WO2011017294A1 - Anticorps anti-rankl humain - Google Patents

Anticorps anti-rankl humain Download PDF

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Publication number
WO2011017294A1
WO2011017294A1 PCT/US2010/044206 US2010044206W WO2011017294A1 WO 2011017294 A1 WO2011017294 A1 WO 2011017294A1 US 2010044206 W US2010044206 W US 2010044206W WO 2011017294 A1 WO2011017294 A1 WO 2011017294A1
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Prior art keywords
antibody
rankl
cdr
fragment
seq
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PCT/US2010/044206
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English (en)
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Diane Hollenbaugh
Raphael Levy
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Schering Corporation
Xoma Technology Ltd.
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Publication of WO2011017294A1 publication Critical patent/WO2011017294A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/70—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
    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • the present invention relates to fully human monoclonal anti-receptor activator of NF- ⁇ B ligand (RANKL) antibodies as well as methods of using the antibodies and methods of producing the antibodies BACKGROUND OF THE INVENTION
  • Osteoporos s is a disorder of impaired bone strength that results in skeletal fragility and increased fracture risk It is a common and costly disorder, and is associated with significant morbidity and mortality About 10 million American adults have osteoporosis and a further 34 million have low bone density Many of these people are at increased risk for fracture More than 1 5 million osteoporotic fractures occur in the USA each year This results in more than half a million hospitalizations more than 800 000 emergency room encounters more than 2,600 000 physician office visits, and the placement of nearly 180,000 individuals in nursing homes
  • RA rheumatoid arthritis
  • synovium lining of your joints (synovium) caus ng swelling that can result in aching and throbbing and eventually deformity
  • Receptor activator of nuclear factor- ⁇ B is a member of the tumor necrosis factor family expressed by osteoclasts and their precursors
  • the interaction of RANK with its ligand (RANKL) has been identified as a pathway through which bone resorption is regulated
  • RANKL controls the different ation, proliferation, and survival of osteoclasts
  • Osteoprotegerin (OPG) is the natural inhibitor of RANKL Mice that are deficient in OPG exh bit osteoporosis, whereas overexpression of OPG in mice results in reduced numbers of osteoclasts and high bone mass RANKL is also expressed by lymphocytes and synovial fibroblasts and may mediate bone loss associated with inflammatory conditions
  • OPG osteoprotegerin
  • RANKL is expressed by synovial cells and secreted by activated T-cells
  • TNF aiso mediates joint destruction in rheumatoid arthritis patients by systemically increasing the number of circulating osteoclast precursors and by promoting their egress from the bone marrow into the peripheral blood and then to the inflamed joints, where it promotes fusion of these cells to osteoclasts along with RANKL and ⁇ nterleuk ⁇ n-1
  • the present invention provides fully human anti- RANKL monoclonal antibodies which, in an embodiment of the invention are useful for treating or preventing diseases mediated by RANKL (e g , bone disorders or
  • the present invention provides an isolated antibody or antigen-binding fragment thereof, which specifically binds to RANKL and comprises one or more properties selected from the group consisting of ( ⁇ ) Ratio, of K 0 for human RANKL binding to K 0 for mouse RANKL binding of about 1/1 to about 10/1 ; ( ⁇ ) Ratio of IC 50 , in a TRAP assay for osteoclastogenesis, induced by human RANKL to IC 50 , in a
  • Ratio of IC 50 in an RANK receptor inhibition assay, wherein signaling is induced by human RANKL to IC 50 in an RANK recaptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 0 25 nM to about 3 3 nM
  • Ratio Ratio, of maximum inhibition, in a TRAP assay, for osteoclastogenesis, induced by human RANKL to maximum inhibition in a TRAP assay, for osteoclastogenesis, induced by mouse RANKL of about 0 9 to about 1 0, and
  • Ratio of maximum inhibition in a RANK receptor inhibition assay, wherein signaling is induced by human RANKL to maximum inhibition, in a RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL of about 0 19 nM to about 1 25 nM
  • Ratio of IC 50 in an RANK receptor inhibition assay, wherein signaling is induced by human RAN
  • the antibody or fragment is linked to a constant chain (e g , a K light chain ⁇ light chain, ⁇ 1 heavy chain, ⁇ 2 heavy chain ⁇ 3 heavy chain or ⁇ 4 heavy chain)
  • a constant chain e g , a K light chain ⁇ light chain, ⁇ 1 heavy chain, ⁇ 2 heavy chain ⁇ 3 heavy chain or ⁇ 4 heavy chain
  • composition comprising any such antibody or fragment and a pharmaceutically acceptable carrier.
  • the present invention also provides a composition comprising any such antibody or fragment in association with one or more further chemotherapeutic agents selected from the group consisting of an antiinflammatory agent an immunosuppressive agent, an anti-cancer agent and a bone condition treating agent.
  • the present invention provides an antibody or fragment of the invent on which is bound to RANKL or a fragment thereof (/ e a complex)
  • the scope of the present invention further encompasses an isolated antibody or antigen binding fragment thereof (e g a monoclonal antibody a labeled antibody a bivalent antibody a polyclonal antibody a bispecific antibody a chimeric antibody a recombinant antibody an anti-idiotypic antibody a humanized antibody a bispecific antibody, a camelized single domain antibody, a diabody an scfv, an scfV dimer a dsfv a (dsfv) 2l a dsFv-dsfv a bispecific ds diabody an Fv an Fab an Fab an F(ab ) 2 or a domain antibody) comprising one or more members selected from the group consisting of (a) XPA12 004 CDR H1 comprising the ammo acid sequence set forth in SEQ ID NO 62 XPA12 004 CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO 67 and XPA12 00
  • the present invention further provides an isolated antibody or antigen-binding fragment thereof (e g , monoclonal antibody, a labeled antibody, bivalent antibody a polyclonal antibody a bispecific antibody a chimeric antibody a recombinant antibody, an anti-idiolypic antibody, a humanized antibody, a bispecific antibody a camelized single domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a (dsfv) 2 , a dsFv dsfv , a bispecific ds diabody, an Fv, an Fab, an Fab , an F(ab') 2 , or a domain antibody) comprising one or more members selected from the group consisting of- (a) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 16, (b) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in S
  • the present invention further provides a monoclonal antibody comprising a member selected from the group consisting of (a) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 1 G, and a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 6 (b) a mature heavy immunoglobulin cha n var able region comprising the amino acid sequence set forth in SEQ ID NO 17 and a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 7, (c) a mature heavy immunoglobulin chain variable region comprising the ammo acid sequence set forth in SEQ ID NO 18 and a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 8 (d) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 19, and a mature light immunoglobulin chain variable region comprising the amino acid saquence set forth in SEQ ID NO 9,
  • the present invention further provdes an isolated hyb ⁇ doma cell producing an antibody of the invention
  • the present invention further includes a method for producing an isolated antibody comprising cultu ⁇ ng the hyb ⁇ doma cell in a culture medium under conditions suitable for expression of said antibody by said hyb ⁇ doma and, optionally, purifying the antibody from the culture medium
  • the scope of the present invention also includes an antibody produced by the method
  • the present invention further includes an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 6-10 and 16-20, along with an isolated polynucleotide encoding the polypeptide, an isolated vector comprising the polynucleotide, and an isolated host cell comprising the vector (e g bacterial (such as E coli) or mammalian)
  • an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOs 1-5 and 11 -15; an isolated vector comprising the polynucleotide, and an isolated host cell comprising the vector (e g , bacterial (such as E coli) or mammalian)
  • the present invention provides a method for treating or preventing a medical condition (e g , psor asis, insulin dependent diabetes, inflammatory bowel disease, multiple sclerosis toxic and septic shock graft versus host disease osteoporosis, osteopenia osteomyelitis, osteonecrosis Paget's disease, hypercalcemia, lytic bane metastases, periodontitis, renal osteodystrophy, osteogenesis imperfecta, hyperparathyroidism, osteomalacia, osteohalisteresis, corticosteroid treatment, childhood idiopathic bone loss, age-related loss of bone mass, rheumatoid arthritis, prosthetic loss of loosening, bone loss due to immobilization or menopause, squamous cell carcinoma, multiple myeloma, melanoma, neuroblastoma, and breast, lung prostate, thyroid, hematologic head and neck and renal cancer) in a subject, wherein said medical condition is mediated by elevated expression or activity of RANK
  • the present invention provides a fully human, monoclonal antibody or antigen- binding fragment thereof (e.g , an antagonist antibody or fragment) which specifically binds to receptor activator of NF- ⁇ B ligand (RANKL), e g , the protein of any one of SEQ ID NOs: 102-104.
  • the antibody or antigen-binding fragment thereof is XPA12.004, XPA12.020, XPA12.039, XPA12 041 or XPA12 042 or any antigen-binding fragment thereof
  • the antibodies and antigen-binding fragments of the invention may be used to inhibit RANKL activity, e g , signaling through receptor activator of NF- ⁇ B (RANK) both in vitro and in vivo
  • RANKL activity e g
  • RANK receptor activator of NF- ⁇ B
  • an "antagonist" anti-RANKL antibody or antigen-binding fragment thereof of the invention inhibits one or more of the following 'RANKL activities" (in vitro and/or in vivo): RANK binding, upregulation of the amount of active NF- ⁇ B that is capable of binding to an NF- ⁇ B DNA binding site promotion of osteoclastogenesi ⁇ [i.e , generation and/or maturation of mononucleated and/or multinucleated osteoclasts); support or promotion of osteoclast (e g , mononucleated and/or multinucleated osteoclasts) differentiation and/or maturation, in a co-culture system of cells capable of expressing RANKL cocultured with osteoclast precursors with factors such as TNF, IL-I b IL-17 or other factors capable of inducing RANKL, or in a co-culture system of osteoblastic stromal cells and spleen cells, in the presence of bone re
  • an anti-RANKL antibody or antigen-binding fragment thereof possesses one or more of the following properties (in vitro or in vivo) inhibits RANK/RANKL binding, and/or preferentially binds to mouse RANKL over that of human RANKL; and/or binds equally to mouse RANKL and human RANKL and/or inhibits the interaction between RANKL and RANK; and/or inhibits upregulation of the amount of active NF- ⁇ B that is capable of binding to an NF- ⁇ B DNA binding site; and/or inhibits osteocla ⁇ togenesis; and/or inhibits osteoclast- mediated or dependent bone resorption
  • antibody or antigen-binding fragment thereof refers to whole antibodies, and fragments, i.e , antigen-binding fragments, thereof Antigen-binding fragments include, e.gr , Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments, single chain Fv antibody fragments and dsFv antibody fragments
  • Th ⁇ terms "RANKL” and "receptor activator of NF- ⁇ B ligand” are well known in the art. RANKL is also indicated by the terms Tumor Necrosis Factor Related Activation-Induced Cytokine (TRANCE) (Wong. ⁇ f a/., J. Exp. Med.
  • RANKL may be from any organism, it is, in an embodiment of the invention, from an animal, such as a mammal (e.g., mouse, rat, rabbit, sheep or dog) for example, a human Two membrane-bound isoforms (RANKL1 and RANKL2) and one soluble isoform (RANKL3) of RANKL have been identified
  • the term "RANKL' encompasses all splice variants and isoforms of RANKL, including RANKL1 , RANKL2 and RANKL3 and the RANKL variants defined by Genbank Accession Nos NIVL003701 and NM 033012
  • the term "RANKL” includes a protein having at least 50%, 60%, 70%
  • RANK and "receptor activator of NF- ⁇ B” are also well known in the art. RANK is also indicated by the term Tumor Necrosis Factor Receptor Superfamily Member 11a. Although RANK may be from any organism, it is, in an embodiment of the invention, from an animal, such as a mammal (e.g., mouse, rat, rabbit, sheep or dog) for example, a human. A nucleotide and amino acid sequonce of a human RANK protein has the Genbank Accession No.
  • RANK includes a protein having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity with an ammo acid sequence selected from the group consisting of SEQ ID NO- 98 and GenBank Accession No NM 003839.
  • said protein has the ability to activate NF- ⁇ B or the ability to bind to TNFR-associated factors TRAF1 , TRAF2, TRAF3, TRAF4, TRAF5 or TRAF6.
