • the present application utilizes soluble RANKL protein as an antigen, and obtains mouse anti-human RANKL monoclonal antibody through a series of screening by immunizing mice and hybridoma fusion technology, and confirms that these murine antibodies not only specifically bind to human RANKL, It also cross-immunizes with RANKL of cynomolgus monkeys and does not immunologically cross-react with murine RANKL.
  • a monoclonal antibody that blocks RANKL/RANK binding and has a blocking ability of not less than or even superior to Denosumab by a competitive binding assay of RANKL to the corresponding receptor RANK.
  • These antibodies can effectively inhibit RANKL-induced osteoclast differentiation in vitro. Based on this, it is inferred that these monoclonal antibodies should effectively inhibit bone resorption and osteoporosis in vivo.
  • These murine antibodies can be made into a clinical pharmacological treatment by human-mouse chimeric or humanized transformation, or by using antibody fragments which bind to the antigen and have a neutralizing effect, and are cultured and expressed by mammalian cells or prokaryotic cells. Monoclonal antibody drugs.
  • Figure 1 shows the nucleotide sequence of the constructed recombinant human RANKL.
  • Figure 2 shows the binding curve of anti-RANKL murine mAb to human RANKL.
  • Figure 3 shows the binding curve of anti-RANKL murine mAb to monkey RANKL.
  • Figure 4 anti-RANKL murine mAb inhibits the binding curve of RANKL to RANK-Fc.
  • Anti-RANKL murine mAb inhibits the activity of human RANKL-induced RAW264.7 cell differentiation.
  • Figure 6 shows a schematic representation of the pFL249-7.19.12 scFv plasmid.
  • Figure 7 shows the electrophoresis map of the first batch of VH mutant library PCR colonies.
  • Figure 8 shows the electrophoresis map of the second batch of VH mutant library PCR colonies.
  • Figure 9 shows a PCR colony screening electrophoresis map of the VL mutant library.
  • Figure 10 shows the results of ELISA screening of the VH library.
  • Figure 11 shows the results of ELISA screening of the VL library.
  • Figure 12 shows a schematic of the H16 heavy chain plasmid map.
  • Figure 13 shows a schematic representation of the plasmid map of the L10Kappa light chain.
  • Figures 14A-14C show a comparison of the binding activity of a humanized antibody to hRANKL.
  • Figures 15A-15C show a comparison of the activity of humanized antibodies to compete for inhibition of hRANKL binding to RANK-Fc.
  • Figures 16A-16D show a comparison of the activity of humanized antibodies to inhibit the differentiation of RAW264.7 cells into osteoclasts.
  • Figure 17 shows the results of humanized antibodies inhibiting hRANKL-induced phosphorylation of ERK1/2 in RAW264.7 cells.
  • immunoglobulin and “antibody” refer to intact immunoglobulins and immunologically active fragments that bind to a desired antigen. Immunoglobulins and their immunologically active (antigen-binding) fragments include epitope binding sites (i.e., sites or epitopes that are capable of specifically binding to the antigen of the recognized antibody).
  • antibody fragments include, for example, Fab, F(v), Fab', F(ab ⁇ ) 2 fragments, "semimolecules” derived from reduced disulfide bonds of immunoglobulins, single-chain immunoglobulins, or other suitable Antigen-binding fragments (see, eg, Bird et al, Science, 242: 423-426 (1988); Huston et al, PMS, (USA), 85: 5879 (1988); Webber et al, Mol. Immunol, 32: 249 (1995)).
  • the antibody or immunologically active fragment thereof can be from an animal (e.g., a rodent such as a mouse or a rat), or a chimeric type (see Morrison et al, PNAS, 81:6851 (1984); Jones et al, Nature, 321:522). (1986)).
  • sequence as used herein (eg, in terms of "immunoglobulin sequence", “antibody sequence”, or “protein sequence”, etc.) is generally understood to include both the relevant amino acid sequence and the encoding of the sequence. Nucleic acid sequence or nucleotide sequence, unless a more limited interpretation is required herein.
  • domain refers to a folded protein domain that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • a domain is responsible for the individual functional properties of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the rest of the protein and/or domain.
  • antibody variable region refers to substantially defined by the art and hereinafter as “complementarity determining region I” or “CDR1”, “complementarity determining region 2" or “CDR2”, and “complementary decision", respectively.
  • the antibody variable region confers specificity to the antigen for the antibody due to its antigen binding site.
  • the specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatchardanalysis, and/or competitive binding assays (eg, radiation). Immunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as their various variations known per se in the art.
  • RIA immunoassays
  • EIA enzyme immunoassays
  • sandwich competition assays as well as their various variations known per se in the art.
  • amino acid residues will be indicated according to standard three-letter or one-letter amino acid codes as are known in the art and agreed upon.
  • amino acid difference refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence compared to a second sequence.
  • substitution will preferably be a conservative amino acid substitution, which means that the amino acid residue is replaced by another amino acid residue of similar chemical structure and its effect on the function, activity or other biological properties of the polypeptide. Smaller or substantially unaffected.
  • conservative amino acid substitutions are well known in the art, for example according to W098/49185, wherein conservative amino acid substitutions are preferably such that one amino acid in groups (i)-(v) below is replaced by another amino acid residue within the same group : (i) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu And Gln; (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, lie, Val and Cys; and (V) aromatic residues Base: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is Gln or His Substituted; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is substituted by Met, Leu or Tyr; Ser is substituted by Thr; Thr is replaced by Ser; Trp is substituted by Tyr; Tyr is replaced by Trp or Phe; Val Replaced by Ile or Leu.
  • Suitable variants of the polypeptides mentioned herein can be determined by those skilled in the art using well-known techniques.
  • one skilled in the art can identify suitable sites for molecules that can be altered but not disruptively active by targeting regions that are not considered important for activity.
  • conservative amino acid substitutions can be made even for biologically active or structurally important regions without disrupting biological activity or adversely affecting polypeptide structure.
  • Those skilled in the art will also be able to analyze three dimensional structures and amino acid sequences associated with structures in similar polypeptides. Based on such information, one skilled in the art can predict the amino acid sequence alignment of the three dimensional structure of the antibody. In some embodiments, one skilled in the art can choose not to make group changes to amino acid residues that are expected to be on the surface of the protein, as such residues are involved in important interactions with other molecules. In addition, one of skill in the art can prepare assay variants that include a single amino acid substitution at each desired amino acid residue. These variants can then be screened using activity assays well known to those skilled in the art. Such variants can also be used to gather information about suitable variants.
  • One method of predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins with a sequence identical to 'I' greater than 30%, or a similarity greater than 40% often have similar structural topologies.
  • the latest growth in protein structure database (PDB) provides predictability of secondary structure enhancement , including the number of possible folds in the structure of the polypeptide or protein. See Holm et al, Nucl. Acid. Res., 27(I): 244-247 (1999). It has been proposed (Brenner et al, Curr. 0p. Struct. Biol., 7(3): 369-376 (1997)), there is a finite number of folds in a given polypeptide or protein, and once a critical number of folds are determined, the structural prediction will suitably become more accurate.
  • Additional methods for predicting secondary structure include “threading method” (Jones, D., Curr. 0 pin. Struct. Biol., 7(3): 377-87 (1997); Sippl et al., Structure, 4(l): 15-19 (1996)), “Graphical Analysis” (Bowie et al, Science, 253: 164170 (1991); Gribskov et al, Meth. Enzym., 183: 146-159 (1990); Gribskov et al, Proc. Nat Acad. Sc1., 84(13): 4355-4358 (1987)), and “evolutionary bonds” (see Holm, supra (1999), and Brenner, supra (1997)).
  • polypeptide refers to a native protein, or a sequence having one or more amino acid deletions, additions and/or substitutions of a native sequence.
  • polypeptide also includes RANKL antibodies or CDRs thereof (described below, SEQ ID NOs: 2-9 and SEQ ID NOs: 10-17), or sequences in which one or more amino acids are deleted, added and/or substituted.
  • the full length heavy chain includes the variable region domain, VH, and three constant region domains, CH1, CH2, and CH3.
  • the VH domain is at the amino terminus of the polypeptide and the CH3 domain is at the carboxy terminus.
  • the term "heavy chain” as used herein includes full length heavy chains and fragments thereof.
  • the full-length light chain includes a variable region domain and a constant region domain, and like the heavy chain, the variable region domain of the light chain is at the amino terminus of the polypeptide.
  • Single-chain antibodies are Fv molecules in which the heavy and light chain variable regions are joined by a flexible linker to form a single-chain polypeptide that forms an antigen-binding region. Single-chain antibodies are discussed in detail in U.S. Patent Nos. 4,946,778 and 5,260,203.
  • a bivalent antibody rather than a “multispecific” or “multifunctional” antibody, is generally understood to have the same binding site.
  • the excess antibody When the excess antibody reduces the amount of receptor that binds to the counterreceptor by at least about 20%, 40%, 60%, 80%, 85%, or more (as determined by in vitro competitive binding assays), the antibody substantially inhibits Adhesion of the ligand to the receptor.
  • epitope includes any polypeptide determinant that specifically binds to an immunoglobulin or T-cell receptor.
  • an epitope determinant comprises a chemically active surface group of a molecule such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and, in some embodiments, may have specific three dimensional structural characteristics, And / or specific charge characteristics.
  • An epitope is a region of an antigen to which an antibody binds.
  • an antibody specifically binds to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • agent refers to a compound, a mixture of compounds, a biomacromolecule, or an extract prepared from a biological material.
  • label refers to the insertion of a detectable label, such as by insertion of a radiolabeled amino acid or attachment of an avidin that can be labeled (eg, which can be detected by optical methods or colorimetric methods). Biotin partial polypeptide detected by fluorescent label or enzymatically active streptavidin). In some embodiments, the label or label can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are well known in the art and can be used.
  • labels for polypeptides include, but are not limited to, the following groups: radioisotopes or radionuclides (eg, 3H, 14C, 15N, 35S, 90Y, 99Tc, lllln, 1251, 1311), fluorescent labels (eg, FI TC, Norden , lanthanide phosphor), enzyme label (eg horseradish peroxidase, galactosidase, luciferase, alkaline phosphatase), chemiluminescent agent, biotinyl, pre-determined second reporter recognition
  • Polypeptide epitopes eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags.
  • the indicia are joined by spacer arms of various lengths to reduce possible steric hindrance.
  • biological sample includes, but is not limited to, any amount of material from a living thing or previously a living thing.
  • living creatures include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals.
  • materials include, but are not limited to, blood, serum, urine, cells, tissues, organs, bones, bone marrow, lymph nodes, and skin.
  • bone-less disease includes, but is not limited to, osteoporosis, osteopenia, Paget's disease, osteolytic metastases, periodontitis, rheumatoid arthritis, and bone loss due to fixation.
  • some cancers are known to increase osteoclast activity and induce bone resorption, such as breast cancer, prostate cancer, and multiple myeloma. These cancers are now known to produce factors that result in overexpression of RANKL in bone and result in increased numbers and activity of osteoclasts.
  • agent or drug refers to a compound or composition that, when properly administered to a patient, induces a desired therapeutic effect.
  • modulator is a compound that alters the activity or function of a molecule.
  • a modulator can cause an increase or decrease in some activity or function of a molecule as compared to an activity or a functional value found in the absence of a modulator.
  • the modulator is an inhibitor that reduces at least one activity or function of the molecule.
  • the activities and functions of some of the exemplified molecules include, but are not limited to, binding affinity, enzymatic activity, and signal transduction.
  • Some exemplified inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules, which are described, for example, in W001/83525.
  • the term "substantially pure” means that the host material is a predominantly present material (e.g., on a molar basis, it is more abundant in the composition than any other various materials).
  • the substantially purified fraction is one wherein the host material comprises at least about 50% (by mole) of all macromolecular species present.
  • the substantially pure composition contains about 80%, 85%, 90%, 95%, or 99% or more of all of the macromolecular species present in the composition.
  • the host material is purified to be substantially homogeneous (the impurity species in the composition cannot be detected by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.
  • patient includes both human and animal subjects.
  • a murine monoclonal antibody against human RANKL is provided.
  • amino acid sequences comprising heavy and light chain immunoglobulins, particularly sequences corresponding to the variable regions, and nucleic acid coding sequences thereof are provided.
  • sequences corresponding to complementarity determining regions (CDRs), particularly from CDR1 to CDR3, are provided.
  • hybridoma cell lines expressing such immunoglobulin molecules and monoclonal antibodies are also provided.
  • the invention provides a humanized antibody derived from the above-described murine monoclonal antibody against human RANKL.
  • humanized antibody mainly refers to an antibody that is re-expressed by a mouse monoclonal antibody by gene cloning and DNA recombination technology, and most of the amino acid sequence is replaced by a human sequence, and the affinity and specificity of the parent mouse monoclonal antibody are substantially retained. It reduces its heterogeneity and is beneficial for the human body.
  • Humanized antibodies include chimeric antibodies, modified antibodies, and fully humanized antibodies. The basic principle of humanization is to alter antigen recognition, ie the CDR domain, specificity in the context of human immunoglobulin ("CDR transplantation", Winter and Milstein). Transcription from animals (usually murine) to humanized antibodies must be compromised between the opposite needs, and the solution varies depending on the situation.
  • the immunoglobulin will retain as many acceptable human sequences as possible. In any event, in order to maintain the original binding properties, the immunoglobulin framework should contain a sufficient number of mutations in an acceptable human sequence to ensure that the conformation of the CDR regions is as similar as possible to the conformation of the CDR regions of the donor murine immunoglobulin. .
  • Naturally occurring antibody building blocks generally include tetramers.
  • Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair having one full length "light chain” (in some embodiments, about 25 kDa) and one full length "heavy chain” (in some In the embodiment, about 50_70 kDa).
  • the amino terminal portion of each chain typically comprises a variable region of about 100 to 110 or more amino acids, which is generally responsible for antigen recognition.
  • the carboxy terminal portion of each chain generally defines a constant region responsible for the function of the effector.
  • Human light chains are generally classified into K and ⁇ light chains.
  • Heavy chains are generally classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and antibody isotypes are defined as IgM, IgD, IgG, IgA, IgE, and IgG, respectively, including several subclasses including, but not limited to, IgG1, IgG2, IgG3, And IgG4.
  • IgM has several subclasses including, but not limited to, IgMl and IgM2.
  • IgA is divided into several subclasses including, but not limited to, IgAl and IgA2.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D” region of about 10 or more amino acids.
  • J variable and constant regions
  • D variable regions of each light/heavy chain pair
  • variable regions generally have the same general structure as the relatively conserved framework regions (FR), passing through three hypervariable regions, also referred to as complementarity determining regions or CDR regions.
  • the CDRs of each pair of two strands are generally arranged by a framework region which can bind to a specific epitope.
  • the light and heavy chain variable regions generally include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the arrangement of amino acids in each domain is generally based on the definition of Kabat Sequences of Proeins of Immunological Interest (Nation Institutes of Health, Bethesda, Md. (1987, 1991)), or Chothia & Lesk J. Mol. Biol. 196: 901-917 ( 1987); Chothia et al, Nature 342: 878-883 (1989).
  • Bispecific and bifunctional antibodies are typically artificial hybrid antibodies having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be prepared by a variety of methods including, but not limited to, fusion of hybridomas or ligation of Fab' fragments. See, for example, Songsivi lai & Lachmann Clin. Exp. Tmmunol. 79: 315-321 (1990), Kostelny et al, J. 1 mmunol. 148: 1547-1553 (1992).
  • the preparation of a murine monoclonal antibody against human RANKL of the invention is a technique well known to those skilled in the art; the DNA sequence of the humanized antibody is operably linked (i.e., positioned in such a manner as to ensure its functionality) Expression is carried out in the host cell after expression control sequences.
  • These vectors are generally capable of being replicated in an host organism as an expression vector or as an integral part of chromosomal DNA.
  • the expression vector comprises an optional marker to allow identification of cells that have been transformed with the DNA sequence of interest.
  • the humanized immunoglobulin of the invention preferably a prokaryotic system, is produced in recombinant form of scFv or in Fab form.
  • coli is one of the prokaryotic hosts that are particularly useful for cloning the DNA sequences of the present invention.
  • a large number of well characterized promoters are available, such as the Lac or trp operon or beta-lactamase or lambda phage. Typically, these promoters control expression and have ribosome binding sites to properly initiate and complete transcription and translation. Conjugation with polyethylene glycol (PEG) makes it possible to increase the half-life of the humanized immunoglobulin of the invention produced in the prokaryotic system.
  • PEG polyethylene glycol
  • Other Single cell organisms, such as yeast can be used for expression.
  • the host of choice is Saccharomyces, utilizing a suitable vector that provides expression control, replication termination, and start sequence.
  • Insect cell cultures can also be used to produce the humanized immunoglobulins of the invention, typically using S2 Drosophila cells transfected in a stable manner or cells of Spodoptera frugiperda having a baculovirus-based expression system (Putlitz et al., 1990). ). Plant and plant cell cultures can be used for the expression of humanized immunoglobulins of the invention (Larrick & Fry, 1991; Benvenuto et al, 1991; Durin et al, 1990; Hiatt et al, 1989).
  • the half-life extending modification of the polypeptide of the invention comprises the attachment of a pharmaceutically acceptable suitable polymer, such as a linear or branched poly(ethylene glycol). (PEG) or a derivative thereof (for example, methoxy poly(ethylene glycol) or mPEG).
  • PEGylation can be used in any suitable form, such as PEGylation in the art for antibodies and antibody fragments including, but not limited to, domain antibodies and scFv fragments; see for example: Cha pman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev.
  • PEG can be linked to a cysteine residue naturally present in the polypeptide of the invention, and the polypeptide of the invention can be modified to suitably introduce one or more cysteines for attachment of PEG A residue, or an amino acid sequence comprising one or more cysteine residues for attachment to PEG, can be fused to the N-terminus and/or C-terminus of a polypeptide of the invention, all of which are used by those skilled in the art
  • an antibody of the invention can be used to detect RANKL in a biological sample. In some embodiments, this allows for the identification of cells or tissues that produce proteins. In some embodiments, an antibody that binds to RANK and blocks interaction with other binding compounds has therapeutic utility in modulating osteoclast differentiation and bone resorption. In some embodiments, an anti-human RANKL antibody can block RANKL binding to ODAR, which can result in blockade in the signal transduction cascade and loss of NF-kB-mediated transcriptional activation. Assays for measuring NF-kB mediated transcriptional activation using, for example, luciferase reporter assays are well known to those skilled in the art.
  • the invention provides an anti-human RANKL antibody comprising a heavy chain and a light chain, wherein: a) the heavy chain comprises the variable region of one of the amino acid sequences of SEQ ID NOs: 2-9; and b) light
  • the strand includes the variable region shown as one of the amino acid sequences of SEQ ID NOS: 10-17; and the antibody binds to RANKL, blocking RANK from interacting with RANKL.
  • the anti-human RANKL antibody is a murine monoclonal antibody, or a single-chain antibody, or a humanized antibody obtained by hybridoma technology.
  • Humanized antibodies include chimeric antibodies, modified antibodies, fully humanized antibodies, and the like.
  • the invention relates to nucleic acids encoding anti-human RANKL antibody molecules and host cells comprising the nucleic acids.
  • the invention further relates to a product or composition comprising at least one anti-human RANKL antibody molecule of the invention and optionally one or more additional components of the composition.
  • the invention also relates to methods of making or producing the anti-human RANKL antibody molecules, nucleic acids, host cells, products, and compositions described herein.
  • the invention provides a humanized antibody against a human RANKL antibody, the humanized antibody specifically binding to human RANKL, and the heavy chain variable region of the humanized antibody is selected from the group consisting of SEQ ID NO: The amino acid sequence shown in 6, 23, 25, 27 or 29, wherein the light chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 14, 31, 33, 35, 37 or 39.
  • the heavy chain constant region of the humanized antibody is from human IgG2, the light chain constant region is from human Kappa; or the heavy chain constant region of the humanized antibody is selected from the group consisting of SEQ ID NO:41
  • the amino acid sequence, the light chain constant region is selected from the amino acid sequence set forth in SEQ ID NO:43.
  • the humanized antibody is an antigen-binding fragment selected from the group consisting of scFv, (scFv) 2 , Fab, Fab' or F(ab') 2 .
  • the entire heavy chain of the humanized antibody is selected from the amino acid sequence set forth in one of SEQ ID NOs: 46-50, and/or the entire light chain is selected from SEQ ID NOs: 51-56 One of the amino acid sequences shown.
  • the entire heavy chain of the humanized antibody is selected from the amino acid sequence set forth in SEQ ID NO: 46, the entire light chain is selected from the amino acid sequence set forth in SEQ ID NO: 51; or the entire heavy chain is selected From the amino acid sequence set forth in SEQ ID NO: 47, the entire light chain is selected from the amino acid sequence set forth in SEQ ID NO: 52, 53, 54 or 55; or the entire heavy chain is selected from the amino acid sequence set forth in SEQ ID NO:48
  • the intact light chain is selected from the amino acid sequence set forth in SEQ ID NO: 52, 53, 54 or 55; or the entire heavy chain is selected from the group consisting of SEQ ID NO: 49
  • the amino acid sequence shown, the entire light chain is selected from the amino acid sequence set forth in SEQ ID NO: 52, 53, 54 or 55; or the entire heavy chain is selected from the amino acid sequence set forth in SEQ ID NO: 50, and the intact light chain is selected from the group consisting of SEQ. ID NO: The amino acid sequence shown in 56.
  • the heavy chain variable region of the humanized antibody is selected from the amino acid sequence set forth in SEQ ID NO:6, and the heavy chain constant region is selected from the amino acid sequence set forth in SEQ ID NO:41, the light chain The variable region is selected from the amino acid sequence set forth in SEQ ID NO: 14, and the light chain constant region is selected from the amino acid sequence set forth in SEQ ID NO: 43; or the heavy chain variable region is selected from the group consisting of SEQ ID NO:
  • the amino acid sequence, the heavy chain constant region is selected from the amino acid sequence set forth in SEQ ID NO: 41, the light chain variable region is selected from the amino acid sequence set forth in SEQ ID NO: 31, and the light chain constant region is selected from the group consisting of SEQ ID NO: 43
  • the amino acid sequence shown; or the heavy chain variable region is selected from the amino acid sequence set forth in SEQ ID NO: 23, the heavy chain constant region is selected from the amino acid sequence set forth in SEQ ID NO: 41, and the light chain variable region is selected from the group consisting of SEQ.
  • the amino acid sequence represented by ID NO: 35, and the light chain constant region is selected from the amino acid sequence shown in SEQ ID NO: 43; or the heavy chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 23, and the heavy chain is constant.
  • the region is selected from the amino acid sequence set forth in SEQ ID NO: 41, and the light chain variable region is selected from the amino acid sequence set forth in SEQ ID NO:33.
  • the light chain constant region is selected from the amino acid sequence set forth in SEQ ID NO: 43; or the heavy chain variable region is selected from the amino acid sequence set forth in SEQ ID NO: 23, and the heavy chain constant region is selected from the group consisting of SEQ ID NO: 41
  • the amino acid sequence, the light chain variable region is selected from the amino acid sequence set forth in SEQ ID NO: 37, and the light chain constant region is selected from the amino acid sequence set forth in SEQ ID NO: 43; or the heavy chain variable region is selected from the group consisting of SEQ ID NO
  • the heavy chain constant region is selected from the amino acid sequence shown in SEQ ID NO: 41
  • the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 31
  • the light chain constant region is selected from the group consisting of The amino acid sequence shown in SEQ ID NO: 43; or the heavy chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 27, the heavy chain constant region is selected from the amino acid sequence shown in SEQ ID NO: 41, and the light chain can be
  • the invention also relates to the use of the anti-human RANKL antibody or the humanized antibody for the preparation of a medicament for treating a bone loss disorder.
  • the bone loss disease includes osteoporosis, bone and joint destruction caused by rheumatoid arthritis, bone destruction caused by tumor bone metastasis, bone destruction caused by growth of giant bone tumor, and others Pathological changes such as loss or destruction of bone formed by RANKL-induced osteoclasts.
  • the present invention provides a method of improving a condition of a patient suffering from a bone loss disease or treating a patient suffering from a bone loss disease, the method comprising administering to the patient a therapeutically effective amount of the anti-human RANKL antibody or the human The antibody is administered or administered to a patient.
  • the bone disease is selected from the group consisting of osteoporosis, bone and joint destruction caused by rheumatoid arthritis, bone destruction caused by tumor bone metastasis, and bone destruction caused by growth of giant bone tumor. And other pathological changes such as bone loss or destruction due to RANKL-induced osteoclasts.
  • the above bone diseases mainly refer to bone loss diseases.
  • methods of treating a rare bone disease comprising administering a pharmaceutically effective amount of the anti-human The RANKL antibody or the humanized antibody or the pharmaceutical composition is administered.
  • a method of treating an inflammatory condition associated with bone loss in a patient comprising administering a pharmaceutically effective amount of the anti-human RANKL antibody or the humanized antibody or administering the pharmaceutical composition is provided.
  • a method of treating an autoimmune disease associated with bone loss in a patient comprising administering a pharmaceutically effective amount of the anti-human RANKL antibody or the humanized antibody or administering the pharmaceutical composition is provided.
  • a method of treating rheumatoid arthritis in a patient comprising administering a pharmaceutically effective amount of the anti-human RANKL antibody or the humanized antibody or administering the pharmaceutical composition is provided.
  • a method of detecting human RANKL levels in a biological sample comprising contacting the sample with an antibody.
  • a method of treating a bone disorder comprising administering a therapeutically effective amount of an anti-human RANKL antibody or a humanized antibody thereof.
  • a method of treating a bone disorder comprising administering a therapeutically effective amount of an anti-human RANKL antibody or a humanized antibody thereof and another therapeutic agent is provided.
  • the additional therapeutic agent is administered in a therapeutically effective amount.
  • the bone disease is a disease characterized by bone loss including, but not limited to, bone scarcity and osteolysis.
  • the treatment with an anti-human RANKL antibody or a humanized antibody thereof inhibits the rate of bone resorption.
  • treatment can be applied to reduce the resorption rate above normal bone resorption rate, or to reduce bone resorption to below normal levels.
  • antibodies can be assayed for the absence or presence of binding to RANKL under OPG and their ability to inhibit RANKL-mediated osteoclastogenesis and/or bone resorption.
  • Conditions that may be treated according to some embodiments include, but are not limited to, the following groups:
  • Osteoporosis including but not limited to primary osteoporosis, endocrine osteoporosis (including but not limited to hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and acromegaly), bone Inherited and innate forms of osteoporosis (including but not limited to osteogenesis imperfecta, homocystinuria, Menkes syndrome, Lai-Dai syndrome), and osteoporosis due to acrosome fixation;
  • Osteomyelitis an infectious bone lesion that causes bone loss
  • Hypercalcemia including but not limited to solid tumors (including but not limited to breast, lung and kidney) and hematological malignancies (including but not limited to multiple myeloma, lymphoma and leukemia), hypercalcemia, Hypercalcemia, and hypercalcemia associated with thyroid function and renal dysfunction;
  • Bone scarcity including but not limited to bone scarcity after surgery, drug-induced bone scarcity, scarcity associated with small bowel and large bowel diseases, and scarcity associated with chronic liver and kidney disease;
  • Osteonecrosis, or bone cell death including but not limited to osteonecrosis associated with creative damage, osteonecrosis associated with Gaucher disease, osteonecrosis associated with sickle cell anemia, osteonecrosis associated with systemic lupus erythematosus, Osteonecrosis associated with rheumatoid arthritis, osteonecrosis associated with periodontal disease, osteonecrosis associated with osteolytic metastasis, osteonecrosis associated with other conditions; and cartilage loss associated with rheumatoid arthritis and Joint erosion.
  • the anti-human RANKL antibody or humanized antibody thereof can be used alone or in combination with at least one additional therapeutic agent for treating bone disease.
  • an anti-human RANKL antibody or humanized antibody thereof is used in combination with a therapeutically effective amount of another therapeutic agent.
  • Exemplary therapeutic agents that can be administered with an anti-human RANKL antibody or a humanized antibody thereof include, but are not limited to, bone morphogenetic factors designated BMP-1 to BMP-12, transforming growth factor- ⁇ (TGF- ⁇ ) and TGF.
  • interleukin -1 (IL-1) inhibition homogeneous U including, but not limited to, IL-1ra and derivatives thereof and Kineret TM, anakinra
  • TNF ⁇ inhibitors including, but not limited to, soluble TNFa receptor, Enbre I TM , Etanercept, anti -TNF ⁇ antibody, Remicade TM, infliximab, and D2E7 antibody; and the like parathyroid hormone; parathyroid hormone related protein and analogs thereof; E series prostaglandins; bisphosphonates (e.g.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • C0X-2 inhibitors such as Celebrex TM, celecoxib, and Vioxx TM, Luo Non-express
  • immunosuppressive agents such as methotrexate or leflunomide, serine protease inhibitors, including but not limited to secretory leukocyte protease inhibitors (SLPI); IL-6 inhibitors (including but not limited to anti-IL -6 antibody), IL-8 inhibitor (including but not limited to anti-IL-8 antibody), IL-18 Inhibitors (including but not limited to IL-18 binding protein and anti-IL-18 antibody), interleukin-1 converting enzyme (ICE) modulator; fibroblast growth factor FGF-1 to FGF-1O and FGF modulator; PAF antagonism Agent; keratinocyte growth factor (KGF), KGF-related molecule, and KGF modulator; matrix metalloproteinase (M
  • an anti-human RANKL antibody or humanized antibody thereof is used with a particular therapeutic agent to treat various inflammatory conditions, autoimmune conditions, or other agents that are associated with bone loss.
  • two, three or more agents can be administered depending on the condition and the desired level of treatment.
  • these agents are provided by inclusion in the same formulation.
  • such an agent and an anti-human RANKL antibody or humanized antibody thereof are provided by inclusion in the same formulation.
  • such agents can be provided together by inclusion in a therapeutic kit.
  • such agents and anti-human RANKL antibodies or humanized antibodies thereof can be provided together by inclusion in a therapeutic kit.
  • such agents can be provided separately.
  • a gene encoding a protein agent and/or an anti-human RANKL antibody when administered by gene therapy, can be included in the same vector. In some embodiments, the gene encoding the protein agent and/or the anti-human RANKL antibody can be under the control of the same promoter region. In some embodiments, the gene encoding the protein agent and/or the anti-human RANKL antibody can be in separate vectors.
  • the invention relates to a therapeutic regimen comprising an anti-human RANKL antibody or a humanized antibody thereof and at least one interleukin-1 (IL-1) inhibitor, and a method of treatment using such a therapeutic regimen.
  • the therapeutic regimen comprises an anti-human RANKL antibody or a humanized antibody thereof and an IL-1 inhibitor and at least one additional molecule described herein.
  • the method of treatment uses an IL-1 inhibitor and/or a TNFa inhibitor in combination with an anti-human RANKL antibody or a humanized antibody thereof.
  • an anti-human RANKL antibody or humanized antibody thereof in combination with an IL-1 inhibitor and/or a TNF a inhibitor can be used to treat conditions such as asthma, rheumatoid arthritis, and multiple sclerosis.
  • Interleukin-1 is an anti-inflammatory cytokine.
  • IL-1 is a mediator in many diseases and medical conditions.
  • macrophage/single cell line cells produce IL-1.
  • interleukin-1 mediated disease is associated with elevated IL-1 levels in body fluids or tissues and/or if cells or tissues from the body produce elevated levels of IL-1 in the culture.
  • interleukin-1 mediated diseases are identified by two other conditions: (I) experimentally using animal mimics and disease or medical conditions by administering IL-1 or upregulating IL-1 expression. Related pathological findings; and (2) treatment by agents that inhibit the action of IL-1 can inhibit or eliminate pathologies induced in experimental animal models in disease or medical conditions.
  • one or more of the above conditions are consistent with an IL-1-mediated disease.
  • all three conditions are consistent with IL-1-mediated disease.