  • osteoclastogenesis includes, for example, promotion of the generation or maturation of mononucleated and/or multinucleated osteoclasts, or, for example, support or promotion of osteoclast differentiation and maturation in a co- culture system of osteoblastic stromal cells or other cells capable of expressing RANKL and spleen cells or other RANKL-responding cells in the presence of bone resorption factors such as active form Vitamin D3 and parathyroid hormone
  • osteoclast includes the large multinucleated cells (MNCs) located on endosteal bone surfaces and the periosteal surface beneath the periosteum as well as mononuclear cells exhibiting osteoclast function
  • Osteoclast precursor includes any cellular entity on the pathway of differentiation between a macrophage and a differentiated and functional osteoclast
  • osteoclast function as used herein includes bone resorption and the processes required for bone resorption
  • an antibody or antigen-binding fragment thereof binds to an antigen 'specifically" if it binds with a K D of about 10 nM or a lower number
  • the amino acid sequences of the variable regions of fully human, monoclonal anti-RANKL antibodies and antigen-binding fragments thereof of the invention are summarized in Table 1
  • Table 1 also includes a summary of the amino acid and nucleotide sequences which correspond to the CDR and FR regions of the antibodies
  • the present invention includes any isolated nucleic acid or isolated polypeptide (e g., antibody or antigen-binding fragment thereof) which comprises one or more (e g 1 , 2 3, 4, 5 6, 7, 8, 9 10, 11 , 12, 13, or 14 (e g , any possible combination thereof)) of any of the immunoglobulin nucleic acids or polypeptides (including mature fragments thereof and CDRs) set forth below in Table 1 along with sequences variants thereof, e g , as discussed here
  • the present invention encompasses any polypeptide (e g., an immunoglobulin polypeptide) comprising 1 , 2 or all 3 CDRs of the heavy or light chain immunoglobulins XPA12 004 XPA12.020, XPA12.039, XPA12 041 or XPA12 042 fused or unfused to another polypeptide such as an immunoglobulin constant chain (e g as discussed herein)
  • Single heavy or light chain polypeptides are useful, for example, as intermediates in the recombinant expression of a full antibody or an antigen binding fragment thereof that comprises both heavy and light chains wherein each chain is individually expressed in a host cell before association and formation of the full antibody or antigen-binding fragment
  • the amino acid and nucleotide sequences are identical between two or more antibodies, only a single sequence is shown
  • the invention also provides isolated polypeptides comprising the VL domains (e.g., SEQ ID No ⁇ .: 6-10) and isolat ⁇ d polypeptides comprising the VH domains (e g , SEQ ID IMos.: 16-20) of the antibodies of the invention having D, 1 , 2, 3, 4, or 5 or more non-conservative amino acid substitutions, while still exhibiting the ability to bind to RANKL.
  • the invention provides an antibody or antigen binding fragment thereof that binds RANKL and has VL domains and VH domains with at least 95%, 90%, 85%, 80%, 75% or 50% sequence homology to one or more of SEQ ID NOs:, and exhibits binding to RANKL,
  • a CDR in an immunoglobulin chain (e.g. , heavy or light chain of XPA12.004; XPA12.020; XPA12.039; XPA12.041 ; and XPA12.042) is altered so as to generate a more stable variant or a variant that is recombinantly expressed at higher levels.
  • the invention encompasses variants wherein the Asp or Asn is changed to GIu or Ala or wherein the GIy is changed to Ala.
  • a benefit of such a change is removal of the potential for isoaspartate formation.
  • the scope of the present invention includes variants wherein the Met is changed to Lys, Leu, Ala or Phe.
  • a benefit of such a change is removal of the potential for methionine oxidation.
  • an Asn is in a CDR of the invention (e.g., in the heavy or light chain of XPA12.004; XPA12.020, XPA12.039, XPA12 041 ; and XPA12.042)
  • the scope of the present invention includes variants wherein Asn is changed to GIn or Ala.
  • a benefit of such a change is removal of the potential for deamidation.
  • an Asn-Pro is in a CDR of the present invention (e.g., in the heavy or light chain of XPA12.004; XPA12.020, XPA12.039; XPA12 041 ; and XPA12.042)
  • the present invention includes variants wherein Asn is changed to GIn or Ala or wherein Pro is changed to Ala.
  • a benefit of such a change is removal of a possible s ⁇ ssile Asn-Pro peptide bond.
  • the scope of the invention includes embodiments wherein the heavy or light chain CDRs of any of XPA12.004; XPA12 020; XPA12.039; XPA12.041 ; and XPA12.042 are independently changed in one or more places as described above.
  • heavy chain immunoglobulin variable regions of the present invention (XPA12.004, XPA12.020; XPA12.039; XPA12.041 ; and XPA12.042) are set forth below. CDRs are underscored and CDR residues of interest, which may be independently altered or left unaltered, are in bold font.
  • the present invention comprises isolated polypeptides (e.g., fused to an immunoglobulin constant chain such as garnma-1 , gamma-2, gamma-2a, gamma-2b, gamma-3 or gamma-1 ), antibodies and antigen-binding fragments thereof comprising these CDRs and/or chains as well as chains comprising any or all of the variants specifically discussed.
  • Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagine in CDR-H1 is mutated to GIn or Ala or left as an unmutated asparagine.
  • Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagine in CDR-H1 (N 1 ) is mutated to GIn or Ala and/or wherein, independently, the Asp-Gly (D 2 G 3 ) in CDR-H3 is mutated such that the Asp is changed to Asn or Ala and/or, independently, wherein the GIy is changed to Ala; and/or independently wherein N 1 is left unmutated and/or wherein, independently, D 2 G 3 are left unmutated.
  • embodiments of the invention comprise those wherein the heavy chain immunoglobulin is as set forth above wherein the residues at the positions of asparagine 1 , a ⁇ partic acid 2 and glycine 3 ⁇ i.e., N 1 , D 2 and G 3 ) are as any of the permutations set forth below:
  • Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein any combination of the asparagines in CDRs -H1 , - -H2 and -H3 (e.g., N 1 , N 2 , N 3 , N 4 , or N 5 ) are independently mutated to GIn or Ala or any one is independently left as an unmutated asparagine.
  • any combination of the asparagines in CDRs -H1 , - -H2 and -H3 e.g., N 1 , N 2 , N 3 , N 4 , or N 5
  • any combination of the asparagines in CDRs -H1 , - -H2 and -H3 are independently mutated to GIn or Ala or any one is independently left as an unmutated asparagine.
  • embodiments of the invention comprise those wherein the heavy chain immunoglobulin is as set forth above wherein the residues at the positions of asparagines 1 , 2, 3, 4 and 5 (i.e., N 1 , N 2 , N 3 , N 4 and N 5 ) are as any of the permutations set forth below:
  • Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagine in CDR-H1 (N 3 ) is mutated to GIn or Ala and/or wherein, independently, the Asp-Gly in CDR-H1 (D 1 G 2 ) is mutated such that the Asp is changed to Asn or Ala and/or wherein the GIy is changed to Ala, and/or independently wherein the N 3 is left unmutated and/or wherein, independently, the D 1 G 2 is left unmutated
  • embodiments of the invention comprise those wherein the heavy chain immunoglobulin is as set forth above wherein the residues at the positions of aspartic acid 1 , glycine 2 and asparagine 3 (i e , D 1 , G 2 and N 3 ) are as any of the permutations set forth below
  • light chain immunoglobulin variable regions of the present invention (XPA12 004 XPA12 020, XPA12 039 XPA12 041 , and XPA12 042) are set forth below CDRs are underscored and CDR residues of interest, which may be independently altered or left unaltered, are in bold font
  • the present invention comprises isolated polypeptides (e g , fused to an immunoglobulin constant chain such as kappa or lambda), antibodies and antigen binding fragments thereof comprising these chains as well as chains comprising any or all of the variants specifically discussed
  • Embodiments of the invention comprise polypeptides, antibodies and antigen- bmding fragments thereof wherein the asparagines in CDR-L1 and/or CDR-L2 (N 1 and N ⁇ ) are independently mutated to GIn or Ala or left as unmutated asparagines
  • embodiments of the invention comprise those wherein the light chain immunoglobulin is as set forth above wherein the residues at the positions ⁇ f asparagines 1 and 2 / e N 1 and N 2 are as an of the ermutations set forth below
  • Embodiments of the invention comprise polypeptides, antibodies and antigen binding fragments thereof wherein the asparagines in CDR-L1 and/or CDR-L2 (N 1 N 2 and N 3 ) are independently mutated to GIn or Ala or left as unmutated asparagines
  • embodiments of the invention comprise those wherein the light chain immunoglobulin is as set forth above wherein the residues at the positions of asparagines 1 , 2 and 3 (f e , N 1 N 2 and N 3 ) are as any of the permutations set forth below
  • Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagines in CDR-L1 and/or CDR-L2 (N 1 and N 2 ) are independently mutated to GIn or Ala or left as unmutated asparagines.
  • embodiments of the invention comprise those wherein the light chain immunoglobulin is as set forth above wherein the residues at the positions of asparagines 1 and 2 [i.e. , N 1 and N 2 ) are as any of the permutations set forth below:
  • Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagines in CDR-L1 and/or CDR-L2 (N 1 , N 2 , N 3 and N 4 ) are independently mutated to GIn or Ala or left as unmutated asparagines.
  • embodiments of the invention comprise those wherein the light chain immunoglobulin is as set forth above wherein the residues at the positions of asparagines 1 , 2, 3 and 4 (/ e., N 1 , N 2 , N 3 and N 4 ) are as any of the permutations set forth below.
  • Embodiments of the invention comprise polypeptides, antibodies and antigen- bmding fragments thereof wherein the asparagines in CDR-L1 (N 1 , N 2 and N 3 ) are independently mutated to GIn or Ala or left as unmutated asparagines
  • ⁇ mbodiments of the invention comprise those wherein the light chain immunoglobulin is as set forth above wherein the residues at the positions of asparagines 1 , 2 and 3 (i e , N 1 , N 2 and N 3 ) are as any of the permutations set forth below
  • the present invention encompasses antibodies and antigen-binding fragments thereof comprising any possible heavy chain/light chain combination of the immunoglobulin CDR variants discussed above or comprising a wild-type heavy chain immunoglobulin and any of the light chain immunoglobulin CDR variants or comprising a wild-type light chain immunoglobulin and any of the heavy chain immunoglobulin CDR variants.
  • an antibody or antigen-binding fragment thereof comprising a CDR variant immunoglobulin of the present invention is characterized such that the antibody or fragment retains any of the biological or binding characteristics of a parental XPA12 004; XPA12.020; XPA12.039; XPA12.041 or XPA12 042 antibody to any degree (e.g., binding to human and/or mouse RANKL).
  • the present invention includes any immunoglobulin polypeptide or polynucleotide set forth in Table 1 along with antibodies and antigen-binding fragments thereof that include one or more CDRs set forth in Table 1 or which include one or more heavy or light chain immunoglobulins set forth in Table 1 or which comprise one or more CDRs taken from the heavy or light chain immunoglobulins set forth in Table 1 ; e.g., as defined by Kabat ef a/., Sequences of Proteins of
  • An XPA12 004 antibody or XPA12 020 antibody or XPA12 041 antibody or XPA12.039 antibody or XPA12.042 antibody comprises light and heavy
  • the present invention includes antibodies and antigen-binding fragments thereof that bind the same RANKL (e.g., mouse and/or human) epitope as the XPA12 004 antibody, XPA12 020 antibody, XPA12.041 antibody, XPA12.039 antibody or XPA12.042 antibody at any detectable level of affinity (e g , at the affinities (KD) set forth in Table 2)
  • the present invention also provides any antibody or antigen-binding fragment thereof that competes with the XPA12 004 antibody, XPA12 020 antibody, XPA12 041 antibody, XPA12 039 antibody or XPA12 042 antibody for binding to RANKL (e.g., mouse or human) to any detectable degree (e g , in vitro), for example, under standard in vitro assay conditions that are well known in the art.
  • the present invention includes antibodies and antigen-binding fragments thereof which include one, two, three, four five, or six complementarity determining regions (CDRs) of the described antibodies and antigen-binding fragments of the invention (e g , 3 light chain CDRs and 3 heavy chain CDRs)
  • CDRs complementarity determining regions
  • the one, two, three, four, five, or six CDR ⁇ may be independently selected from the described CDR sequences of the antibodies and antigen-binding fragments of the invention.
  • the one, two, three, four, five, or six CDRs may be selected from the CDR sequences of a single described antibody or antigen-binding fragment of the invention.
  • CDR-L1 includes, e g , amino acids 23-35 of SEQ ID NO: 6, ammo acids 23-36 of SEQ ID NO: 7, amino acids 23-36 of SEQ ID NO: 8, amino acids 23-35 of SEQ ID NO: 9, or amino acids 23-35 of SEQ ID NO: 10
  • CDR-L2 includes, e.g. amino acids 21-27 of SEQ ID NO: 6, amino acids 52-58 of SEQ ID NO. 7, amino acids 52-58 of SEQ ID NO- 8, amino acids 51-57 of SEQ ID NO- 9, or amino acids 51 -57 of SEQ ID NO 10.
  • CDR-L3 includes, e.g., amino acids 90-100 of SEQ ID NO: 6, amino acids 91 -103 of SEQ ID NO: 7, amino acids 91-100 of SEQ ID NO: 8, amino acids 90-100 of SEQ ID NO: 9, or amino acids 90-100 of SEQ ID NO. 10.
  • CDR-H1 includes, e.g , amino acids 26-35 of SEQ ID NO: 16, amino acids 26-35 of SEQ ID NO: 17, ammo acids 26-35 of SEQ ID NO- 18, amino acids 26-35 of SEQ ID NO: 19, or ammo acids 26-35 of SEQ ID NO.
  • CDR-H2 includes, e g amino acids 50-66 of SEQ ID NO 16, amino acids 50-66 of SEQ ID NO 17, amino acids 50-66 of SEQ ID NO 18, ammo acids 50-66 of SEQ ID NO 19, or ammo acids 50-66 of SEQ ID NO 20
  • CDR-H3 includes, e g , amino acids 99-109 of SEQ ID NO 16 amino acids 99-11 1 of SEQ ID NO 17, ammo acids 99-106 of SEQ ID NO 18, amino acids 99-110 of SEQ ID NO 19 or amino acids 99-109 of SEQ ID NO 20
  • the present invention also includes antibodies and antigen-binding fragments which include one, two three four five, six, seven, or eight framework regions of the described antibodies and antigen-binding fragments of the invention
  • the one, two, three, four five, six, seven, or eight framework regions may be independently selected from the FR sequences of the described antibodies and antigen-binding fragments of the invention
  • the one, two three, four, five, six, seven or eight framework regions may be selected from the FR sequences of a single described antibody or antigen binding fragment of the invention.
  • the present invention also includes antibodies and ant gen-binding fragments which include one or two variable regions of the antibodies and antigen-binding fragments of the invention
  • the one or two variable regions may be independently selected from the variable regions of the d ⁇ sc ⁇ bed antibodies and antigen-binding fragments of the invention Alternatively, the one or two variable regions may be selected from the variable regions of a single described antibody or antigen-binding fragment of the invention
  • a mature light chain variable region of the invention, which lacks the signal peptide may comprise or consist of an ammo acid sequence selected from the group consisting of SEQ ID NOs 6-10 or may be encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs 1-5
  • a mature heavy chain variable region of the invention, which lacks the signal peptide may comprise or consist of an ammo acid sequence selected from the group consisting of SEQ ID NOs 16-20, or may be encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO3 11-15.
  • a polypeptide or protein comprises two or more amino acids.
  • isolated protein is a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1 ) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, (4) was isolated or purified e.g., by a technician and/or (5) does not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • Non-limiting examples of isolated antibodies include an anti-RANKL antibody that has been affinity purified using RANKL, recombinant ⁇ expressed antibodies and antigen-binding fragments thereof, an anti-RANKL antibody that has been synthesized by a hybridoma or other cell line in vitro, and a human anti-RANKL antibody derived from a transgenic mouse or a human immunoglobulin library (e.g., phage display library).
  • a “polynucleotide”, “nucleic acid “ or “nucleic acid molecule” includes double- stranded and single-stranded DNA and RNA.
  • An amino acid sequence comprises two or more amino acids.
  • a "coding sequence” or a sequence “encoding” an expression product, such a ⁇ an RNA or polypeptide, is a nucleotide sequence that, when expressed, results in production of the product.
  • Amplification ' of DNA as used herein includes the use of polymerase chain reaction (PCR) to increase the co ⁇ ce ⁇ traton of a particular DNA sequence within a rn xture of DNA sequences
  • PCR polymerase chain reaction
  • the present invention includes a nucleic acid, which encodes an anti-RANKL antibody, an anti-RANKL antibody heavy or light chain an anti-RANKL antibody heavy or light chain variable region an anti-RANKL antibody heavy or light chain constant chain or an anti-RANKL antibody CDR (e g CDR L1 CDR-L2, CDR-L3 CDR-H1 CDR-H2 or CDR-H3) which can be amplified by PCR
  • oligonucleotide refers to a nucleic acid, generally of at least 10 (e g 10, 1 1 12, 13 or 14), for example at least 15 (e g , 15, 16, 17 18 or 19) or at least 20 nucleotides (e g , 20, 21, 22, 23, 24, 25, 26, 27 28, 29 or 30), or no more than 100 nucleotides (e g , 40, 50, 60 70 80 or 90), thai may be hyb ⁇ dizable to a genomic DNA molecule a cDNA molecule or an mRNA molecule encoding a gene mRNA, cDNA, or other nucleic acid of interest Oligonucleotides can be labeled e g by incorporation of 32 P-nucleot ⁇ des, 3 H-nucleot ⁇ des 14 C-nucle ⁇ t ⁇ des, 35 S-nucleot ⁇ des or nucleotides to which a label such as biotin has been covalently
  • any nucleic acid may be sequenced by any method known in the art (e g , chemical sequencing or enzymatic sequencing).
  • “Chemical sequencing” of DNA may denote methods such as that of Maxam and Gilbert (1977) (Proc Nafl Acad Sa USA 74 560) in which DNA is randomly cleaved using individual base-specific reactions
  • “Enzymatic sequencing” of DNA may denote methods such as that of Sanger (Sanger, et al , (1977) Proc. Natl. Acad. Sa. USA 74:5463)
  • nucleic acids herein may b ⁇ flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ⁇ bosorne entry sites (IRES) and other ⁇ bosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
  • promoters include promoters, internal ⁇ bosorne entry sites (IRES) and other ⁇ bosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
  • IVS internal ⁇ bosorne entry sites
  • a “promoter” or 'promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence
  • a promoter sequence is, in general, bounded at its 3' terminus by the transcription initiation site and extends upstream (5 r direction) to include the minimum number of bases or elements necessary to initiate transcription at any level.
  • a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • the promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences or with a nucleic acid of the invention (e.g., SEQ ID NO 1 -5, 11-15, 21-34, 49-61 , 75-79, or 85-90).
  • Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U S. Patent Nos.
  • a coding sequence is "under the control of”, “functionally associated with” or “operably associated with” transcriptional and translational control sequences in a cell when the sequences direct RNA polymerase mediated transcription of the coding sequence into RNA, e g , mRNA, which then may be trans-RNA spliced (if it contains introns) and optionally translated into a protein encoded by the coding sequence
  • express and "expression” mean allowing or causing the information in a gene, RNA or DNA sequence to become manifest for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene
  • a DNA sequence is expressed in or by a cell to form an ' expression product' such as an RIMA (e g mRNA) or a protein (e g , immunoglobulin chain of an antibody XPA12 004, XPA12 020 XPA12 039, XPA12 041 or XPA12 042 or a fragment thereof)
  • the expression product itself may also be said to be "expressed by the cell
  • vector means the vehicle (e g , a plasmid) by which a DNA or RNA sequence can be introduced into a host cell so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence
  • transformation means the introduction of a nucleic acid into a cell These terms may refer to the introduction of a nucleic acid encoding an anti-RANKL antibody or fragment thereof into a cell
  • the introduced gene or sequence may be called a 'clone"
  • a host cell that receives Ihe introduced DNA or RNA has been "transformed” and is a 'transformant” or a “clone”
  • the DNA or RNA introduced to a host cell can come from any source including cells of the same genus or species as the host cell, or cells of a different genus or species
  • host cell means any cell of any organism that is selected, modified, transfected, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression or replication by the cell, of a gene a DNA or RNA sequence a protein or an enzyme Isolated host cells, e g , comprising a polynucleotide or polypeptide of the present invention, form part of the present invention
  • Isolated host cells e g , comprising a polynucleotide or polypeptide of the present invention, form part of the present invention
  • host cells include eukaryotic cells such as Chinese hamster ovary cells, yeast cells (e g , Pichia, Pichia pastons, S.cerevisiae) and bacteria such as E coll (e g , BL21 , BL21 (DE3) or HB101)
  • expression system means a host cell and compatible vector which, under suitable conditions, can express one or more proteins or nucleic acids of the present invention (e.g., as set forth in Table 1 ) which is carried by the vector and introduced to the host cell.
  • proteins or nucleic acids of the present invention e.g., as set forth in Table 1
  • Common expression systems include E.
  • RANKL or an antibody or antigen binding fragment of the invention or any polypeptide or nucleic acid of the present invention may be expressed in human embryonic kidney cells (HEK293)
  • suitable cells include CHO (Chinese hamster ovary) cells, HeLa cells and NIH 3T3 cells and NSO cells (non-lg-producing murine myeloma cell line)
  • the nucleic acid molecules, vectors and host cells may be used to make mutated anti-RANKL antib ⁇ di ⁇ s.
  • mutated anti-RANKL antibodies and antigen-binding fragments thereof are discussed in detail above
  • Other mutated immunoglobulin chains are included within the scope of the present invention
  • the antibodies may be mutated in the variable domains of the heavy and/or light chains e g , to alter a binding property of the antibody
  • a mutation may be made in one or more of the CDR regions to increase or decrease the K 0 of the antibody for RANKL, to increase or decrease k off , or to alter the binding specificity of the antibody.
  • the mutations may be made in a CDR region or a framework region of a variable domain, or in a constant domain. In one embodiment, the mutations are made in a variable domain
  • the framework region is mutated so that the resulting framework reg ⁇ on(s) have the amino acid sequence of the corresponding germline gene
  • the present invention encompasses polypeptides comprising the light chain CDRs of the present invention in any light chain germ line framework (e g , vk A30 A27 012) and polypeptides comprising the heavy chain CDRs of the present invention in any heavy chain germ line framework (e.g , VH DP47, DP35, DP-71 or VIV-4) along with antibodies and antigen-binding fragments thereof comprising such polypeptides
  • a mutation may be made in a framework region or constant domain to increase the half-life of the anti-RANKL antibody.
  • a mutation in a framework region or constant domain also can be made to alter the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molecule, or to alter such properties as complement fixation, FcR binding and antibody-dependent cell mediated cytotoxicity (ADCC).
  • a single antibody may have mutations n any one or more of the CDRs or framework regions of the variable domain or in the constant domain
  • any of the mutations can be conservative amino acid substitutions
  • the present invention also contemplates any superfical or slight modification to the amino acid or nucleotide sequences which correspond to the antibodies or ant gen-binding fragments of the invention
  • sequence conservative variants of the nucleic ac ds which encode the ant bodies or antigen b nding fragments of the invention sequence conservative variants of the nucleic ac ds which encode the ant bodies or antigen b nding fragments of the invention
  • sequence-conservative variants of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon results in no alteration in the amino acid encoded at that position
  • “Function-conservative variants” are those in which one or more amino acid residues in a protein have been changed ablating the function of the polypeptide including but, by no means limited to, replacement of an ammo acid with one having similar properties
  • Amino acids with similar properties are well known in the art
  • polar/hydrophilic amino acids which may b ⁇ interchangeable include asparagine, glutamine, serine cysteine, threonine lysine, arginine histidine aspartic acid and glutamic acid nonpolar/hydrophobic ammo acids which may be interchangeable include glycine alanine valine leucine isoleucine, proline, tyrosine phenylalanine, tryptophan and methionine
  • acidic amino acids which may be interchangeable include aspartic acid and glutamic acid and basic amino acids which may be interchangeable include histidine, lysine and arginine
  • IL-I b IL-17 or other factors capable of inducing RANKL, or in a co-culture system of osteoblastic stromal cells and spleen cells, in the presence of bone resorption factors such as active form Vitamin D3 and parathyroid hormone; induction of bone resorption).
  • bone resorption factors such as active form Vitamin D3 and parathyroid hormone; induction of bone resorption.