  • Acute and chronic interleukin-1 (IL-1)-mediated diseases include, but are not limited to, the following groups: acute pancreatitis; amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease); Alzheimer's disease; cachexia/anorexia , including but not limited to AIDS-induced cachexia; asthma and other lung diseases; atherosclerosis; autoimmune vasculitis; chronic fatigue syndrome, Clostridium-related diseases, including but not limited to Clostridium-associated diarrhea Coronary conditions and indications, including but not limited to congestive heart failure, coronary restenosis, myocardial infarction, myocardial dysfunction (eg, associated with sepsis), and coronary artery bypass grafting; cancer, including but not limited to Leukemia, including but not limited to multiple myeloma leukemia and myeloid leukemia (eg AML and CML), and tumor metastasis; diabetes (including but not limited to insulin dependent diabetes); endometriosis; fever; fibroids;
  • the invention relates to a therapeutic regimen comprising an anti-human RANKL antibody or a humanized antibody thereof and at least one TNFa inhibitor, and a method of using the same.
  • the therapeutic regimen comprises an anti-human RANKL antibody or humanized thereof Antibody and TNFa inhibitors and at least one additional molecule described herein.
  • TNF-mediated disease includes, but is not limited to, diseases associated with elevated levels of TNF in body fluids or tissues and/or diseases in which cells or tissues are derived from an individual that produces high levels of TNF in culture or Medical condition.
  • (I) is capable of experimentally mimicking pathological findings associated with a disease or medical condition in an animal by administering or upregulating TNF expression and/or (2) inhibiting or destroying by treatment with an agent that inhibits TNF action
  • the pathology induced in an experimental animal model of a disease or medical condition is considered to be a TNF-mediated disease.
  • Some diseases and medical conditions are TNF mediated and can be classified as inflammatory conditions.
  • TNF-mediated diseases include, but are not limited to, cachexia and anorexia; cancers including, but not limited to, leukemia; chronic fatigue syndrome; coronary conditions and/or indications including, but not limited to, hyperemia Heart failure, coronary restenosis, myocardial infarction, myocardial dysfunction (including but not limited to such conditions associated with sepsis), and coronary artery bypass grafting; depression; diabetes, including but not limited to, adolescent onset Type I diabetes, diabetes, and insulin resistance (including but not limited to insulin resistance associated with obesity); endometriosis, endometritis, and related conditions; fibroids and analgesia; graft resistance Host rejection; hyperalgesia; inflammatory bowel disease, including but not limited to Crohn's disease and Clostridium difficile-associated diarrhea; ischemia, including but not limited to, brain injury as a result of trauma, epilepsy, hemorrhage, and/or stroke Lung disease, including but not
  • a TNF inhibitor acts by downregulating or inhibiting TNF production, binding to free THF, interfering with binding of TNF to its receptor, and interfering with at least one of TNF signaling regulation upon binding to its receptor.
  • TNF inhibitor includes, but is not limited to, soluble TNF receptors including, but not limited to, soluble tumor necrosis factor receptor type I (sTNF-RI; also known as p55 receptor), soluble tumor necrosis factor receptor type II (also known as p75 receptor), and EnbreI TM, etanercept; anti-TNF antibody, including but not limited to, Remicade TM, infliximab and D2E7 (see, e.g. U.S. Patent No.
  • an anti-human RANKL antibody or humanized antibody thereof can be administered with at least one therapeutic agent for inflammation. In some embodiments, an anti-human RANKL antibody or humanized antibody thereof can be administered with at least one therapeutic agent for an immune disease.
  • Exemplary therapeutic agents for inflammatory and immune diseases include, but are not limited to, corticosteroids including, but not limited to, prednisolone; non-steroidal anti-inflammatory drugs (NSAIDs) including, but not limited to, cyclooxygenase type I ( C0X-1) and cyclooxygenase type 2 (C0X-2) inhibitors; disease modifying anti-rheumatic drugs (DMARDs) including, but not limited to, methotrexate, hydroxychloroquine, chloroquine, cyclosporin, Gold compounds (eg, acesulfame, gold thiosuccinate, and glucosinolate), leflunomide; type IV phosphodiesterase inhibitors, including but not limited to, cyclopentaphenone and ketone cocoa Alkali; tacrolimus (FK-506); sirolimus (rapamycin); mycophenolic acid; 5_lipoxygenase inhibitors, including but not limited to, albino
  • Some exemplary therapeutic agents for inflammation are described, for example, in CADinarello and LLMoldawer Proinfla.mma.torv and Ant1-1nflammatorv Cytokines in Rheuma toid Arthritis: A Primer for Clinicians Third Edition (2001) Amgen Inc. Thousand Oaks, CA .
  • Exemplary therapeutic agents for inflammatory and autoimmune diseases including, but not limited to, interferon-Y (IFN- ⁇ ) modulators; modulators of 0X40/0X40L (including soluble form of 0X40); 4-1BB/4 -1BB ligand modulator (including soluble form of 4-1BB); and B cell-T cell co-stimulatory pathway modulator;
  • IFN- ⁇ interferon-Y
  • modulators of 0X40/0X40L including soluble form of 0X40
  • 4-1BB/4 -1BB ligand modulator including soluble form of 4-1BB
  • B cell-T cell co-stimulatory pathway modulator including interferon-Y
  • an anti-human RANKL antibody or humanized antibody thereof is used to treat bone loss, including but not limited to, malignant or Loss of bone caused by osteolytic destruction of bone caused by metastasis.
  • an anti-human RANKL antibody or humanized antibody thereof can be used to treat bone loss associated with cancer.
  • Exemplary cancers include, but are not limited to, breast cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, esophageal cancer, rectal cancer, bladder cancer, cervical cancer, ovarian cancer, and liver cancer, as well as gastrointestinal cancer.
  • an anti-human RANKL antibody or humanized antibody thereof can be used to treat bone loss associated with some hematological malignancies including, but not limited to, multiple myeloma and lymphoma, including Hodgkin's disease .
  • the anti-human RANKL antibody or humanized antibody thereof is administered alone. In some embodiments, the anti-human RANKL antibody or humanized antibody thereof is administered with at least one other therapeutic agent, including, but not limited to, at least one other cancer therapeutic agent.
  • Exemplary cancer therapeutics include, but are not limited to, radiation therapy and chemotherapy.
  • chemotherapy can include treatment with one or more of the following drugs: anthracycline, paclitaxel, tamoxifen, doxorubicin, 5-fluorouracil, and other drugs known in the art.
  • the cancer therapeutic is a luteinizing hormone releasing hormone (LHRH) antagonist.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of an anti-human RANKL antibody or a humanized antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant .
  • the invention provides a therapeutically effective amount of an anti-human RANKL antibody or a humanized antibody thereof, and a therapeutically effective amount of at least one additional therapeutic agent, and a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsified
  • a pharmaceutical composition of a preservative, a preservative and/or an adjuvant A pharmaceutical composition of a preservative, a preservative and/or an adjuvant.
  • the at least one additional therapeutic agent is selected from the group consisting of bone morphogenetic factor, transforming growth factor-beta (TGF-beta), interleukin-1 (IL-1) inhibitor, including but not limited to IL -1ra and derivatives thereof and Kineret TM, anakinra; TNF ⁇ inhibitors, including, but not limited to, soluble TNF [alpha] receptor, EnbreI TM, etanercept, anti -TNF ⁇ antibody, Remicade TM, infliximab, and D2E7 antibody; parathyroid hormone And analogues thereof; parathyroid hormone-related proteins and their analogues; E-series prostaglandins; bisphosphonates (such as alendronate and others); bone-enhancing minerals such as fluoride and calcium; non-steroidal anti-inflammatory drugs (the NSAIDs), including but not limited to C0X-2 inhibitors such as Celebrex TM, celecoxib and Vioxx TM, rof
  • acceptable formulation materials are preferably such that the dosage and concentration employed are not toxic to the recipient.
  • the pharmaceutical composition may contain, for example, change, maintain or retain, for example, pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, Formulation material for absorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (eg, ascorbic acid, sodium sulfite or sub- Sodium bisulfate); buffer (eg borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acid); filler (eg mannitol or glycine); chelating agent (eg ethylene) Aminetetraacetic acid (EDTA)); complexing agents (eg caffeine, polyvinylpyrrolidone, cyclodextrin or hydroxypropyl cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (eg glucose, mannose) Or dextrin); protein (eg serum albumin, gelatin or immunoglobulin); colorants, flavors and diluents; emulsifiers; hydrophilic polymers (eg,
  • an anti-human RANKL antibody or humanized antibody and/or therapeutic molecule thereof is linked to a half-life extending excipient known in the art.
  • excipients include, but are not limited to, polyethylene glycol and dextran.
  • excipients are described, for example, in the US application for registration No. N0.09/428082 and the published PCT application No. WO99/25044, which is incorporated herein by reference.
  • one skilled in the art will determine an optimized pharmaceutical composition based on, for example, the desired route of administration, the mode of delivery, and the desired dosage. See, for example, Remington's Pharmaceutical Science, 18th Edition, edited by A.R. Gennaro, Mack Publish Company (1990).
  • such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibodies of the invention.
  • the primary excipient or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature.
  • a suitable excipient or carrier can be water for injection, physiological saline or artificial cerebrospinal fluid, possibly supplemented with other materials commonly used in parenteral compositions.
  • neutral buffered saline or saline mixed with serum albumin are other exemplary excipients.
  • the pharmaceutical composition contains a Tris buffer of about pH 7.0-8.5, or an acetate buffer of about pH 4.0-5.5, which may further comprise sorbitol or a suitable substitute thereof.
  • a composition comprising an anti-human RANKL antibody or a humanized antibody thereof, with or without at least one additional therapeutic agent can be prepared by combining a selected composition having a desired degree of purity with an optional formulation reagent Mix to prepare. Furthermore, in some embodiments, a composition comprising an anti-human RANKL antibody or a humanized antibody thereof, with or without at least one additional therapeutic agent, can be formulated as a lyophilizate using a suitable excipient such as sucrose.
  • the pharmaceutical compositions of the invention may be selected for parenteral administration.
  • the pharmaceutical composition can be selected for inhalation or by the digestive tract, such as oral delivery.
  • the preparation of such pharmaceutically acceptable compositions is well known in the art.
  • the therapeutic composition when administered parenterally, can be pyrogen-free, pharmaceutically acceptable, parenterally acceptable, containing the desired anti-human RANKL antibody or humanized antibody thereof , in the form of an aqueous solution with or without additional therapeutic agents.
  • the excipient for parenteral injection is sterile distilled water, wherein the anti-human RANKL antibody or humanized antibody thereof, with or without at least one additional therapeutic agent, is formulated into a sterile isotonic solution , save as appropriate.
  • the preparation involves formulating the desired molecules and agents, such as injectable microspheres, biodegradable particles, polymeric compounds (eg, polylactic acid or polyglycolic acid), beads or liposomes, which can provide products Controlled or sustained release, then delivered via a delayed injection.
  • agents such as injectable microspheres, biodegradable particles, polymeric compounds (eg, polylactic acid or polyglycolic acid), beads or liposomes, which can provide products Controlled or sustained release, then delivered via a delayed injection.
  • hyaluronic acid can also be used, with the effect of promoting retention in the circulation.
  • an implantable drug delivery device can be used to introduce the desired molecule.
  • the pharmaceutical composition can be formulated into a formulation for inhalation.
  • an anti-human RANKL antibody, or a humanized antibody thereof, with or without at least one additional therapeutic agent can be formulated into a dry powder for inhalation.
  • an inhalation solution of an anti-human RANKL antibody or a humanized antibody thereof, with or without at least one additional therapeutic agent may also be formulated with a propellant for aerosol delivery.
  • the solution can be sprayed. Pulmonary administration is further described in PCT Application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.
  • formulations are contemplated that can be administered orally.
  • anti-humans administered in this manner, with or without at least one additional therapeutic agent may be formulated with or without those carriers in solid dosage forms such as those conventionally used in the combination of tablets and capsules.
  • RANKL antibody or humanized antibody thereof may be formulated with or without those carriers in solid dosage forms such as those conventionally used in the combination of tablets and capsules.
  • RANKL antibody or humanized antibody thereof a capsule of the active portion of the point release formulation in the digestive tract when bioavailability is greatest and pre-systemic degradation is minimal can be designed.
  • at least one additional agent may be included to facilitate absorption of the anti-human RANKL antibody or its humanized antibody and/or any additional therapeutic agent.
  • diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablets, disintegrating agents, and binders may also be employed.
  • the pharmaceutical composition may contain an effective amount of an anti-human RANKL antibody or a humanized antibody thereof, with or without at least one additional therapeutic agent, in a non-toxic excipient mixture suitable for tablet preparation.
  • a single dosage form solution can be prepared by dissolving the tablet in sterile water, or other suitable vehicle.
  • suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or gum arabic; Or a lubricant such as magnesium stearate, stearic acid or talc.
  • sustained or controlled release delivery formulations containing an anti-human RANKL antibody or a humanized antibody thereof, with or without at least one additional therapeutic agent.
  • sustained or controlled release delivery formulation techniques such as liposome carriers, biodegradable microparticles or porous beads and extended-effect injections, are well known to those skilled in the art. . See, for example, PCT Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions.
  • the sustained release formulation may contain a semi-permeable form in the form of a tangible product
  • a permeable polymer matrix such as a film, or a microcapsule.
  • Sustained release matrices may include polyesters, hydrogels, polylactides (US3773919 and EP058481), copolymers of L-glutamic acid and Y-ethyl-L-glutamic acid (Sidman et al, Biopolymers, 22:547) -556 (1983)), poly(2-light ethyl-methacrylate) (Langer et al, J. Biomed. Mater. Res. 15: 167-277 (1981) and Langer, Chem.
  • the sustained release composition can also include liposomes, which can be prepared by several methods well known in the art. See, for example, Eppstein et al, Proc. Natl. Acad. Sc1. USA, 82: 3688-3692 (1985); EP 036676; EP 088046 and EP 143949.
  • compositions for general administration in vivo are sterile. In some embodiments, this can be achieved by filtration through a sterile filtration membrane. In some embodiments, where the composition is lyophilized, it can be sterilized using this method either before or after lyophilization and reconstitution. In some embodiments, compositions for parenteral administration can be stored in lyophilized form or in solution. In some embodiments, the parenteral compositions are typically placed in a container having a sterile outlet, such as an IV fluid bag or vial with a hypodermic needle that can be perfused with a hypodermic needle.
  • the pharmaceutical composition once the pharmaceutical composition is formulated, it can be stored as a solution, suspension, gel, emulsion, solid in a sterile vial, or as a dehydrated or lyophilized powder in a sterile vial. in. In some embodiments, such formulations may be stored in ready-to-use form or in a reconstituted form prior to administration (eg, lyophilized).
  • kits for preparing single dose dosing units can include a first container containing dried protein and a second container containing an aqueous formulation.
  • kits comprising single or multi-chamber pre-filled syringes eg, liquid injectors and dissolving syringes are included.
  • the effective amount of a pharmaceutical composition comprising an anti-human RANKL antibody or a humanized antibody thereof, with or without at least one additional therapeutic agent, to be used therapeutically depends, for example, on the therapeutic content and the subject. It will be apparent to those skilled in the art that, according to some embodiments, the appropriate dosage level of the treatment is, therefore, based in part on the delivered molecule, using an indication of an anti-human RANKL antibody or a humanized antibody thereof with or without at least one additional therapeutic agent, The route of administration differs from the size of the patient (weight, body surface area or organ size) and/or condition (age and general health). In some embodiments, the physician can adjust the dosage and alter the route of administration to achieve optimal therapeutic effects.
  • a typical dosage range may range from about 0.1 micrograms per kilogram to at most about 100 milligrams per kilogram or more, depending on the factors mentioned above. In some embodiments, the dosage can be from 0.1 micrograms/kg to up to about 100 mg/kg; or from 1 microgram/kg to up to about 100 mg/kg; or from 5 micrograms/kg to up to about 100 mg/kg.
  • the frequency of administration will take into account the pharmacokinetic parameters of the anti-human RANKL antibody or its humanized antibody and/or any additional therapeutic agent in the formulation used.
  • the physician applies the composition until a dose that achieves the desired effect is achieved. In some embodiments, it may therefore be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implant device or catheter
  • the pharmaceutical composition of the invention is administered.
  • a suitable dose can be determined by applying appropriate dose response data.
  • the route of administration of the pharmaceutical composition is a well-known method, for example, oral, intravenous, intraperitoneal, intracerebral (intrinsic), intraventricular, intramuscular, intraocular, intraarterial, intrahepatic , or intralesional route; through a sustained release system or through an implanted device.
  • the composition can be administered by bolus or continuous infusion or by an implant device.
  • the composition can be topically applied by implanting a film, sponge, or other suitable material onto which the desired molecule is adsorbed or the desired molecule is encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, release of the drug mass over time, or continuous administration.
  • a pharmaceutical composition comprising an anti-human RANKL antibody or a humanized antibody thereof, with or without at least one additional therapeutic agent, in an ex vivo manner.
  • the cells, tissues and/or organs taken from the patient are contacted with a pharmaceutical composition comprising an anti-human RANKL antibody or a humanized antibody thereof, with or without at least one additional therapeutic agent, and then these cells are then , tissue and / or organ transplanted back to the patient.
  • an anti-human RANKL antibody or an anti-human RANKL humanized antibody and/or any additional therapeutic agent can be delivered by implanting the genetically engineered cells to express and secrete the polypeptide using the methods described herein.
  • such cells can be animal cells or human cells and can be autologous, heterologous, or xenogeneic.
  • the cells can multiply indefinitely.
  • the cells can be encapsulated to avoid infiltration of surrounding tissue.
  • the capsule material is typically a biocompatible semipermeable polymeric outer shell or membrane that releases the protein product but prevents the patient's immune system or other harmful factors from surrounding tissue from damaging the cells.
  • CHO cells were stably transfected, and a cell line capable of stably expressing human RANKL was selected.
  • the cell strain was cultured on a large scale, and the cell supernatant was collected, and the human RANKL protein was purified by nickel column and used for mouse immunization, cloning screening and functional identification in Examples 2, 3, 4, 5, 6 and 7.
  • CHO cells were purchased from Invitrogen, RAW264.7 cells were purchased from the Cell Bank of Shanghai Academy of Sciences, and the expression vector was provided by the company.
  • T4 DNA ligase, protein molecular weight standard Marker, restriction endonuclease and the like were purchased from NEB; Invitrogen; 302 medium, trypsin and FBS were purchased from Invitrogen; Phenyl sepharose 6FF-low sub, SP-Sepharose FF, Ni-NTA Sepharose FF gel was purchased from GE; disodium hydrogen phosphate, sodium dihydrogen phosphate, Sodium chloride, Tris, citric acid, trisodium citrate, imidazole and MTX were purchased from Sigma, goat anti-human IgG-HRP was purchased from Jackson, TMB Substrate, purchased from Cell Signaling, RANK-Fc, TRANC, M-CSF TGF- ⁇ was purchased from R&D, TRAP chromogenic system was purchased from Sigma, DMEM medium
  • the multi-function microplate reader is Molecular Devices SpectraMax M5 Multi-mode Microplate Reader is TECAN HydroFlex Plate Washer, the ultra-clean workbench brand is Sujing, the specification model SW-CJ-2F/T, the carbon dioxide incubator is branded Thermo, Specification model Forma 311.
  • the human RANKL target sequence was synthesized by genetic engineering (Fig. 1).
  • the sequence starts from 136 Gly of natural human RANKL, and 182 amino acids (SEQ ID NO: 1) to 317 Asp, and 10 His at the N-terminus. It is combined with nickel chloride in a nickel column to be purified by ion affinity chromatography, and two restriction sites of NotI and PmeI are added at both ends.
  • the recombinant human RANKL and the expression vector were digested with NotI and PmeI, and the human RANKL target fragment and expression vector fragment were recovered, ligated, transformed, positive clones were identified by PCR and restriction enzyme digestion, and finally the expression vector was verified by sequencing. Correctness.
  • the plasmid was extracted using a plasmid extraction kit for stable transfection.
  • CHO cells Forty-eight hours prior to transfection, CHO cells were subcultured in 302 serum-free medium at a seeding density of 3 x 10 5 /ml. The total number of cells on the day of transfection should be greater than 1.5 ⁇ 10 7 and the cell viability should be above 95%. The conversion was carried out using a Bio-rad electrorotator with a transfection voltage of 300 V and a capacitance of 900 ⁇ F. The cells should be passaged every 2 to 3 days, and the culture medium should be replaced with fresh medium until the growth of the cells returns to normal. When the cell viability is above 90%, MTX pressure screening is performed.
  • the concentration of MTX was gradually increased by 25 nM, 50 nM, 100 nM, 250 nM, 500 nM.
  • the plate was divided according to the limiting dilution method.
  • the Dot-blot assay was performed. The supernatant of the 5 ⁇ l 96-well plate was taken and spotted on a nitrocellulose membrane. After air-drying, 5% skim milk powder was added to the room temperature.
  • the cell supernatant was loaded at a flow rate of 30 ml/h, washed with Binding buffer for 10-20 column volumes, and then eluted with 10 mM imidazole, 70 mM, 100 mM and 500 mM imidazole, respectively, and the eluate was collected, human RANKL.
  • the main elution peak appeared in the elution condition of 70 mM imidazole.
  • the purified human RANKL protein was identified by reduction SDS-PAGE electrophoresis, and its molecular weight was about 34 KDa, which was consistent with the theoretical molecular weight and the purity was greater than 90%.
  • RANK-Fc was prepared by gradient dilution from 10 ⁇ g/ml, incubated for 1 hour at room temperature, and a 1:200 diluted secondary antibody (goat anti-human IgG-Fc) was added, incubated for 1 hour at room temperature, and developed using a TMB color development system.
  • the absorbance of each well in a 96-well plate was measured on a multi-function microplate reader at a measurement wavelength of 450 nm and a reference wavelength of 630 nm.
  • the absorbance (OD) of each well was OD450nm-OD630nm.
  • the binding activity of human RANKL to RANK-Fc was identified.
  • the results showed that the recombinant human RANKL coated on the ELISA plate bound to its receptor RANK-Fc, and its binding was RANK-Fc concentration-dependent and saturated, reaching a 50% maximal binding RANK-Fc concentration of 10.9 ng/ml.
  • the combination is consistent with R&D's commercially available RANKL standard, which achieves a 50% maximum binding RANK-Fc concentration of 5.6 ng/ml.
  • RAW264.7 cells were cultured in DMEM + 10% FBS medium and passaged every 3-4 days.
  • the cell culture medium in the logarithmic growth phase was changed to ⁇ -MEM medium + 10% FBS to prepare a cell suspension.
  • the 96-well plate was inoculated at 2000 cells/100 ⁇ l, and cultured at 37 ° C for 1 hour in a 5% CO 2 incubator.
  • a double dilution of human RANKL starting at 800 ng/ml, a double dilution of the commercially available hRANKL standard and fixed concentrations of M-CSF and TGF[beta] starting at 200 ng/ml were added.
  • recombinant and purified human RANKL not only retains its ability to bind to the corresponding receptor RANK-Fc, but also has the biological activity by agonizing the differentiation of osteoclasts through the receptor.
  • Recombinant human RANKL was used as an antigen for the immunization procedures described herein and was used as a material for the screening of positive clones for coating proteins and antibody immunological and cytological functions as an ELISA method.
  • the immunizing antigen (recombinant human RANKL) was from Example 1, and the BALB/c mouse was purchased from Beijing Tonglihua Experimental Animal Technology Co., Ltd. Monoclonal antibodies against RANKL were obtained by multiple immunizations of BALB/c mice.
  • the immunization route was a plantar injection with an immunization dose of 10 ⁇ g/50 ⁇ l/mouse, 25 ⁇ l per sole. A total of 10 mice were immunized.
  • mice were subjected to the fifth (20th) immunization and the ninth (Day 40) immunization, respectively, and blood was collected from the eye, and the titer of the anti-human RANKL antibody in the serum of the immunized mice was determined by ELISA.
  • human RANKL was diluted with coating buffer (0.1 M coating buffer, pH 9.6 NaHCO 38.4 g/L) to 1 ⁇ g/ml, 100 ⁇ l/well coated in 96-well ELISA plate (Corning, Acton, MA), 4 Overnight.
  • coating buffer 0.1 M coating buffer, pH 9.6 NaHCO 38.4 g/L
  • the plates were washed 3 times with 1 x PBST (0.05% Tween 20 in 1 x PBS), and 200 ul/well was added to blocking solution (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in 1x PBS) for 1 hour at room temperature.
  • the plate was washed 3 times, the mouse serum was diluted 3-fold from 1:100 with 0.5% BSA/PBS, the blank well was 0.5% BSA/PBS, 100 ⁇ l/well was added to the ELISA plate, incubated for 2 hours at room temperature, and the plate was washed 3 times.
  • Sheep anti-mouse IgG Fc-HRP was added at a final concentration of 1 ⁇ g/ml and incubated for 1 hour at room temperature. Wash the plate 3 times and add TMB (BioFx BSTP-0100-01) The color developing solution was developed at room temperature for 10-20 minutes, and the stop solution was added and read at 450 nm. A 2-fold greater OD value than a blank well was defined as a positive clone, and a higher OD value at the highest dilution factor indicates a stronger immunoreactivity to human RANKL.
  • the serum titer detection data is shown in Table 1.
  • the serum titer range of mice was 1:62500 except for 4# and 8#, and the other mice reached 1:312500.
  • the serum titer range of mice was 1:312500 except for 1# and 4#, and the serum titers of the other mice reached 1:1562500.
  • mice The immunized mice were euthanized with CO 2 , then the cervical vertebrae were dislocated, lymph nodes were isolated, and lymph nodes derived from different mice were combined.
  • the cells were placed in DMEM medium for grinding, the supernatant was collected, and lymphocytes were obtained by centrifugation and counted using a hemocytometer.
  • the B cells obtained above were washed and mixed with non-secretory myeloma cells P3X63Ag8.653 (ATCC, Cat# CRL1580) at 1:1, and the cell mixture was centrifuged at 800 g, and the supernatant was gently removed, and 2-4 ml was added.
  • Chain enzyme protease solution (CalBiochem, cat.
  • a sterile medium-containing hybridoma medium (DMEM (JRH Biosciences), 15% FBS (Hyclone), supplemented with L-glutamine, pen/strep, OPI. (oxaloacetate, pyruvate, bovine insulin) (all from Sigma) and IL-6 (Boehringer Mannheim) in a centrifuge tube, incubate at 37 degrees for 15-30 minutes, then centrifuge at 400g (1000rpm) for 5 minutes, gently place the cells Resuspend in a small amount of hybridoma screening medium (Hybridoma Culture Medium supplemented with 0.5x HA (Sigma, cat.
  • Human RANKL was diluted to 1 ⁇ g/ml with coating buffer (0.1M Carbonate Buffer, pH 9.6, NaHCO 38.4g/L), coated at 50 ⁇ l/well, 4° overnight, the next day, with washing solution (0.05% Tween) Wash in 3 times with PBS), and add 200 ⁇ l/well to the blocking solution (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in 1x PBS) for 1 hour at room temperature, wash the plate 3 times, and add 50 ⁇ l of hybridoma cell supernatant to each well.
  • coating buffer 0.1M Carbonate Buffer, pH 9.6, NaHCO 38.4g/L
  • washing solution 0.05% Tween
  • positive and negative controls (positive control is human RANKL immunized mouse serum, negative control is pre-immune BALB/c mouse serum), incubate for 2 hours at room temperature, wash plate 3 times, add 1:2000 dilution of sheep per well Anti-mouse IgG-HRP (Jackson Lab, Cat. No: 115-035-062) Incubate for 1 hour at room temperature, wash the plate 3 times, add 100 ⁇ l of TMB color solution (BioFX Lab. Cat. No.TMSK-0100-01) to each well for 10 minutes at room temperature, and add 50 ⁇ l of stop solution per well. , reading at 450 nm. As a result, after a first round of screening, a total of 190 96-well cell culture plates were screened for 435 positive clones (OD values greater than 0.5) capable of binding to human RANKL.
  • the results of the primary screening ELISA were positive for RANKL antibody.
  • the medium of the positive clone was removed, fresh hybridoma medium was added, and the clone was transferred to 24-well plate culture. After 2 days, the 24-well plate clone was subjected to a second round of ELISA screening.
  • the experiments included the same direct ELISA assay, competitive inhibition assay and immunological cross-reactivity with mouse RANKL. Among them, the immune cross-reaction of mouse RANKL replaced the coating protein with mouse RANKL, the coating concentration was 1 ⁇ g/ml, and the test sample was the original cell supernatant, one concentration point, and the other experimental conditions were consistent with the primary screening ELISA method.
  • the competition inhibition experiment was a single point test, and RANK-Fc was coated with a coating solution to 1 ⁇ g/ml for coating at 4 degrees overnight.
  • the original cell supernatant was mixed with an equal volume of human RANKL at a concentration of 600 ng/ml, added to an ELISA plate, incubated for 2 hours at room temperature, washed 3 times, and added 1:10000 diluted rabbit anti-human His-HRP (purchased from Abcam antibody was incubated for 1 hour at room temperature, washed 4 times, and developed using a TMB color development system.
  • the second round of ELISA results showed that 249 clones had an OD value greater than 0.5, 41 clones had the activity of inhibiting the binding of human RANKL to RANK-Fc, and 9 clones had an immunological cross-reaction with murine RANKL.
  • the above 50 clones were subcloned, the cell strain was frozen, and the cell supernatant was purified for functional identification of Examples 4, 5, 6, and 7.
  • Human RANKL protein was diluted to 1 ⁇ g/ml with 0.05 M carbonate buffer, pH 9.6, and 100 ⁇ l per well was added to a 96-well ELISA plate and allowed to stand overnight at 4 °C. The plate was washed 3 times with PBST, blocked with PBST + 2% BSA for 1 hour, and then washed 3 times with PBST. The antibody to be tested was serially diluted 10 times from 10 ⁇ g/ml to 1 ⁇ 10 5 ⁇ g/ml with PBST, and 100 ⁇ l per well was added to the ELISA plate. The sample was double-replicated, and 5 samples were prepared for each 96-well ELISA plate. A total of 10 96-well ELISA plates were made.
  • the plate was washed 3 times with PBST. 1:10000 diluted HRP-labeled goat anti-mouse IgG (purchased from Jackson) was added to the wells of the test antibody, 100 ⁇ l per well, and incubated for 1 hour at room temperature, and then washed 4 times with PBST washing solution. 100 ⁇ l of TMB chromogenic solution (purchased from Cell Signaling) was added to each well, and allowed to stand at room temperature for 7 minutes in the dark, and 50 ⁇ l of 1 M H 2 SO 4 was added to each well to terminate the color reaction.
  • TMB chromogenic solution purchased from Cell Signaling
  • the absorbance of each well in a 96-well plate was measured on a Molecular Devices SpectraMax M5 Multi-mode Microplate Reader at a measurement wavelength of 450 nm and a reference wavelength of 630 nm.
  • the absorbance (OD) of each well was OD450nm-OD630nm.
  • the absorbance per well measured was plotted on the ordinate and plotted by Sigmoidal dose-response (variable slope) (GraphPad Prism software) to obtain an antigen-antibody binding curve (Fig. 2).
  • the results showed that the majority of anti-RANKL murine monoclonal antibodies were concentration-dependently bound to human RANKL coated on the solid surface of the ELISA plate and reached saturation.
  • the EC50 was between 10-20 ng/ml. See Table 2 for detailed data.
  • the direct ELISA method was used to detect the cross-reaction of anti-human RANKL murine monoclonal antibody with cynomolgus RANKL and mouse RANKL.
  • the experimental method was identical to that of Example 4. Human RANKL, monkey RANKL and mouse RANKL were used for coating, and both monkey RANKL and mouse RANKL were purchased from R&D.
  • the antibody concentration was plotted on the abscissa, and the absorbance per well was measured on the ordinate.