  • such a mutant or variant exhibits one or more of the following characteristics
  • RANKL to IC 50 , in a TRAP assay for osteoclastogenesis, induced by mouse RANKL, of about 0 19 nM to about 1.25 nM;
  • IC 50 in an RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 1.5 nM to about 2.2 nM,
  • RANKL of about 0.6 nM to about 7.3 nM
  • Ratio of maximum inhibition, in a TRAP assay, for osteoclastogenesis, induced by human RANKL to maximum inhibition, in a TRAP assay, for ⁇ steoclastogenesis, induced by mouse RANKL, of about 0.9 to about 1.0;
  • the present invention includes anii-RANKL antibodies and fragments thereof comprising V L or V H immunoglobulins or CDRs thereof which are encoded by nucleic acids as described in Table 1 as well as nucleic acids which hybridize thereto
  • the nucleic a ⁇ ds hybridize under low stringency conditions, or under moderate stringency conditions or under high stringency conditions
  • a nucleic acid molecule is "hyb ⁇ dizable 1 to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sarnbrook, et al., supra).
  • Stringent hybridization conditions include: pr ⁇ hybr ⁇ d ⁇ zat ⁇ on for 2 hours at 60 0 C in 6X SSC, 0 5% SDS, 5X Denhardt's solution, and 100 ⁇ g/ml heat denatured salmon sperm DNA; hybridization for 18 hours at 60 0 C; washing twice in 4X SSC, 0.5% SDS, 0.1 % sodium pyrophosphate for 30 minutes at 6O 0 C, and twice in 2X SSC, 0 1 % SDS for 30 minutes at 6O 0 C; or, for example, hybridization in 4X SSC at 65 0 C, followed by a washing in 0.1X SSC at 65 0 C for one hour, or hybridization in 50% formamide, 4X SSC at 42°C.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency under which the nucleic acids may hybridize. For hybrids of greater than 100 nucleotides in length, equations for calculating the melting temperature have been derived (see Sambrook, ef a/., supra, 9.50-9.51 ).
  • oligonucleotides For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook, ef a/., supra, 11.7-11.8).
  • nucleic acids comprising nucleotide sequences and polypeptides comprising amino acid sequences which are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e g., 95%, 95%, 97%, 98%, 99%, 100%) to the reference immunoglobulin nucleotide and amino acid sequences of Table 1 when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.
  • Polypeptides comprising amino acid sequences which are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the reference immunoglobulin amino acid sequences of Table 1 (e.g., SEQ ID NOs: 6-10, 16-20, 35-48, 62-74, 80-84, and 91-96) when the comparison is performed with a BLAST algorithm wherein the parameters of the algorithm are selected to give the largesi match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention
  • Sequence identity refers to exact matches between the nucleotides or ammo acids of two sequences which are being compared
  • Sequence similarity refers to both exact matches between the ammo acids of two polypeptides which are being compared in addition to matches between nonidentical, biochemically related amino acids Biochemically related amino acids which share similar properties and may be interchangeable are discussed above
  • antibody or antigen-binding fragments thereof includes monoclonal antibodies, polyclonal antibodies, labeled antibodies, bivalent antibodies, bispecific antibodies, recombinant antibodies, anti-idiotypic antibodies, camehzed single domain antibodies, diabodies, Fab antibody fragments Fab' antibody fragments, F(ab) 2 antibody fragments, Fv antibody fragments (e.g., V H or VL), single chain Fv antibody fragments, nanobodies, single chain Fv antibody fragment dimers, dsFv antibody fragments, (dsFv) 2 antibody fragments, dsFv-ctefv' dimers, bispecific ds diabodies and domain antibodies.
  • antibodies oi the invention may be fully human antibodies or chimeric or humanized antibodies.
  • the antibodies are monoclonal, fully human antibodies.
  • the antibodies of the invention are XPA12.004, XPA12 020, XPA12.039, XPA12.041 or XPA12.042.
  • the antibodies and antigen-binding fragments thereof include one or more of the variable regions and/or CDRs whose amino acid and nucleotide sequences are set forth in Table 1.
  • CDRs from immunoglobulin variable regions set forth herein may, in an embodiment of the invention, be defined as described in Kabat ef a/., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987).
  • the scope of the present invention includes complexes comprising an anti- RANKL antibody or antigen-binding fragmont thereof (e.g., an isolated or unisolated antibody or fragment) of the invention bound to RANKL or a fragment thereof (e g , an isolated or unisolated RANKL or fragment); wherein the complex is in vivo or in vitro.
  • the present invention includes complexes between an isolated anti- RANKL antibody or fragment bound to an endogenous RANKL in the body of a subject.
  • the present invention includes any antibody or antigen-binding fragment thereof comprising a CDR selected from those set forth below in Table 1a.
  • the present invention includes immunoglobulin chain polypeptides as well as antibodies and antigen-binding fragments thereof comprising one or more CDRs (e g 3 light chain CDRs and/or 3 heavy chain CDRs) which independently have at least about 85% (e g , 85%, 86% 87% 88%, 89%, 90%, 91 % 92%, 93%, 94%, 95%, 96% 97%, 98%, 99% or 100%) amino acid sequence similar ty or identity to the sequence in any of SEQ ID NOs 35-48 and 62-73
  • the scope of th ⁇ present invention includes embodiments wherein the antibody or antigen-binding fragment comprises the following arrangement
  • CDRH3, or CDRH1-CDRH2 with > 85% similar or identical to any of SEQ ID NOs 67-69- CDRH3, or
  • CDRL1-CDRL2 with > 85% similar or identical to any of SEQ ID NOs- 39-43- CDRL3, and, optionally,
  • CDRL1-CDRL2 -CDRL3 with > 85% similar or identical to any of SEQ ID NOs 44- 48 and, optionally,
  • CDRL1 with > 85% similar or identical to any of SEQ ID NOs: 35-38-CDRL2 - CDRL3
  • CDRL1 -CDRL2 with > 85% similar or identical to any of SEQ ID NOs: 39-43-
  • CDRL1-CDRL2 -CDRL3 with > 85% similar or identical to any of SEQ ID NOs- 44-48; or
  • CDRL1 with > 85% similar or identical to any of SEQ ID NOs: 35-38-CDRL2 -
  • CDRL1 -CDRL2 with > 85% similar or identical to any of SEQ ID NOs: 39-43-
  • CDRL3 or CDRL1-CDRL2 -CDRL3 with > 85% similar or identical to any of SEQ ID NOs
  • CDRL1 with > 35% similar or identical to any of SEQ ID NOs 35-38-CDRL2 -
  • CDRL1 -CDRL2 with > 85% similar or identical to any of SEQ ID NOs: 39-43-
  • CDRL1-CDRL2 -CDRL3 with > 85% similar or identical to any of SEQ ID NOs 44-48.
  • Isolated polypeptides comprising any of the foregoing individual heavy or light chains, fused or unfused to an immunoglobulin constant chain, form part of the present invention.
  • an antibody of fragment described above (1-6) retains one or more RANKL antagonist activities (e g , the ability to inhibit one or more of the following ' RANKL activities" (in vitro and/or in vivo) including RANK binding, upregulation of the amount of active NF- ⁇ B that is capable of binding to an NF-icB DNA binding site; promotion of
  • osteoclastogenesis ⁇ i.e , generation and/or maturation of mononucleated and/or multinucleated osteoclasts
  • support or promotion of osteoclast e.g., mononucleated and/or multinucleated osteoclasts
  • osteoclast e.g., mononucleated and/or multinucleated osteoclasts
  • the antibody or fragment described above (1 -6) exhibits one or more of the following characteristics
  • Ratio of IC 5 O, in a TRAP assay for osteoclastogenesis, induced by human RANKL, to ICso, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL, of about 0 19 nM to about 1 25 nM;
  • IC 50 in a TRAP assay for osteoclastogenesis, induced by human RANKL, of about
  • RANKL of about 0 6 nM to about 7.3 nM
  • RANKL of about 97% (xxi) maximum inhibition, in a TRAP assay for osteoclastogenesis induced by mouse RANKL of about 100%,
  • an isolated polypeptide (unfused or fused to an immunoglobulin constant chain) or antibody or antigen-binding fragment thereof of the invention comprises any of the following amino acid sequences.
  • a mature heavy immunoglobulin chain va ⁇ able region which has at least about 85% (e g , 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence similarity or identity to the amino acid sequence of SEQ ID NO 16, optionally, with any of the light chain sequences described below,
  • a mature heavy immunoglobulin chain variable region which has at least about 85% (e g , 85%, 86%, 87% 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100%) ammo acid sequenca similarity or identity to the amino acid sequence of SEQ ID NO 17, optionally, with any of the light chain sequences described below;
  • a mature heavy immunoglobulin chain variable region which has at least about 85% (e g 85%, 86% 87%, 88% 89% 90%, 91 %, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence similarity or identity to the amino acid sequence of SEQ ID NO- 18, optionally, with any of the light chain sequences described below;
  • a mature heavy immunoglobulin chain variable region which has at least about 85% (e.g , 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or 100%) amino acid sequence similarity or identity to the amino acid sequence of SEQ ID NO: 19, optionally, with any of the light chain sequences described below,
  • a mature heavy immunoglobulin chain variable region which has at least about 85% ( ⁇ g , 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or 100%) amino acid sequence similarity or identity to the amino acid sequence of SEQ ID NO 20, optionally with any of the light chain sequences described below
  • a mature light immunoglobulin chain variable region which has at least about 85% (e g , 85% 86% 87% 88% 89% 90% 91%, 92% 93%, 94% 95%, 96%, 97%, 98%, 99% or 100%) ammo acid sequence similarity or identity to the amino acid sequence of SEQ ID NO 6, optionally, wilh any of the heavy chain sequences described above,
  • a mature light immunoglobulin chain variable region which has at least about 85% (e g , 85%, 86% 87% 88%, 89%, 90%, 91%, 92%, 93% 94% 95%, 96% 97%, 98%, 99% or 100%) amino acid sequence similarity or identity to the amino acid sequence of SEQ ID NO 7, optionally, with any of the heavy chain sequences described above,
  • a mature light immunoglobulin chain variable region which has at least about 85% (e g , 85%, 86%, 87% 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence similarity or identity to the amino acid sequence of SEQ ID NO 8 optionally, with any of the heavy chain sequences described above
  • a mature light immunoglobulin chain variable region which has at least about 85% (e g , 85%, 86%, 87%, 88% 89%, 90%, 91 %, 92% 93%, 94%, 95%, 96% 97% 98%, 99% or 100%) ammo acid sequence similarity or identify to the amino acid sequence of SEQ ID NO: 9, optionally, with any of the heavy chain sequences described above;
  • an antibody of fragment described above retains one or more RANKL antagonist activities (e g , the ability to inhibit one or more of the following RANKL activities" (in vitro and/or in vivo) including RANK binding, upregulation of the amount of active NF- ⁇ B that is capable of binding to an NF- ⁇ B DNA binding site, promotion of osteoclastogenesis (/ e , generation and/or maturation of mononudeated and/or multinucleated osteoclasts), support or promotion of osteoclast (e.g.
  • th ⁇ antibody or fragment described above exhibits one or more of the following characteristics ( ⁇ ) Ratio, of K D for human RANKL binding to K 0 for mouse RANKL binding, of about 1/1 to about 10/1 ,
  • IC 50 in a TRAP assay for osteoclastogenesis, induced by mouse RANKL of about 0 1 nM to about 1.6 nM
  • ( ⁇ x) IC 50 in an RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 0 6 nM to about 7 3 nM;
  • any anti-RANKL aniibody or antigen- bi ⁇ ding fragment thereof of the invention binds to an extracellular domain of RANKL
  • the scope of the present invention includes antibody variable regions of the present invention (e g , any mature variable region indicated in Table 1 or an unprocessed version thereof) linked to any immunoglobulin constant chain. If a light chain variable region is linked to a constant chain, in one embodiment of the invention it is a lambda or kappa chain If a heavy chain variable region is linked to a constant chain, in certain embodiments of the invention, it is a gamma-1 , gamma-2, gamma-3 or gamma-4 chain
  • the anti-RANKL antibodies and antigen- bmding fragments thereof of the invention recognize human RANKL
  • the present invention includes antibody molecules which recognize RANKL from different species, e.g., mammals (e g , mouse, rat, rabbit, sheep or dog).