  • the antigen-antibody binding curve was obtained by Sigmoidal dose-response (variable slope) mapping (GraphPad Prism software). The results indicated that all murine monoclonal antibodies that bind to human RANKL bind to monkey RANKL, and their binding is concentration-dependent and can reach saturation with an EC50 between 10-20 ng/ml ( Figure 3, Table 3).
  • Figure 3, Table 3 Nine of the antibodies tested cross-reacted with mouse RANKL. The binding is concentration dependent and can be saturated with an EC50 between 10-20 ng/ml.
  • Example 6 Purified murine monoclonal antibody inhibits the binding activity of human RANKL to RANK-Fc
  • the anti-human RANKL murine mAb was tested for its neutralizing activity against the binding of human RANKL to RANK-Fc using a competition ELISA assay and compared to the commercial product Denosumab.
  • the RANK-Fc protein was diluted to 1 ⁇ g/ml with 0.05 M carbonate buffer, pH 9.6, and 100 ⁇ l per well was added to a 96-well ELISA plate and allowed to stand overnight at 4 °C. The plate was washed 3 times with PBST, blocked with PBS + 2% BSA for 1 hour, and then washed 3 times with PBST.
  • the antibody to be tested and Denosumab were serially diluted 4-fold from 20 ⁇ g/ml to 0.0024 ⁇ g/ml with PBS+2% BSA, and mixed with an equal volume of human RANKL (concentration: 0.6 ⁇ g/ml), 100 ⁇ l per well.
  • the sample was double-replicated, and 5 samples were made for each ELISA plate, and each of the ELISA plates contained Denosumab control, a total of 10 ELISA plates. After incubating for 2 hours at room temperature, the plate was washed 3 times with PBST.
  • a 1:10000 diluted HRP-labeled goat anti-His-tagged secondary antibody (purchased from Abcam) was added to an ELISA plate, 100 ⁇ l per well, and incubated for 1 hour at room temperature, and then washed 4 times with PBST washing solution. 100 ⁇ l of TMB chromogenic solution (purchased from Cell Signal) was added to each well, and the mixture was allowed to stand at room temperature for 15 minutes in the dark, and 50 ⁇ l of 1 M H 2 SO 4 was added to each well to terminate the color reaction.
  • the absorbance of each well in a 96-well plate was measured on a multi-function microplate reader at a measurement wavelength of 450 nm and a reference wavelength of 630 nm. The absorbance (OD) of each well was OD450nm-OD630n.
  • the measured absorbance value is plotted on the ordinate, and the Sigmoidal dose-response (variable slope) method is used for mapping (GraphPad Prism software) to obtain a curve in which the antibody competitively inhibits the binding of the receptor to the ligand.
  • Sigmoidal dose-response variable slope
  • the EC50 value of Denosumab was 0.67 ⁇ g/ml, and the EC50 values of the 8 antibodies measured under the same experimental conditions were between 0.2-1.0 ⁇ g/ml.
  • Figure 4 shows the results of experiments in which 8 samples inhibited the binding of RANKL to RANK-Fc. See Table 4 for details.
  • Example 7 murine monoclonal antibody inhibits human RANKL-induced differentiation of RAW264.7 cells
  • Human RANKL can induce RAW264.7 cells (purchased from Shanghai University of Chinese Academy of Sciences) to differentiate into osteoclasts.
  • the purpose of this experiment was to evaluate whether anti-human RANKL murine monoclonal antibody capable of neutralizing RANKL and RANK-Fc can inhibit human RANKL.
  • the cell differentiation activity of RAW264.7 mouse mononuclear macrophage leukemia cells, purchased from the Shanghai Library of Chinese Academy of Sciences
  • Denosumab There is a characteristic enzyme-anti-tartaric acid phosphatase in osteoclasts.
  • the pNPP chromogenic system can reflect the activity of tartaric acid phosphatase by the high and low OD405nm readings, thus reflecting the degree of differentiation of RAW264.7 cells into osteoclasts.
  • RAW264.7 cells were cultured using DMEM + 10% FBS (purchased from Invitrogen) medium and passaged every 3-4 days. When the cell activity assay was performed, the cell culture medium in the logarithmic growth phase was changed to ⁇ -MEM medium + 10% FBS (purchased from Invitrogen) to prepare a cell suspension.
  • the 96-well plate was inoculated at 2000 cells/100 ⁇ l, and cultured for 1 hour at 37 ° C in a 5% CO 2 incubator (Thermo Corporation, specification model Forma 311).
  • a double-diluted sample to be tested 22 antibodies in total, 7 96-well cell culture plates) and Denosumab, and human RANKL and MCSF, TGF ⁇ (purchased from R&D) were added.
  • the final concentrations of the antibodies to be tested were 2000, 1000, 500, 250, 125 and 62.5 ng/ml
  • the final concentration of human RANKL was 150 ng/ml
  • the final concentrations of MCSF and TGF ⁇ were 20 ng/ml and 2 ng/ml, respectively.
  • the measured absorbance was plotted on the ordinate and plotted by Sigmoidal dose-response (variable slope) (GraphPad Prism software) to obtain a curve in which the antibody inhibited the differentiation of RAW264.7 cells into osteoclasts.
  • anti-RANKL murine mAb can completely inhibit the differentiation of human RANKL-induced RAW264.7 cells into osteoclasts, which is dose-dependent, except for 114-6.6.7/114-6.6.18/114-6.6.29
  • the sample has a poor inhibitory effect, and the other 19 antibodies have a strong inhibitory activity and a very low EC50 value.
  • Denosumab has an EC50 value of 334.4 ng/ml, while the eight antibodies measured under the same experimental conditions have an EC50 value between 200-800 ng/ml.
  • the smaller the EC50 value the stronger the activity of the antibody against RANKL-induced RAW264.7 cell differentiation, and 3 of the 8 antibodies obtained were superior to Denosumab, and 2 antibody activities were comparable to those of Denosumab.
  • Figure 5 shows the results of inhibition of human RANKL-induced differentiation of RAW264.7 cells into osteoclasts by 8 samples. See Table 5 for details.
  • Table 5 EC50 and other relevant data of murine monoclonal antibody inhibiting human RANKL-induced RAW264.7 cell differentiation
  • the collected 22 samples were sent to Nanjing Kingsray Biotechnology Co., Ltd. for determination of the nucleotide sequence of the CDR regions.
  • the variable region sequences of the heavy and light chains were amplified by RACE method, and the obtained variable region sequences were constructed into a sequencing vector, and five positive clones were selected for each variable region sequence for nucleotide sequence. Determination.
  • the nucleotide sequence is translated into an amino acid sequence. By sequence alignment, the final heavy and light chains each yielded 8 unique CDR sequences.
  • mice were immunized with recombinant human RANKL (hRANKL) and hybridoma technology was used to obtain a series of murine anti-human RANKL monoclonal antibodies.
  • Some antibodies with neutralizing activity were screened by competitive ELISA and cytology experiments, which blocked the binding of RANKL to its receptor RANK-Fc and inhibited the differentiation of RAW264.7 cells into osteoclasts.
  • monoclonal antibody 7.19.12 is one of the antibodies with high binding ability and neutralizing activity, and the heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences of 7.19.12 have been patented in China. Published in the public (patent number CN201310753972.3).
  • the scFv construction of 7.19.12 is to use the PCR technique to link 7.19.12VH (SEQ ID NO: 6) and VL (SEQ ID NO: 14) with a linker region (Gly4Ser)3 to form a single-chain antibody (scFv) gene.
  • the T vector was then ligated into the T vector to obtain a 7.19.12 scFv (SEQ ID NO: 21) recombinant plasmid.
  • VH5'NcoI and VH3'XhoI link as primers, and 7.19.12VH plasmid as a template
  • pfu DNA polymerase purchased from Beijing Quanjin Biotech Co., Ltd.
  • pfu DNA polymerase was used to amplify NcoI and XhoI-containing sites by PCR.
  • the VH and VL gene fragments obtained by PCR were used as templates, and VH5'NcoI and VL3'NotI- ⁇ were used as primers, and PCR-amplified with pfu DNA polymerase to obtain a 7.19.12 scFv gene fragment.
  • the obtained 7.19.12 scFv product was subjected to extension at 72 ° C for 1 hour by rTaq polymerase (purchased from TAKARA Co., Ltd.) so that the 3' end of the scFv product was added with A. After adding A, the scFv product was purified by gel recovery and then treated with T4 DNA ligase (purchased from NEB).
  • the 18-T vector was ligated (16 ° C, 1 hour).
  • the heat shock method (42 ° C, 90 seconds) was used to transform Escherichia coli, and the bacterial solution was plated (LBAmp medium) and cultured at 37 ° C overnight.
  • the clones were picked for colony PCR (general primers M13-47 and M13-48) for identification screening, and clones positive for PCR were subjected to sequencing analysis, and a 7.19.12 scFv recombinant plasmid was obtained by screening. 7.19.12 scFV construction primers are shown in Table 7.
  • the phagemid vector pFL249 was provided by Nanjing Jinsrui Co., Ltd., NcoI and NotI were used as insertion sites of scFv gene, pelB was signal peptide (directing scFv secretion into periplasmic cavity), gIII249 was phage recognition region, and ampicillin resistance gene was used. To screen and maintain the role of phagemids.
  • the scFv gene is ligated to the c-myc tag and the his tag, wherein the his tag can be used for affinity purification, and the c-myc tag can be recognized by the anti-c-myc antibody for detection purposes.
  • a succinic acid terminator (TAG) is present between the his tag and gIII, and the phagemid DNA is partially inhibited by the inhibitory E. coli TG1 (purchased from Lucigen Corporation No. 60502-1) by the action of helper phage.
  • the scFv is displayed on the surface of the phage.
  • the pMD18-7.19.12 scFv plasmid was digested with NcoI and NotI (purchased from NEB) restriction enzymes to obtain a 7.19.12 scFv gene fragment with sticky ends, and ligated with the pFL249 vector subjected to the same double digestion treatment.
  • the ligation, transformation and identification methods were the same as in Example 9, and the clones were subjected to sequencing analysis to obtain wild-type pFL249-7.19.12 scFv phagemid (see Fig. 6).
  • the human VH and VL ligand sequences with the highest homology to the 7.19.12 antibody framework regions were determined by the IgBlast search database (IMGT human V genes (F+ORF+in-frame P)).
  • the FR sequences were examined to determine different amino acid residues between the murine and human sequences, and the importance of each different residue for antigen binding was evaluated. Finally, it will be difficult to determine the importance of antigen binding, and both murine and human copies of human murine residues are incorporated into humanized antibody sequences.
  • Murine monoclonal antibody 7.19.12VH humanized library mutation strategy (fixed 7.19.12 VL, mutation of 9.19.12VH FR region) is shown in Table 8
  • murine monoclonal antibody 7.11.12VL humanized library mutation strategy (fixed 7.19.12VH, The mutation in the FR region of 7.19.12 VL is shown in Table 9.
  • the theoretical design diversity of VH humanized library is 1 ⁇ 10 5
  • the theoretical design diversity of VL humanized library is 9216.
  • the 7.19.12 scFv humanized library is synthesized by degenerate base method (degenerate base representation: N Represents A/G/T/C, W stands for A/T, B stands for G/T/C, D stands for G/A/T, R stands for A/G, Y stands for C/T, and V stands for A/G/C , M stands for A/C, S stands for G/C, H stands for A/C/T, and K stands for G/T).
  • N degenerate base representation: N Represents A/G/T/C
  • W stands for A/T
  • B stands for G/T/C
  • D stands for G/A/T
  • R stands for A/G
  • Y stands for C/T
  • V stands for A/G/C
  • M stands for A/C
  • S stands for G/C
  • H stands for A/C/T
  • K stands for G/T
  • the construction of the anti-hRANKL murine monoclonal antibody 7.19.12 scFv library was outsourced to Nanjing Kingsray Biotechnology Co., Ltd.
  • the idea of library synthesis was to separate VH and VL into 4 oligonucleotide sequences of about 60-75 bp in size, and the fragments overlap by about 20 bp.
  • the VL-immobilized VH mutant library and VH-fixed were synthesized by overlap extension PCR. VL mutant library.
  • GenScript standard operating procedures the synthetic V H, V L primer (see Table 10) were mixed in amounts equimolar annealing, extending and amplifying the complete V H, V L fragment was purified by gel extraction and cloned into sequencing vectors, respectively 15, 20 random clones were sent to test PCR products were analyzed diversity, the final selection of clone ORF normal percentage 60%, PCR product was cloned and ORF normal V H percentage purified in two batches of 66% 75% of the VL purified PCR product was used for library construction.
  • VH PCR product was digested with Sfi I and Xho I, ligated with pFI249 ⁇ 7.19.12scFv vector digested with Sfi I and Xho I, and TG1 was electroporated to construct a VL-immobilized VH mutant library;
  • the VL PCR product was digested with Sac I and Not I, ligated with the pFL249 ⁇ 7.19.12 scFv vector digested with Sac I and Not I, and TG1 was electrotransformed to construct a VH-immobilized VL mutant library.
  • the library construction was first tested by ligation and electroporation, and a large number of ligation and electroporation were performed according to the optimized conditions.
  • the results showed that the positive rate was 98.6% (142/144, Figure 7); the second batch of VH mutant library had a storage capacity of 1.04 ⁇ 10 7 (Table 12), and 96 primers were randomly selected with primer M13R ( ⁇ 48) and M13F ( ⁇ 47) was used for PCR colony screening.
  • the PCR colony screening showed a positive rate of 99% (95/96, Figure 8).
  • the total storage capacity of the VH mutant library was about 2.04 ⁇ 10 7 , and the concentration of the two batches of bacteria was combined. It is 6.7 ⁇ 10 9 cfu/ml.
  • the VL mutant library had a reservoir size of 2.8 ⁇ 10 8 and a bacterial concentration of 2 ⁇ 10 11 cfu/ml (Table 12). 144 transformants were randomly picked and PCR primers were used for primers M13R ( ⁇ 48) and M13F ( ⁇ 47).
  • a transformant with a ⁇ 1500 bp DNA band indicates that the VH insert is contained, and the transformant without the DNA band is the background of the vector.
  • PCR colony screening showed a positive rate of 96.5%, (139/144, Figure 9).
  • the VH/VL mutant library randomly selected 100 clones that were positive for PCR colony screening for sequencing analysis. The sequencing results showed that 58/70 clones could be translated normally and matched the mutation design, and the diversity and the small test results were equivalent.
  • the first batch of VH mutant libraries obtained about 1.5 ⁇ 10 6 transformants by single electrotransformation, and the second batch was about 1.3 ⁇ 10 6 , and 9 and 8 electrotransformations were performed respectively, with a total storage capacity of about 2.4 ⁇ 10 7 .
  • a single electrotransformation of the VL mutant library yielded about 2.8 ⁇ 10 7 transformants, and a total of 10 electrotransformations were performed with a total storage capacity of about 2.8 ⁇ 10 8 .
  • the library was amplified, and a certain amount of VH mutant library glycerol solution was taken to 370 ml of 2xYT medium (hereinafter referred to as 2xYT-AG) containing 100 ⁇ g/ml Amp and 2% glucose, so that the OD600 was between 0.05 and 0.1 at 37. Incubate at °C, 200 rpm/min until the OD600 reaches 0.3 to 0.4. M13KO7 helper phage (purchased from NEB, item number N0315S) was added to the culture broth for 20 times, mixed, and then inoculated at 37 ° C for 30 minutes, and then shaken at 37 ° C, 200 rpm / minute for 1 hour. .
  • 2xYT-AG 2xYT medium
  • M13KO7 helper phage purchased from NEB, item number N0315S
  • 2xYT-AK 2xYT medium
  • the supernatant was taken, and a 1/4 supernatant volume of PEG/NaCl solution (20% (w/v) polyethylene glycol (PEG) 8000, 2.5 M NaCl) was added, thoroughly mixed, and precipitated on ice for 1 hour. After centrifugation at 8000 rpm/min for 30 minutes at 4 ° C, the supernatant was removed and the pellet was resuspended in 2.2 ml of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.2-7.4).
  • PBS 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.2-7.4
  • the supernatant (this is a recombinant phage library displaying scFv) was stored at 4 ° C until use (10 ⁇ l was titrated with a gradient of 10 -2 to 10 -10 to determine the titer).
  • hRANKL was diluted to 10 ⁇ g/ml with PBS, and added to an Immunotube tube (purchased from NUNC, Cat. No. 470319) at 1 ml/tube, and coated overnight at 4 °C.
  • the VL mutant library enriched the panning process with the VH library.
  • Table 13 shows the amount of recombinant phage input and the total number of clones after panning.
  • Example 13 To screen for positive clones that specifically bind to hRANKL, the clones selected in Example 13 were induced to express scFv by IPTG and detected by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • 5 ⁇ 96 clones were randomly selected from the third and fourth rounds of the SOBAG cloned plates and inoculated into sterile 96 deep well plates containing 400 ⁇ l of 2 ⁇ YT-AG medium. Incubate overnight at 30 ° C, shaking at 200 rpm.
  • the deep-well plate solution was transferred to a new 96-deep well plate containing 400 ⁇ l of 2xYT-AG fresh medium at a ratio of 1:50, and cultured at 37 ° C, shaking at 200 rpm until the OD600 was about 0.8 (about 3 hours). Collect the cells by centrifugation at 1500g for 10 minutes, The culture supernatant was transferred and discarded, and the cells were resuspended in 400 ⁇ l of sterile fresh 2xYT-AI (I refers to IPTG, final concentration of 100 mM) medium, and cultured overnight at 30 ° C with shaking at 200 rpm (i.e., induced expression of scFv overnight).
  • I refers to IPTG, final concentration of 100 mM
  • the deep well plates in which the scFv supernatant was induced to be induced overnight were centrifuged at 3000 g for 20 minutes, and 300 ⁇ l of the supernatant was transferred to a clean 96-well cell plate for use.
  • hRANKL was diluted to 1 ⁇ g/ml with 1 ⁇ PBS (pH 7.4), and added to a 96-well microtiter plate (purchased from NUNC, Cat. No. 442404) at 50 ⁇ l/well for a total of 10 plates, and coated at 4 ° C overnight. After washing the plate three times with PBST (containing 0.05% Tween 20), it was blocked with a blocking solution (2% (w/v) BSA in PBS) for 1 to 1.5 hours at room temperature.
  • PBST containing 0.05% Tween 20
  • the induced scFv supernatant was transferred to a closed enzyme plate, 50 ⁇ l/well, and the well containing only 2xYT-AI medium was used as a negative control, and the wild type 7.19.12 scFv supernatant was positive. Control, incubate for 2 hours at room temperature.
  • the plate was washed 3 times with PBST, then a 1:5000 dilution of an anti-c-myc HRP-labeled secondary antibody (purchased from Bethyl Corporation, Cat. No. A190-104P) was added and incubated for 1 hour at room temperature.
  • TMB substrate purchased from Cell Signaling, Cat. No. 7004
  • 50 ⁇ l of 2 M HCl was added to each well to stop color development, and the OD450nm reading was detected using an M5 instrument.
  • a third round of 33 clones with OD450 > 1.0 was selected, and a fourth round of 114 clones with OD450 > 1.5, a total of 147 clones.
  • the three heavy chain variable regions H16 (SEQ ID NO: 23), H16u (SEQ ID NO: 25) and H114 (SEQ ID NO: 27) obtained by the phage panning platform were constructed into IgG2 full-length heavy chains, respectively.
  • the obtained four light chain variable regions L10 (SEQ ID NO: 31), L10u (SEQ ID NO: 33), L37 (SEQ ID NO: 35), and L37u (SEQ ID NO: 37) were respectively constructed into Kappa.
  • the full-length light chain, full-length heavy and light chains were constructed into the eukaryotic expression vector pCDNA3.1, respectively.
  • FR framework regions of the heavy light chain variable region
  • FR1 framework regions of the heavy light chain variable region
  • FR2 framework regions of the heavy light chain variable region
  • FR3 and FR4 were searched through the IgBlast search database (IMGT human V genes (F+ORF+in -frame P)) alignment, obtaining the human FR region with the highest homology, and combining the human FR region with the wild type 7.19.12 CDRs to obtain a new VH (designated H16 (hFR), SEQ ID NO: 29) And VL (designated WT-VL (hFR), SEQ ID NO: 39) sequence.
  • IMGT human V genes F+ORF+in -frame P
  • variable region gene was obtained by a whole gene synthesis method, and constructed into a full-length heavy chain and a light chain by the same method as above, and ligated to the expression vector pCDNA3.1.
  • Table 14 shows the amino acid sequence alignment of the heavy chain variable region of the anti-hRANKL humanized antibody
  • Table 15 shows the amino acid sequence alignment of the light chain variable region of the anti-hRANKL humanized antibody
  • 7.19.12 is the original murine sequence.
  • H16 heavy chain variable region DNA sequence (SEQ ID NO: 22)
  • H16 heavy chain variable region amino acid sequence (SEQ ID NO: 23)
  • H16u heavy chain variable region DNA sequence (SEQ ID NO: 24)
  • H16u heavy chain variable region amino acid sequence (SEQ ID NO: 25)
  • H114 heavy chain variable region DNA sequence (SEQ ID NO: 26)
  • H114 heavy chain variable region amino acid sequence (SEQ ID NO: 27)
  • H16 (hFR) heavy chain variable region DNA sequence SEQ ID NO: 28
  • H16 (hFR) heavy chain variable region amino acid sequence (SEQ ID NO: 29)
  • WT-VL (hFR) light chain variable region DNA sequence SEQ ID NO: 38
  • WT-VL (hFR) light chain variable region amino acid sequence SEQ ID NO: 39
  • the H16 heavy chain variable region VH was amplified by PCR; the human IgG2 heavy chain obtained by gene synthesis
  • the human region IgG2 heavy chain constant region (SEQ ID NO: 41) gene fragment was amplified by PCR using the constant region plasmid as a template; the H16 heavy mammalian expression signal peptide (SEQ ID NO: 45) was amplified by overlapping PCR technique.
  • the chain gene VH-CH (SEQ ID NO: 47); the H16 full-length heavy chain gene was constructed into pCDNA3.1 (+) (purchased from Invitrogen, Cat. No. V790-20) vector to obtain an H16 full-length heavy chain expression vector.
  • the H16VH and IgG2 type CH gene fragments were amplified using pfu DNA polymerase (purchased from Beijing Quanjin Biotechnology Co., Ltd.).
  • pfu DNA polymerase purchased from Beijing Quanjin Biotechnology Co., Ltd.
  • a VH gene fragment containing partial eukaryotic expression signal peptide was amplified by PCR;
  • IgG2-CH-F and IgG2-CH- R is a primer and a human IgG2 plasmid is used as a template.
  • a CH gene fragment containing a TAG stop codon and a NotI restriction site is amplified by a PCR method, and an overlapping sequence of at least 20 bp is present between VH and CH.
  • the PCR conditions were: 95 ° C for 2 minutes, [95 ° C for 20 seconds, 55 ° C for 20 seconds, 72 ° C for 40 seconds] 30 cycles, 72 ° C for 5 minutes.
  • the H16 heavy chain gene was amplified with pfu DNA polymerase. Using the VH and CH gene fragments obtained by the above PCR as a template, using Whole-SP-F and IgG2-CH-R as primers, PCR-amplified the EcoRI and NotI restriction sites, and the eukaryotic expression signal peptide sequence H16 Heavy chain gene. PCR conditions: 95 ° C for 2 minutes, [95 ° C for 20 seconds, 55 ° C for 20 seconds, 72 ° C for 100 seconds] 35 cycles, 72 ° C for 5 minutes.
  • the H16 heavy chain gene obtained in the above manner was treated according to the PCR product of Example 9 and purified by gel, and then purified by T4 DNA ligase (purchased from NEB).
  • the 19-T simple vector was ligated (16 ° C, 1 hour), heat shocked (42 ° C, 90 sec) was transformed into E. coli, plated (LB Amp medium) and cultured overnight at 37 ° C, and clones were picked for colony PCR ( Universal primers M13-47 and M13-48) were screened for identification.
  • the clones positive for PCR were further subjected to plasmid extraction, double digestion with EcoRI and NotI, and the H16 heavy chain fragment of about 1500 bp was recovered by cleavage, and the pCDNA3.1(+) vector recovered by the same double digestion treatment was used. connection.
  • the H16 IgG2 heavy chain clone was confirmed by sequencing, and the recombinant plasmid was 6800 bp in length and contained H16 heavy chain variable region and human IgG2 heavy chain constant region (Fig. 12 is H16 heavy chain plasmid map).
  • IgG2 heavy chain constant region DNA sequence (SEQ ID NO: 40)
  • IgG2 heavy chain constant region amino acid sequence (SEQ ID NO: 41)
  • the L10 light chain variable region VL was amplified by PCR, and the human Kappa light chain was obtained by gene synthesis.
  • the Kapa light chain constant region (SEQ ID NO: 43) gene fragment was PCR-amplified by the template, and the L10 containing the mammalian expression signal peptide sequence (SEQ ID NO: 45) was amplified by overlapping PCR.
  • Light chain gene VL-CL (SEQ ID NO: 52).
  • the L10 light chain gene was constructed into the pCDNA3.1 (+) vector to obtain an L10 light chain expression vector.
  • the L10VL and Kappa light chain CL gene fragments were amplified using pfu DNA polymerase (purchased from Beijing Quanjin Biotechnology Co., Ltd.). Using Whole-1L-F and Whole-1L-R as primers and pFL249-L10 plasmid as template, VL gene fragment containing partial eukaryotic expression signal peptide was amplified by PCR; CL-F and CL-R were used as primers.
  • the human Kappa light chain plasmid is used as a template, and a CL gene fragment containing a TAG stop codon and a NotI restriction site is amplified by a PCR method, and VL and CL have an overlapping sequence of at least 20 bp.
  • the PCR conditions were: 95 ° C for 2 minutes, [95 ° C for 20 seconds, 55 ° C for 20 seconds, 72 ° C for 40 seconds] 30 cycles, 72 ° C for 5 minutes.
  • the L10 light chain gene was amplified with pfu DNA polymerase. Using the VL and CL gene fragments obtained by the above PCR as a template, using Whole-1L-F and CL-R as primers, PCR-amplified L10 light chain containing EcoRI and NotI cleavage sites, and eukaryotic expression signal peptide sequences. gene. PCR conditions: 95 ° C for 2 minutes, [95 ° C for 20 seconds, 55 ° C for 20 seconds, 72 ° C for 100 seconds] 35 cycles, 72 ° C for 5 minutes.
  • the L10 light chain gene obtained in the above manner was treated according to the PCR product of Example 9 and purified by gel, and then purified by T4 DNA ligase (purchased from NEB).
  • the 19-T simple vector was ligated (16 ° C, 1 hour), heat shocked (42 ° C, 90 sec) was transformed into E. coli, plated (LB Amp medium) and cultured overnight at 37 ° C, and clones were picked for colony PCR ( Universal primers M13-47 and M13-48) were screened for identification.
  • the positive clones identified by PCR were subjected to plasmid extraction, double-digested with EcoRI and NotI, and the L10 light chain gene fragment of about 750 bp was recovered by cleavage, and the pCDNA3.1(+) vector recovered by the same double digestion treatment was used. connection.
  • the L10 light chain clone was confirmed by sequencing, and the recombinant plasmid was 6123 bp in length and contained the L10 light chain variable region and the human Kappa light chain constant region (Fig. 13 is the L10Kappa light chain plasmid map).
  • Table 16 shows the construction of primers for light and heavy chain vectors of anti-hRANKL humanized antibodies, the remaining three heavy chains (H16u, H114 and H16 (hFR)), and four light chains (L10u, L37, L37u and WT-VL (hFR)
  • the plasmid construction method is similar and will not be described in detail herein.
  • Recombinant antibodies by anti hRNAKL FreeStyle TM 293-F cells (Invitrogen Corp., Cat. No. R790-07) produced.
  • the DNA sequences encoding the entire heavy and light chains have been cloned into mammalian expression vectors as described in Example 15.
  • Anti-hRNAKL antibodies were generated by co-transfection of 293F cells mediated by a cationic polymer PEI (purchased from Polysciences, Cat. No. 23966-2) by vectors expressing the entire heavy and intact light chains.
  • PEI cationic polymer
  • a vector expressing the entire heavy chain (amino acid sequence number SEQ ID NO: 46) is co-transfected into a 293F cell with a vector expressing the entire light chain (amino acid sequence number SEQ ID NO: 51);
  • G2 antibody, a vector expressing the entire heavy chain (amino acid sequence number SEQ ID NO: 47) and a vector expressing the entire light chain (amino acid sequence number SEQ ID NO: 52) were co-transfected into 293F cells; for expression of G3 antibody
  • the vector of the entire heavy chain (amino acid sequence number SEQ ID NO: 47) was co-transfected into 293F cells with a vector expressing the entire light chain (amino acid sequence number SEQ ID NO: 53); to generate the G4 antibody, the entire heavy chain was expressed (
  • the vector having the amino acid sequence number SEQ ID NO: 47) is co-transfected with the vector expressing the entire light chain (amino acid sequence number SEQ ID NO: 54) into
  • a vector expressing the entire heavy chain (amino acid sequence number SEQ ID NO: 48) is co-transfected into a 293F cell with a vector expressing the entire light chain (amino acid sequence number SEQ ID NO: 54);
  • G9 antibody a vector expressing the entire heavy chain (amino acid sequence number SEQ ID NO: 48) and a vector expressing the entire light chain (amino acid sequence number SEQ ID NO: 55) were co-transfected into 293F cells; for expression of G10 antibody
  • the vector of the entire heavy chain (amino acid sequence number SEQ ID NO: 49) was co-transfected into 293F cells with a vector expressing the entire light chain (amino acid sequence number SEQ ID NO: 52); to generate the G11 antibody, the entire heavy chain was expressed (
  • the vector having the amino acid sequence number SEQ ID NO: 49) is co-transfected with the vector expressing the entire light chain (amino acid sequence number SEQ ID NO: 53) into 293
  • 293-F cells were passaged at a density of 6 to 7 ⁇ 10 5 /mL, and cultured at 37 ° C, 8% CO 2 , and 135 rpm. On the next day, the cell density and viability were measured using a hemocytometer to ensure cell viability greater than 95%, and the cell density was adjusted to 1 ⁇ 10 6 /mL for transient experiments.
  • rProtein A available from GE Inc., Best Chrom
  • Centrifugal concentration was carried out using a 10 KD ultrafiltration tube (purchased from Millipore, UFC901008), and resuspended with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.2-7.4). .
  • the modified BCA protein concentration assay kit (purchased from Shanghai Shenggong Biotech Co., Ltd.) was used to determine the protein concentration, and the filter was sterilized by filtration using a 0.22 ⁇ M filter (purchased from Millipore Corporation under the trade number SLGP033RB) and stored at -80 °C.
  • Example 17 ELISA method for detecting the binding ability of humanized antibody to hRNAKL
  • hRANKL was diluted to 1 ⁇ g/ml with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.2-7.4), and 100 ⁇ l/well was added to a 96-well microtiter plate (purchased from NUNC). Company), 4 ° C coated overnight. The plate was washed 3 times with PBST (0.05% Tween 20 in PBS), and blocked with a blocking solution (PBS containing 2% (w/v) BSA, PBS-2% BSA for short) for 1 to 1.5 hours at room temperature.
  • PBS 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.2-7.
  • Humanized antibodies G2 to G14, mouse anti-G1 and positive control Prolia purchased from Amgen, batch number 1021139 were diluted 3-fold to 0.0002 ⁇ g with sample dilution (PBS-1% BSA) from 1 ⁇ g/ml, respectively. /ml, 100 ⁇ l/well was added to the plate, and incubated for 2 hours at room temperature. The plate was washed 3 times with PBST, and HRP-labeled goat anti-human IgG Fc antibody (Jackson, Cat. No. 109-035-098) was diluted 1:10,000 with PBS-1% BSA, 100 ⁇ l/well was added to the plate, and incubated for 1 hour at room temperature. .