  • mammals e.g , mouse, rat, rabbit, sheep or dog.
  • the anti-RANKL antibodies and antigen-binding fragments thereof of the invention recognize RANKL from human and from mouse (e.g., preferential binding to mouse RANKL over human RANKL or approximately equally to both).
  • the present invention also includes anti-RANKL antibodies or fragments thereof or variants thereof (e g , as discussed herein) which are complexed with RANKL or any fragment thereof, or with any cell or cell membrane which is expressing RANKL or any portion or fragment thereof on the cell surface
  • anti-RANKL antibodies or fragments thereof or variants thereof e g , as discussed herein
  • Such complexes may be made by contacting the antibody or antibody fragment with RANKL or a RANKL fragment
  • Fully human monoclonal antibodies directed against RANKL are generated using transgenic mice carrying parts of the human immune system rather than the mouse system
  • transgenic mice which may be referred to, herein, as "HuMAb” mice, contain a human immunoglobulin gene mi ⁇ iloci that encodes unrearranged human heavy ( ⁇ and ⁇ ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and K chain loci (Lonberg, N., et al , (1994) Nature 368(6474) 856- 859).
  • mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human lgG ⁇ monoclonal antibodies (Lonberg, N , et at. , (1994), supra; reviewed in Lonberg, N.
  • HuMab mice (1995) Intern. Rev. Immunol. 13:65-93, and Harding, F., et al., (1995) Ann N. Y Acad Sa 764'536-546).
  • the preparation of HuMab mice is commonly known in the art and is described, for example, in Taylor, L., et al., (1992) Nucleic Acids Research 20:6287-6295; Chen, J , et al , (1993) International immunology 5 647-656, Tuaillo ⁇ , ⁇ t al. , (1993) Proc Natl.
  • HuMab mice can be immunized with an antigenic RANKL polypeptide as described by Lonberg, N , et a! , (1994) Nature 368(6474)- 856-859; Fishwild, D., ef al., (1996) Nature
  • Hyb ⁇ doma cells which produce the monoclonal, fully human anti-RANKL antibodies may be produced by methods which are commonly known in the art. These methods include, but are not limited to, the hyb ⁇ doma technique originally developed by Kohler, ef a/ , (1975) (Nature 256 495-497), as well as the trioma technique (He ⁇ ng, et al , (1988) Biomed. Biochim. Acta 47:211-216 and Hagiwara, et a/., (1993) Hum. Antibod.
  • Hyb ⁇ domas 4:15) the human B-cell hybridoma technique (Kozbor, et al , (1983) Immunology Today 4:72 and Cote, et al., (1983) Proc. Natl. Acad Sci U.S.A 80:2026-2030), and the EBV-hyb ⁇ doma technique (Cole, et a/., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985).
  • mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols The resulting hyb ⁇ domas may then be screened for the production of antigen-specific antibodies
  • Nucleic acids encoding an antibody or antigen-binding fragment of the invention, or an immunoglobulin chain or CDR thereof or any polypeptide of the present invention or RANKL or a soluble fragment thereof may be expressed at high levels in an £.eo///T7 expression system as disclosed in U.S. Patent Nos. 4,952,496, 5,693,489 and 5,869,320 and in Davanloo, P., ef a/., (1984) Proc. Natl. Acad. ScL USA 81 , 2035-2039; Studier, F. W., et aL , (1986) J. MoI Biol. 189" 113-130;
  • the present invention also includes a method tor the synthesis of an antibody or antigen-binding fragment thereof in a yeast diploid cell (e.g., Pichia, Pichia methanolica, Pichia angusta, Pichia pastoria, Saccharomyces cerevisiae), the method comprising : transforming a first yeast haploid cell with a first expression vector, said expression vector comprising a first immunoglobulin chain, operably linked to a first yeast promoter; transforming a second yeast haploid cell with a second expression vector, said expression vector comprising a second immunoglobulin chain, operably linked to a second yeast promoter; generating a diploid cell from said first and second yeast haploid cells, e.g., by mating; cult
  • the anti-RANKL antibodies and antigen-binding fragments thereof of the present invention may also be produced recombinantly (e g , in an E coli/T7 expression system as discussed above).
  • nucleic acids encoding the antibodies and antigen-binding fragments thereof of the invention e.g., V H or V L
  • V H or V L may be inserted into a pET-based plasm id and expressed in the E.coWll system.
  • the invention comprises a method comprising (a) preparing a DNA sequence encoding a heavy and/or light chain immunoglobulin variable region of XPA12.004, XPA12.020, XP A12.039, XPA12.041 or XPA12.042 or an antigen- binding fragment thereof; (b) inserting the sequence into a replicable expression vactor operably linked to a suitable promoter compatible with a host cell; (c) transforming the host cell with the vector of (b); (d) culturing the host cell; and (e) recovering the immunoglobulin chain(s) from the host cell culture. Transformation can be by any known method for introducing polynucleotides into a host cell.
  • Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfecti ⁇ n, calcium phosphate precipitation, poiybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleot ⁇ de(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei.
  • nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216; 4,912,040, 4,740,461 and 4,959 455.
  • Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and a number of other cell lines
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells.
  • Cell lines of particular utility can be selected by determining which cell lines have high expression levels
  • Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.
  • insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody or fragment in the host cells or, e.g., secretion of the antibody into the culture medium in which the host cells are grown.
  • antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other.
  • all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies. Accordingly, the scope of the present invention includes antibodies and antigen-binding fragments thereof that are aglycosylated, glycosylated or only partially glycosylated, e.g., lacking O-linked and/or N-linked glycosylation.
  • the term "monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al , (1975) Nature 256 495
  • a polyclonal antibody is an antibody which was produced among or in the presence of one or more other, non-identca! antibodies
  • polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B- lyrnphocytes which produced non-identical antibodies
  • polyclonal antibodies are obtained directly from an immunized animal
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites Bispecific antibodies can be produced by a variety of methods including fusion of hyb ⁇ domas or linking of Fab 1 fragments See e g , Songsivilai, et a/ , (1990) Clin Exp Immunol 79 315-321 , Kostelny, et al , (1992) J Immunol.
  • bispecific antibodies may be formed as "diabodies” (Holliger, ⁇ i al , (1993) PNAS USA 90 6444-6448) or as "Janusins ' (Traunecker, et al , (1991 ) EMBO J 10 3655-3659 and Traunecker, et af , (1992) lnt J Cancer Suppl. 7 51 52)
  • Fully human antibody refers to an antibody which comprises human immunoglobulin protein sequences only A fully human antibody may contain murine carbohydrate chains If produced in a mouse in a mouse cell or In a hybridoma derived from a mouse cell Similarly, “mouse antibody” refers to an antibody which comprises mouse immunoglobulin sequences only
  • Fully human antibodies possess certain therapeutic advantages over antibodies from mouse or other species in the treatment of humans When immunocompetent human subjects are administered a dose of non-human antibodies, the subjecis may produce antibodies against the non-human
  • HAMA human anti-mouse antibodies
  • HAMA human anti-mouse antibodies
  • HAMA may neutralize therapeutic antibodies and may induce acute toxicity (/ e , a HAMA response)
  • Use of fully human antibodies which lack any foreign (e g , mouse) ammo acid sequences averts the HAMA response.
  • the present invention also includes "chimeric antibodies"- an antibody which comprises a variable region of the present invention fused or chime ⁇ zed with an antibody region (e g , constant chain) from another non-human species (e g , mouse, horse, rabbit, dog, cow or chicken).
  • an antibody region e g , constant chain
  • another non-human species e g , mouse, horse, rabbit, dog, cow or chicken.
  • the present invention also includes "antigen-binding fragments ' An antigen- bi ⁇ ding portion may compete with the intact antibody for specific binding See generally, Fundamental Immunology. Ch. 7 (Paul, W., ed , 2nd ed. Raven Press N Y (1989)) (incorporated by reference in its entirety for all purposes) Antigen binding fragments may be produced by, for example, recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies
  • Single-chain Fv or “sFv” antibody fragments have the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain
  • the sFv polypeptide further comprises a polypeptide linker between the V H and Vt domains which enables the sFv to form the desired structure for antigen binding
  • Techniques described for the production of single chain antibodies can be adapted to produce anti- RANKL-sp ⁇ cific single chain antibodies
  • sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol 113, Rosenburg and Moore eds. Springer Verlag, N Y , pp. 269 315 (1994).
  • Disulfide stabilized Fv fragments' and “dsFv' refer to antibody molecules comprising a variable heavy chain (V H ) and a variable light chain (V L ) which are linked by a disulfide bridge
  • Antibody fragments within the scope of the present invention also include F(ab)i fragments which may be produced by enzymatic cleavage of an IgG by, for example, pepsin Fab fragments may be produced by, for example, papain digestion of an IgG antibody.
  • a Fab fragment is a V L chain appended to a V H chain by a disulfide bridge.
  • a F(ab) 2 fragment includes two Fab fragments and a portion of the hinge region which, in turn, are appended by four disulfide bridges
  • An Fv fragment is a VL or V H domain.
  • immunoglobulins can be assigned to different classes. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM and several of these may be further divided into subclasses (isotypes), e g lgG-1 , lgG-2, lgG-3 and lgG-4, IgA 1 and lgA-2. Each of theee types of anti-RANKL antibody or antigen-binding fragment thereof is within the scope of the present invention.
  • VHH domains that comprise one or more (e g , 3) CDRs of the present invention as well as humanized versions thereof (wher ⁇ in the framework regions between the CDRs have been replace with human framework sequences)
  • antigen-binding fragments may be referred to as nanobodies
  • the anti-RANKL antibody molecules of the invention may also be conjugated to a chemical moiety
  • the chemical moiety may be, inter alia a polymer, a radionuclide or a cytotoxic factor
  • the chemical moiety is a polymer which increases the half-life of the antibody molecule in the body of a subject
  • Suitable polymers include but are not limited to, polyethylene glycol (PEG) (e g , PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol (mPEG) Lee, et a/ , (1999) (Bioconj Chem 10 973-981) discloses PEG conjugated single-chain antibodies Wen, et a! , (2001 ) (Bioconj Chem 12.545-553) disclose conjugating antibodies with PEG which is attached to a
  • the antibodies and antigen-binding fragments thereof of the invention may also be conjugated with labels such as sa Tc, 90 Y, 111 In, 32 P, 14 C, 125 I 3 H, 131 I, 11 C 15 O, n N, 18 F, 35 S, 51 Cr, 67 To, 22 Va, 60 Co, 59 Fe, 67 Se, 152 Eu, 67 CU, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th 1 and 40 K, 157 Gd 55 Mn, 52 Tr and 56 Fe
  • labels such as sa Tc, 90 Y, 111 In, 32 P, 14 C, 125 I 3 H, 131 I, 11 C 15 O, n N, 18 F, 35 S, 51 Cr, 67 To, 22 Va, 60 Co, 59 Fe, 67 Se, 152 Eu, 67 CU, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th 1 and 40 K, 157 Gd 55
  • the antibodies and antigen-binding fragments thereof of the invention may also be conjugated with fluorescent or chemilluminescent labels including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives isothiocya ⁇ ate phyc ⁇ eryth ⁇ n, phycocyanin, allophycocyanin, o- phthaladehyde, fluorescamine, 152 Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label an aromatic acridinium ester label, an imidazole label, an ac ⁇ dimium salt label, an oxalate ester label, an aequo ⁇ n label, 2,3-d ⁇ hydrophthalaz ⁇ ned ⁇ ones, biotin/avidin, spin labels and stable free radicals
  • fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives isothioc
  • the antibodies and antigen-binding fragments thereof of the invention may be conjugated to a cytotoxic factor such as dipthe ⁇ a toxin, Pseudomonas aeruginosa exotoxin A chain , ⁇ cin A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleuntes fordii proteins and compounds (e.g , fatty acids), dianthin proteins, Phytolacca amencana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, rest ⁇ ctocin, phenomycin, and enomy ⁇ n Any method known in the art for conjugating the antibody molecules of the invention to the various moieties may be employed, including those methods described by Hunter, ⁇ t al.
  • K O fl refers to the off-rate constant for dissociation of the antibody from an antibody/antigen complex.
  • K 0n refers to the rate at which the antibody associates with the antigen .
  • K 0 refers to the dissociation constant of a particular antibody/antigen interaction.