  • the plate was washed 6 times with PBST and then added with TMB substrate (Cell Signaling, Cat. No. 7004) for 10 minutes at room temperature, 100 ⁇ l of 2 M HCl was added to each well to stop color development, and the OD450nm reading was detected using an M5 instrument.
  • TMB substrate Cell Signaling, Cat. No. 7004
  • Example 18 Using a Competitive ELISA Method to Detect the ability of Humanized Antibodies to Inhibit Binding of RANK-Fc to hRANKL
  • the ability of the anti-hRANKL humanized antibody to inhibit binding of the receptor RANK-Fc to the ligand hRANKL was evaluated using a competition ELISA assay and compared to Prolia.
  • RANK-Fc was diluted to 2 ⁇ g/ml with PBS, 100 ⁇ l/well was added to a 96-well microtiter plate, and coated at 4 ° C overnight.
  • the humanized antibody (G2 ⁇ G14), the mouse anti-(G1) and the positive control Prolia were diluted with the sample diluent (PBS-1% BSA), and the final concentrations were 30, 10, 3.3, 1.1, 0.37, respectively.
  • the above antibody at 0.12, 0.04, 0.01, 0.005, 0.0015, 0.0005 ⁇ g/ml was mixed with 0.25 ⁇ g/ml of hRANKL and preincubated overnight at 4 °C.
  • the plate was washed 3 times with PBST (PBS containing 0.05% Tween 20), and then blocked with a blocking solution (PBS-2% BSA) for 1 to 1.5 hours at room temperature, and the plate was washed 3 times with PBST.
  • the pre-incubated mixture was added to the sealed plate at 100 ⁇ l/well, in which only PBS-1% BSA wells were used as negative controls, and only 0.25 ⁇ g/ml hRANKL wells were used as positive controls.
  • the reaction was carried out for 1 hour at room temperature.
  • the plate was washed 3 times with PBST, and an HRP-labeled anti-His tag antibody (purchased from Abcam, catalog No. ab1187) was diluted 1:5,000 in PBS containing 1% BSA and 0.05% Tween, and 100 ⁇ l/well was added to the plate. Incubate for 1 hour at room temperature.
  • the plate was washed 6 times with PBST and then added with TMB substrate (Cell Signaling, Cat. No. 7004) for 10 minutes at room temperature, 100 ⁇ l of 2 M HCl was added to each well to stop color development, and the OD450nm reading was detected using an M5 instrument.
  • TMB substrate Cell Signaling, Cat. No. 7004
  • IC50 value of Prolia was 0.13 ⁇ g/ml, and the IC50 value of 13 humanized antibodies was between 0.1 and 0.4 ⁇ g/ml, which was equivalent to the inhibition of G1 and Prolia.
  • the IC50 values of each humanized antibody competitively inhibiting the binding of hRANKL to RANK-Fc are shown in Table 19.
  • Example 19 Humanized antibody inhibits hRANKL-induced differentiation of RAW264.7 cells
  • hRANKL can induce mouse mononuclear macrophage leukemia cell RAW264.7 (purchased from Shanghai Library of Chinese Academy of Sciences) to differentiate into osteoclasts in vitro.
  • the aim of this study was to evaluate the in vitro induction of RAW264.7 cell differentiation by hRANKL by humanized anti-hRANKL antibody and compare it with Prolia.
  • the pNPP chromogenic system can react with tartaric acid phosphatase activity by high and low OD405nm readings, thus reflecting the differentiation of RAW264.7 cells into osteoclasts. degree.
  • RAW264.7 cells were cultured using DMEM + 10% FBS (purchased from Invitrogen) medium and passaged every 3-4 days.
  • FBS purchased from Invitrogen
  • cells in the logarithmic growth phase were resuspended using ⁇ -MEM medium + 10% FBS (purchased from Invitrogen) to prepare a cell suspension.
  • 96-well cell culture plates were seeded at 2000/100 ⁇ l/well, and cultured for 1 hour at 37 ° C in a 5% CO 2 incubator (Thermo Corporation, specification model Forma311).
  • the humanized antibody (G2 ⁇ G14), the mouse anti-(G1) and the positive control Prolia were diluted in a serum-free ⁇ -MEM medium with an equal volume of hRANKL and M-CSF, TGF- ⁇ .
  • the ⁇ -MEM medium was mixed, and 100 ⁇ l/well was added to the cell culture plate so that the final concentrations of each antibody were 4, 2, 1, 0.5, 0.25, and 0.125 ⁇ g/ml, respectively, hRANKL, M-CSF, and TGF- ⁇ .
  • Final concentrations were 150, 20 and 2 ng/ml, respectively.
  • Negative control wells containing only 20 ng/ml M-CSF + 2 ng/mL TGF- ⁇ mixture, and 20 ng/ml M-CSF + 2 ng/mL TGF- ⁇ + 150 ng/ml hRANKL mixture were positive control wells. All are three holes. Incubate at 37 ° C, 5% CO 2 incubator for 5 days, discard the cell supernatant, add 100 ⁇ l of cell lysate (Citrate Buffer pH 5.0 + 0.5% Triton X-100) per well, and lyse for 10 minutes in a refrigerator at 4 ° C. .
  • the cell lysate was diluted 10-fold, and an equal volume of 100 ⁇ l of pNPP chromogenic solution (purchased from Sigma) was added to each well, incubated at 37 ° C for 30 minutes, 50 ⁇ l of stop solution (0.5 M NaOH) was added to each well, and OD 405 nm was measured by M5 instrument. reading.
  • the logarithm of the antibody concentration is plotted on the abscissa, and the absorbance is measured on the ordinate.
  • the image is labeled with "log(inhibitor) vs.response--Variable slope" (GraphPad Prism software) to obtain antibody inhibition of RAW264.7 cell differentiation. It is the curve of osteoclasts (Fig. 16). From the graph of Fig. 16A, the inhibitory effects of G9 and G13 were weak, and the inhibitory effects of G8 and G12 antibodies were better, which was close to that of prolia. From the analysis of the graph of Fig.
  • Example 20 Inhibitory effect of humanized antibody on hRANKL-induced phosphorylation of ERK1/2 in RAW264.7 cells
  • RAW264.7 cells in the logarithmic growth phase were trypsinized, resuspended in DMEM + 10% FBS medium (purchased from Invitrogen), and the cell density was adjusted, and 6-well cell culture plates were seeded at 3.0 ⁇ 10 5 /well. The adherent was cultured at 37 ° C in a 5% CO 2 incubator. After the cells were attached, the medium was changed to ⁇ -MEM + 0.1% FBS (purchased from Invitrogen), and cultured for 24 hours under starvation.
  • the antibody and hRANKL were diluted with ⁇ -MEM+0.1% FBS, and the antibody (G4, G8, G10, G12, G14) with a final concentration of 250 ng/ml was pre-mixed with 150 ng/ml of hRANKL for 2 hours at 37 ° C, and discarded 6
  • the supernatant in the well cell culture plate was added to the premix at 1 ml/well, in which prolia was the positive control, only ⁇ -MEM + 0.1% FBS was added as the negative control, and incubation was carried out for 30 minutes at 37 ° C in a 5% CO 2 incubator. .
  • the culture solution was aspirated, and the reaction was terminated by washing once with 4 ° C pre-cooled PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4), and 120 ⁇ l of LDS (purchased from Invitrogen) was added. Company, item number: NP0007), placed on ice and quickly collected cell lysate, stored at -80 ° C for later use.
  • PBS 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4
  • LDS purchased from Invitrogen
  • the collected cell lysate was subjected to electrophoresis under reducing conditions containing a final concentration of 50 mM dithiothreitol (purchased from Sangon, Cat. No. D0281), and the electrophoresed gel was subjected to electrotransfer (300 mA, 80 minutes).
  • the method was transferred to a NC membrane (purchased from Pall Corporation, article number: S80209).
  • 5% skim milk powder purchasedd from Sangon, article number: NB0669
  • the present invention humanizes the murine antibody 7.19.12 by a humanization modification technique, thereby obtaining a humanized antibody with an increased degree of human origin and maintaining antigen-antibody affinity.
  • the present application humanizes the mouse antibody 7.19.12 by humanized transformation technology, and after serial screening, a series of humanized antibodies are obtained, and the binding characteristics of these humanized antibodies are confirmed by immunological and cytological experiments.
  • the murine antibody is consistent and still has neutralizing activity, and can inhibit the competitive binding of RANKL to the corresponding receptor RANK, and can effectively inhibit RANKL-induced osteoclast differentiation in vitro. Based on this, it is inferred that these monoclonal antibodies should effectively inhibit bone resorption and osteoporosis in vivo.
  • humanized antibodies are prepared by cultured expression in mammalian cells or prokaryotic cell systems, and can be used as a clinical pharmacological treatment for monoclonal antibody drugs, which can be used for the treatment of osteoporosis and rheumatoid arthritis by intravenous or subcutaneous administration.
  • monoclonal antibody drugs which can be used for the treatment of osteoporosis and rheumatoid arthritis by intravenous or subcutaneous administration.
  • the antibody shown in the present application is a humanized antibody, and the nucleic acid and amino acid sequences of the variable region and the constant region have no similarities compared to the fully human antibody Prolia obtained by immunization with a transgenic mouse.
  • the sequences and structures of the humanized antibodies shown herein are unique and novel.

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Abstract

本发明提供一种抗人RANKL抗体，其人源化抗体和它们的药物组合物和用途，所述抗人RANKL抗体可特异性结合如SEQ ID NO:1所述的氨基酸序列，并且其重链包括SEQ ID NO:2-9的氨基酸序列中之一所示的可变区，轻链包括SEQ ID NO:10-17的氨基酸序列中之一所示的可变区。所述人源化抗体可特异性地与人RANKL结合，并且其重链可变区选自SEQ ID NO:6、23、25、27或29所示的氨基酸序列，轻链可变区选自SEQ ID NO:14、31、33、35、37或39所示的氨基酸序列。

Description

一种抗人RANKL抗体、其人源化抗体及它们的药物组合物和用途
本申请要求2013年12月31日提交的中国申请号201310753972.3的优先权。
背景技术
人体骨骼是动态的不断变化的组织，破骨细胞吸收骨质后，成骨细胞形成骨质，完成一个正常的骨重塑循环。成年人骨重塑率在每年2-10％。正常情况下骨吸收与骨形成(骨代谢)保持动态平衡，破骨细胞与成骨细胞之间的平衡打破后会造成骨质过多(骨质硬化)或骨质过少(骨质疏松)。
破骨细胞的分化信号是由成骨/间充质干细胞传递的。各种刺激骨吸收的因子将诱导破骨细胞形成的信号传递给成骨/间充质干细胞，诱导其膜上表达破骨细胞分化因子-1(Osteoclast differentiation factor,ODF-1)，又称细胞核因子-κB受体活化因子配基(Receptor activator of nuclear factor κB ligand,RANKL)。RANKL可与破骨前体细胞膜上的破骨细胞分化和活化受体(Osteoclast differentiation and activation receptor,ODAR),又称细胞核因子-κB受体活化因子(Receptor activator of nuclear factor κB,RANK)直接结合，将信号传入破骨前体细胞，引起级联反应，刺激破骨细胞形成、分化和成熟。在骨代谢过程中，OPG/RANKL/RANK系统是发挥重要调节作用的关键信号通路，已有文献报道发现一些全身性代谢疾病如骨质疏松、类风湿性疾病、肿瘤以及骨折愈合等疾病都存在骨改建活性的增强,与RANK/RANKL/OPG系统有密切关系。
许多疾病通过增加破骨细胞的数量和(或)增强破骨细胞的活性而引起骨量减少，如绝经后和老年性骨质疏松症、恶性肿瘤并发体液性高钙血症、肿瘤转移、Paget‘s骨病、类风湿性关节炎、甲状旁腺功能亢进、假体周边骨自溶症等。绝经后雌激素水平下降，IL21、IL26、TNF-α的基因表达增加，促进破骨细胞的增殖、分化、融合，抑制其凋亡，使骨吸收增加，骨代谢偶联失衡，从而导致了骨质疏松。
骨质疏松疾病的治疗主要从两方面着手：(一)促进成骨细胞的骨形成作用；(二)抑制破骨细胞的骨吸收作用。阻断RANKL/RANK信号传递能抑制破骨细胞形成和活性，阻断骨吸收，从而治疗骨质疏松症，已证实是一条可行的途径。美国Amgen公司出品的Prolia为通过免疫人IgG转基因小鼠XenoMouse获得的全人源抗人RANKL(human RANKL,或hRANKL)单抗隆抗体，能阻断RANKL/RANK信号传递，有效抑制破骨细胞形成和活性，阻断骨吸收和骨质破坏。2010年被US FDA批准治疗妇女绝经后骨质疏松。欧洲还批准了用于前列腺癌相关的骨丢失。Prolia抗体的临床有效性使得RANKL作为单克隆抗体药物治疗一系列由于破骨细胞活动增强导致的骨丢失的骨代谢疾病的靶点得到最终验证。
单克隆抗体类药物具有特异性强，副作用小，疗效显著等优点，已经成为对抗肿瘤，传染病，自身免疫病等病患的重要工具。目前细胞融合与杂交瘤技术，仍然是最为可靠的制备单克隆抗体的方法，常用来免疫的动物有小鼠，大鼠，羊，兔子及其他动物，其中最常用的是鼠类包括大鼠与小鼠。利用该方法得到的单克隆抗体是动物源的，开发动物源单克隆抗体成为抗体类药物应用于人体，必须要进行人源化改造，以减少异源抗体所引起的人抗动物抗体反应(Human Anti-Animal Antibodies,HAAA)，同时也可以更加有效的激活人体免疫系统，降低抗体类药物的清除速度，并延长半衰期。
抗体互补决定区(Complementary Determining Regions,CDRs)是在抗体框架区(Framework Regions,FRs)的支持下与抗原分子上的表位氨基酸相互作用的区域。CDR区与抗原表位精密的分子对接是抗体亲和力与特异性的分子基础，而天然的亲本抗体的CDRs区构象代表了最高的亲和力与最好的抗原结合特异性。理论上FRs区氨基酸的改变，会引起CDRs区构象上发生变化使亲和力下降。有研究证明，在FRs区的所有氨基酸中，大多数的氨基酸的人源化替换对CDRs区构象只产生轻微的影响，不会对抗体亲和力产生严重影响，但也存在着少数几个关键氨基酸，一旦这些关键氨基酸被替换成人的相应氨基酸后，会引起CDRs区构象上很大的改变，进而引起抗体亲和力的严重下降。
发明内容
本发明所要解决的技术问题是提供抗人RANKL抗体、其人源化抗体以及它们的药物组合物，用途。
第一方面，本发明提供抗人RANKL抗体，其可特异性结合如SEQ ID NO:1所述的氨基酸序列。
在一些实施方案中，所述抗人RANKL抗体，包括重链和轻链，其中:a)重链包括SEQ ID NO:2-9的氨基酸序列中之一所示的可变区；并且b)轻链包括SEQ ID NO:10-17的氨基酸序列中之一所示的可变区；并且所述抗体与RANKL结合，阻断RANK与RANKL相互作用。
在一些实施方案中，所述抗人RANKL抗体为单链抗体或人源化抗体，或通过杂交瘤技术获得的鼠源单克隆抗体。
本发明还涉及编码抗人RANKL抗体分子的核酸以及含有所述核酸的宿主细胞。
本发明还涉及制备或生成本文所述抗人RANKL抗体分子、核酸、宿主细胞、产物及组合物的方法。
第二方面，本发明提供抗人RANKL抗体的人源化抗体，所述人源化抗体可特异性地与人RANKL结合，并且所述人源化抗体的重链可变区选自SEQ ID NO:6、23、25、27或29所示的氨基酸序列，轻链可变区选自SEQ ID NO:14、31、33、35、37或39所示的氨基酸序列。
在一些实施方案中，所述人源化抗体的重链恒定区来自人IgG2，轻链恒定区来自人Kappa；或所述人源化抗体的重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链恒定区选自SEQ ID NO:43所示的氨基酸序列。
在一些实施方案中，所述人源化抗体为选自scFv、(scFv)2、Fab、Fab’或F(ab’)2的抗原结合片段。
在一些实施方案中，所述人源化抗体的完整重链选自SEQ ID NO:46-50中之一所示的氨基酸序列，和/或完整轻链选自SEQ ID NO:51-56中之一所示的氨基酸序列。
第三方面，本发明提供一种药物组合物，其活性成分为所述抗人RANKL抗体或所述人源化抗体。
第四方面，本发明提供所述抗人RANKL抗体或所述人源化抗体在制备治疗骨损失疾病药物中用途。
第五方面，本发明提一种改善患有骨损失疾病的患者的状况或治疗患有骨损失疾病的患者的方法，所述方法包括给所述予患者治疗有效量的所述抗人RANKL抗体或所述人源化抗体或给予所述患者所述药物组合物。
自然人RANKL蛋白在体内存在三种形式，全长的跨膜性质蛋白，不含胞内区的膜结合蛋白和可溶性的蛋白(从136位Gly开始，到317位Asp,共182个氨基酸)。研究表明，生理条件下，RANKL主要以跨膜形态存在于成骨细胞表面，但在病理状态下，基质金属蛋白酶14(MMP14)和金属蛋白酶10(ADAM10)将跨膜形态的RANKL从细胞表面切割下来，释放出可溶性RANKL蛋白，导致循环中可溶性RANKL明显增高，破骨细胞活性增强。因此，本申请利用可溶性RANKL蛋白作为抗原，通过免疫小鼠和杂交瘤融合技术，经过系列筛选，获得鼠抗人RANKL单克隆抗体，并证实这些鼠源性抗体不仅特异性地与人RANKL结合，还与食蟹猴的RANKL有交叉免疫反应，并与鼠源RANKL没有免疫交叉反应。重要的是通过RANKL与相应受体RANK的竞争结合实验，筛选获得了可以阻断RANKL/RANK结合，且阻断能力不低于，甚至优于Denosumab的单克隆抗体。这些抗体可以在体外有效地抑制RANKL诱导的破骨细胞的分化。据此推断，这些单克隆抗体在体内应有效地抑制骨吸收和骨质疏松。这些鼠源抗体经过人-鼠嵌合或人源化改造，或者利用其与抗原结合并有中和作用的抗体片段，利用哺乳类动物细胞或原核细胞系统培养表达制备，可以成为适合临床药理治疗单克隆抗体药物。
附图说明
图1显示了构建的重组人RANKL的核苷酸序列。
图2anti-RANKL鼠单抗与human RANKL结合曲线。
图3anti-RANKL鼠单抗与monkey RANKL结合曲线。
图4anti-RANKL鼠单抗抑制RANKL与RANK-Fc结合曲线。
图5anti-RANKL鼠单抗抑制human RANKL诱导的RAW264.7细胞分化的活性。
图6显示了pFL249-7.19.12scFv质粒的示意图。
图7显示了第一批VH突变文库PCR菌落筛选电泳图。
图8显示了第二批VH突变文库PCR菌落筛选电泳图。
图9显示了VL突变文库PCR菌落筛选电泳图。
图10显示了VH文库的ELISA筛选结果。
图11显示了VL文库的ELISA筛选结果。
图12显示了H16重链质粒图谱示意图。
图13显示了L10Kappa轻链的质粒图谱示意图。
图14A～14C显示了人源化抗体与hRANKL的结合活性比较。
图15A～15C显示了人源化抗体竞争抑制hRANKL与RANK-Fc结合的活性比较。
图16A～16D显示了人源化抗体抑制RAW264.7细胞分化为破骨细胞的活性比较。
图17显示了人源化抗体抑制hRANKL诱导的RAW264.7细胞ERK1/2磷酸化的结果。
具体实施方式
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook等人，“Molecular Cloning:A Laboratory Manual，，(第2版)，第1-3卷,Cold Spring Harbor Laboratory Press(1989)；Lewin,“Genes IV”，Oxford University Press,NewYork,(1990)；及Roitt等人，“Immunology”(第2版)，Gower Medical Publishing,London,New York(1989),以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献
除非另有说明，否则术语“免疫球蛋白”及“抗体”指完整免疫球蛋白及可结合所需抗原的免疫活性片段。免疫球蛋白与其免疫活性(抗原结合性)片段包括表位结合位点(亦即能被识别抗体的抗原特异性结合的位点或表位)。抗体片段实例包括例如Fab、F(v)、Fab`、F(ab`)2片段，由还原免疫球蛋白的二硫键而衍生的"半分子"，单链免疫球蛋白、或其他合适的抗原结合性片段(参见例如Bird等人，Science,242:423—426(1988)；Huston等人，PMS，(USA)，85:5879(1988)；Webber等人，Mol.Immunol,32:249(1995))。抗体或其免疫活性片段可来自动物(例如啮齿类，如小鼠或大鼠)、或嵌合型(参见Morrison等人，PNAS，81:6851(1984)；Jones等人，Nature,321:522(1986))。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用的术语(多肽或蛋白的)“域”/“区”是指折叠蛋白结构域，其能够独立于蛋白的其余部分维持其三级结构。一般而言，域负责蛋白的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白而不损失蛋白的其余部分和/或域的功能。
如本文所用的术语“抗体可变区”是指基本上由本领域及下文中分别称为为“互补决定区I”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3"的三个“互补决定区”或“CDR”。抗体可变区正是因具有抗原结合位点而赋予抗体对抗原的特异性。
抗原结合蛋白对抗原或表位的特异性结合可以以本身已知的任何适合方式来测定，包括例如本文所述的分析、斯卡查德分析(Scatchardanalysis)和/或竞争性结合分析(例如放射免疫分析(RIA)、酶免疫分析(EIA)及夹心式竞争性分析(sandwich competition assay))及本领域中本身已知的其不同变化形式。
氨基酸残基将根据如本领域中公知，且达成一致的标准三字母或一字母氨基酸码加以指示。在比较两个氨基酸序列时，术语“氨基酸差异”是指相比于第二序列，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如根据W098/49185，其中保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代:(i)较小脂族非极性或弱极性残基:Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺:Asp、Asn、Glu及Gln；(iii)极性带正电残基:His、Arg及Lys；(iv)较大脂族非极性残基:Met、Leu、lie、Val及Cys；及(V)芳族残基:Phe、Tyr及Trp。特别优选的保守氨基酸取代如下:Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His 取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
本领域技术人员应用公知技术能确定这里提到的多肽的合适的变体。在一些实施方案中，本领域技术人员可以鉴定通过导向于不认为对活性是重要的区而可以改变但是不破坏活性的分子的合适的部位。在一些实施方案中，人们可以鉴定相似多肽之间保守的分子的残基和部分。在一些实施方案中，即使对于生物活性或者对结构重要的区也可以进行保守氨基酸取代而不破坏生物活性或者不会不利地影响多肽结构。
另外，本领域技术人员能综述结构功能研究，鉴定对于活性或结构重要的相似多肽中的残基。考虑这样一种比较，人们能预知蛋白质中氨基酸残基的重要性，相当于对相似蛋白质中对活性或结构重要的氨基酸残基。本领域技术人员对于这样预测的重要的氨基酸残基可以选择化学相似氨基酸取代作用。
本领域技术人员还能分析与相似多肽中的结构相关的三维结构和氨基酸序列。根据这样的信息，本领域技术人员可以预测抗体三维结构的氨基酸序列比对。在一些实施方案中，本领域技术人员可以选择对预计在蛋白质表面上的氨基酸残基不进行基团变化，因为这样的残基在与其他分子的重要的相互作用中涉及。此外，本领域技术人员可以制备在各个期望的氨基酸残基处包含单一氨基酸取代作用的试验变体。然后可以应用本领域技术人员公知的活性测定来对这些变体进行筛选。这样的变体也可以被用来收集关于合适的变体的信息。例如，如果有人发现特定氨基酸残基的变化导致破坏的不期望的降低的，或者不合适的活性，则可以避免这样的变化。换句话说，以从这样的常规实验获得的信息为基础，本领域技术人员容易确定哪里应该避免进一步的取代作用，不管是单独的或者是与其他突变一起的。
关于二级结构的预测已经出版了大量科学出版物。参见MoultJ.,Curr.0p.1nBiotech.，7(4):422-427(1996)，Chou等，Biochemistry，13(2):222-245(1974)；Chou等，Biochemistry,113(2):211-222(1974)；Chou等，Adv.Enzymol.Relat.Areas Mol.Biol.,47:45-148(1978)；Chou等，Ann.Rev.Biochem.,47:251-276和Chou等，Biophys.J.,26:367-384(1979)。此外，目前可获得计算机程序辅助预测二级结构。预测二级结构的一种方法以同源性模拟为基础。例如，序列同一'I"生大于30％,或相似性大于40％的两种多肽或蛋白质经常具有相似结构拓扑学。蛋白质结构数据库(PDB)的最新增长提供对二级结构增强的可预测性，包括多肽或蛋白质结构中可能的折叠数目。参见Holm等，Nucl.Acid.Res.，27(I):244-247(1999)。有人提议(Brenner等，Curr.0p.Struct.Biol.,7(3):369-376(1997))，在给定多肽或蛋白质中存在有限数目的折叠，并且一旦确定临界数目的折叠，结构预测将合适地变得更精确。
预测二级结构的另外的方法包括"穿线法"(Jones,D.,Curr.0pin.Struct.Biol.，7(3):377-87(1997)；Sippl等，Structure，4(l):15-19(1996))，"图形分析"(Bowie等，Science,253:164170(1991)；Gribskov等，Meth.Enzym.,183:146-159(1990)；Gribskov等，Proc.Nat Acad.Sc1.,84(13):4355-4358(1987))，和"进化的键"(参见Holm，上文(1999)，和Brenner，上文(1997))。
术语“多肽”作为遗传术语指天然蛋白质，或者具有天然序列的一个或多个氨基酸缺失，添加和/或取代的序列。术语“多肽”还包括RANKL抗体或其CDR(如下所述，SEQ IDNO:2-9和SEQ ID NO:10-17)，或它们的一个或多个氨基酸缺失，添加和/或取代的序列。
全长重链包括可变区结构域，VH，和三个恒定区结构域，CH1，CH2，和CH3。VH结构域在多肽的氨基末端，CH3结构域在羧基末端。这里使用的术语"重链"包括全长重链及其片段。
全长轻链包括可变区结构域和恒定区结构域，和重链一样，轻链的可变区结构域在多肽的氨基末端。术语"轻链"，如这里使用的，包括全长轻链及其片段。单链抗体是Fv分子，其中重链和轻链可变区通过柔性接头连接，形成单链多肽，其形成抗原-结合区。W088/01649和美国专利Nos.4，946，778和5，260,203详细讨论单链抗体。
在一些实施方案中，二价抗体而不是"多特异性"或"多功能"抗体，一般理解其每个结合位点是相同的。
当过量抗体将与反受体结合的受体的量减少至少大约20％，40％，60％，80％，85％，或更多(根据体外竞争结合分析测定的)时，抗体基本上抑制配体与受体的粘附。
术语"表位"包括能与免疫球蛋白或T-细胞受体特异性结合的任何多肽决定簇。在一些实施方案中，表位决定簇包括象氨基酸这样的分子的化学活性表面基团，糖侧链，磷酰基，或磺酰基，并且，在一些实施方案中，可以具有特异的三维结构特征，和/或特异的电荷特征。表位是抗体结合的抗原的区。在一些实施方案中，当它优先识别蛋白质和/或大分子的复杂混合物中的其靶抗原，就说抗体特异性结合抗原。
本文使用的术语“试剂”指化合物，化合物的混合物，生物大分子，或从生物材料制备的提取物。
本文使用的，术语“标记”或“标记的”指插入可检测标记物，例如通过插入放射标记的氨基酸或连接能被标记的抗生物素蛋白(例如能被光学方法或比色法检测的含有荧光标记物或酶活性的链霉抗生物素蛋白)检测的生物素部分多肽。在一些实施方案中，标记或标记物也可以是治疗性的。标记多肽和糖蛋白的各种方法是本领域公知的并且可以使用。多肽的标记的例子包括但不限于下组:放射性同位素或放射性核素(例如3H，14C，15N，35S，90Y,99Tc，lllln，1251，1311)，荧光标记物(例如FI TC，诺丹明，镧系磷光剂)，酶标记(例如辣根过氧化物酶，半乳糖苷酶，荧光素酶，碱性磷酸酶)，化学发光剂，生物素基，预先确定的第二报道分子识别的多肽表位(例如亮氨酸拉链对序列，二抗的结合位点，金属结合结构域，表位标记)。在一些实施方案中，标记通过各种长度的间隔臂连接以减小可能的空间位阻。
本文使用的术语“生物样品”包括但不限于任何量的来自活物或以前是活物的物质。这样的活物包括但不限于人，小鼠，猴，大鼠，兔，和其他动物。这样的物质包括但不限于血液，血清，尿液，细胞，组织，器官，骨，骨髓，淋巴结和皮肤。
术语“骨质稀少疾病”包括但不限于骨质疏松，骨质稀少，佩吉特氏病，溶骨性转移灶，牙周炎，类风湿性关节炎和由于固定导致的骨损失。除了这些骨疾病之外，已知一些癌症提高破骨细胞活性并且诱导骨再吸收，例如乳腺癌，前列腺癌和多发性骨髓瘤。现在已知这些癌症产生导致骨中RANKL过量表达的因子，并且导致增加的破骨细胞数目和活性。
术语“药剂或药物”指当适当地对患者施用时能诱导期望的治疗效果的化合物或组合物。
术语“调节剂”是改变分子的活性或功能的化合物。例如，与不存在调节剂下发现的活性或功能值相比，调节剂可以引起分子的一些活性或功能值的提高或降低。在一些实施方案中，调节剂是抑制剂，降低分子的至少一种活性或功能值。一些例示的分子的活性和功能包括但不限于结合亲和性，酶活性，和信号转导。一些例示的抑制剂包括但不限于蛋白质，肽，抗体，肽体，碳水化合物或小有机分子，肽体描述于例如W001/83525。
术语“基本上纯”指主体物质是占优势存在的物质(例如以摩尔为基础，它在组合物中比任何其他各种物质更丰富)。在一些实施方案中，基本上纯化的级分是其中主体物质包括占存在的全部大分子物质至少大约50％(摩尔比)的组合物。在一些实施方案中，基本上纯的组合物含有组合物中存在的所有的大分子物质的大约80％，85％，90％，95％，或99％以上。在一些实施方案中，主体物质被纯化至基本上均质(通过常规检测方法不能检测到组合物中的杂质物质)，其中组合物基本上由单一大分子物质组成。
术语“患者”包括人和动物受试者。
在本发明的一些实施方案中，提供了抗人RANKL的鼠源单克隆抗体。在一些实施方案中，提供了包括重链和轻链免疫球蛋白，特别是相应于可变区的序列的氨基酸序列及其编码核苷酸序列。在一些实施方案中，提供了相应于互补决定区(CDR)，特别是从CDRl至CDR3的序列。根据一些实施方案，也提供了表达这样的免疫球蛋白分子和单克隆抗体的杂交瘤细胞系。在一些实施方案中，本发明提供了来自于上述抗人RANKL的鼠源单克隆抗体的人源化抗体。
术语“人源化抗体”主要指鼠源单克隆抗体以基因克隆及DNA重组技术改造，重新表达的抗体，其大部分氨基酸序列为人源序列取代，基本保留亲本鼠单克隆抗体的亲和力和特异性，又降低了其异源性，有利应用于人体。人源化抗体包括嵌合抗体、改型抗体和全人源化抗体等几类。人源化作用的基本原理为改变抗原识别，即CDR结构域，在人免疫球蛋白的环境中的特异性(“CDR移植”，Winter和 Milstein)。由动物(一般是鼠)到人源化的抗体的转录必需在相反的需求之间妥协，其解决方法根据情况而改变。为了使免疫原性最小化，免疫球蛋白将保持尽可能多的可接受的人序列。在任何情况下，为了保持最初的结合特性，免疫球蛋白构架在可接受的人序列中应当包含足够数目的突变以保证CDR区的构象尽可能与供体鼠免疫球蛋白的CDR区的构象相似。具体可参照Maeda等，1991；Singer等，1993；Tempest等，1994；Kettleborough等，1991；Hsiao等，1994；Baca等，1997；Leger等，1997；Ellis等，1995；Sato等，1994；Jones等，1986；Benhar等，1994；Sha and Xiang,1994；Shearman等，1991；Rosok等，1996；Gussow&Seemann,1991；Couto等，1994；Kashmiri等，1995；Baker等，1994；Riechmann等，1988；Gorman等，1991；Verhoeyen等，1988；Foote&ffinter,1992；Lewis&Crowe,1991；Co等，1991；Co等，1991；Verhoeyen等，1991；Eigenbrot等，1994；Hamilton等，1997；Tempest等，1995；Verhoeyen等，1993；Cook等，1996；Poul等，1995；Co等，1992；Graziano等，1995；Presta等，1993；Hakimi等，1993；Roguska等，1996；Adair等，1994；Sato等，1993；Tempest等，1991；Sato等，1996；Kolbinger等，1993；Zhu和Carter,1995；Sims等，1993；Routledge等，1991；Roguska等，1994；Queen等，1989；Carter等，1992；专利EP 592106；EP 1709076等
天然存在的抗体结构
天然存在的抗体结构单元一般包括四聚体。每一个这样的四聚体一般由两个相同的多肽链对组成，每一对具有一条全长“轻链”(在一些实施方案中，大约25kDa)和一条全长“重链”(在一些实施方案中，大约50_70kDa)。每条链的氨基末端部分一般包括大约100至110或更多氨基酸的可变区，其一般负责抗原识别。每条链的羧基末端部分一般定义负责效应物功能的恒定区。人轻链一般分类为K和λ轻链。重链一般分类为μ，δ，γ，α，或ε，抗体同种型分别定义为IgM,IgD,IgG,IgA,IgE,IgG有几个亚类，包括但不限于IgGl，IgG2，IgG3，和IgG4。IgM有几个亚类，包括但不限于IgMl和IgM2。类似地，IgA分为几个亚类，包括但不限于IgAl和IgA2。一般情况下，全长轻链和重链中，可变区和恒定区通过大约12个或更多个氨基酸的“J”区连接，重链也包括大约10个以上氨基酸的“D”区。参见，例如，Fundamental Immunology第七章(Paul,W.编著，第二版，Raven Press,N.Y.(1989))(为所有的目的全文引作参考)。各个轻/重链对的可变区一般形成抗原结合位点。
可变区一般具有相同的相对保守的构架区(FR)的一般结构，通过三个超可变区，也称作互补决定区或CDR区。各对两个链的CDRs—般通过构架区排列，其可以与特异性表位结合。从N-末端至C-末端，轻链和重链可变区一般包括结构域FR1，CDR1，FR2，CDR2，FR3,CDR3,和FR4。氨基酸在每个结构域的排列一般根据Kabat Sequences of Proeins of Immunological Interest的定义(Nation Institutes of Health,Bethesda,Md.(1987，1991)),或Chothia&Lesk J.Mol.Biol.196:901-917(1987)；Chothia等，Nature342:878-883(1989)。
双特异性和双功能抗体
双特异性和双功能抗体一般是具有两个不同的重链/轻链对和两个不同的结合位点的人工杂合抗体。通过各种方法可以制备双特异性抗体，包括但不限于杂交瘤的融合或Fab’片段的连接。参见，例如，Songsivi lai&Lachmann Clin。Exp.Tmmunol.79:315-321(1990)，Kostelny等，J.1mmunol.148:1547-1553(1992)。
本发明抗人RANKL的鼠源单克隆抗体的制备为本领域技术人员所公知的技术；其人源化抗体的DNA序列在可操作性地结合(即以这种确保其功能性的方式定位)至表达控制序列上后在宿主细胞中进行表达。这些载体一般能够在宿主生物中作为表达载体或者作为染色体DNA的整合部分进行复制。一般，所述表达载体包含可选的标记以允许鉴定已经用目标DNA序列转化的细胞。以scFv重组形式或者以Fab形式生产本发明的人源化免疫球蛋白，优选原核系统。大肠杆菌是对于克隆本发明的DNA序列特别有用的原核宿主之一。而且，现有大量的经充分表征的启动子，例如Lac或trp操纵子或β-内酰胺酶或λ噬菌体。一般，这些启动子控制表达并且具有核糖体结合位点，以便正确启动并完成转录和翻译。通过与聚乙二醇(PEG)缀合有可能提高在原核系统中生产的本发明的人源化免疫球蛋白的半衰期。其它 单细胞生物,如酵母,可以用于表达。选择的宿主是酵母属(Saccharomyces),利用提供了表达控制、复制终止和起点序列的合适载体。昆虫细胞培养物也可以用来生产本发明的人源化免疫球蛋白，一般使用以稳定方式转染的S2果蝇细胞或具有基于杆状病毒的表达系统的Spodoptera frugiperda的细胞(Putlitz等，1990)。植物和植物细胞培养物可以用于本发明的人源化免疫球蛋白的表达(Larrick&Fry,1991；Benvenuto等，1991；Durin等，1990；Hiatt等，1989)。
根据另一实施方案，本发明的多肽的半衰期延长修饰(此修饰亦降低多肽的免疫原性)包含连接药理学上可接受的合适的聚合物，例如直链或支链聚(乙二醇)(PEG)或其衍生物(例如甲氧基聚(乙二醇)或mPEG)。一般而言，可使用任何适合形式的聚乙二醇化，例如本领域中用于抗体及抗体片段(包括但不限于域抗体及scFv片段)的聚乙二醇化；参考例如:Cha pman,Nat.Biotechnol.,54,531-545(2002)；Veronese及Harris,Adv.Drug Deliv.Rev.54,453-456(2003)；Harris及Chess,Nat.Rev.Drug.Discov.2(2003)；及W02004/060965。用于使多肽聚乙二醇化的各种试剂也是市售的。优选使用(特别是通过半胱氨酸残基的)定点聚乙二醇化(参见例如Yang等人，Protein Engineeringl6，761-770(2003))。例如，出于此目的，PEG可连接至天然存在于本发明的多肽中的半胱氨酸残基，本发明的多肽可经修饰以便适当引入一个或多个用于连接PEG的半胱氨酸残基，或包含一个或多个用于连接PEG的半胱氨酸残基的氨基酸序列可融合至本发明的多肽的N-末端和/或C-末端，以上均使用对本领域技术人员而言本身已知的蛋白工程的技术。
根据一些实施方案，本发明的抗体可用于检测生物样品中的RANKL。在一些实施方案中，这使得可以鉴定产生蛋白质的细胞或组织。在一些实施方案中，与RANK结合并且阻断与其它结合化合物相互作用的抗体在调节破骨细胞分化和骨再吸收中具有治疗性用途。在一些实施方案中，抗人RANKL抗体可以阻断RANKL结合ODAR，这可以导致信号转导级联中的阻断和NF-kB介导的转录激活作用的丧失。应用例如荧光素酶报道分子测定测量NF-kB介导的转录激活作用的分析是本领域技术人员公知的。
一方面，本发明提供抗人RANKL抗体，其包括重链和轻链，其中:a)重链包括SEQ ID NO:2-9的氨基酸序列中之一所示的可变区；并且b)轻链包括SEQ ID NO:10-17的氨基酸序列中之一所示的可变区；并且所述抗体与RANKL结合，阻断RANK与RANKL相互作用。
在一些实施方案中，所述抗人RANKL抗体为通过杂交瘤技术获得的鼠源单克隆抗体、或单链抗体、或人源化抗体。人源化抗体包括嵌合抗体、改型抗体和全人源化抗体等。
在一些实施方案中，本发明涉及编码抗人RANKL抗体分子的核酸以及含有所述核酸的宿主细胞。
本发明还涉及含有本发明的至少一种抗人RANKL抗体分子及任选所述组合物的一或多种其它组份的产物或组合物。
本发明还涉及制备或生成本文所述抗人RANKL抗体分子、核酸、宿主细胞、产物及组合物的方法。
另一方面，本发明提供抗人RANKL抗体的人源化抗体，该人源化抗体可特异性地与人RANKL结合，并且所述人源化抗体的重链可变区选自SEQ ID NO:6、23、25、27或29所示的氨基酸序列，轻链可变区选自SEQ ID NO:14、31、33、35、37或39所示的氨基酸序列。
在一些实施方案中，所述人源化抗体的重链恒定区来自人IgG2，轻链恒定区来自人Kappa；或所述人源化抗体的重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链恒定区选自SEQ ID NO:43所示的氨基酸序列。
在一些实施方案中，所述人源化抗体为选自scFv、(scFv)2、Fab、Fab’或F(ab’)2的抗原结合片段。
在一些实施方案中，所述人源化抗体的完整重链选自SEQ ID NO:46-50中之一所示的氨基酸序列，和/或完整轻链选自SEQ ID NO:51-56中之一所示的氨基酸序列。
在一些实施方案中，所述人源化抗体的完整重链选自SEQ ID NO:46所示的氨基酸序列，完整轻链选自SEQ ID NO:51所示的氨基酸序列；或完整重链选自SEQ ID NO:47所示的氨基酸序列，完整轻链选自SEQ ID NO:52、53、54或55所示的氨基酸序列；或完整重链选自SEQ ID NO:48所示的氨基酸序列，完整轻链选自SEQ ID NO:52、53、54或55所示的氨基酸序列；或完整重链选自SEQ ID NO:49所 示的氨基酸序列，完整轻链选自SEQ ID NO:52、53、54或55所示的氨基酸序列；或完整重链选自SEQ ID NO:50所示的氨基酸序列，完整轻链选自SEQ ID NO:56所示的氨基酸序列。
在一些实施方案中，所述人源化抗体的重链可变区选自SEQ ID NO:6所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:14所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:23所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:31所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:23所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:35所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:23所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:33所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:23所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:37所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:27所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:31所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:27所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:35所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:27所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:33所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:27所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:37所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:25所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:31所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:25所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:35所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:25所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:33所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:25所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:37所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；或重链可变区选自SEQ ID NO:29所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:39所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列。再一方面本发明提供一种药物组合物，其活性成分为所述抗人RANKL抗体或所述人源化抗体。
本发明还涉及所述抗人RANKL抗体或所述人源化抗体在制备治疗骨损失疾病药物中用途。
在一些实施例中所述骨损失疾病包括骨质疏松症、类风湿性关节炎引起的骨关节骨质破坏、肿瘤骨转移造成的骨质破坏、巨骨细胞瘤生长导致的骨质破坏和其它由于RANKL诱导的破骨亢进所形成的骨质丢失或破坏等病理改变。
再一方面本发明提供一种改善患有骨损失疾病的患者的状况或治疗患有骨损失疾病的患者的方法，所述方法包括给予患者治疗有效量的所述抗人RANKL抗体或所述人源化抗体或给予患者所述药物组合物。
在一些实施例中所述所述骨疾病选自骨质疏松症、类风湿性关节炎引起的骨关节骨质破坏、肿瘤骨转移造成的骨质破坏、巨骨细胞瘤生长导致的骨质破坏和其它由于RANKL诱导的破骨亢进所形成的骨质丢失或破坏等病理改变。上述骨疾病主要指骨损失疾病。
在本发明的一些实施方案中，提供了治疗骨质稀少疾病的方法，包括施用药学有效量的所述抗人 RANKL抗体或所述人源化抗体或施用所述药物组合物。
在一些实施方案中，提供了治疗患者的伴随骨损失的炎症状况的方法，包括施用药学有效量的所述抗人RANKL抗体或所述人源化抗体或施用所述药物组合物。
在一些实施方案中，提供了治疗患者的伴随骨损失的自身免疫疾病的方法，包括施用药学有效量的所述抗人RANKL抗体或所述人源化抗体或施用所述药物组合物。
在一些实施方案中，提供了治疗患者的类风湿性关节炎的方法，包括施用药学有效量的所述抗人RANKL抗体或所述人源化抗体或施用所述药物组合物。
在本发明的实施方案中，提供了检测生物样品中人RANKL水平的方法，包括使样品接触抗体。
在一些实施方案中，提供了治疗骨病的方法，包括施用治疗有效量的抗人RANKL抗体或其人源化抗体。在一些实施方案中，提供了治疗骨病的方法，包括施用治疗有效量的抗人RANKL抗体或其人源化抗体和另一种治疗剂。在一些这样的实施方案中，以治疗有效量施用所述另外的治疗剂。在一些实施方案中，所述骨病是特征在于骨损失的疾病，包括但不限于骨质稀少和骨质溶解。在一些实施方案中，使用抗人RANKL抗体或其人源化抗体的治疗抑制骨再吸收速度。因此，在一些实施方案中，为了补偿骨形成低于正常水平，可以应用治疗来降低再吸收速度高于正常的骨再吸收速度，或者将骨再吸收降低至低于正常水平。在一些实施方案中，可以对抗体测定不存在或存在OPG下与RANKL的结合，并且检查它们抑制RANKL介导的破骨细胞生成和/或骨再吸收的能力。
根据一些实施方案可以治疗的状况包括但不限于下组:
骨质疏松症,包括但不限于原发性骨质疏松症，内分泌骨质疏松症(包括但不限于甲状腺功能亢进，甲状旁腺功能亢进，库欣综合征，和肢端肥大症)，骨质疏松症的遗传和先天形式(包括但不限于成骨不全，高胱氨酸尿症，门克斯综合征，赖-戴综合征)，和由于肢端固定的骨质疏松症；
成年人和少年骨佩吉特氏病(畸形性骨炎)；
骨髓炎，即导致骨损失的骨感染性病变；
高钙血症，包括但不限于实体肿瘤(包括但不限于乳房，肺和肾)和血液恶性病变(包括但不限于多发性骨髓瘤，淋巴瘤和白血病)导致的高钙血症，特发性高钙血症，和与甲状腺功能九进和肾功能失调相关的高钙血症；
骨质稀少，包括但不限于手术之后的骨质稀少，施用药物诱导的骨质稀少，与小肠和大肠疾病相关的骨质稀少，和与慢性肝和肾病相关的骨质稀少；
骨坏死，即骨细胞死亡，包括但不限于与创作性损伤有关的骨坏死，与戈谢病有关的骨坏死，与镰状细胞贫血有关的骨坏死，与系统性红斑狼疮有关的骨坏死，与类风湿性关节炎有关的骨坏死，与牙周病有关的骨坏死，与溶骨性转移有关的骨坏死，与其它状况有关的骨坏死；和与类风湿性关节炎有关的软骨损失和关节侵蚀。
在一些实施方案中，可以单独地或者与至少一种用于治疗骨病的另外的治疗剂一起使用抗人RANKL抗体或其人源化抗体。在一些实施方案中，与治疗有效量的另外一种治疗剂结合使用抗人RANKL抗体或其人源化抗体。可以与抗人RANKL抗体或其人源化抗体一起施用的举例的治疗剂包括但不限于指定为BMP-1至BMP-12的骨形态发生因子，转化生长因子_β(TGF-β)和TGF-β家族成员；白介素-1(IL-1)抑制齐U，包括但不限于IL-1ra及其衍生物和KineretTM,anakinra；TNFα抑制剂,包括但不限于可溶性TNFa受体，Enbre ITM,etanercept,抗-TNFα抗体，RemicadeTM,英夫单抗,和D2E7抗体；甲状旁腺素及其类似物；甲状旁腺素相关蛋白及其类似物；E系列前列腺素；双磷酸盐(例如阿仑特罗和其他)；骨增强矿物例如氟化物和钙；非甾族抗炎药物(NSAIDs)，包括但不限于C0X-2抑制剂，例如CelebrexTM，塞来西布，和VioxxTM，罗非克西；免疫抑制剂，例如甲氨蝶呤或来氟米特，丝氨酸蛋白酶抑制剂，包括但不限于分泌性白细胞蛋白酶抑制剂(SLPI)；IL-6抑制剂(包括但不限于抗IL-6抗体)，IL-8抑制剂(包括但不限于抗IL-8抗体)，IL-18抑制剂(包括但不限于IL-18结合蛋白和抗IL-18抗体)，白介素-1转化酶(ICE)调节剂；成纤维细胞生长因子FGF-1至FGF-1O和FGF调节剂；PAF拮抗剂；角质形成细胞生长因子(KGF)，KGF-相关分子，和KGF调节剂；基质金属蛋白酶(MMP)调节剂；一氧化氮合 酶(NOS)调节剂，包括但不限于，诱导型NOS的调节剂；糖皮质激素受体调节剂；谷氨酸受体调节剂；脂多糖(LPS)水平调节剂；和去甲肾上腺素和调节剂及其模拟物。
在一些实施方案中，抗人RANKL抗体或其人源化抗体与特定治疗剂一起使用来治疗各种炎症状况，自身免疫状况，或伴随骨损失的其他试剂。在一些实施方案中，根据状况和期望的治疗水平，可以施用两种，三种或更多试剂。在一些实施方案中，通过包含在相同配方中一起提供这些试剂。在一些实施方案中，通过包含在相同配方中一起提供这样的试剂和抗人RANKL抗体或其人源化抗体。在一些实施方案中，通过包含在治疗试剂盒中可以一起提供这样的试剂。在一些实施方案中，通过包含在治疗试剂盒中可以一起提供这样的试剂和抗人RANKL抗体或其人源化抗体。在一些实施方案中，可以分开提供这样的试剂。在一些实施方案中，当通过基因治疗施药时，相同载体中可以包含编码蛋白质试剂和/或抗人RANKL抗体的基因。在一些实施方案中，编码蛋白质试剂和/或抗人RANKL抗体的基因可以处于相同启动子区的控制下。在一些实施方案中，编码蛋白质试剂和/或抗人RANKL抗体的基因可以在分开的载体中。
在一些实施方案中，本发明涉及包括抗人RANKL抗体或其人源化抗体和至少一种白介素-1(IL-1)抑制剂的治疗方案，和应用这样的治疗方案的治疗方法。在一些实施方案中，治疗方案包括抗人RANKL抗体或其人源化抗体和IL-1抑制剂和至少一种这里描述的另外的分子。在一些实施方案中，治疗方法与抗人RANKL抗体或其人源化抗体联合使用IL-1抑制剂和/或TNFa抑制剂。在一些实施方案中，抗人RANKL抗体或其人源化抗体与IL-1抑制剂和/或TNF a抑制剂联合可以用于治疗象哮喘，类风湿性关节炎和多发性硬化这样的状况。
白介素-1(IL-1)是抗炎细胞因子。在一些例子中，IL-1是很多疾病和医学状况中的介导物。在一些例子中，巨噬细胞/单细胞系细胞生产IL-1。在一些例子中，IL-1以两种形式产生＝IL-1a(IL-1a)和IL-1β(IL-1β)。
如果自发的或实验性疾病或医学状况与体液或组织中升高的IL-1水平有关和/或如果来自身体的细胞或组织在培养物中产生升高水平的IL-1，则认为这种疾病或医学状况是“白介素-1介导的疾病”。在一些实施方案中，通过下面另外两种状况识别这样的白介素-1介导的疾病:(I)通过施用IL-1或上调IL-1的表达能从实验上用动物模拟与疾病或医学状况相关的病理发现；和(2)通过用抑制IL-1作用的试剂治疗能抑制或消除疾病或医学状况中实验动物模型中诱导的病理。在一些实施方案中，上述状况中的一项或多项符合IL-1-介导的疾病。在一些实施方案中，所有三种状况都符合IL-1-介导的疾病。急性和慢性白介素-1(IL-1)-介导的疾病包括但不限于下组:急性胰腺炎；肌萎缩侧索硬化(ALS，或Lou Gehrig’s病)；阿尔茨海默病；恶病质/厌食，包括但不限于AIDS-诱导的恶病质；哮喘和其它肺病；动脉粥样硬化；自身免疫性脉管炎；慢性疲劳综合征，梭状芽孢杆菌相关疾病，包括但不限于梭状芽孢杆菌相关腹泻，冠状动脉状况和适应症，包括但不限于充血性心衰，冠状动脉再狭窄，心肌梗塞，心肌功能障碍(例如与脓毒症有关)，和冠状动脉旁路移植；癌症，包括但不限于白血病，包括但不限于多发性骨髓瘤白血病和髓细胞白血病(例如AML和CML)，和肿瘤转移；糖尿病(包括但不限于胰岛素依赖性糖尿病)；子宫内膜异位；发热；纤维肌瘤；肾小球肾炎；移植物抗宿主疾病和/或移植物排斥；出血性休克；痛觉过敏；炎性肠病；关节炎症状况，包括但不限于骨关节炎，牛皮癣关节炎，和类风湿性关节炎；炎性眼病，包括但不限于例如与角膜移植有关的那些；局部缺血，包括但不限于脑局部缺血(包括但不限于作为例如创伤，癫痫，出血或中风的结果的脑损伤，这些的每一种情况都可以导致神经变性)；Kawasaki’s病；学习障碍；肺病(包括但不限于急性呼吸窘迫综合症，或ARDS)；多发性硬化；肌病(例如肌肉蛋白质代谢，包括但不限于脓毒症中的肌肉蛋白质代谢)；神经毒性(包括但不限于HIV诱导的这样的状况)；骨质疏松症；疼痛，包括但不限于与癌症相关的疼痛；帕金森氏病；牙周病；早产；牛皮癣；再灌注损伤；脓毒性休克；放射治疗副作用；颞颌关节病；睡眠障碍；葡萄膜炎；从例如由劳损，扭伤，软骨损伤，创伤，整形术，感染，或其它疾病过程引起的炎症。
在一些实施方案中，本发明涉及包括抗人RANKL抗体或其人源化抗体和至少一种TNFa抑制剂的治疗方案，和应用该治疗方案的方法。在一些实施方案中，治疗方案包括抗人RANKL抗体或其人源化 抗体和TNFa抑制剂和至少一种这里描述的另外的分子。一些疾病和医学状况是由TNF介导的并且归类为炎症状况。如这里使用的“TNF-介导的疾病”包括但不限于与体液或组织中升高水平的TNF相关的和/或其中细胞或组织取自在培养物中产生高水平TNF的个体的疾病或医学状况。在一些实施方案中，如果(I)通过施用或上调TNF表达在动物中能实验性模拟与疾病或医学状况相关的病理发现和/或(2)通过用抑制TNF作用的试剂治疗能抑制或破坏疾病或医学状况的实验动物模型中诱导的病理，则认为疾病是TNF-介导疾病。一些疾病和医学状况是TNF介导的，并且可以分类为炎症状况。如这里使用的，“TNF介导的疾病”包括但不限于:恶病质和厌食；癌症，包括但不限于，白血病；慢性疲劳综合征；冠状动脉状况和/或适应症，包括但不限于，充血性心衰，冠状再狭窄，心肌梗塞，心肌功能障碍(包括但不限于与脓毒症有关的这样的状况)，和冠状动脉旁路移植；抑郁症；糖尿病，包括但不限于，青少年起病I型糖尿病，糖尿病，和胰岛素抗性(包括但不限于与肥胖有关的胰岛素抗性)；子宫内膜异位，子宫内膜炎，和相关的状况；纤维肌瘤和痛觉缺失；移植物抗宿主排斥；痛觉过敏；炎性肠病，包括但不限于克隆氏病和艰难梭菌-相关腹泻；局部缺血，包括但不限于，作为创伤，癫痫，出血，和/或中风结果的脑损伤；肺病，包括但不限于成人呼吸窘迫综合征，哮喘，肺纤维化；多发性硬化；神经炎性疾病；眼部疾病和状况，包括但不限于角膜移植，眼退化和葡萄膜炎；疼痛，包括但不限于，与癌相关的疼痛；胰腺炎，牙周病；毛发红糠疹(PRP)；前列腺炎，包括细菌性和非细菌性前列腺炎，和相关状况；牛皮癣和相关状况；肺纤维化；再灌注损伤；风湿病，包括但不限于类风湿性关节炎，骨关节炎，青少年关节炎(包括但不限于青少年类风湿性关节炎)，血清反应阴性多关节炎，强直性脊柱炎，Reiter’s综合征和反应性关节炎，Still’s病，牛皮癣关节炎，肠病关节炎，多肌炎，皮肌炎，硬皮病，系统性硬化，脉管炎(例如Kawasaki’s病)，脑脉管炎，赖姆病，葡萄球菌诱导(“脓毒性”)的关节炎，斯耶格伦综合征，风湿热，多软骨炎和风湿性多肌病和巨细胞动脉炎)；脓毒性休克；放疗副作用；系统性红斑狼疮(SLE)；颞颌关节病；甲状腺炎；和组织移植和/或例如由劳损，扭伤，软骨损伤，创伤，整形术，感染(例如HIV，艰难梭菌和相关物种)或其他疾病过程引起的炎症。
在一些实施方案中，TNF抑制剂通过下调或抑制TNF产生，结合游离THF，干扰TNF与其受体结合，和干扰与其受体结合之后TNF信号传递调节中的至少一种而发挥作用。术语“TNF抑制剂”包括但不限于可溶性TNF受体，包括但不限于，可溶性肿瘤坏死因子受体I型(sTNF-RI；也称作p55受体)，可溶性肿瘤坏死因子受体II型(也称作p75受体)，和EnbreITM,etanercept；抗TNF抗体,包括但不限于，RemicadeTM,英夫单抗和D2E7(参见,例如美国专利N0.6090382和6258562)；抗TNF受体抗体；sTNF-RI(参见，例如，W098/24463)，Etanercept(EnbrelTM)；TNF-α转化酶(TACE)的抑制剂；和影响TNF活性的其他分子。
在一些实施方案中，抗人RANKL抗体或其人源化抗体可以与至少一种用于炎症的治疗剂一起施用。在一些实施方案中，抗人RANKL抗体或其人源化抗体可以与至少一种用于免疫疾病的治疗剂一起施用。用于炎症和免疫疾病的举例的治疗剂包括但不限于皮质类固醇，包括但不限于，泼尼松龙；非留族抗炎药物(NSAIDs)，包括但不限于，环加氧酶I型(C0X-1)和环加氧酶2型(C0X-2)抑制剂；疾病修饰抗风湿药物(DMARDs)，包括但不限于，甲氨蝶呤，羟基氯奎，氯奎，环孢菌素，金化合物(例如醋硫葡金，金硫丁二酸盐，和葡糖硫金)，来氟米特；IV型磷酸二酯酶抑制剂，包括但不限于，环戊苯吡酮和己酮可可碱；他克莫司(FK-506)；西罗莫司(瑞帕霉素)；霉酚酸；5_脂肪氧化酶抑制剂，包括但不限于，弃白通；白介素-6(IL-6)调节剂；38kDa促细胞分裂剂激活蛋白激酶的小分子调节剂(P38-MAPK)；炎症途径中涉及的胞内分子的小分子调节剂，其中这样的胞内分子包括但不限于，jnk，IKK,NF-Kb,ZAP70，和lck。用于炎症的一些举例的治疗剂描述于，例如，C.A.Dinarello和L.L.Moldawer Proinfla.mma.torv andAnt1-1nflammatorv Cytokines in Rheuma toid Arthritis:A Primer for Clinicians第三版(2001)Amgen Inc.Thousand Oaks，CA。用于炎症和自身免疫疾病的举例的治疗剂，包括但不限于，干扰素-Y(IFN-Y)调节剂；0X40/0X40L的调节剂(包括可溶形式的0X40)；4-1BB/4-1BB配体的调节剂(包括可溶形式的4-1BB)；和B细胞-T细胞共同刺激途径的调节剂等；
在一些实施方案中，抗人RANKL抗体或其人源化抗体被用来治疗骨损失，包括但不限于，恶性或 转移肿瘤引起的骨的溶骨性破坏产生的骨损失。在一些实施方案中，抗人RANKL抗体或其人源化抗体可以被用来治疗与癌症相关的骨损失。举例的癌症包括但不限于乳房癌，前列腺癌，甲状腺癌，肾癌，肺癌，食道癌，直肠癌，膀胱癌，子宫颈癌，卵巢癌和肝癌，以及胃肠道癌。在一些实施方案中，抗人RANKL抗体或其人源化抗体可以被用来治疗与一些血液恶病变相关的骨损失，包括但不限于，多发性骨髓瘤和淋巴瘤，包括何杰金氏病。
在一些实施方案中，单独施用抗人RANKL抗体或其人源化抗体。在一些实施方案中，抗人RANKL抗体或其人源化抗体与至少一种其他治疗剂一起施用，包括但不限于，至少一种其他癌症治疗剂。举例的癌症治疗剂包括但不限于放疗和化疗。在一些实施方案中，化疗可以包括用一种或几种下面的药物治疗:蒽环霉素，紫杉醇，他莫西芬，阿霉素，5-氟尿嘧啶，和本领域公知的其他药物。在一些实施方案中，癌症治疗剂是黄体激素释放激素(LHRH)拮抗剂。