  • K 0 K 0n ZK 0n .
  • the present invention provides a bispecific antibody or antigen-binding fragment thereof comprising a first antigen binding domain that specifically binds to a RANKL epitope; and, a second antigen binding domain that specifically binds to a distinct epitope.
  • the two epitopes bound by the bispecific antibody can both be on RANKL or one can be on RANKL and the other can be on another protein.
  • a bispecific antibody or antigen-binding fragment thereof of the invention comprises a CDR-H/CDR-L pair comprising 1 or more CDRs set forth herein (e g., 3 heavy and 3 light chain CDRs) (e.g , a set of CDRs: CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and CDR-L3 specific for one epitope; and, another set of CDRs' CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and CDR-L3 specific for a distinci epitope).
  • CDR-H/CDR-L pair comprising 1 or more CDRs set forth herein (e.g., 3 heavy and 3 light chain CDRs) (e.g , a set of CDRs: CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and CDR
  • the present invention encompasses bispecific antibodies and antigen-binding fragments thereof that include an anti-RANKL antibody or antigen-binding fragment thereof or 1 or more CDRs (e.g., 3 light chain CDRs or 3 heavy chain CDRs) of such anii-RANKL antibodies or fragments that are set forth herein.
  • CDRs e.g., 3 light chain CDRs or 3 heavy chain CDRs
  • Bispecific antibodies and antigen-binding fragments thereof comprise binding specificity for RANKL as well as any of several other antigens including, but not limited to: VEGF (vascular epidermal growth factor), HGF (hepatocyte growth factor), CD40, IL-10, TNF (tumor necrosis factor), IL17, APRIL (also called TRDL, TNFSF13 and TALL-2), IL-23 (e.g.
  • IL-23 receptor IL-23R or IL-12Rbeta1 subunit
  • IGF1 R insulin-like growth factor-1 receptor
  • MDL-1 IL-1 beta
  • IL-1 RAcP IL-1 Receptor Accessory Protein
  • IL-1 RA IL- 1 receptor antagonist
  • the present invention encompasses a bispecific antibody including an immunoglobulin that binds specifically to RANKL associated with CTLA4-lg (e g , abatacept)
  • any of the anti-RANKL antibodies or antigen-binding fragments (including bispecific antibodies and fragments) thereof are provided in association with a further chennotherapeutic agent which is an antibody or antigen-binding fragment thereof that specifically binds to an antigen selected from VEGF (vascular epidermal growth factor ⁇ , HGF (hepatocyte growth factor), CD40 IL- 10, TNF (tumor necrosis factor), IL17, APRIL (also called TRDL, TNFSF13 and TALL-2), IL-23 (e g p19 or p40 subunit) IL-23 receptor (IL-23R or IL-12Rbeta1 subunit), IGF1 R (insulin-like growth factor-1 receptor) MDL-1 IL-1 beta, IL-1 RAcP (IL-1 Receptor Accessory Protein), IL-1 receptor antagonist (IL-1 RA), PD-1
  • VEGF vascular epidermal growth factor ⁇ , HGF (hepatocyte growth factor), CD40 IL- 10, TNF (tumor
  • DEC205 programmed death-1
  • EGFR epidermal growth factor receptor
  • DEC205/EGFR epidermal growth factor receptor
  • e g comprising an immunoglobulin sequence set forth herein.
  • bispecific' antibodies and antigen-binding fragments thereof includes whole bispecific antibodies as well as antigen binding fragments such as bispecific diabody, ds diabody or dsFv-dsFv' or a bispecific F(ab') 2 , wherein at least one epitope recognized is RANKL
  • bispecific antibody refers to a full antibody wherein the V H and V L on one arm binds one epitope and the VH and V L on the other arm bind a distinct epitope.
  • the general shape and size of a bispecific antibody resembles that of a monospecific antibody.
  • a bispecific diabody refers a molecule comprising to two chains V H i-V LZ and
  • a bispecific diabody comprises two chains wherein each chain comprises a V H domain connected to a VL domain using a linker is too short to allow pairing between domains on the same chain, thus driving the pairing between complementary domains on different chains to recreate two separate antigen-binding sites
  • a bispecific ds diabody resembles a bispecific diabody except that a disulfide bridge tethers the VH-J ⁇ /LZ and V 1 ⁇ VH? chains together (e.g., disulfide bridge between V H i and V L1 )
  • a dsFv-dsFv' comprises a V H i-V L2 molecule wherein the variable regions are linked, e.g , by a peptide linker, and wherein the V m disulfide bridges and pairs with an independent Vu to form one binding site and wherein the V L2 disulfide bridges and pairs with a V H2 to form a distinct binding site.
  • a bispecific F(ab') 2 refers to a F(ab') 2 wherein the V H and V L on one arm binds one epitope and the V H and V L on the other arm bind a distinct epitope.
  • a bispecific antibody or antigen-binding fragment thereof comprises the immunoglobulin chain or a variable region thereof or one or more CDRs thereof of the anti-tumor necrosis factor alpha (TNF ⁇ ) antibody adalimumab, infliximab or g ⁇ limumab
  • TNF ⁇ anti-tumor necrosis factor alpha
  • the anti-TNFalpha portion of the bispecific antibody or antigen-binding fragment comprises a heavy or light chain immunoglobulin selected from the group below or an immunoglobulin chain comprising one or more CDRs thereof (e g , all 3 light or heavy chain CDRs):
  • the anti-IL-23R portion of the bispecific antibody or antigen-binding fragment comprises a heavy or light chain immunoglobulin selected from the group below or an immunoglobulin chain comprising one or more CDRs thereof (e g., all 3 light or heavy chain CDRs):
  • the ant ⁇ -IL-23 portion of the bispecific antibody or antigen-binding fragment comprises a 3 light or 3 heavy chain CDRs selected from:
  • a bispecific antibody or antigen-binding portion thereof comprises CTLA4-lg associated with an immunoglobulin that binds specifically to RANKL [e.g., as set forth herein) wherein the CTLA4-lg comprises the sequence set forth below or a fragment thereof:
  • the anti-IL-23 p19 portion of the bispecific antibody or antigen-binding fragment comprises a heavy or light chain
  • immunoglobulin selected from the group below or an immunoglobulin chain comprising one or more CDRs thereof (e.g , all 3 light or heavy chain CDRs):
  • Light chain :
  • the a ⁇ ti-IL23 p19 portion comprises the following CDRs:
  • the ant ⁇ -IL23 p19 binding portion comprises any of the immunoglobulins variable regions thereof or CDRs thereof which are set forth in any of U S palent nos 7 247 711 or 7 491 391 , published U S application no US 2007/0218064 or US 2008/0095775, or published PCT application no WO 2007/024846
  • the ant ⁇ -IL-17 portion of the b specific antibody or antigen-binding fragment comprises a heavy or light chain immunoglobulin selected from the group below or an immunoglobulin chain comprising one or more CDRs thereof (e g , all 3 light or heavy chain CDRs) Light chain
  • the ant ⁇ -IL-10 portion of the bispecific antibody or antigen-binding fragment comprises a heavy or light chain immunoglobulin selected from the group below or an immunoglobulin chain comprising one or more CDRs thereof (e g all 3 light or heavy chain CDRs)
  • the anti-EGFR portion of the bispecific antibody or antigen-binding fragment comprises a heavy or light chain immunoglobulin selected from the group below or an immunoglobulin chain comprising one or more CDRs thereof (e g , all 3 light or heavy chain CDRs)
  • a bispecific antibody or antigen-binding portion thereof comprises a heavy or light immunoglobulin chain or a variable region thereof or one or more CDRs thereof of an anti-hepatocyte growth factor antibody (e.g. , antibody a or b or c).
  • an anti-hepatocyte growth factor antibody e.g. , antibody a or b or c
  • the CDR sequences are as set forth below:
  • the anti-IGF1 R portion of the bispecific antibody or antigen-binding fragment comprises a mature heavy or light chain immunoglobulin ⁇ i.e., lacking signal sequence (dotted underscored text)) selected from the group below; or an immunoglobulin chain comprising one or m ⁇ re CDRs thereof (e.g., all 3 light or heavy chain CDRs):
  • an antibody or antigen-binding fragment of the invention can be incorporated into a pharmaceutical composition, along with a pharmaceutically acceptable carrier, suitable for administration to a subject in vivo.
  • a pharmaceutically acceptable carrier suitable for administration to a subject in vivo.
  • the scope of the present invention includes pharmaceutical compositions which may be administered to a subject by any route (e.g., oral, ocular, topical or pulmonary (inhalation)), for example, by a parenteral route such as intratumoral injection, intravenous injection, subcutaneous injection or intramuscular injection, in one embodiment of the invention, the pharmaceutical compositions of the invention comprise one or more of antibodies XPA12 004, XPA12 020, XPA12 039, XPA12 041 and XPA12 042 or antigen-binding fragments of any of said antibodies and a pharmaceutically acceptable carrier
  • compositions are conventional and very well known in the art Examples include aqueous and nonaqueous carriers, stabilizers antioxidants, solvents, dispersion media, coatings, antimicrobial agents, buffers serum proteins, isotonic and absorption delaying agents, and the like that are physiologically compatible
  • the carrier is suitable for injection into a subject s body
  • aqueou ⁇ and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof vegetable oils such as olive oil, and injectable organic esters, such as ethyl oleate
  • polyols such as glycerol, propylene glycol, polyethylene glycol and the like
  • suitable mixtures thereof vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate
  • Proper fluidity can be maintained for example by the use of coating materials such as lecithin by the maintenance of the required particle s ze in the case of dispersions, and by the use of surfactants
  • Stabilizers such as ⁇ ⁇ -trehalose dihydrate may be included for stabilizing the antibodies and antigen-binding fragments thereof of the invention from degrading effects of desiccation or freeze-drying
  • antioxidants examples include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite sodium sulfite and the like, and oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and metal chelating agents, such as citric acid ethylenediamine tetraacetic acid (EDTA), sorbitol tartaric acid, phosphoric acid, and the like.
  • EDTA citric acid ethylenediamine tetraacetic acid
  • sorbitol tartaric acid phosphoric acid
  • Suitable buffers which may be included in the pharmaceutical compositions of the invention include L-histidine based buffers, phosphate based buffers ( ⁇ g , phosphate buffered saline, pH ⁇ 7), sorbate based buffers or glycine-based buffers
  • Serum proteins which may be included in the pharmaceutical compositions of the invention may include human serum albumin
  • Isotonic agents such as sugars ethanol, polyalcohols (e g , glycerol, propylene glycol, liquid polyethylene glycol man ⁇ itol or sorbitol) sodium citrate or sodium chloride (e.g., buffered saline) may also be included in the pharmaceutical compositions of the invention.
  • polyalcohols e.g , glycerol, propylene glycol, liquid polyethylene glycol man ⁇ itol or sorbitol
  • sodium citrate or sodium chloride e.g., buffered saline
  • Prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and/or gelatin
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions
  • Sterile injectable solutions can be prepared by incorporating the antibody or antigen-binding fragment of the invention in the required amount in an appropnat ⁇ solvent, optionally with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration
  • dispersions are prepared by incorporating the aniibody or antigen-binding fragment thereof into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above
  • methods of preparation include vacuum drying and freeze-dr/ing (lyophilization) that yield a powder of the active ingredient plus any additional, desired ingredient from a previously sterile-filtered solution thereof Further chetnotherapeutic agents and therapeutic procedures
  • the pharmaceutical composition can be administered by combination therapy.
  • the present invention includes, within its scope, compositions or combinations (e g., kits) comprising an anti-RANKL antibody or antigen-binding fragment thereof in association with one or more further chernotherapeutic agents.
  • Methods for combination therapies are also within the scope of the present invention.
  • a combination therapy includes, in an embodiment of the invention, administration of an anti-RANKL antibody or antigen-binding fragment thereof in association with one or more further chernotherapeutic agents Further
  • the combination therapy includes a pharmaceutical composition of the present invention in association with another RANKL inhibitor, e g , OPG, an OPG derivative, or denosumab (Bekker et al . J Bone Miner Res 15:348-360 (2001 )) or any other antagonist anti-RANKL antibody or antigen-binding fragment thereof
  • another RANKL inhibitor e g , OPG, an OPG derivative, or denosumab
  • Bone condition treating agents are known in the art, and include, but are not limit ⁇ d to, anti-r ⁇ orptiv ⁇ drugs, bone-forming agents, osteoclast stimulators and estrogen receptor antagonists.