药物组合物
在一些实施方案中，本发明提供含有治疗有效量的抗人RANKL抗体或且人源化抗体和药学可接受稀释剂，载体，增溶剂，乳化剂，防腐剂和/或佐剂的药物组合物。
在一些实施方案中，本发明提供含有治疗有效量的抗人RANKL抗体或其人源化抗体和治疗有效量的至少一种另外的治疗剂，和药学可接受稀释剂，载体，增溶剂，乳化剂，防腐剂和/或佐剂的药物组合物。在一些实施方案中，所述至少一种另外的治疗剂选自:骨形态发生因子，转化生长因子-β(TGF-β)，白介素-1(IL-1)抑制剂，包括但不限于IL-1ra及其衍生物和KineretTM，anakinra；TNFα抑制剂，包括但不限于可溶性TNFα受体，EnbreITM,etanercept,抗-TNFα抗体,RemicadeTM,英夫单抗,和D2E7抗体；甲状旁腺素及其类似物；甲状旁腺素相关蛋白及其类似物；E系列前列腺素；双磷酸盐(例如阿仑特罗和其他)；骨增强矿物例如氟化物和钙；非留族抗炎药物(NSAIDs)，包括但不限于C0X-2抑制剂，例如CelebrexTM,塞来西布和VioxxTM，罗非克西；免疫抑制剂，例如甲氨蝶呤或来氟米特，丝氨酸蛋白酶抑制剂，包括但不限于分泌性白细胞蛋白酶抑制剂(SLPI)；IL-6抑制剂(包括但不限于抗IL-6抗体)，IL-8抑制剂(包括但不限于抗IL-8抗体)，IL-18抑制剂(包括但不限于IL-18结合蛋白和抗IL-18抗体)，白介素-1转化酶(ICE)调节剂；成纤维细胞生长因子FGF-1至FGF-10和FGF调节剂；PAF拮抗剂；角质形成细胞生长因子(KGF)，KGF-相关分子，和KGF调节剂；基质金属蛋白酶(MMP)调节剂；一氧化氮合酶(NOS)调节剂，包括但不限于，诱导型NOS的调节剂；糖皮质激素受体调节剂；谷氨酸受体调节剂；脂多糖(LPS)水平调节剂；和去甲肾上腺素和调节剂及其模拟物。
在一些实施方案中，可接受的配方材料优选是使用的剂量和浓度对接受者没有毒性。在一些实施方案中，所述药物组合物可以含有用于改变，保持或保留例如pH，渗透压，粘度，澄清度，颜色，等张性，气味，无菌，稳定性，溶解或释放速度，组合物的吸收或渗透的配方材料。在一些实施方案中，合适的配方材料包括但不限于，氨基酸(例如甘氨酸，谷氨酰胺，天冬酰胺，精氨酸或赖氨酸)；抗微生物剂；抗氧化剂(例如抗坏血酸，亚硫酸钠或亚硫酸氢钠)；缓冲液(例如硼酸盐，碳酸氢盐，Tris-HCl，柠檬酸盐，磷酸盐或其他有机酸)；填充剂(例如甘露糖醇或甘氨酸)；螯合剂(例如乙二胺四乙酸(EDTA))；络合剂(例如咖啡因，聚乙烯吡咯烷酮，环糊精或羟丙基环糊精)；填料；单糖；二糖；和其他碳水化合物(例如葡萄糖，甘露糖或糊精)；蛋白质(例如血清白蛋白，明胶或免疫球蛋白)；着色剂，矫味剂和稀释剂；乳化剂；亲水性聚合物(例如聚乙烯吡咯烷酮)；低分子量多肽；成盐抗衡离子(例如钠)；防腐剂(例如苯扎氯铵，苯甲酸，水杨酸，硫汞撒，苯乙醇，羟苯甲酸甲酯，羟苯甲酸丙酯，氯己定，山梨酸或过氧化氢)；溶剂(例如甘油，丙二醇或聚乙二醇)；糖醇(例如甘露糖醇或山梨糖醇)；悬浮剂；表面活性剂或湿润剂(例如普流罗尼、PEG、脱水山梨糖醇酯，polysorbates,如polysorbate20,polysorbate80，卵磷脂，胆甾醇，四丁酚醛)，稳定增强剂(例如蔗糖或山梨糖醇)；张力增强剂(例如碱金属卤化物，优选氯化钠或氯化钾，甘露糖醇，山梨糖醇)；送递赋形剂；稀释剂；赋形剂和/或药物佐剂(Remington’s Pharmaceutical Science,18版，A.R.Gennaro编著，Mack Publish Company(1990))。
在一些实施方案中，抗人RANKL抗体或其人源化抗体和/或治疗分子与本领域公知的半衰期延长赋形剂连接。这样的赋形剂包括但不限于聚乙二醇和葡聚糖。这样的赋形剂描述于，例如，美国申请登记 号N0.09/428082和公开的PCT申请N0.W099/25044，这里为了任何目的引作参考。
在一些实施方案中，本领域技术人员根据，例如，想要的给药途径，送递方式和期望的剂量，来确定优化的药物组合物。例如，参见Remington’s Pharmaceutical Science,18版，A.R.Gennaro编著，Mack Publish Company(1990)。在一些实施方案中，这样的组合物可以影响本发明的抗体的物理状态，稳定性，体内释放速度和体内清除速度。
在一些实施方案中，药物组合物中的主要赋形剂或载体性质上可以是含水或不含水的。例如，在一些实施方案中，合适的赋形剂或载体可以是注射用水，生理盐水或人工脑脊液，可能补充有肠胃外施用组合物中常用的其他材料。在一些实施方案中，中性缓冲盐水或与血清白蛋白混合的盐水是其他举例的赋形剂。在一些实施方案中，药物组合物含有大约pH7.0-8.5的Tris缓冲液，或大约pH4.0-5.5的乙酸盐缓冲液，其可以进一步含有山梨糖醇或其合适的取代物。在一些实施方案中，含有抗人RANKL抗体或其人源化抗体，有或没有至少一种另外的治疗剂的组合物，可以通过将具有期望程度纯度的选择的组合物与任选的配方试剂混合来制备。此外，在一些实施方案中，使用合适的赋形剂例如蔗糖，可以将含有抗人RANKL抗体或其人源化抗体，有或没有至少一种另外的治疗剂的组合物配制成冻干物。
在一些实施方案中，可以选择本发明的药物组合物用于肠胃外给药。在一些实施方案中，可以选择药物组合物用于吸入或者通过消化道例如口服送递。这样的药学可接受组合物的制备是本领域公知的。
在一些实施方案中，当涉及肠胃外给药时，治疗组合物可以是药学可接受赋形剂中无热原的，肠胃外可接受的，含有期望的抗人RANKL抗体或其人源化抗体，有或没有另外的治疗剂的水溶液形式。在一些实施方案中，用于肠胃外注射的赋形剂是无菌蒸馏水，其中将有或没有至少一种另外的治疗剂的抗人RANKL抗体或其人源化抗体配制成无菌等张溶液，适当保存。在一些实施方案中，制备涉及配制期望的分子和试剂，例如可注射微球，可生物降解的颗粒，聚合化合物(例如聚乳酸或聚乙醇酸)，珠或脂质体，它可以提供产品的可控或持续释放，然后通过延效型注射送递。在一些实施方案中，也可以使用透明质酸，有在循环中促进保持的作用。在一些实施方案中，可以使用可植入的药物送递装置引入期望的分子。
在一些实施方案中，药物组合物可以配制成用于吸入的制剂。在一些实施方案中，可以将有或没有至少一种另外的治疗剂的抗人RANKL抗体或其人源化抗体配制成用于吸入的干燥粉末。在一些实施方案中，也可以用用于气溶胶送递的推进剂配制含有有或没有至少一种另外的治疗剂的抗人RANKL抗体或其人源化抗体的吸入用溶液。在一些实施方案中，溶液可以喷雾。肺部给药进一步描述于PCT申请N0.PCT/US94/001875,它描述了化学修饰蛋白质的肺部递送。
在一些实施方案中，涉及可以口服给药的制剂。在一些实施方案中，可以用固体剂型例如片剂和胶囊的复合中常规使用的那些载体或者不用那些载体，可以配制以这种方式给药的有或没有至少一种另外的治疗剂的抗人RANKL抗体或其人源化抗体。在一些实施方案中，可以设计当生物利用率最大并且预先全身性降解最小的消化道中的点释放制剂的活性部分的胶囊。在一些实施方案中，可以含有至少一种另外的试剂以有利于抗人RANKL抗体或其人源化抗体和/或任何另外的治疗剂的吸收。在一些实施方案中，还可以使用稀释剂，矫味剂，低熔点蜡，植物油，润滑剂，悬浮剂，片剂，崩解剂和粘合剂。
在一些实施方案中，药物组合物在适合片剂制备的无毒性赋形剂混合物中可以含有有效量的抗人RANKL抗体或其人源化抗体，有或没有至少一种另外的治疗剂。在一些实施方案中，通过将片剂溶解于无菌水中，或其他合适的赋形剂中，可以制备单剂型的溶液。在一些实施方案中，合适的赋形剂包括但不限于惰性稀释剂，例如碳酸钙，碳酸钠或碳酸氢钠，乳糖，或磷酸钙；或粘合剂，例如淀粉，明胶，或阿拉伯胶；或润滑剂，例如硬脂酸镁，硬脂酸或滑石。
另外的药物组合物对于本领域技术人员是明显的，包括涉及持续或控制释放送递配方中含有抗人RANKL抗体或其人源化抗体，有或没有至少一种另外的治疗剂的制剂。在一些实施方案中，各种各样的其他持续或控制释放送递方式的配制技术，例如脂质体载体，可生物降解微颗粒或多孔珠和延效型注射剂，是本领域技术人员公知的。参见例如，PCT申请N0.PCT/US93/00829，它描述了用于送递药物组合物的多孔聚合物微颗粒的控制释放。在一些实施方案中，持续释放制剂可以含有有形产物形式的半渗 透性聚合物基质，例如膜，或微胶囊。持续释放的基质可以包括聚酯，水凝胶，聚交酯(US3773919和EP058481)，L-谷氨酸和Y-乙基-L-谷氨酸的共聚物(Sidman等，Biopolymers,22:547-556(1983)),聚(2-轻乙基-甲基丙烯酸酯)(Langer等，J.Biomed.Mater.Res.15:167-277(1981)和Langer,Chem.Tech.，12:98-105(1982))，乙酸亚乙基乙烯酯(Langer等，上文)或聚-D(-)-3-羟基丁酸(EP133988)。在一些实施方案中，持续释放的组合物还可以包括脂质体，它可以通过本领域公知的几种方法制备。参见，例如Eppstein等，Proc.Natl.Acad.Sc1.USA,82:3688-3692(1985)；EP036676；EP088046和EP143949。
一般体内施用的药物组合物是无菌的。在一些实施方案中，可以通过经无菌过滤膜过滤来实现。在一些实施方案中，在组合物被冻干的情况下，可以在冻干和重新配置之前或之后利用该方法灭菌。在一些实施方案中，肠胃外给药的组合物可以以冻干形式或溶液贮存。在一些实施方案中，一般将肠胃外组合物放到有灭菌出口的容器中，例如有皮下注射针头可穿孔的盖的静脉输液液袋或小瓶。
在一些实施方案中，一旦配制了药物组合物，它可以作为溶液，混悬液，凝胶，乳状液，固体保存在无菌小瓶中，或者作为脱水或冻干的粉末剂保存在无菌小瓶中。在一些实施方案中，这样的制剂可以以即用形式或者以给药之前重新配制的形式(例如冻干的)贮存。
在一些实施方案中，本发明涉及制备单剂量给药单位的试剂盒。在一些实施方案中，每个试剂盒可以包括盛有干燥蛋白质的第一容器和盛有含水配方的第二容器。在本发明的一些实施方案中，包括包含单腔或多腔预填充注射器(例如液体注射器和溶解注射器)的试剂盒。
在一些实施方案中，治疗上要使用的含有有或没有至少一种另外的治疗剂的抗人RANKL抗体或其人源化抗体的药物组合物的有效量取决于，例如，治疗内容和主体。本领域技术人员明白根据一些实施方案，治疗的适当剂量水平因此部分地根据送递的分子，使用有或没有至少一种另外的治疗剂的抗人RANKL抗体或其人源化抗体的适应症，给药途径，和患者尺寸(体重，体表面积或器官大小)和/或状况(年龄和一般健康状况)而不同。在一些实施方案中，医师可以调整剂量和改变给药途径，以获得最佳治疗效果。在一些实施方案中，根据上面提到的因素，一般剂量范围可以是大约0.1微克/千克至最多大约100毫克/千克或更多。在一些实施方案中，剂量可以是0.1微克/千克至最多大约100毫克/千克；或者I微克/千克至最多大约100毫克/千克；或者5微克/千克至最多大约100毫克/千克。
在一些实施方案中，给药频率要考虑使用的制剂中抗人RANKL抗体或其人源化抗体和/或任何另外的治疗剂的药物动力学参数。在一些实施方案中，医师施用组合物，直到达到实现期望的效果的剂量。在一些实施方案中，因此可以以单一剂量，或随时间作为两个或多个剂量给药(其可以含有或不含有相同量的期望的分子)，或者作为通过植入装置或导管连续输注来施用本发明药物组合物。本领域技术人员按常规确定精确的合适的剂量，并且这是平常他们完成的常规任务。在一些实施方案中，通过应用合适的剂量反应数据可以确定合适的剂量。
一些实施方案中，药物组合物的给药途径是公知的方法，例如，口服，静脉内注射,腹膜内，脑内(实质内)，脑室内，肌内，眼内，动脉内，肝门内，或病变内途径；通过持续释放系统或者通过植入装置。在一些实施方案中，可以通过浓注或连续输液或者通过植入装置施用组合物。
在一些实施方案中，通过植入其上吸附期望的分子或者将期望的分子制成胶囊的膜，海绵状物或者其它合适的材料可以局部施用组合物。在一些实施方案中，施用植入装置的情况下，可以将装置植入任何合适的组织或器官，期望的分子的送递可以通过扩散，随时间释放药团，或连续给药。
在一些实施方案中，期望以离体的方式使用含有抗人RANKL抗体或其人源化抗体，有或没有至少一种另外的治疗剂的药物组合物。在这样的情况下，取自患者的细胞，组织和/或器官接触含有抗人RANKL抗体或其人源化抗体，有或没有至少一种另外的治疗剂的药物组合物，然后接着将这些细胞，组织和/或器官植回患者。
在一些实施方案中，通过使用这里描述的方法植入经基因工程处理的细胞来表达和分泌多肽，能送递抗人RANKL抗体或抗人RANKL人源化抗体和/或任何另外的治疗剂。在一些实施方案中，这样的细胞可以是动物细胞或人细胞，并且可以是自体的，异源的，或异种的。在一些实施方案中，细胞可以无限繁殖。在一些实施方案中，为了减少免疫应答机会，可以将细胞制成胶囊以避免周围组织浸润。在一 些实施方案中，胶囊材料一般是生物相容的半渗透性聚合物外壳或膜，它使得蛋白质产物释放，但是阻止患者免疫系统或者来自周围组织的其它有害因子破坏细胞。
以下结合具体实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1、human RANKL的制备
通过构建human RANKL表达载体，稳定转染CHO细胞，筛选能够稳定高表达human RANKL的细胞株。大规模培养该细胞株，收集细胞上清，通过镍柱纯化制备human RANKL蛋白，用于实施例2、3、4、5、6和7中小鼠免疫、克隆筛选及功能鉴定。
CHO细胞购自Invitrogen公司，RAW264.7细胞购自上海中科院细胞库，表达载体为本公司提供，T4DNA连接酶、蛋白分子量标准Marker、限制性内切酶等购自NEB公司；胶回收试剂盒购自Invitrogen；302培养基、胰蛋白酶和FBS购自Invitrogen公司；Phenyl sepharose 6FF-low sub,SP-Sepharose FF,Ni-NTA Sepharose FF凝胶购自GE公司；磷酸氢二钠，磷酸二氢钠，氯化钠，Tris，柠檬酸，柠檬酸三钠，咪唑和MTX购自Sigma公司，羊抗人IgG-HRP购自Jackson，TMB Substrate，购自Cell Signaling公司，RANK-Fc,TRANC,M-CSF和TGF-β购自R&D公司，TRAP显色系统购自Sigma公司，DMEM培养基、α-MEM培养基和胎牛血清购自Invitrogen公司。
PCR仪(杭州朗基科学仪器生物有限公司)，摇床CLIMO-SHAKER 1SFS-X(瑞士科耐Kuhner公司)。多功能酶标仪为Molecular Devices SpectraMax M5Multi-mode Microplate Reader洗板机为TECAN HydroFlex Plate Washer,超净工作台品牌为苏净，规格型号SW-CJ-2F/T，二氧化碳培养箱的品牌为Thermo，规格型号Forma 311.
(1)human RANKL表达载体的构建
首先通过基因工程手段合成human RANKL目标序列(图1)，序列从自然人RANKL的136位Gly开始，到317位Asp共182个氨基酸(SEQ ID NO:1)，在N端增加10个His，可以与镍柱中的氯化镍结合，从而可以通过离子亲和层析进行纯化，并在两端增加NotI和PmeI两个酶切位点。将合成的human RANKL和表达载体均通过NotI和PmeI进行双酶切，回收human RANKL目的片段和表达载体片段，进行连接，转化，通过PCR和酶切方法鉴定阳性克隆，最后通过测序验证表达载体的正确性。采用质粒提取试剂盒抽提质粒用于稳定转染。
(2)稳定转染CHO细胞及克隆筛选
转染前48小时，在302无血清培养基中传代培养CHO细胞，接种密度为3×105/ml。转染当天细胞总数应大于1.5×107，细胞活力在95％以上。采用Bio-rad的电转仪进行转化，转染电压为300V，电容为900μF。细胞每2～3天需传代一次，更换新鲜的培养基继续培养，直至细胞生长恢复正常。当细胞活力在90％以上，进行MTX加压筛选。MTX的浓度按照25nM，50nM，100nM，250nM，500nM逐步增加。MTX压力达到500nM时开始按照有限稀释法分板，待克隆长出后进行Dot-blot检测，取5μl 96孔板细胞上清，点至硝酸纤维素膜上，风干后加入5％脱脂奶粉中室温封闭30分钟，加入1:1000稀释的RANK-Fc，室温震荡孵育1小时，TBST中洗涤3次，每次5分钟，加入1:10000稀释的HRP标记的羊抗人IgG-Fc(购自Sigma公司)中室温震荡孵育1小时，采用DAB显色系统进行显色。筛选表达水平较高的克隆，共有120个克隆进行首次筛选，选择表达水平最高的一个克隆再次进行亚克隆，从而保证获得的表达human RANKL的克隆为单克隆。
(3)human RANKL的纯化
收集大规模培养的细胞上清，离心后采用镍柱进行纯化，镍柱中的氯化镍能够跟His结合，也能够与咪唑进行结合。首先将样品按一定流速流过镍柱，使得样品中的human RANKL与镍柱结合，然后加 入不同浓度的咪唑，与human RANKL竞争结合镍柱，从而将human RANKL洗脱下来。取2ml Ni2+Sepharose 6FF填装层析柱，Binding buffer(20mM PB+0.15M NaCl，pH 7.4)洗10～20个柱体积。细胞上清液按照30ml/h的流速上样，用Binding buffer洗涤10-20个柱体积后，10mM咪唑洗去杂蛋白，70mM,100mM和500mM咪唑分别洗脱，收集洗脱液，human RANKL的主要洗脱峰出现在70mM咪唑的洗脱条件。纯化的人RANKL蛋白经还原SDS-PAGE电泳鉴定，其分子量约为34KDa，与理论分子量一致，纯度大于90％。
(4)human RANKL的鉴定
包被human RANKL和市售hRANKL标准品，样品稀释至1μg/ml，100μl/孔加入，4度孵育过夜。加入从10μg/ml开始梯度稀释的RANK-Fc，室温孵育1小时，加入1:20000稀释的二抗(羊抗人IgG-Fc)，室温孵育1小时，采用TMB显色系统进行显色。在多功能酶标仪上以测定波长450nm，参比波长630nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD630nm。鉴定human RANKL与RANK-Fc的结合活性。结果表明，包被于ELISA板上的重组human RANKL与其受体RANK-Fc结合，其结合具有RANK-Fc浓度依赖性并可达到饱和，达到50％最大结合的RANK-Fc浓度为10.9ng/ml，其结合与R&D公司市售RANKL标准品一致，后者达到50％最大结合的RANK-Fc浓度为5.6ng/ml。
RAW264.7细胞使用DMEM+10％FBS的培养基进行培养，每3-4天传代一次。进行样品活性检测时，将对数生长期的细胞培养基更换为α-MEM培养基+10％FBS，制成细胞悬液。以2000cell/100μl接种96孔板，放入37℃,5％CO2培养箱中培养1小时。加入从800ng/ml开始对倍稀释human RANKL，从200ng/ml开始对倍稀释的市售hRANKL标准品和固定浓度的M-CSF和TGFβ。置于37℃,5％CO2培养箱中培养5天，弃细胞上清，每孔加入100μl细胞裂解液(Citrate Buffer pH 5.0+0.5％Triton X-100)，4℃冰箱中裂解10min，每孔加入100μl pNPP显色液，37℃孵育30min，再每孔加入50μl终止液(0.5M NaOH)，450/570nm读数。经human RANKL诱导RAW264.7细胞分化的活性实验鉴定，重组human RANKL能够诱导RAW264.7细胞分化为破骨细胞，并具有明显的剂量依赖性，其细胞活性与R&D的产品相似，重组human RANKL的EC50为113.4ng/ml，市售hRANKL标准品的EC50略小一些，为35.7ng/ml。
本实施例表明，重组且经纯化的human RANKL，不仅保持了与相应受体RANK-Fc的结合能力，且能通过受体激动破骨细胞的分化，具有其生物学活性。重组human RANKL作为抗原用于本申请所描述的免疫程序，并且用来作为ELISA法筛选阳性克隆的包被蛋白和抗体免疫学和细胞学功能鉴定的材料。
实施例2、小鼠免疫和滴度测定
免疫用抗原(重组human RANKL)来自实施例1，BALB/c小鼠购自北京通利华实验动物技术有限公司。抗RANKL的单克隆抗体通过多次免疫BALB/c小鼠获得。免疫途径为足底注射，免疫剂量为10μg/50μl/只小鼠，每只足底25μl。共免疫了10只小鼠。
首次免疫将10μg human RANKL与等体积的TiterMax
Figure PCTCN2014095236-appb-000001
(Sigma,Oakville,ON)混合，随后的10多次免疫将10μg human RANKL与等体积的100μg明矾(Sigma,Oakville,ON)和10μl无热源的含CpG的D-PBS混合，不含佐剂。BALB/c小鼠的免疫分别发生在第0,5,10,15,20,25,30,35,40and 44天，第44天取四只最高血清滴度的小鼠进行融合。
10只小鼠分别在第五次(第20天)免疫和第九次(第40天)免疫后，眼球采血，通过ELISA的方法测定免疫小鼠血清中抗人RANKL抗体的滴度。首先，human RANKL用包被缓冲液稀释(0.1M包被缓冲液,pH 9.6NaHCO38.4g/L)至1μg/ml，100μl/孔包被于96孔ELISA板(Corning,Acton,MA)，4度过夜。次日，用1ⅹPBST(0.05％Tween 20in 1x PBS)洗板3次，200ul/孔加入封闭液(0.5％BSA,0.1％Tween 20,0.01％Thimerosal in 1x PBS)室温封闭1小时。洗板3次，小鼠血清用0.5％BSA/PBS从1:100开始3倍稀释，空白孔为0.5％BSA/PBS，100μl/孔加入ELISA板中，室温孵育2小时，洗板3次，加入终浓度为1μg/ml的羊抗鼠IgG Fc-HRP，室温孵育1小时。洗板3次，加入TMB(BioFx BSTP-0100-01) 显色液室温显色10-20分钟，加入终止液，于450nm读数。OD值大于空白孔的2倍定义为阳性克隆，血清在最高稀释倍数时，OD值越高，表明对human RANKL的免疫反应性越强。血清滴度检测数据如表1所示。第五次免疫后，小鼠血清滴度范围除了4#和8#为1:62500，其余小鼠均达到1:312500。第九次免疫后，小鼠血清滴度范围除了1#和4#为1:312500，其余小鼠血清滴度均达到了1:1562500。
表1
Figure PCTCN2014095236-appb-000002
实施例3、抗human RANKL鼠单克隆的产生
免疫小鼠用CO2安乐死，然后颈椎脱臼，分离获得淋巴结，合并不同小鼠来源的淋巴结。放入DMEM培养基中进行研磨，收集上清，离心获得淋巴细胞，并采用血球计数板计数。
将上述获得的B细胞洗涤，与非分泌性骨髓瘤细胞P3X63Ag8.653(ATCC,Cat#CRL1580)按1:1进行混合，细胞混合液于800g离心，轻轻地去掉上清，加入2-4ml链酶蛋白酶溶液(CalBiochem,cat.#53702；0.5mg/ml in PBS)，作用不超过2分钟，加入3-5ml胎牛血清终止酶反应，加入细胞电融合液ECFS(0.3M Sucrose,Sigma,Cat#S7903,0.1mM Magnesium Acetate,Sigma,Cat#M2545,0.1mM Calcium Acetate,Sigma,Cat#C4705)调整体积为40ml，离心，去掉上清，重新将细胞悬于40ml ECFS中，洗涤一次，加入ECFS，调整细胞密度为2x106个/ml.采用电融合仪(ECM2001,BTX,Harvard Apparatus,Holliston,MA进行融合)。融合室选择2.0ml，参数设置为：
Alignment condition:voltage:50V,time:50s
Membrane breaking at:voltage:3000V,time:30usec
Post-fusion holding time:3sec
电融合完成后，轻轻地取出细胞上清，转入无菌的含等体积杂交瘤培养基(DMEM(JRH Biosciences),15％FBS(Hyclone),supplemented with L-glutamine,pen/strep,OPI(oxaloacetate,pyruvate,bovine insulin)(all from Sigma)and IL-6(Boehringer Mannheim))的离心管中，于37度孵育15-30分钟，然后400g(1000rpm)离心5分钟，轻轻地将细胞重悬于少量的杂交瘤筛选培养基(Hybridoma Culture Medium supplemented with 0.5x HA(Sigma,cat.#A9666)),中调整细胞密度，轻柔混匀，每块96孔细胞培养板接种5x106个B细胞，200μl/孔。在第7或10天去掉一半的培养上清，每孔再加入100μl筛选培养基。
细胞培养14天后，用ELISA法测定杂交瘤上清中特异性结合RANKL的抗体筛选阳性杂交瘤。human RANKL用包被缓冲液(0.1M Carbonate Buffer,pH 9.6,NaHCO38.4g/L)稀释至1μg/ml，50μl/孔进行包被，4度过夜，次日，用洗板液(0.05％Tween 20in PBS)洗涤3次，200μl/孔加入封闭液(0.5％BSA,0.1％Tween 20,0.01％Thimerosal in 1x PBS)室温封闭1小时，洗板3次，每孔加入50μl杂交瘤细胞上清，或者阳性和阴性对照(阳性对照为human RANKL免疫的小鼠血清，阴性对照为免疫前的BALB/c小鼠血清)，室温孵育2小时，洗板3次，每孔加入1:2000稀释的羊抗鼠IgG-HRP(Jackson Lab,Cat.No: 115-035-062)室温孵育1小时，洗板3次，每孔加入100μl TMB显色液(BioFX Lab.Cat.No.TMSK-0100-01)室温显色10分钟，每孔加入50μl终止液，于450nm读数。结果，经过第一轮初筛，共190块96孔细胞培养板，筛选到435个能够与human RANKL结合的阳性克隆(OD值大于0.5)。
初筛ELISA检测结果呈RANKL抗体阳性者，去掉阳性克隆的培养基，加入新鲜的杂交瘤培养基，将克隆转至24孔板培养，2天后，对24孔板克隆进行第二轮ELISA筛选验证实验，包括同上的直接ELISA检测，竞争抑制实验和与mouse RANKL的免疫交叉反应。其中，mouse RANKL的免疫交叉反应将包被蛋白更换为mouse RANKL，包被浓度为1μg/ml，检测样品为原倍的细胞上清，一个浓度点，其余实验条件与初筛ELISA实验方法一致。结果显示有9个克隆与mouse RANKL具有免疫交叉反应。竞争抑制实验为单点测试，RANK-Fc用包被液稀释至1μg/ml进行包被，4度过夜。原倍的细胞上清与等体积的浓度为600ng/ml的human RANKL混合，加入ELISA板中，室温孵育2小时，洗板3次，加入1:10000稀释的兔抗人His-HRP(购自Abcam公司)抗体，室温孵育1小时，洗板4次，采用TMB显色系统进行显色。室温显色15分钟，加入1M H2SO4终止显色。在多功能酶标仪(Molecular Devices SpectraMax M5Multi-mode Microplate Reader)上以测定波长450nm，参比波长630nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD630nm。筛选出具有竞争活性的克隆，将其细胞上清再做3倍梯度稀释重复实验，选择竞争活性相对较强的克隆再进行一轮亚克隆，扩增、冻存细胞株，并纯化细胞上清获得一定量的抗体用于功能鉴定。
第二轮ELISA结果显示：有249个克隆OD值大于0.5，有41个克隆具有抑制human RANKL与RANK-Fc结合的活性，有9个克隆具有与鼠RANKL的免疫交叉反应。将上述50个克隆进行亚克隆，冻存细胞株，并纯化细胞上清用于实施例4、5、6和7的功能鉴定。
实施例4、纯化的鼠单抗与human RANKL的免疫反应性
上述41个具有中和活性的克隆和9个与鼠有交叉反应的克隆进行扩增培养，在T125方瓶中加入100ml杂交瘤培养基，于37度，5％CO2培养箱中培养10天左右，收集上清，采用ProteinA进行抗体纯化，用pH＝3.0的柠檬酸缓冲液进行洗脱，收集样品，调pH至6.0左右。A280进行抗体浓度测定。每个抗体均得到mg级的蛋白。采用直接ELISA法检测上述50个抗human RANKL鼠单抗与免疫原human RANKL的结合能力。用pH 9.6的0.05M碳酸盐缓冲液将Human RANKL蛋白稀释至1μg/ml，100μl每孔加入到96孔ELISA板中，4℃放置过夜。PBST洗板3次，用PBST+2％BSA封闭1小时，之后用PBST洗板3次。分别将待测抗体用PBST从10μg/ml起进行10倍系列稀释至1×105μg/ml，每孔100μl加入ELISA板中，样品做双复孔，每块96孔ELISA板做5个样品，共做10块96孔ELISA板。抗原抗体室温孵育1小时后，用PBST洗板3次。将1：10000稀释HRP标记的羊抗鼠IgG(购自Jackson公司)加入待测抗体孔中，每孔100μl，室温孵育1小时后，用PBST洗涤液洗板4次。每孔加入100μlTMB显色液(购自Cell Signaling公司)，室温避光放置7分钟，每孔加入50μl 1M H2SO4终止显色反应。在多功能酶标仪(Molecular Devices SpectraMax M5Multi-mode Microplate Reader)上以测定波长450nm，参比波长630nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD630nm。
以抗体浓度为横坐标，测得的每孔吸光值为纵坐标，用Sigmoidal dose-response(variable slope)方式作图(GraphPad Prism软件)，得到抗原-抗体结合曲线(图2)。结果表明绝大多数anti-RANKL鼠单抗与包被于ELISA板固相表面的human RANKL呈浓度依赖型结合并可达到饱和，其EC50在10-20ng/ml之间，详细数据见表2。
表2：鼠单抗与human RANKL结合的EC50及其他相关数据比较
Figure PCTCN2014095236-appb-000003
实施例5、纯化的鼠单抗与monkey RANKL和mouse RANKL的种属交叉反应
采用直接ELISA的方法检测抗human RANKL鼠单抗与食蟹猴RANKL和小鼠RANKL的种属交叉反应，实验方法与实施例4完全相同。分别采用human RANKL,monkey RANKL和mouse RANKL进行包被，monkey RANKL和mouse RANKL均购自R&D公司。
以抗体浓度为横坐标，测得的每孔吸光值为纵坐标，用Sigmoidal dose-response(variable slope)方式作图(GraphPad Prism软件)，得到抗原-抗体结合曲线。结果表明所有与human RANKL有结合能力的鼠单抗均能与monkey RANKL结合，其结合呈浓度依赖型结合并可达到饱和，EC50在10-20ng/ml之间(图3，表3)。所检测抗体中有9个抗体与小鼠RANKL有交叉反应。其结合呈浓度依赖型并可达到饱和，EC50在10-20ng/ml之间。续的实验证明，这9个抗体均不能抑制RANKL与RANK-Fc的结合。
表3：鼠单抗与monkey RANKL结合的EC50及其他相关数据比较
Figure PCTCN2014095236-appb-000004
实施例6、纯化的鼠单抗抑制human RANKL与RANK-Fc的结合活性
采用竞争ELISA实验评价抗human RANKL鼠单抗是否具有抑制human RANKL与RANK-Fc结合的中和活性，并与市售品Denosumab进行比较。用pH 9.6的0.05M碳酸盐缓冲液将RANK-Fc蛋白稀释至1μg/ml，100μl每孔加入到96孔ELISA板中，4℃放置过夜。PBST洗板3次，用PBS+2％BSA封闭1小时，之后用PBST洗板3次。分别将待测抗体和Denosumab用PBS+2％BSA从20μg/ml起进行4倍系列稀释至0.0024μg/ml，与等体积的human RANKL(浓度为0.6μg/ml)混匀，每孔100μl加入ELISA板中，样品做双复孔，每块ELISA板做5个样品，且每块ELISA板中均有Denosumab对照品，共10块ELISA板。室温孵育2小时后，用PBST洗板3次。将1：10000稀释HRP标记的羊抗人His标签的二抗(购自Abcam公司)加入ELISA板中，每孔100μl，室温孵育1小时后，用PBST洗涤液洗板4次。每孔加入100μl TMB显色液(购自Cell signaling公司)，室温避光放置15分钟，每孔加入50μl 1M H2SO4终止显色反应。在多功能酶标仪上以测定波长450nm，参比波长630nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD630n。
以抗体浓度为横坐标，测得的吸光值为纵坐标，用Sigmoidal dose-response(variable slope)方式作图(GraphPad Prism软件)，得到抗体竞争抑制受体与配体结合的曲线。9个与鼠RANKL有交叉反应的抗体不具备抑制受体与配体结合的活性，其余41个抗体中，有8个抗体失去中和活性，另外的33个抗体具有明显的中和活性，能够抑制RANKL与RANK-Fc的结合，且呈浓度依赖性。实验测得Denosumab的EC50值为0.67μg/ml，而同一实验条件下测得的8个抗体的EC50值位于0.2-1.0μg/ml之间。抗体的EC50值越小，表示其阻断RANKL与RANK-Fc结合的活性越强，所得的8个抗体中有5个抗体的活性明显优于Denosumab。图4示为8个样品抑制RANKL与RANK-Fc结合的实验结果，详细信息见表4。
表4：鼠单抗抑制RANKL与RANK-Fc结合的EC50及其他相关数据比较
Figure PCTCN2014095236-appb-000005
实施例7、鼠单抗抑制human RANKL诱导的RAW264.7细胞分化实验
Human RANKL可以诱导RAW264.7细胞(购自上海中科院细胞库)分化为破骨细胞，本实验目的为评价能够中和RANKL与RANK-Fc结合的抗human RANKL鼠单抗是否具有抑制human RANKL诱 导的RAW264.7(小鼠单核巨噬细胞白血病细胞，购自中科院上海细胞库)细胞分化活性，并与市售品Denosumab进行比较。破骨细胞中有一种特征性的酶-抗酒石酸酸性磷酸酶，pNPP显色系统可以通过OD405nm读数的高低反应抗酒石酸酸性磷酸酶活性的大小，从而反应RAW264.7细胞分化为破骨细胞的程度。RAW264.7细胞使用DMEM+10％FBS(购自Invitrogen公司)的培养基进行培养，每3-4天传代一次。进行细胞活性检测时，将对数生长期的细胞培养基更换为α-MEM培养基+10％FBS(购自Invitrogen公司)，制成细胞悬液。以2000cell/100μl接种96孔板，放入37℃,5％CO2培养箱中(Thermo公司，规格型号Forma 311)培养1小时。加入对倍稀释的待测样品(共22个抗体，7块96孔细胞培养板)和Denosumab，以及human RANKL和MCSF,TGFβ(购自R&D公司)。使得待测抗体的终浓度分别为2000,1000,500,250,125和62.5ng/ml，human RANKL的终浓度为150ng/ml，MCSF和TGFβ的终浓度分别为20ng/ml和2ng/ml。置于37℃,5％CO2培养箱中培养5天，弃细胞上清，每孔加入100μl细胞裂解液(Citrate Buffer pH 5.0+0.5％Triton X-100)，4℃冰箱中裂解10min，每孔加入100μl pNPP显色液(购自Sigma公司)，37℃孵育30min，再每孔加入50μl终止液(0.5M NaOH)，450/570nm读数。以抗体浓度为横坐标，测得的吸光值为纵坐标，用Sigmoidal dose-response(variable slope)方式作图(GraphPad Prism软件)，得到抗体抑制RAW264.7细胞分化为破骨细胞的曲线。anti-RANKL鼠单抗能完全抑制human RANKL诱导的RAW264.7细胞分化为破骨细胞，称剂量依赖性，所测抗体中除114-6.6.7/114-6.6.18/114-6.6.29样品抑制作用较差，其余19个抗体其抑制活性很强，具有很低的EC50值。市售品Denosumab的EC50值为334.4ng/ml，而同一实验条件下测得的8个抗体的EC50值位于200-800ng/ml之间。EC50值越小，表示抗体抑制RANKL诱导的RAW264.7细胞分化的活性越强，所得的8个抗体中有3个抗体活性优于Denosumab，有2个抗体活性与Denosumab相当。图5所示为8个样品抑制human RANKL诱导的RAW264.7细胞分化为破骨细胞的结果。详细信息见表5。
表5：鼠单抗抑制human RANKL诱导的RAW264.7细胞分化的EC50及其他相关数据
Figure PCTCN2014095236-appb-000006
实施例8、抗human RANKL鼠单抗的核酸CDR区核苷酸序列测定
根据上述鼠单抗抑制human RANKL与RANK-Fc结合实验和RAW264.7细胞分化实验结果，选择具有中和活性的抗体进行CDR区核苷酸序列的测定。共选择22个样品，采用杂交瘤培养基进行细胞培养，收集1ⅹ106个细胞，850rpm离心5分钟，去掉上清，加入450μl RNAlater(购自Sigma公司)重悬，冻存于-80度冰箱。具体编号信息如下：
114-5.7.11 114.5.7.14 114-5.7.29 114-5.16.15 114-5.16.21 114-2.222
114-5.17.12 114-5.17.11 114-5.17.3 114-6.6.29 114-6.6.18 114-2.402
114-7.38.7 114-7.38.29 114-7.38.48 114-7.37.26 114-7.37.36  
114-7.19.12 114-7.19.17 114.5.9.4 114-6.6.7 114-5.16.3  
将收集好的22个样品送南京金斯瑞生物科技有限公司进行CDR区核苷酸序列的测定。首先采用
Figure PCTCN2014095236-appb-000007
Plus RNA Purification System(Invitrogen,Cat.No.:15596-026)进行总RNA的提取，然后通过RT-PCR技术，反转录获得cDNA。以cDNA为模板，通过RACE方法扩增重链和轻链的可变区序列，将获得的可变区序列构建到测序载体中，每个可变区序列挑选5个阳性克隆进行核苷酸序列测定。并将核苷酸序列翻译为氨基酸序列。通过序列比对，最终重链和轻链均分别得到8个独特的CDR序列。其中114-5.7.11、114-5.7.14、114-5.7.29、114-5.16.15、114-5.16.3、114-5.16.21、114-5.17.12、114-5.17.11和114-5.17.3共9个样品的氨基酸序列完全一致，114-6.6.29、114-6.6.18和114-6.6.7共3个样品的氨基酸序列完全一致，114-7.38.7、114-7.38-29和114-7.38.48共3个样品的氨基酸序列完全一致，114-7.37.36和114-7.37.26的氨基酸序列完全一致，114-7.19.12和114-7.19.17的氨基酸序列完全一致。具体氨基酸 序列信息如表6：
表6：CDR的氨基酸序列
重链的CDR序列
Figure PCTCN2014095236-appb-000008
轻链的CDR序列
Figure PCTCN2014095236-appb-000009
Figure PCTCN2014095236-appb-000010
实施例9、7.19.12scFv(单链抗体single—chain antibody fragment)重组质粒的构建
用重组人RANKL(hRANKL)免疫小鼠，采取杂交瘤技术，获得一系列鼠抗人RANKL单抗。通过竞争ELISA实验和细胞学实验筛选出一些具有中和活性的抗体，它们能够阻断RANKL和其受体RANK-Fc结合，抑制RAW264.7细胞分化为破骨细胞。其中单克隆抗体7.19.12是具有较高的结合能力和中和活性的抗体之一，7.19.12的重链可变区(VH)和轻链可变区(VL)氨基酸序列已在中国专利中公开发表(专利号CN201310753972.3)。7.19.12的scFv构建是利用PCR技术，将7.19.12VH(SEQ ID NO:6)和VL(SEQ ID NO:14)，用linker区域(Gly4Ser)3连接，形成单链抗体(scFv)基因，再通过T4连接酶连入T载体，得到7.19.12scFv(SEQ ID NO:21)重组质粒。
首先以VH5'NcoI和VH3'XhoI link为引物，7.19.12VH质粒为模板，用pfu DNA聚合酶(购自北京全式金生物公司)通过PCR方法扩增得到含NcoI、XhoI酶切位点和部分linker序列的VH基因片段；然后以VL5'linkSacI-κ和VL3'NotI-κ为引物，7.19.12VL质粒为模板，用pfu DNA聚合酶通过PCR方法扩增得到含部分linker序列、SacI和NotI酶切位点的VL基因片段。以PCR得到的VH和VL基因片段为模板，VH5'NcoI和VL3'NotI-κ为引物，用pfu DNA聚合酶PCR扩增得到7.19.12scFv基因片段。通过rTaq聚合酶(购自TAKARA公司)对获得的7.19.12scFv产物进行72℃延伸1小时，使得scFv产物3’端加上A。加A后scFv产物经胶回收纯化后，在T4DNA连接酶(购自NEB公司)作用下，与
Figure PCTCN2014095236-appb-000011
18-T载体进行连接(16℃，1小时)。热休克法(42℃，90秒)转化大肠杆菌，菌液涂平板(LBAmp培养基)，于37℃培养过夜。挑取克隆进行菌落PCR(通用引物M13-47和M13-48)鉴定筛选，并将PCR验证为阳性的克隆进行测序分析，筛选得到7.19.12scFv重组质粒。7.19.12scFV构建引物见表7。
表7：7.19.12scFv构建引物
Figure PCTCN2014095236-appb-000012
7.19.12重链可变区DNA序列(SEQ ID NO:18)
CAGGTTCAGCTCCAGCAGTCTGGGGCTGAACTGGCGAGTCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACTGGCTGCAGTGGGTAAAGCAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTTATCCTGGACCTGGTAATACTAAATACACTCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGATAAATCCGCCAGCACAGCCTACATGCAACTCAACAGCTTGACATCTGAAGACTCTGCGGTCTATTACTGCGCAAGGAGGGGATCACGACGGGGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA
7.19.12轻链可变区DNA序列(SEQ ID NO:19)
AACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGCCAGAGGGCCACCATTTCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAGAAGTTTTATGCACTGGTACCAGCAGAGACCAGGACAGCCACCCACACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGTCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAGATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
7.19.12scFv DNA序列(SEQ ID NO:20)
CAGGTTCAGCTCCAGCAGTCTGGGGCTGAACTGGCGAGTCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACTGGCTGCAGTGGGTAAAGCAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTTATCCTGGACCTGGTAATACTAAATACACTCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGATAAATCCGCCAGCACAGCCTACATGCAACTCAACAGCTTGACATCTGAAGACTCTGCGGTCTATTACTGCGCAAGGAGGGGATCACGACGGGGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCTCGAGTGGTGGTGGCGGTTCAGGCGGTGGTGGCTCTGGTGGCGGTGGGAGCTCTAACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGCCAGAGGGCCACCATTTCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAGAAGTTTTATGCACTGGTACCAGCAGAGACCAGGACAGCCACCCACACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGTCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAGATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
7.19.12scFv氨基酸序列(SEQ ID NO:21)
QVQLQQSGAELASPGASVKLSCKASGYTFTTYWLQWVKQRPGQGLEWIGAIYPGPGNTKYTQKFKDKATLTADKSASTAYMQLNSLTSEDSAVYYCARRGSRRGIAYWGQGTLVTVSAASSGGGGSGGGGSGGGGSSNIVLTQSPASLAVSLGQRATISCRASESVDSYGRSFMHWYQQRPGQPPTLLIYLASNLESGVPVRFSGSGSRTDFTLTIDPVEADDAATYYCQQDNEDPYTFGGGTKLEIK
实施例10野生型7.19.12scFv噬菌粒的构建
噬菌粒载体pFL249由南京金斯瑞公司提供，NcoI和NotI作为scFv基因的插入位点，pelB为信号肽(指导scFv分泌到周质腔)，gIII249为噬菌体识别区域，氨苄青霉素抗性基因起到筛选和维持噬菌粒的作用。scFv基因连接c-myc tag和his tag，其中his tag可用作亲和纯化，c-myc tag可被抗c-myc抗体识别达到检测目的。在his tag和gIII之间存在琥珀酸终止子(TAG)，噬菌粒DNA在抑制型E.coli TG1(购自Lucigen公司货号为60502-1)中产生部分抑制作用，通过辅助噬菌体的作用，将scFv展示在噬菌体表面。
pMD18-7.19.12scFv质粒用NcoI和NotI(购自NEB)限制性内切酶进行双酶切，获得具有粘性末端的7.19.12scFv基因片段，并与进行同样双酶切处理的pFL249载体连接，具体连接、转化及鉴定方法同实施例9，克隆经测序分析，获得野生型pFL249-7.19.12scFv噬菌粒(见图6)。
实施例117.19.12scFv人源化库的设计
通过IgBlast搜索数据库(IMGT human V genes(F+ORF+in-frame P))，确定与7.19.12抗体框架区(Framework Regions,FRs)同源性最高的人VH和VL配系基因序列。检查FR序列，确定鼠、人序列之间不同的氨基酸残基，评价每个不同残基对抗原结合的重要性。最后将难以确定对抗原结合的重要性，且人鼠源残基不同的鼠源、人源副本均编入人源化抗体序列。鼠单抗7.19.12VH人源化库突变策略(固定7.19.12VL，对7.19.12VH的FR区进行突变)见表8，鼠单抗7.19.12VL人源化库突变策略(固定7.19.12VH，对7.19.12VL的FR区进行突变)见表9。VH人源化库理论设计多样性为1×105，VL人源化库理论设计多样性为9216。7.19.12scFv人源化库以简并碱基方式合成(简并碱基表示方式：N代表A/G/T/C，W代表A/T，B代表G/T/C，D代表G/A/T，R代表A/G，Y代表C/T，V代表A/G/C，M代表A/C，S代表G/C，H代表A/C/T，K代表G/T)。
表8：鼠抗7.19.12VH人源化库突变策略
Figure PCTCN2014095236-appb-000013
Figure PCTCN2014095236-appb-000014
表9：鼠抗7.19.12VL人源化库突变策略
Figure PCTCN2014095236-appb-000015
Figure PCTCN2014095236-appb-000016
实施例127.19.12scFv人源化库的构建
抗hRANKL鼠单抗7.19.12scFv文库构建工作外包给南京金斯瑞生物科技有限公司完成。文库合成思路为：分别将VH和VL拆分为大小约60～75bp的4个寡核苷酸序列，片段之间重叠约20bp，采用重叠延伸PCR方法合成VL固定的VH突变文库和VH固定的VL突变文库。根据金斯瑞标准操作流程，将合成的VH、VL引物(见表10)分别按等摩尔的量混合、退火、延伸并扩增出完整的VH、VL片段，经胶回收纯化后克隆到测序载体中，分别随机送测15、20个克隆进行PCR产物多样性分析，最终选定ORF正常克隆百分比为60％、66％的两批VH纯化后PCR产物及ORF正常克隆百分比为75％的VL纯化后PCR产物用于文库构建。
纯化后VH PCR产物用Sfi I和Xho I进行双酶切，与经Sfi I及Xho I双酶切的pFL249‐7.19.12scFv载体进行连接，电转化TG1，构建VL固定的VH突变文库；纯化后的VL PCR产物用Sac I和Not I进行双酶切，与经Sac I及Not I双酶切的pFL249‐7.19.12scFv载体进行连接，电转化TG1，构建VH固定的VL突变文库。文库构建先经过连接及电转化测试，根据优化后条件进行大量连接及电转化。取100μl电转化文库复苏菌进行10倍比梯度稀释，取10-3～10‐5三个稀释梯度各100μl菌液涂布含100μg/ml氨卞青霉素的2×YT平板，于37℃过夜培养。次日通过菌落计数计算文库库容；余下文库复苏菌涂布
Figure PCTCN2014095236-appb-000017
的含100μg/ml氨卞青霉素和2％葡萄糖的2×YT平板，37℃过夜培养，次日将菌苔从平板刮下收集文库菌体，重悬后加甘油至40％(V/V)终浓度，文库甘油菌保存于‐80℃。
统计结果显示，第一批VH突变文库的库容大小为1.35×107(表11)，随机挑取144个转化子用引物M13R(‐48)及M13F(‐47)进行PCR菌落筛选，，电泳图中出现～1500bp DNA条带(箭头所示)的转化子表明含VH插入片段，无DNA条带的转化子为酶切载体背景。结果显示阳性率为98.6％(142/144，图7)；第二批VH突变文库的库容大小为1.04×107(表12)，随机挑取96个转化子用引物M13R(‐48)及M13F(‐47)进行PCR菌落筛选，PCR菌落筛选显示阳性率为99％(95/96，图8)，VH突变文库总库容约为2.04×107，两个批次菌液合并后菌浓度为6.7×109cfu/ml。VL突变文库的库容大小为2.8×108，菌浓度为2×1011cfu/ml(表12)，随机挑取144个转化子用引物M13R(‐48)及M13F(‐47)进行PCR菌落筛选，电泳图中出现～1500bp DNA条带(箭头所示)的转化子表明含VH插入片段，无DNA条带的 转化子为酶切载体背景。PCR菌落筛选显示阳性率为96.5％，(139/144，图9)。VH/VL突变文库分别随机挑取100个PCR菌落筛选为阳性的克隆进行测序分析，测序结果显示分别有58/70个克隆能正常翻译且符合突变设计，多样性和小试结果相当。
表10：hRANKL鼠抗7.19.12scFv人源化库VH、VL引物
Figure PCTCN2014095236-appb-000018
表11.VH突变文库库容统计结果
Figure PCTCN2014095236-appb-000019
注：第一批VH突变文库单个电转化获得约1.5×106个转化子，第二批约1.3×106，分别进行了9个及8个电转化，总库容约2.4×107。
表12.VL突变文库库容统计结果
Figure PCTCN2014095236-appb-000020
注：VL突变文库单个电转化获得约2.8×107个转化子，共进行了10个电转化，总库容约2.8×108。
实施例13噬菌体展示文库淘选
先进行文库扩增，取一定量的VH突变文库甘油菌液到370ml含100μg/ml Amp和2％葡萄糖的2xYT培养基(下文简称2xYT-AG)，使得OD600在0.05～0.1之间，在37℃，200rpm/分钟振荡培养至OD600达到0.3～0.4。向培养菌液中加入20倍菌体总数的M13KO7辅助噬菌体(购自NEB,货号为N0315S)，混匀后于37℃静置侵染30分钟，然后于37℃，200rpm/分钟振荡培养1小时。2000g离心10分钟，去上清，然后将菌体重悬于含100μg/ml Amp和50μg/ml卡那霉素的370ml 2xYT培养基(下文简称2xYT-AK)中，在30℃，200rpm/分钟振荡培养过夜(至少15小时)。转移过夜培养菌液到500ml离心瓶中，4℃，10,000g离心15分钟。取上清，加入1/4上清体积的PEG/NaCl溶液(20％(W/V)聚乙二醇(PEG)8000，2.5M NaCl)，充分混匀后置冰上沉淀1小时。4℃，8000rpm/分钟离心30分钟，去上清，将沉淀重悬于2.2ml PBS(137mM NaCl，2.7mM KCl，10mM Na2HPO4，1.8mMKH2PO4，pH7.2～7.4)。4℃，10000g离心2分钟，取上清(此为展示了scFv的重组噬菌体库)于4℃保存备用(取10μl采用10-2～10-10梯度稀释来测定滴度)。
通过培养稳定表达hRANKL的CHO细胞株(C219)，收集细胞上清，经Ni柱纯化获得hRANKL蛋白(见专利CN201310753972.3)。hRANKL用PBS稀释到10μg/ml，按照1ml/管加入Immunotube管(购自NUNC，货号为470319)，4℃包被过夜。用5ml PBST(含0.05％(V/V)Tween 20的PBS)洗管3次，向管中加入5ml封闭液(含10％(W/V)脱脂奶粉的PBS溶液)，室温(20～25℃)封闭2小时(用封闭液封闭另一新Immunotube管作为阴性对照)。用5ml PBST分别洗Immunotube管3次。取制备好的重组噬菌体加到Immunotube管，于水平低速摇床上60rpm，室温孵育2小时。用5ml PBST和PBS分别洗涤10次(第二轮、第三轮和第四轮淘选时增加洗涤次数至15次、25次和35次)。洗涤完后，向Immunotube管中加入1ml OD600为0.3的E.coli TG1，37℃静置1小时。取20μl被侵染的TG1菌液进行10倍比梯度稀释，取10-2～10-4三个稀释梯度各100μl菌液涂布
Figure PCTCN2014095236-appb-000021
的含100μg/ml氨卞青霉素和2％葡萄糖的SOB平板(下文简称SOBAG)，37℃过夜培养，次日通过菌落计数计算每一轮淘选后的库容；余下被侵染TG1菌液涂布
Figure PCTCN2014095236-appb-000022
SOBAG平板，37℃过夜培养，次日将菌苔从平板上刮下收集菌体，重悬后加甘油至20％(V/V)终浓度。按照上述文库扩增方法进行扩增进入下一轮淘选(淘选方法同上，包被抗原hRANKL量逐轮递减，第一到四轮依次为10、1、0.05和0.01μg/ml)。VL突变文库富集淘选过程同VH库。表13为每轮淘选重组噬菌体投入量和淘选后克隆总数。通过数据可以看出，随着抗原量逐轮递减，展示了scFv抗体的克隆总数在减少，即非特异性结合的scFv克隆降低、特异性结合hRANKL的scFv克隆得以富集。
表13.每轮淘选重组噬菌体投入量和淘选后克隆总数
Figure PCTCN2014095236-appb-000023
实施例14采用ELISA方法筛选与hRANKL特异性结合的scFv
为筛选能与hRANKL特异性结合的阳性克隆，实施例13淘选的克隆经IPTG诱导表达scFv，再用酶联免疫吸附(简称ELISA)方法检测。对于VH、VL库均从第三、四轮淘选得到的SOBAG克隆平板上，随机各挑取5×96个克隆接种到无菌96深孔板中，每孔含400μl 2xYT-AG培养基，于30℃、200rpm振荡培养过夜。次日按1:50比例转接深孔板菌液到含400μl 2xYT-AG新鲜培养基的新的96深孔板中，于37℃，200rpm振荡培养到OD600约为0.8(约3小时)。通过1500g离心10分钟收集菌体，将 培养上清转移废弃，并用400μl无菌新鲜的2xYT-AI(I指IPTG，终浓度为100mM)培养基重悬菌体，于30℃，200rpm振荡培养过夜(即过夜诱导表达scFv)。将过夜诱导表达scFv上清的深孔板于3000g离心20分钟，转移300μl上清到干净的96孔细胞板中备用。
hRANKL用1xPBS(pH7.4)稀释到1μg/ml，按照50μl/孔加入96孔酶标板(购自NUNC,货号为442404)中，共10块板，4℃包被过夜。用PBST(含0.05％Tween20)洗板3次后，用封闭液(含2％(W/V)BSA的PBS溶液)室温封闭1～1.5小时。PBST洗板3次后将诱导的scFv上清转移到封闭好的酶标板中，50μl/孔，仅含2xYT-AI培养基的孔作为阴性对照，含野生型7.19.12scFv上清孔作为阳性对照，室温孵育2小时。PBST洗板3次，然后加入1:5000稀释的抗c-myc的HRP标记的二抗(购自Bethyl公司,货号为A190-104P)，室温孵育1小时。PBST洗板6次后加入TMB底物(购自Cell Signaling公司，货号为7004)室温显色10分钟，每孔加入50μl的2M HCl终止显色，使用M5仪器检测OD450nm读数。
以OD450>0.4为阳性显色。结果显示，VH库共获得534个可与hRANKL结合的阳性克隆，阳性率为94％(见图10)，选取OD450>1.2共179个克隆送上海美吉生物用引物M13R(‐48)测序，经序列分析，其中有3个VH序列的人源化突变程度较高，为11个突变，满足人源化程度要求，这3个克隆编号为pFL249-H16，pFL249-H16u和pFL249-H114；VL共获得486个可与hRANKL结合的阳性克隆，阳性率为86％(见图11)，选取第三轮OD450>1.0的33个克隆，第四轮OD450>1.5的114个克隆，共147个克隆送上海美吉生物用引物M13R(‐48)测序，经序列分析，其中有4个VL序列的人源化突变程度较高，为9个突变，满足人源化程度要求，这4个克隆编号为pFL249-L10，pFL249-L10u，pFL249-L37和pFL249-L37u。
实施例15抗hRANKL人源化抗体重链和轻链表达载体的构建
将通过噬菌体淘选平台获得的3个重链可变区H16(SEQ ID NO:23)、H16u(SEQ ID NO:25)及H114(SEQ ID NO:27)分别构建成IgG2型全长重链，将获得的4个轻链可变区L10(SEQ ID NO:31)、L10u(SEQ ID NO:33)、L37(SEQ ID NO:35)及L37u(SEQ ID NO:37)分别构建成Kappa型全长轻链，全长重链和轻链分别构建到真核表达载体pCDNA3.1上。根据噬菌体淘选得到的阳性克隆测序序列，将重轻链可变区的框架区(Framework，下文简称FR)FR1、FR2、FR3和FR4通过IgBlast搜索数据库(IMGT human V genes(F+ORF+in-frame P))比对，获得同源性最高的人FR区，并将人FR区与野生型7.19.12 CDRs组合，得到新的VH(命名为H16(hFR)，SEQ ID NO:29)和VL(命名为WT-VL(hFR)，SEQ ID NO:39)序列。可变区基因通过全基因合成方法获得，按同前的方法构建成全长重链和轻链，连接到表达载体pCDNA3.1上。表14为抗hRANKL人源化抗体的重链可变区氨基酸序列比对，表15为抗hRANKL人源化抗体的轻链可变区氨基酸序列比对，7.19.12为原始鼠源序列。
H16重链可变区DNA序列(SEQ ID NO:22)
CAGGTTCAGCTCGTGCAGTCTGGGGCTGAACTGAAGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACTGGCTGCAGTGGGTAAGGCAGGCTCCTGGACAGGGTCTGGAATGGATGGGGGCTATTTATCCTGGACCTGGTAATACTAAATACACTCAGAAGTTCAAGGACAGATTCACAATTACTGCAGATAAGTCCAAGAGCACAGCCTACATGCAACTCAACAGCTTGAAGTCTGAAGACTCTGCGGTCTATTACTGCGCAAGGAGGGGATCACGACGGGGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA
H16重链可变区氨基酸序列(SEQ ID NO:23)
QVQLVQSGAELKKPGASVKLSCKASGYTFTTYWLQWVRQAPGQGLEWMGAIYPGPGNTKYTQKFKDRFTITADKSKSTAYMQLNSLKSEDSAVYYCARRGSRRGIAYWGQGTLVTVSA
H16u重链可变区DNA序列(SEQ ID NO:24)
CAGGTTCAGCTCGTGCAGTCTGGGGCTGAACTGAAGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACTGGCTGCAGTGGGTAAGGCAGGCTCCTGGACAGGGTCTGGAATGGATGGGGGCTATTTATCCTGGACCTGGTAATACTAAATACACTCAGAAGTTCAAG GACAGATTCACAATTACTGCAGATACGTCCAAGAGCACAGCCTACATGCAACTCAACAGCTTGAAGTCTGAAGACTCTGCGGTCTATTACTGCGCAAGGAGGGGATCACGACGGGGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA
H16u重链可变区氨基酸序列(SEQ ID NO:25)
QVQLVQSGAELKKPGASVKLSCKASGYTFTTYWLQWVRQAPGQGLEWMGAIYPGPGNTKYTQKFKDRFTITADTSKSTAYMQLNSLKSEDSAVYYCARRGSRRGIAYWGQGTLVTVSA
H114重链可变区DNA序列(SEQ ID NO:26)
CAGGTTCAGCTCGTGCAGTCTGGGGCTGAACTGGTGCAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACTGGCTGCAGTGGGTAAGGCAGACTCCTGGACAGGGTCTGGAATGGATGGGGGCTATTTATCCTGGACCTGGTAATACTAAATACACTCAGAAGTTCAAGGACAGGGTCACAATTACTGCAGATACATCCACCAGCACAGCCTACATGCAACTCAACAGCTTGAGATCTGAAGACTCTGCGGTCTATTACTGCGCAAGGAGGGGATCACGACGGGGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA
H114重链可变区氨基酸序列(SEQ ID NO:27)
QVQLVQSGAELVQPGASVKLSCKASGYTFTTYWLQWVRQTPGQGLEWMGAIYPGPGNTKYTQKFKDRVTITADTSTSTAYMQLNSLRSEDSAVYYCARRGSRRGIAYWGQGTLVTVSA
H16(hFR)重链可变区DNA序列(SEQ ID NO:28)
CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCACCTACTGGCTGCAGTGGGTAAGGCAGGCTCCTGGACAGGGTCTGGAATGGATGGGGGCTATTTATCCTGGACCTGGTAATACTAAATACACTCAGAAGTTCAAGGACAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAGGGGATCACGACGGGGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA
H16(hFR)重链可变区氨基酸序列(SEQ ID NO:29)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWLQWVRQAPGQGLEWMGAIYPGPGNTKYTQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARRGSRRGIAYWGQGTLVTVSA
L10轻链可变区DNA序列(SEQ ID NO:30)
AACATTGTGCTGACCCAATCTCCAGCTTCTTTGTCTGTGTCTCTAGGCCAGAGGGCCACCATTTCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAGAAGTTTTATGCACTGGTACCAGCAGAGACCAGGACAGGCTCCCAGACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAGCAGTGTGGAGGCTGAAGATGAGGCAACCTATTACTGTCAGCAAGATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
L10轻链可变区氨基酸序列(SEQ ID NO:31)
NIVLTQSPASLSVSLGQRATISCRASESVDSYGRSFMHWYQQRPGQAPRLLIYLASNLESGVPARFSGSGSRTDFTLTISSVEAEDEATYYCQQDNEDPYTFGGGTKLEIK
L10u轻链可变区DNA序列(SEQ ID NO:32)
AACATTGTGCTGACCCAATCTCCAGCTTCTTTGTCTGTGTCTCTAGGCCAGAGGGCCACCATTTCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAGAAGTTTTATGCACTGGTACCAGCAGAGACCAGGACAGGCTCCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAGCAGTGTGGAGGCTGAAGATGAGGCAACCTATTACTGTCAGCAAGATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
L10u轻链可变区氨基酸序列(SEQ ID NO:33)
NIVLTQSPASLSVSLGQRATISCRASESVDSYGRSFMHWYQQRPGQAPKLLIYLASNLESGVPARFSGSGSRTDFTLTISSVEAEDEATYYCQQDNEDPYTFGGGTKLEIK
L37轻链可变区DNA序列(SEQ ID NO:34)
AACATTGTGCTGACCCAATCTCCAGCTTCTTTGTCTGTGTCTCTAGGCCAGAGGGCCACCATTTCC TGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAGAAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGGCACCCAAGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTTCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAGCGCAGTGGAGGCTGAAGATGCGGCAACCTATTACTGTCAGCAAGATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
L37轻链可变区氨基酸序列(SEQ ID NO:35)
NIVLTQSPASLSVSLGQRATISCRASESVDSYGRSFMHWYQQKPGQAPKLLIYLASNLESGVPSRFSGSGSRTDFTLTISAVEAEDAATYYCQQDNEDPYTFGGGTKLEIK
L37u轻链可变区DNA序列(SEQ ID NO:36)
AACATTGTGCTGACCCAATCTCCAGCTTCTTTGTCTGTGTCTCTAGGCCAGAGGGCCACCATTTCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAGAAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGGCACCCAAGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTTCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAGCAGAGTGGAGGCTGAAGATGCGGCAACCTATTACTGTCAGCAAGATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
L37u轻链可变区氨基酸序列(SEQ ID NO:37)
NIVLTQSPASLSVSLGQRATISCRASESVDSYGRSFMHWYQQKPGQAPKLLIYLASNLESGVPSRFSGSGSRTDFTLTISRVEAEDAATYYCQQDNEDPYTFGGGTKLEIK
WT-VL(hFR)轻链可变区DNA序列(SEQ ID NO:38)
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAGAAGTTTTATGCACTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCCTCACCATCAATAGCCTGGAAGCTGAAGATGCTGCAACGTATTACTGTCAGCAAGATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
WT-VL(hFR)轻链可变区氨基酸序列(SEQ ID NO:39)
DIVMTQSPDSLAVSLGERATINCRASESVDSYGRSFMHWYQQKPGQAPRLLIYLASNLES
GVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQDNEDPYTFGGGTKLEIK
Figure PCTCN2014095236-appb-000024
H16全长重链表达载体的构建
以pFL249-H16质粒为模板，PCR扩增出H16重链可变区VH；以基因合成得到的人IgG2型重链 恒定区质粒为模板PCR扩增出人IgG2型重链恒定区(SEQ ID NO:41)基因片段；通过重叠PCR技术，扩增出含哺乳动物表达信号肽(SEQ ID NO:45)的H16重链基因VH-CH(SEQ ID NO:47)；构建H16全长重链基因到pCDNA3.1(+)(购自Invitrogen,货号为V790-20)载体，得到H16全长重链表达载体。