  • a "bone condition treating agent” is a substance which, when administered to a subject, treats or prevents a bone disorder, e g , an osteopenic disorder such as osteoporosis, in the subject's body.
  • bone condition treating agents may include bisphosphonates (e g , alendronate, risedronate, ibandro ⁇ ate and zoledronate), estrogen or estrogQn analogues, selective estrogen receptor modulators (SERM; e g., tamoxifen), dietery calcium and other supplements (e.g., strontium, magnesium, vitamin K, boron silicon, zinc, copper and ascorbic acid (vitamin C)), tibolone, calcitonin, calcitriol, parathyroid hormone (PTH) or a peptide fragment thereof, PTH-related protein (PTHrp), bone
  • bisphosphonates e g , alendronate, risedronate, ibandro ⁇ ate and zoledronate
  • SERM selective estrogen receptor modulators
  • dietery calcium and other supplements e.g., strontium, magnesium, vitamin K, boron silicon, zinc, copper and ascorbic acid (vitamin C)
  • tibolone calcitonin
  • statins e g , simvastatin, rosuvastatin, lovastatn atorvastatin
  • vitamin D or a derivative or mimic thereof
  • raloxifene raloxifene
  • raloxifene raloxifene
  • lasofoxifene raloxifene
  • Anti-inflammatory agents are useful for the treatment of rheumatoid arthritis and other inflammatory disorders
  • An anti-inflammatory therapeutic agent is a substance which, when administered to a subject, treats or prevents an inflammatory response
  • Such agents include, but are not limited to, steroidal anti-inflammatory compounds, non-steroidal anti-inflammatory compounds, and anti-inflammatory compounds isolated from natural sources.
  • Steroidal anti-inflammatory compounds include, but are not limited to, 21 acetoxypregnenolone, alclometasone, algesto ⁇ e, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolo ⁇ e, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort flucloronide, flumethasone, flu ⁇ isolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolo ⁇ e, fluorometholone, fluperolone acetate, fluprednidene acetate, flu
  • paramethasone prednicarbate, prednisolone, prednisolone 25-d ⁇ ethylam ⁇ no-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, and triamcinolone hexacetonide.
  • Non-steroidal anti-inflammatory compounds include, but are not limited to, arninoarylcarboxylic acid derivatives (e.g , enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g.
  • aceclofenac acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etod ⁇ lac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, progiumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbuty ⁇ c acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorol
  • Anti-inflammatory compounds isolated from natural sources include, but are not limited to, candelilla wax, alpha bisabolol, aloe vera, Manjistha, Guggal kola extract, chamomile, sea whip extract, glycyrrhetic acid, glycyrrhizic acid, oil soluble licorice extract, monoammonium glycyrrhizinate, monopotassium glycyrrhizinate, dipotassium glycyrrhizinate, 1-beta-glycyrrhet ⁇ c ac ⁇ d, stearyl glycyrrhetinate, and 3- stearyloxy-glycyrrhetmic acid, and disodium 3-suc ⁇ nyloxy-beta-glycyrrhet ⁇ nate
  • Immunosuppressive agents include, but are not limited to, interferon, ⁇ nterleuk ⁇ n-2, cyclosporin A, diftitox, demleuki ⁇ , levamisole, azathiopnne, brequmar guspe ⁇ mus, 6-mercaptopu ⁇ ne, mizo ⁇ bine, rapamycin tacrolimus (FK-506), folic acid analogs (e g , denopte ⁇ n, edatrexate, methotrexate, pi ⁇ trexirn, pteropterin, Tomudex, and trimetrexate), purine analogs (e g , cladribine, fludarabine 6-mercaptopur ⁇ ne, thiamip ⁇ ne and thiaguanine), pyrimidine analogs (e g , ancitabine, azacitidine, 6- azaundine, carmofur, cytarabine, doxifluridine, emitefur,
  • Antibody therapies which may be administered in conjunction with the antibodies or antigen-binding fragments of the invention include denosumab (Bekker ⁇ t at , J Bone Miner Res 16.348-360 (2001)), trastuzumab (e g , herceptin) (see, for example, Sliwkowski, ef a/ , (1999) Sem ⁇ n. Oncol. 26(4 Suppl 12) 60-70), vitaxin and rituximab
  • the compositons of the invention may also be administered in association with one or more anti-osteopenic immunosuppressive antiinflammatory or anti cancer therapeutic procedures
  • An anti osteopenic therapeutic procedure' is a process that is performed on a subject which treats or reduces the incidence or symptoms of osteopenia in the subject e g , applying low level high frequency stress to bone
  • An 'anti-cancer therapeutic procedure is a process that is performed on a subject which treats or reduces the incidence of cancer in the subject e g , radiation therapy or surgical tumorectomy
  • compositions of the invention e g , anti-RANKL antibody or antigen-binding fragment thereof along with denosumab
  • components of a composition of the invention can be formulated into a single composition for simultaneous delivery or formulated separately into two or more compositions (e g a kit)
  • each component can be administered to a subject at a different time than when the other component is administered for example, each administration may be given non simultaneously (e g separately or sequentially) at several intervals over a given period of time Moreover, the separate components may be administered to a subj ⁇ ct by the same or by a different route (e g , wherein an anti- RANKL antibody is administered parenterally and rofecoxib is administered orally)
  • an anti-RANKL antibody or antigen-binding fragment thereof of the invention is administered to a subject at a 'therapeutically effective dosage" which, e g inhibits a disease or condition which is mediated by RANKL (e g osteoporosis or rheumatoid arthritis) to any detectable degree
  • a therapeutically effective dosage is in an embod ment of the invention, a dosage which reduces, to any detectable degree an immune or inflammatory response or any symptom thereof in an individual suffering from or susceptible to a condition characterized by immune or inflammatory responses or which reduces, to any detectable degree, the rate or extent of bone loss in an individual suffering from or susceptible to a condition characterized by bone loss or any symptom thereof
  • a therapeut cally effective dosage of an antibody or antigen-binding fragment thereof is, in an embodiment of the invention, a dosage which reduces joint inflammation stiffness or pain or which increases joint mobil ty in a subject with rheumatoid arthritis
  • therapeutically effective dosage of an antibody or antigen-binding fragment thereof is a dosage which r ⁇ ducQS the rate or likelihood of bone fracture (e.g., of the pelvis, lumbar spine, hip trochanter, femoral neck or distal radius); which increases or slows any decreases in bone mineral density (BMD), bone strength, bone mass or bone quality (as characterized, e.g., by bone architecture, bone turnover, bone damage accumulation (e.g., microfractures) and bone mineralization); or which slows osteoclast maturation; in a subject with osteoporosis or any osteopenic disorder or rheumatoid arthritis characterized by joint destruction which is mediated by bone loss (e g., RANKL mediated bone loss).
  • an agent e.g., a further chennotherapeutic agent (e g , as discussed herein) is, when possible, done according to the schedule listed in the product information sheet of the approved agents, in the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November
  • a "therapeutically effective dosage" of any anti-RANKL antibody or antigen-binding fragment thereof of the invention is between about 0.01 mg/kg and about 50 mg/kg, dosed once, or repeatedly, at a schedule of weekly, biweekly, monthly quarterly, half-yearly or yearly.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies or the particular circumstances or requirements of the therapeutic situation. For example, dosage may be determined or adjusted, by a practitioner of ordinary skill in the art (e.g., physician or veterinarian) according to the patient's age, weight, height, past medical history, present medications and the potential for cross-reaction, allergies, sensitivities and adverse side-effects.
  • a practitioner of ordinary skill in the art e.g., physician or veterinarian
  • the effectiveness of a given dose or treatment regimen of an antibody or antigen-binding fragment thereof or a combination thereof of the invention, in connection with treatment of osteoporosis or any osteopenic disorder can be determined, for example, by dual X-ray absorptiometry (DXA) to measure bone mineral density (density of minerals, such as calcium, in bones), peripheral dual- ⁇ nergy X-ray absorptiometry (P-DEXA) to measure bone mineral density, dual photon absorptiometry (DPA) to measure bone mineral density, e.g., of the spine or hips, ultrasound analysis, quantitative computed tomography (QCT) to measure bone mineral density, e.g., of the vertebra, and blood tests for osteoporosis markers
  • Blood markers of osteoporosis include elevated bone-specific alkaline phosphatase (bone ALP or BALP, e g , elevated 3X or more), elevated osteocalcin, elevated urinary N- telo
  • the effectiveness of a given dose or treatment regimen of an antibody or antigen-binding fragment thereof or a combination thereof of the invention, in connection with treatment of an inflammatory disorder, such as rheumatoid arthritis (RA), can be determined, for example, by patient interview to detect joint stiffness or lack of mobility, radiological analysis of affected joints (e g., to detect erosion), joint fluid level evaluation (e g., to detect fluid appearance, clotting properties and white blood cell content), u ⁇ nanaly ⁇ is ⁇ e.g., to detect hematuria or proteinuria), blood test ⁇ to measure C-reactive protein, blood tests to measure erythrocyte sedimentation rate, hernoglobin/hematocrit levels, liver function, platelet levels, rheumatoid factor, white blood cell count, anticyclic citrullinated peptide antibody, antinuclear antibody, complement levels and immunoglobulin levels. Based on the results of these tests, a physician or clinician can adjust dosage of an antibody or antigon-binding
  • compositions comprising the antibodies or antigen-binding fragments of the invention may be used for treating or preventing any disease or condition in a subject which is mediated by elevated expression or activity of RANKL or by elevated expression or activity of RANK or decreased expression or activity of osteoprotegerin (OPG; e.g., RANKL binding), and which may be treated or prevented, for example by inhibition of said RANKL activity or RANK activity or an increase of OPG activity.
  • OPG osteoprotegerin
  • the antibodies or antigen-binding fragments of the invention and pharmaceutical compositions comprising the antibodies or antigen-binding fragments of the invention may also be used for treating or preventing any disease or condition in a subject which is mediated by an imbalance of bone formation and bone resorption such that bone resorption and/or osteoclastogene ⁇ is is favored
  • the disease or condition is treated or prevented by decreasing RANKL receptor binding, activity or expression.
  • the disease or condition may, in an embodiment of the invention, be an immune or inflammatory condition, including, but not limited to, rheumatoid arthritis, psoriasis, insulin dependent diabetes, inflammatory bowel disease, multiple sclerosis, and toxic and septic shock, and graft versus host disease.
  • an immune or inflammatory condition including, but not limited to, rheumatoid arthritis, psoriasis, insulin dependent diabetes, inflammatory bowel disease, multiple sclerosis, and toxic and septic shock, and graft versus host disease.
  • the disease or condition may, in an embodiment of the invention, be a condition characterized by bone loss, including, but not limited to, osteopenic disorders, osteoporosis, osteomyelitis, osteonecrosis, Paget's disease,
  • the pharmaceutical composition of the invention may be used to treat, e g., multiple myeloma, melanoma, neuroblastoma, and breast, lung, prostale, thyroid, hematologic, head and neck, and renal cancer
  • Osteopenic disorders are disorders characterized by lower than normal bone mineral density (BMD). Osteopenia can be a pre-cursor to osteoporosis.
  • BMD bone mineral density
  • Osteopenia can be a pre-cursor to osteoporosis.