用pfu DNA聚合酶(购自北京全式金生物公司)扩增H16VH和IgG2型CH基因片段。以Whole-1H-F和Whole-1H-R为引物，pFL249-H16质粒为模板，PCR方法扩增得到含部分真核表达信号肽的VH基因片段；以IgG2-CH-F和IgG2-CH-R为引物，人IgG2质粒为模板，PCR方法扩增得到含TAG终止密码子和NotI酶切位点的CH基因片段，VH与CH存在至少20bp的重叠序列。PCR条件为：95℃2分钟，【95℃20秒，55℃20秒，72℃40秒】30个循环，72℃5分钟。
用pfu DNA聚合酶扩增H16重链基因。以上述PCR得到的VH和CH基因片段为模板，以Whole-SP-F和IgG2-CH-R为引物，PCR扩增得到含EcoRI和NotI酶切位点，及真核表达信号肽序列的H16重链基因。PCR条件：95℃2分钟，【95℃20秒，55℃20秒，72℃100秒】35循环，72℃5分钟。
按实施例9中PCR产物加A方法处理上述获得的H16重链基因，经胶回收纯化后，在T4DNA连接酶(购自NEB公司)作用下，与
Figure PCTCN2014095236-appb-000025
19-T simple载体进行连接(16℃，1小时)，热休克法(42℃，90秒)转化大肠杆菌，涂平板(LB Amp培养基)后37℃培养过夜，挑取克隆进行菌落PCR(通用引物M13-47和M13-48)鉴定筛选。PCR鉴定为阳性的克隆再进行质粒抽提，用EcoRI和NotI进行双酶切，切胶回收约1500bp的H16重链片段，与同样双酶切处理后切胶回收的pCDNA3.1(+)载体连接。H16IgG2重链克隆经测序确认，重组质粒全长6800bp，包含H16重链可变区和人IgG2重链恒定区(图12为H16重链质粒图谱)。
IgG2重链恒定区DNA序列(SEQ ID NO:40)
GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAATACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
IgG2重链恒定区氨基酸序列(SEQ ID NO:41)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
哺乳动物表达信号肽DNA序列(SEQ ID NO:44)
ATGGAGTTGGGACTGTCTTGGATTTTCCTGTTGGCTATTCTGAAAGGTGTGCAGTGT
哺乳动物表达信号肽氨基酸序列(SEQ ID NO:45)
MELGLSWIFLLAILKGVQC
L10轻链表达载体的构建
以pFL249-L10质粒为模板，PCR扩增出L10轻链可变区VL，以基因合成得到的人Kappa轻链恒 定区质粒为模板PCR扩增出人Kappa轻链恒定区(SEQ ID NO:43)基因片段，再通过重叠PCR技术，扩增出含哺乳动物表达信号肽序列(SEQ ID NO:45)的L10轻链基因VL-CL(SEQ ID NO:52)。构建L10轻链基因到pCDNA3.1(+)载体，得到L10轻链表达载体。
用pfu DNA聚合酶(购自北京全式金生物公司)扩增L10VL和Kappa轻链CL基因片段。以Whole-1L-F和Whole-1L-R为引物，pFL249-L10质粒为模板，PCR方法扩增得到含部分真核表达信号肽的VL基因片段；以CL-F和CL-R为引物，人Kappa轻链质粒为模板，PCR方法扩增得到含TAG终止密码子和NotI酶切位点的CL基因片段，VL与CL存在至少20bp的重叠序列。PCR条件为：95℃2分钟，【95℃20秒，55℃20秒，72℃40秒】30个循环，72℃5分钟。
用pfu DNA聚合酶扩增L10轻链基因。以上述PCR得到的VL和CL基因片段为模板，以Whole-1L-F和CL-R为引物，PCR扩增得到含EcoRI和NotI酶切位点，及真核表达信号肽序列的L10轻链基因。PCR条件：95℃2分钟，【95℃20秒，55℃20秒，72℃100秒】35个循环，72℃5分钟。
按实施例9中PCR产物加A方法处理上述获得的L10轻链基因，经胶回收纯化后，在T4DNA连接酶(购自NEB公司)作用下，与
Figure PCTCN2014095236-appb-000026
19-T simple载体进行连接(16℃，1小时)，热休克法(42℃，90秒)转化大肠杆菌，涂平板(LB Amp培养基)后37℃培养过夜，挑取克隆进行菌落PCR(通用引物M13-47和M13-48)鉴定筛选。PCR鉴定为阳性克隆在进行质粒抽提，用EcoRI和NotI进行双酶切，切胶回收约750bp的L10轻链基因片段，与同样双酶切处理后切胶回收的pCDNA3.1(+)载体连接。L10轻链克隆经测序确认，重组质粒全长6123bp，包含L10轻链可变区和人Kappa轻链恒定区(图13为L10Kappa轻链质粒图谱)。表16为抗hRANKL人源化抗体的轻重链载体构建引物表，其余3个重链(H16u，H114及H16(hFR))、4个轻链(L10u，L37，L37u及WT-VL(hFR))质粒构建方法类似，在此不做详细说明。
Kappa轻链恒定区DNA序列(SEQ ID NO:42)
GGTACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT
Kappa轻链恒定区氨基酸序列(SEQ ID NO:43)
GTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
表16.抗hRANKL人源化抗体的轻重链载体构建引物表
Figure PCTCN2014095236-appb-000027
Figure PCTCN2014095236-appb-000028
实施例16抗hRNAKL人源化抗体的表达及纯化
重组抗hRNAKL抗体通过FreeStyleTM 293-F细胞(购自Invitrogen公司,货号为R790-07)产生。编码完整重链和轻链的DNA序列已克隆到哺乳动物表达载体上，详情见实施例15。通过将表达完整重链和完整轻链的载体在阳离子聚合物PEI(购自Polysciences,货号为23966-2)介导下共转染到293F细胞，产生抗hRNAKL抗体。例如，为产生G1抗体，表达完整重链(氨基酸序列号为SEQ ID NO:46)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:51)的载体共转染293F细胞；为产生G2抗体，表达完整重链(氨基酸序列号为SEQ ID NO:47)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:52)的载体共转染293F细胞；为产生G3抗体，表达完整重链(氨基酸序列号为SEQ ID NO:47)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:53)的载体共转染293F细胞；为产生G4抗体，表达完整重链(氨基酸序列号为SEQ ID NO:47)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:54)的载体共转染293F细胞；为产生G5抗体，表达完整重链(氨基酸序列号为SEQ ID NO:47)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:55)的载体共转染293F细胞；为产生G6抗体，表达完整重链(氨基酸序列号为SEQ ID NO:48)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:52)的载体共转染293F细胞；为产生G7抗体，表达完整重链(氨基酸序列号为SEQ ID NO:48)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:53)的载体共转染293F细胞；为产生G8抗体，表达完整重链(氨基酸序列号为SEQ ID NO:48)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:54)的载体共转染293F细胞；为产生G9抗体，表达完整重链(氨基酸序列号为SEQ ID NO:48)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:55)的载体共转染293F细胞；为产生G10抗体，表达完整重链(氨基酸序列号为SEQ ID NO:49)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:52)的载体共转染293F细胞；为产生G11抗体，表达完整重链(氨基酸序列号为SEQ ID NO:49)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:53)的载体共转染293F细胞；为产生G12抗体，表达完整重链(氨基酸序列号为SEQ ID NO:49)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:54)的载体共转染293F细胞；为产生G13抗体，表达完整重链(氨基酸序列号为SEQ ID NO:49)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:55)的载体共转染293F细胞；为产生G14抗体，表达完整重链(氨基酸序列号为SEQ ID NO:50)的载体与表达完整轻链(氨基酸序列号为SEQ ID NO:56)的载体共转染293F细胞。表17为抗hRANKL抗体的重链和轻链组合表。
表17.抗hRANKL抗体的重链和轻链组合表
抗体 重链可变区+重链恒定区 完整重链
G1 SEQ ID NO:6+SEQ ID NO:41 SEQ ID NO:46
G2 SEQ ID NO:23+SEQ ID NO:41 SEQ ID NO:47
G3 SEQ ID NO:23+SEQ ID NO:41 SEQ ID NO:47
G4 SEQ ID NO:23+SEQ ID NO:41 SEQ ID NO:47
G5 SEQ ID NO:23+SEQ ID NO:41 SEQ ID NO:47
G6 SEQ ID NO:27+SEQ ID NO:41 SEQ ID NO:48
G7 SEQ ID NO:27+SEQ ID NO:41 SEQ ID NO:48
G8 SEQ ID NO:27+SEQ ID NO:41 SEQ ID NO:48
G9 SEQ ID NO:27+SEQ ID NO:41 SEQ ID NO:48
G10 SEQ ID NO:25+SEQ ID NO:41 SEQ ID NO:49
G11 SEQ ID NO:25+SEQ ID NO:41 SEQ ID NO:49
G12 SEQ ID NO:25+SEQ ID NO:41 SEQ ID NO:49
G13 SEQ ID NO:25+SEQ ID NO:41 SEQ ID NO:49
G14 SEQ ID NO:29+SEQ ID NO:41 SEQ ID NO:50
抗体 轻链可变区+轻链恒定区 完整轻链
G1 SEQ ID NO:14+SEQ ID NO:43 SEQ ID NO:51
G2 SEQ ID NO:31+SEQ ID NO:43 SEQ ID NO:52
G3 SEQ ID NO:35+SEQ ID NO:43 SEQ ID NO:53
G4 SEQ ID NO:33+SEQ ID NO:43 SEQ ID NO:54
G5 SEQ ID NO:37+SEQ ID NO:43 SEQ ID NO:55
G6 SEQ ID NO:31+SEQ ID NO:43 SEQ ID NO:52
G7 SEQ ID NO:35+SEQ ID NO:43 SEQ ID NO:53
G8 SEQ ID NO:33+SEQ ID NO:43 SEQ ID NO:54
G9 SEQ ID NO:37+SEQ ID NO:43 SEQ ID NO:55
G10 SEQ ID NO:31+SEQ ID NO:43 SEQ ID NO:52
G11 SEQ ID NO:35+SEQ ID NO:43 SEQ ID NO:53
G12 SEQ ID NO:33+SEQ ID NO:43 SEQ ID NO:54
G13 SEQ ID NO:37+SEQ ID NO:43 SEQ ID NO:55
G14 SEQ ID NO:39+SEQ ID NO:43 SEQ ID NO:56
转染前24小时，将293-F细胞按6～7×105个/mL密度进行传代，于37℃，8％CO2，135rpm的培养条件下进行培养。次日，采用血球计数板测定细胞密度和活力，确保细胞活力大于95％，将细胞密度调整至1×106/mL进行瞬转实验。用离心管(管A)将轻链质粒和重链质粒各15μg加入OptiPRO SFM(购自Invitrogen公司)中，使终体积为600μL，轻轻混匀；在新的离心管(管B)中加入90μg PEI转染试剂到OptiPRO SFM，使终体积为600μL，轻轻混匀。即刻将管B中液体吸取至管A中，轻轻混匀。将A和B的混合液在室温放置20分钟后，逐滴加入到30ml 293-F细胞中。将转染后的细胞置于37℃，8％CO2，135rpm的摇床中进行培养。转染后6～7天，测定转染细胞活力，当细胞活力低于50％时收集细胞上清进行纯化。
9000rpm离心30分钟收集细胞上清，用rProtein A(购自GE公司,Best Chrom)重力柱进行抗体纯化。用5-10个柱体积的平衡液(20mM Tris-HCl+0.15M NaCl pH＝7.4)进行平衡，上样流速控制在≤1ml/ 分钟，上样完毕再用5-10个柱体积的平衡液进行平衡，用100mM柠檬酸(pH3.0)进行洗脱，收集的洗脱液用1M Tris-HCl(pH9.0)中和，调节pH为6.0左右。用10KD超滤管(购自Millipore公司,货号为UFC901008)进行离心浓缩，用PBS(137mM NaCl，2.7mM KCl，10mM Na2HPO4，1.8mM KH2PO4，pH7.2～7.4)重悬换液。改良型BCA蛋白浓度测定试剂盒(购自上海生工生物公司)测定蛋白浓度，用0.22μM滤膜(购自Millipore公司,货号为SLGP033RB)过滤除菌后分装保存在-80℃。
实施例17采用ELISA方法检测人源化抗体与hRNAKL的结合能力
hRANKL用PBS(137mM NaCl，2.7mM KCl，10mM Na2HPO4，1.8mM KH2PO4，pH7.2～7.4)稀释到1μg/ml，100μl/孔加入96孔酶标板中(购自NUNC公司)，4℃包被过夜。用PBST(含0.05％Tween20的PBS溶液)洗板3次后，用封闭液(含2％(W/V)BSA的PBS溶液，简称PBS-2％BSA)室温封闭1～1.5小时。将人源化抗体G2～G14，鼠抗G1和阳性对照Prolia(购自Amgen公司,批号为1021139)用样品稀释液(PBS-1％BSA)分别从1μg/ml开始进行3倍稀释至0.0002μg/ml，100μl/孔加入酶标板中，室温孵育2小时。PBST洗板3次，用PBS-1％BSA按1:10,000稀释HRP标记的羊抗人IgG Fc抗体(Jackson,货号109-035-098)，100μl/孔加入酶标板中，室温孵育1小时。PBST洗板6次后加入TMB底物(Cell Signaling，货号7004)室温显色10分钟，每孔加入100μl的2M HCl终止显色，采用M5仪器检测OD450nm的读数。
以抗体浓度的对数为横坐标，测得的每孔吸光值为纵坐标，用log(agonist)vs.response--Variable slope方式拟合作图(GraphPad Prism软件)，得到抗体与hRANKL的结合曲线(图14A/14B/14C)。结果表明13个人源化抗体均能与hRANKL结合，呈浓度依赖型并可达到饱和，达到最大结合程度50％时的抗体浓度在1-4ng/ml之间，结合能力与G1和Prolia相当，各人源化抗体与hRANKL结合，达到最大结合程度50％时的抗体浓度见表18。
表18.人源化抗体与hRANKL结合达到最大结合程度50％时的抗体浓度比较
Figure PCTCN2014095236-appb-000029
实施例18采用竞争ELISA方法检测人源化抗体抑制RANK-Fc与hRANKL结合的能力
采用竞争ELISA实验评价抗hRANKL人源化抗体抑制受体RANK-Fc与配体hRANKL结合的能力，并与Prolia进行比较。RANK-Fc用PBS稀释到2μg/ml，100μl/孔加入96孔酶标板中，4℃包被过夜。用样品稀释液(PBS-1％BSA)对人源化抗体(G2～G14)、鼠抗(G1)和阳性对照Prolia分别进行稀释，将终浓度分别为30、10、3.3、1.1、0.37、0.12、0.04、0.01、0.005、0.0015、0.0005μg/ml的上述抗体与0.25μg/ml的hRANKL混匀，4℃预孵育过夜。PBST(含0.05％Tween20的PBS溶液)洗板3次后，用封闭液(PBS-2％BSA)室温封闭1～1.5小时，PBST洗板3次。将预孵育后的混合液按100μl/孔加入封闭好的酶标板中，其中仅含有PBS-1％BSA孔为阴性对照，仅含有0.25μg/ml hRANKL孔为阳性对照。室温反应1小时。PBST洗板3次，用含1％BSA和0.05％Tween的PBS溶液按1:5,000稀释HRP标记的anti-His tag抗体(购自Abcam,货号为ab1187)，100μl/孔加入酶标板中，室温孵育1小时。PBST洗板6次后加入TMB底物(Cell Signaling，货号7004)室温显色10分钟，每孔加入100μl的2M HCl终止显色，采用M5仪器检测OD450nm的读数。
以抗体浓度的对数为横坐标，测得的吸光值为纵坐标，用"log(inhibitor)vs.response--Variable slope"方式作图(GraphPad Prism软件)，得到抗体竞争抑制受体与配体结合的曲线(见图15A/15B/15C)。结果表明13个人源化抗体均有明显的抑制活性，能够抑制RANK-Fc与hRANKL结合，且呈浓度依赖性。实 验测得Prolia的IC50值为0.13μg/ml，13个人源化抗体的IC50值在0.1～0.4μg/ml之间，与G1和Prolia的抑制作用相当。各人源化抗体竞争抑制hRANKL与RANK-Fc结合的IC50值见表19。
表19.人源化抗体竞争抑制RANK-Fc与hRANKL结合的IC50值比较
Figure PCTCN2014095236-appb-000030
实施例19人源化抗体抑制hRANKL诱导的RAW264.7细胞分化实验
hRANKL在体外可以诱导小鼠单核巨噬细胞白血病细胞RAW264.7(购自中科院上海细胞库)分化为破骨细胞。本实验目的为评价人源化抗hRANKL抗体抑制hRANKL在体外诱导RAW264.7细胞分化活性，并与Prolia进行比较。破骨细胞中有一种特征性的酶——抗酒石酸酸性磷酸酶，pNPP显色系统可以通过OD405nm读数的高低反应抗酒石酸酸性磷酸酶活性的大小，从而反应RAW264.7细胞分化为破骨细胞的程度。RAW264.7细胞使用DMEM+10％FBS(购自Invitrogen公司)培养基进行培养，每3-4天传代一次。进行细胞活性检测时，使用α-MEM培养基+10％FBS(购自Invitrogen公司)重悬对数生长期的细胞，制成细胞悬液。以2000个/100μl/孔接种96孔细胞培养板，放入37℃，5％CO2培养箱中(Thermo公司，规格型号Forma311)培养1小时。用不含血清的α-MEM培养基对人源化抗体(G2～G14)、鼠抗(G1)和阳性对照Prolia进行倍比稀释，与等体积的含hRANKL和M-CSF、TGF-β的α-MEM培养基混匀，100μl/孔加入细胞培养板中，使得各抗体的终浓度分别为4、2、1、0.5、0.25、0.125μg/ml，hRANKL、M-CSF、TGF-β的终浓度分别为150、20和2ng/ml。仅含20ng/ml M-CSF+2ng/mL TGF-β混合液的为阴性对照孔，含20ng/ml M-CSF+2ng/mL TGF-β+150ng/ml hRANKL混合液的为阳性对照孔，均为三复孔。置于37℃，5％CO2培养箱中培养5天，弃细胞上清，每孔加入100μl细胞裂解液(Citrate Buffer pH5.0+0.5％Triton X-100)，4℃冰箱中裂解10分钟。将细胞裂解液稀释10倍，每孔加入等体积的100μl pNPP显色液(购自Sigma公司)，37℃孵育30分钟，每孔加入50μl终止液(0.5M NaOH)，通过M5仪器测定OD 405nm读数。
以抗体浓度的对数为横坐标，测得的吸光值为纵坐标，用"log(inhibitor)vs.response--Variable slope"方式作图(GraphPad Prism软件)，得到抗体抑制RAW264.7细胞分化为破骨细胞的曲线(图16)。从图16A曲线图上分析，G9和G13抑制作用较弱，G8和G12抗体的抑制作用较好，与prolia接近；从图16B曲线图上分析，G4和G10抗体的抑制作用较好，与prolia接近；从图16C曲线图上分析，G14抗体的抑制作用与prolia相当。通过图16A、B、C三组实验数据，选出5个抑制活性较好的人源化抗体，分别是G4，G8，G10，G12及G14。将上述5个人源化抗体与Prolia在同等实验条件下进行比较，结果显示Prolia的IC50值为635ng/ml，5个抗体的IC50值介于500～1100ng/ml之间，其中有3个抗体的抑制活性与prolia相当，分别为G8、G10及G14。图16D所示为5个人源化抗体抑制RANKL诱导的RAW264.7分化为破骨细胞的结果。各人源化抗体的抑制活性大小比较见表20。
表20.人源化抗体在RAW264.7细胞中的IC50值比较
抗体名称 G4 G8 G10 G12 G14 Prolia
IC50(ng/ml) ～1081 ～572.9 695.3 ～1060 ～517.4 635
实施例20人源化抗体对hRANKL诱导的RAW264.7细胞ERK1/2磷酸化的抑制作用
胰酶消化处于对数生长期的RAW264.7细胞，用DMEM+10％FBS培养基(购自Invitrogen公司)重悬，调整细胞密度，按3.0×105个/孔接种6孔细胞培养板。于37℃,5％CO2培养箱中培养贴壁。细胞 贴壁后将培养基更换成α-MEM+0.1％FBS(购自Invitrogen公司)，饥饿培养24小时。用α-MEM+0.1％FBS稀释抗体和hRANKL，将终浓度为250ng/ml的抗体(G4，G8，G10，G12，G14)与150ng/ml的hRANKL在37℃预先混合孵育2小时，弃6孔细胞培养板中的上清，按1ml/孔加入预混合液，其中prolia为阳性对照，只加α-MEM+0.1％FBS为阴性对照，37℃,5％CO2培养箱中孵育30分钟。吸去培养液，用4℃预冷的PBS(137mM NaCl，2.7mM KCl，10mM Na2HPO4，1.8mM KH2PO4，pH7.4)洗涤一次后终止反应，添加120μl LDS(购自Invitrogen公司，货号：NP0007)，冰上放置并迅速收集细胞裂解液，-80℃保存备用。
收集的细胞裂解液在含终浓度为50mM的二硫苏糖醇(购自Sangon公司，货号：D0281)的还原条件下进行电泳,将电泳后的凝胶通过电转移(300mA，80分钟)的方法转移至NC膜上(购自Pall公司，货号：S80209)。5％脱脂奶粉(购自Sangon公司,货号：NB0669)封闭后分别加入1:1000稀释的兔抗p-ERK1/2(购自Cell Signaling Technology公司,货号：4370),1:1000稀释的兔抗ERK1/2(购自Cell Signaling Technology公司,货号：4695),1:5000稀释的兔抗GAPDH(Cell Signaling Technology公司,货号：5174)，4℃孵育过夜。用1×TBST洗三遍，洗膜后再加入1:10000稀释的HRP标记的羊抗兔抗体(购自MERCK公司，货号：401315)，用1×TBST洗三遍，加入ECL(购自PerkinElmer公司，货号：NEL104001EA)显色，胶片曝光记录信号(购自Kodak公司，货号：FF057)。分析检测抗体对hRANKL诱导的RAW264.7细胞ERK1/2磷酸化的影响。
图17的结果显示，与不加hRANKL的空白组相比，hRANKL诱导了RAW264.7细胞中ERK1/2磷酸化的上调，G4，G8，G10，G12，G14能够抑制hRANKL对RAW264.7细胞中ERK1/2磷酸化的上调作用，其抑制作用与Prolia一致。
本发明通过人源化改造技术对鼠源抗体7.19.12进行人源化，从而获得人源程度提升且保持抗原抗体亲和力的人源化抗体。本申请通过人源化改造技术将鼠源抗体7.19.12进行人源化，经过系列筛选，获得了一系列人源化抗体，通过免疫学和细胞学实验证实这些人源化抗体的结合特性与鼠源抗体一致，仍具有中和活性，能够抑制RANKL与相应受体RANK的竞争结合，可以在体外有效地抑制RANKL诱导的破骨细胞的分化。据此推断，这些单克隆抗体在体内应有效地抑制骨吸收和骨质疏松。这些人源化抗体利用哺乳类动物细胞或原核细胞系统培养表达制备，可以成为适合临床药理治疗单克隆抗体药物，通过静脉或皮下给药，可用于治疗骨质疏松症、类风湿性关节炎引起的骨关节骨质破坏、肿瘤骨转移造成的骨质破坏、巨骨细胞瘤生长导致的骨质破坏和其他由于RANKL诱导的破骨亢进所形成的骨质丢失或破坏等病理改变。
本申请所展示的抗体为人源化抗体，与利用转基因鼠免疫获得的全人源抗体Prolia相比，其可变区和恒定区的核酸及氨基酸序列无相同之处。本申请所展示的人源化抗体的序列和结构为独特的和新颖的。

Claims (19)

  1. 一种抗人RANKL抗体，其特征在于其可特异性结合如SEQ ID NO:1所述的氨基酸序列。
  2. 如权利要求1所述的抗人RANKL抗体，其特征在于所述抗人RANKL抗体，包括重链和轻链，其中:a)重链包括SEQ ID NO:2-9的氨基酸序列中之一所示的可变区；并且b)轻链包括SEQ ID NO:10-17的氨基酸序列中之一所示的可变区；并且所述抗体与RANKL结合，阻断RANK与RANKL相互作用。
  3. 如权利要求2所述的抗人RANKL抗体，其特征在于所述抗人RANKL抗体，包括重链和轻链，重链为含SEQ ID NO:2的氨基酸序列所示的可变区，轻链为含SEQ ID NO:10的氨基酸序列所示的可变区；或重链为含SEQ ID NO:3的氨基酸序列所示的可变区，轻链为含SEQ ID NO:11的氨基酸序列所示的可变区；或重链为含SEQ ID NO:4的氨基酸序列所示的可变区，轻链为含SEQ ID NO:12的氨基酸序列所示的可变区；或重链为含SEQ ID NO:5的氨基酸序列所示的可变区，轻链为含SEQ ID NO:13的氨基酸序列所示的可变区；或重链为含SEQ ID NO:6的氨基酸序列所示的可变区，轻链为含SEQ ID NO:14的氨基酸序列所示的可变区；或重链为含SEQ ID NO:7的氨基酸序列所示的可变区，轻链为含SEQ ID NO:15的氨基酸序列所示的可变区；或重链为含SEQ ID NO:8的氨基酸序列所示的可变区，轻链为含SEQ ID NO:16的氨基酸序列所示的可变区；或重链为含SEQ ID NO:9的氨基酸序列所示的可变区，轻链为含SEQ ID NO:17的氨基酸序列所示的可变区。
  4. 如权利要求2所述的抗人RANKL抗体，其特征在于所述抗人RANKL抗体，包括重链和轻链，重链为含SEQ ID NO:6的氨基酸序列所示的可变区，轻链为含SEQ ID NO:14的氨基酸序列所示的可变区。
  5. 一种抗人RANKL抗体，其特征在于所述抗人RANKL抗体，包括重链和轻链，其中:a)重链包括具有一个或多个氨基酸缺失、添加和/或取代的SEQ ID NO:2-9的氨基酸序列中之一所示的可变区；并且b)轻链包括具有一个或多个氨基酸缺失、添加和/或取代的SEQ ID NO:10-17的氨基酸序列中之一所示的可变区；并且所述抗体与RANKL结合，阻断RANK与RANKL相互作用。
  6. 如权利要求5所述的抗人RANKL抗体，其特征在于所述抗人RANKL抗体，包括重链和轻链，重链为含SEQ ID NO:2的氨基酸序列所示的可变区，轻链为含SEQ ID NO:10的氨基酸序列所示的可变区；或重链为含SEQ ID NO:3的氨基酸序列所示的可变区，轻链为含SEQ ID NO:11的氨基酸序列所示的可变区；或重链为含SEQ ID NO:4的氨基酸序列所示的可变区，轻链为含SEQ ID NO:12的氨基酸序列所示的可变区；或重链为含SEQ ID NO:5的氨基酸序列所示的可变区，轻链为含SEQ ID NO:13的氨基酸序列所示的可变区；或重链为含SEQ ID NO:6的氨基酸序列所示的可变区，轻链为含SEQ ID NO:14的氨基酸序列所示的可变区；或重链为含SEQ ID NO:7的氨基酸序列所示的可变区，轻链为含SEQ ID NO:15的氨基酸序列所示的可变区；或重链为含SEQ ID NO:8的氨基酸序列所示的可变区，轻链为含SEQ ID NO:16的氨基酸序列所示的可变区；或重链为含SEQ ID NO:9的氨基酸序列所示的可变区，轻链为含SEQ ID NO:17的氨基酸序列所示的可变区。
  7. 如权利要求5所述的抗人RANKL抗体，其特征在于所述抗人RANKL抗体，包括重链和轻链，重链为含SEQ ID NO:6的氨基酸序列所示的可变区，轻链为含SEQ ID NO:14的氨基酸序列所示的可变区。
  8. 如权利要求1-7中任一项所述抗人RANKL抗体，其特征在于所述抗人RANKL抗体为单链抗体或人源化抗体，或通过杂交瘤技术获得的鼠源单克隆抗体。
  9. 一种权利要求1-7中任一项所述的抗人RANKL抗体的人源化抗体，其特征在于所述人源化抗体可特异性地与人RANKL结合，并且所述人源化抗体的重链可变区选自SEQ ID NO:6、23、25、27或29所示的氨基酸序列，轻链可变区选自SEQ ID NO:14、31、33、35、37或39所示的氨基酸序列。
  10. 如权利要求9所述的人源化抗体，其特征在于所述人源化抗体的重链恒定区来自人IgG2，轻链恒定区来自人Kappa；或所述人源化抗体的重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链恒定区选自SEQ ID NO:43所示的氨基酸序列。
  11. 如权利要求9或10所述的人源化抗体，其特征在于所述人源化抗体为选自scFv、(scFv)2、Fab、Fab’或F(ab’)2的抗原结合片段。
  12. 如权利要求9或10所述的人源化抗体，其特征在于所述人源化抗体的完整重链选自SEQ ID NO:46-50中之一所示的氨基酸序列，和/或完整轻链选自SEQ ID NO:51-56中之一所示的氨基酸序列。
  13. 如权利要求12所述的人源化抗体，其特征在于所述人源化抗体的完整重链选自SEQ ID NO:46所示的氨基酸序列，完整轻链选自SEQ ID NO:51所示的氨基酸序列；或完整重链选自SEQ ID NO:47所示的氨基酸序列，完整轻链选自SEQ ID NO:52、53、54或55所示的氨基酸序列；或完整重链选自SEQ ID NO:48所示的氨基酸序列，完整轻链选自SEQ ID NO:52、53、54或55所示的氨基酸序列；或完整重链选自SEQ ID NO:49所示的氨基酸序列，完整轻链选自SEQ ID NO:52、53、54或55所示的氨基酸序列；或完整重链选自SEQ ID NO:50所示的氨基酸序列，完整轻链选自SEQ ID NO:56所示的氨基酸序列。
  14. 如权利要求9或10所述的人源化抗体，其特征在于所述人源化抗体的重链可变区选自SEQ ID NO:6所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:14所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:23所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:31所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:23所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:35所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:23所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:33所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:23所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:37所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:27所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:31所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:27所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:35所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:27所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:33所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:27所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:37所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:25所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:31所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:25所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:35所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:25所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:33所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:25所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:37所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列；
    或重链可变区选自SEQ ID NO:29所示的氨基酸序列，重链恒定区选自SEQ ID NO:41所示的氨基酸序列，轻链可变区选自SEQ ID NO:39所示的氨基酸序列，和轻链恒定区选自SEQ ID NO:43所示的氨基酸序列。
  15. 一种药物组合物，其活性成分为权利要求1-7中任一项所述的抗人RANKL抗体或权利要求9-14任一项所述的人源化抗体。
  16. 如权利要求1-7中任一项所述抗人RANKL抗体或权利要求9-14任一项所述的人源化抗体在制备治疗骨损失疾病药物中的用途。
  17. 如权利要求16所述的用途，其中所述骨损失疾病包括骨质疏松症、类风湿性关节炎引起的骨关节骨质破坏、肿瘤骨转移造成的骨质破坏、巨骨细胞瘤生长导致的骨质破坏和其它由于RANKL诱导的破骨亢进所形成的骨质丢失或破坏等病理改变。
  18. 一种改善患有骨损失疾病的患者的状况或治疗患有骨损失疾病的患者的方法，所述方法包括给予所述患者治疗有效量的如权利要求1-7中任一项所述抗人RANKL抗体或权利要求9-14任一项所述的人源化抗体或给予所述患者权利要求15所述的药物组合物。
  19. 如权利要求18所述的方法，其中所述骨损失疾病选自骨质疏松症、类风湿性关节炎引起的骨关节骨质破坏、肿瘤骨转移造成的骨质破坏、巨骨细胞瘤生长导致的骨质破坏和其它由于RANKL诱导的破骨亢进所形成的骨质丢失或破坏等病理改变。
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