  • DXA dual-energy X-ray absorptiometry
  • Normal A value of BMD within 1 standard deviation of the young adult reference mean (T-score ⁇ -1 );
  • Low bone mass (osteopenia) A value of BMD more than 1 standard deviation below the young adult mean, but less than 2 standard deviations below this value (T-score ⁇ 1 and > -2 5)
  • Osteoporosis' A value of BMD 2.5 standard deviations or more below the young adult mean (T-score ⁇ -2 5); Severe osteoporosis (established osteoporosis) A value of BMD 2 5 standard deviations or more below the young adult mean in the presence of one or more fragility fractures (see Prevention and Management
  • bone mineral is measured, in an embodiment of the invention, at appendicular sites, such as the heel or wrist SXA is widely available for forearm mineral measurements, and is more precise than singlephoton absorptiometry (SPA), which also has the disadvantage of requiring the use of isotopes such as 125 I Use of SPA, however, to determine BMD is within the scope of the present invention
  • Dual-energy absorptiometry measures bone mineral at sites such as For example, the spine and hip; it can also measure total body bone mineral SPA and SXA typically cannot be used for these sites. DXA can also be used for measurements at appendicular sites
  • bone cancer e g , osteosarcoma, Ewing's sarcoma chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma or chordoma
  • bone metastases e g , osteosarcoma, Ewing's sarcoma chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma or chordoma
  • Subjects who have a disease characterized by an inflammatory response or bone loss can be identified by those having ordinary skill in the art by well known diagnostic means and criteria Individuals who are susceptible to certain conditions characterized by an immune or inflammatory response or by bone loss may be identified by those having ordinary skill in the art based upon family medical history and/or the presence of genetic markers or genes associated with said conditions. Diagnostic techniques are discussed above in the context of therapy monitoring and dosage adjustment
  • the present invention includes methods for reducing the incidence or probability of bone fracture or breakage, and/or increasing or stabilizing bone mineral density (BMD, e g , of the pelvis, lumbar spine, hip trochanter, femoral neck or distal radius) and/or bone strength, and/or bone mass, and/or bone quality , ⁇ n a subject, by administering to the subject a therapeutically effective amount of an anti- RANKL antibody or antigen-binding fragment thereof of the invention
  • a ' subject is any mammal, e g , rat mouse, dog, cat, rabbit for example, a human.
  • a subject is any mammal possessing a RANKL gene
  • the antibodies and antigen-binding fragments of the invention and pharmaceutical compositions thereof are administered by an invasive route such as by injection Administration by a no ⁇ -mvasive route is also within the scope of the present invention.
  • compositions can be administered with medical devices known in the art
  • a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle
  • the present invention includes any medical device such as an injection device, e.g a hypodermic needle and syringe comprising an anti-RANKL antibody or antigen- bmding fragment thereof of the invention or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier
  • the present invention also includes a container such as a vial (e.g. glass or plastic vial) comprising an anti- RANKL antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof comprising a pharmaceut cally acceptable carrier
  • Examples of well-known implants and modules useful in the present invention include.
  • U S Patent No 4,447,224 which discloses a variable flow implantable infusion apparatus for continuous drug delivery;
  • U S. Patent. No. 4,439,196 which discloses an osmotic drug delivery system having multi-charnber compartments.
  • Many other such implants, delivery systems, and modules are well known to those skilled in the art.
  • anti-RANKL antibodies and antigen-binding fragments thereof of the invention may be used in various experimental or diagnostic assays.
  • the anti-RANKL antibodies or antigen-binding fragments thereof may be used to detect RANKL in a biological sample in vitro or in vivo (see, for example, Zola, Monoclonal Antibodies A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987))
  • the anti-RANKL antibodies or fragments may be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or irnmunoprecipitation.
  • the invention provides a method for detecting RANKL or an antigenic fragment thereof in the biological sample comprising contacting the biological sample with an anti-RANKL antibody or antigen-binding fragment thereof of the invention and detecting the anti-RANKL antibody or antigen-binding fragment thereof bound to RANKL or an antigenic fragment thereof, thereby indicating the presence of the RANKL or an antigenic fragment thereof in the biological sample.
  • the anti-RANKL antibody or antigen-binding fragment thereof is directly labeled with a detectable label and may be detected directly.
  • the anti-RANKL antibody or antigen-binding fragment thereof (the first/primary antibody) is unlabeled and a secondary antibody or other molecule that can bind the anti-RANKL antibody or fragment is labeled.
  • a secondary antibody is chosen that is able to specifically bind the specific species and class of the primary antibody.
  • the primary anti-RANKL antibody or antigen-binding fragment thereof is a human IgG
  • the secondary antibody should be an anti-human-lgG antibody.
  • the presence of an anti-RANKL/RANKL complex in the biological sample can be detected by detecting the presence of the labeled secondary antibody.
  • the bound secondary antibody can be developed and detected based on, e g , chemilluminescence of the label (e g , by autoradiography or scintillation counting)
  • Labels for the anti-RANKL antibody or antigen-binding fragment thereof or the secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials
  • Examples of such enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ - galactosidase, or acetylcholinesterase, examples of prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of fluorescent materials include urnbelliferone, fluorescein, fluorescein isoihiocyanate, rhodamine,
  • RANKL or an antigenic fragment thereof can be assayed in a biological sample by a competition immunoassay utilizing RANKL standards labeled with a detectable substance and an unlabeled anti-RANKL antibody or antigen-binding fragment thereof.
  • the biological sample the labeled RANKL standards and the anti-RANKL antibody or fragment are combined and the amount of labeled RANKL standard bound to the unlabeled antibody or antigen-binding fragment thereof is determined
  • the amount of RANKL or antigenic fragment thereof in the biological sample is inversely proportional to the amount of labeled RANKL standard bound to the anti-RANKL antibody or antigen-binding fragment thereof
  • the anti-RANKL antibodies or antigen binding fragments thereof may be used to detect RANKL or antigenic fragments thereof in cells in cell culture or in a sample.
  • the antibodies and antigen-binding fragments of the present invention may be used in vivo to locate tissues and organs that express RANKL.
  • the fully human anti-RANKL antibodies and antigen-binding fragments thereof of the present invention will generate less of an immune response upon administration than non-human antibodies
  • the method comprises the steps of administering a detectably labeled anti-RANKL antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof to a patient in need of such a diagnostic test and subjecting the patient to imaging analysis to determine the location of the antibody or fragment bound-RANKL-expressing tissues.
  • Imaging analysis is well known in the medical art, and includes, without limitation, X-ray analysis, magnetic resonance imaging (MRI) or computed tomography (CT).
  • a biopsy is obtained from the patient to determine whether a tissue of interest expresses RANKL or an antigenic fragment thereof rather than subjecting the patient to imaging analysis.
  • the anti- RANKL antibodies or antigen-binding fragments thereof are labeled with a detectable agent that can be imaged in a patient.
  • the antibody or antigen-binding fragment thereof may be labeled with a contrast agent, such as barium, which can be used for X-ray analysis, or a magnetic contrast agent, such as a gadolinium chelate, which can be used for MRI or CE.
  • Other labeling agents include, without limitation, radioisotopes, such as 99 Tc; or other labels discussed herein.
  • the binding affinities of anti-RANKL antibodies XPA.12.004, XPA.12.020, XPA.12 039, XPA.12.041 and XPA.12.042 against human and murine RANKL were determined using a Proteon XPR36 (Bio-Rad) instrument.
  • a GLM chip Bio-Rad was prepared with a high density of Protein A/G using a standard amine coupling method.
  • the anti-RANKL antibodies were diluted into the running buffer
  • the antigen human or murine RANKL
  • the antigen was then diluted into running buffer to produce a 5 point serial dilution of antigen and a blank control
  • These samples were then injected over the captured antibody. Association of the antigen to the antibodies and dissociation of the antigen from the antibodies was monitored. The cycles were repeated two to three times for each antibody/antigen pairing This data was double referenced against the empty reference spots and the zero concentration flow cell and then fit to a 1 1 interaction model using the Proteon software (Bio-Rad).
  • TRAP thyroid-resistant acid phosphatase
  • RAW264 7 mouse macrophage-like cells were cultured in DMEM growth medium with 10% FBS
  • Non-tissue culture-treated tissue culture flasks were used to improve ease of detachment and viability of detached cells.
  • the cells were harvested by rinsing once with 1X PBS, then incubating with non-enzymatic dissociation buffer for 15-20 minutes at 37°C
  • the harvested cells were transferred to a fresh 50 mL conical tube and counted.
  • Ceil density was adjusted to 10 s cells/ml Dy dilution into pre-warm ⁇ d growth medium.
  • Recombinant murine or human RANKL (constant final concentration of 50 or 100 ng/ml) and anti-RANKL antibodies (final concentrations ranging from 6.1 pM to 100 ⁇ M) were diluted to 4X final concentration in pre-warmed growth medium and combined in an assay plate by addition of 25 ⁇ l of each to appropriate wells in a 96-well tissue culture dish 50 ⁇ l of cell suspension were immediately added to all assay wells, resulting in a final cell density of approximately 5000 cells/well The assay plate was placed in 37°C incubator with 5% CO 2 for 4 days.
  • TRAP assay buffer 59 mM sodium citrate, 41 mM citric acid, 0.1% Triton X-100, pH 5.2
  • 100 ⁇ l of TRAP assay buffer containing 1 mg/ml p-n ⁇ trophenolphos ⁇ hate (pNPP) and 10% (33 5 mM) tartrate salt were added to each assay well.
  • 50 ⁇ l of 05 M NaOH were added to each well to develop the substrate color and stop the reaction.
  • Sample absorbances were read at 405 nm.
  • Ba/F3 assay employs a Ba/F3 cell line stably expressing a RANK-Fas fusion protein Signaling by human or murine RANKL through this receptor fusion leads to cell death mediated by the Fas signaling pathway
  • Cells were cultured overnight at 37°C and 5% CO 2 with a constant concentration of either human or murine recombinant RANKL (100 ng/ml) and varying concentrations of anti RANKL ant bodies (6 1 pM to 100 ⁇ M) Subsequently cells were incubated with Alamar Blue, a redox sensitive dye For determining cell viability for 6 hours at 37°C and 5% CO 2 Absorbance was read at 570 nm and 600 nm

Abstract

La présente invention porte sur des anticorps contre l'activateur de récepteur humain du ligand NF-B (RANKL). Les anticorps sont utiles par exemple pour traiter ou prévenir des troubles osseux et des états immuns/inflammatoires dans un sujet. L'invention porte également sur des procédés d'utilisation et de production des anticorps de l'invention.
PCT/US2010/044206 2009-08-07 2010-08-03 Anticorps anti-rankl humain WO2011017294A1 (fr)

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WO2013176469A1 (fr) * 2012-05-21 2013-11-28 이화여자대학교 산학협력단 Anticorps capable de supprimer la différenciation des ostéoclastes
WO2014159430A1 (fr) * 2013-03-14 2014-10-02 Apexigen, Inc. Anticorps anti-rankl et leurs procédés d'utilisation
WO2015030701A1 (fr) * 2013-08-24 2015-03-05 R-Pharm Overseas, Inc. Anticorps entièrement humains contre le rankl humain
WO2015033144A1 (fr) * 2013-09-05 2015-03-12 Queen Mary University Of London Anticorps bispécifique contre le tnf-alpha et le système vasculaire synovial de patients souffrant d'arthrite
WO2014210545A3 (fr) * 2013-06-27 2015-06-25 The University Of Chicago Compositions et procédés associés à des anticorps recombinants dirigés contre des modifications d'histone post-traductionnelles
EP2878670A4 (fr) * 2012-06-08 2016-03-02 Univ Kinki Anticorps dirigé contre un transporteur et son utilisation
EP2914292A4 (fr) * 2012-10-30 2016-05-04 Univ Emory Stimulation de la formation osseuse par inhibition de la costimulation de cd28
EP3091030A4 (fr) * 2013-12-31 2017-11-29 Genor Biopharma Co., Ltd Anticorps anti-rankl humain, anticorps humanisé, leurs compositions pharmaceutiques et leurs utilisations
CN108064249A (zh) * 2014-11-05 2018-05-22 伊莱利利公司 抗tnf/抗il-23双特异性抗体
WO2018223182A1 (fr) * 2017-06-05 2018-12-13 The Council Of The Queensland Institute Of Medical Research Combinaison de, ou molécule de liaison bispécifique à, un antagoniste de molécule de point de contrôle immunitaire et d'un antagoniste rank l (ligand nf-kb) pour la thérapie ou la prophylaxie du cancer et utilisations correspondantes
CN109072303A (zh) * 2016-02-05 2018-12-21 詹森生物科技公司 用于治疗或预防1型糖尿病的抗tnf抗体、组合物、方法和用途
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WO2020183270A1 (fr) * 2019-03-14 2020-09-17 Janssen Biotech, Inc. Procédés de production de compositions d'anticorps anti-tnf
WO2020183269A1 (fr) * 2019-03-14 2020-09-17 Janssen Biotech, Inc. Procédés de fabrication pour la production de compositions d'anticorps anti-tnf
EP3576782A4 (fr) * 2017-02-02 2020-12-30 Silverback Therapeutics, Inc. Compositions de produit de recombinaison-peptide et leurs méthodes d'utilisation
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