[go: up one dir, main page]

WO2011017294A1 - Human anti-rankl antibodies - Google Patents

Human anti-rankl antibodies Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
rankl
cdr
fragment
seq
Prior art date
Application number
PCT/US2010/044206
Other languages
French (fr)
Inventor
Diane Hollenbaugh
Raphael Levy
Original Assignee
Schering Corporation
Xoma Technology Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Schering Corporation, Xoma Technology Ltd. filed Critical Schering Corporation
Publication of WO2011017294A1 publication Critical patent/WO2011017294A1/en

Links

Classifications

    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention antibodies against human receptor activator of NF- B ligand (RANKL). The antibodies are useful, for example, for treating or preventing bone disorders and immune/inflammatory conditions in a subject. Also included are methods of using and producing the antibodies of the invention.

Description

HUMAN ANTI-RANKL ANTIBODIES
This application claims the benefit of LJ S provisional patent application no 61/232,221 , filed August 7, 2009, which is incorporated by reference in its entirety
FIELD OF THE INVENTION
The present invention relates to fully human monoclonal anti-receptor activator of NF-κB 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
In addition rheumatoid arthritis (RA) is an inflammatory form of arthritis that causes sometimes debilitating joint pain and damage Rheumatoid arthritis attacks the lining of your joints (synovium) caus ng swelling that can result in aching and throbbing and eventually deformity
Receptor activator of nuclear factor-κB (RANK) 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 By binding to its receptor RANK on osteoclastic precursors 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 The discovery of the RANK/RANKL/OPG pathway and its implications in the pathogenesis of osteoporosis provides a molecular target for new therapies to improve bone health
In inflamed joints, RANKL is expressed by synovial cells and secreted by activated T-cells These sources of RANKL appear to be responsible, at least in part, for mediating the joint destruction in patients with rheumatoid arthritis 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 ιnterleukιn-1
SUMMARY OF THE INVENTION
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
immune/inflammatory conditions)
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 (ι) Ratio, of K0 for human RANKL binding to K0 for mouse RANKL binding of about 1/1 to about 10/1 ; (ιι) Ratio of IC50, in a TRAP assay for osteoclastogenesis, induced by human RANKL to IC50, in a
TRAP assay for osteoclastogenesis, induced by mouse RANKL, of about 0 19 nM to about 1 25 nM, (in) Ratio, of IC50 in an RANK receptor inhibition assay, wherein signaling is induced by human RANKL to IC50 in an RANK recaptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 0 25 nM to about 3 3 nM (ιv) 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 (v) 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 95 to about 1 1 The present invention slso 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- (i) K0 for binding to human RANKL of about 0.4 nM, (iι) KD for binding to mouse RANKL of about 0 2 nM; (in) IC50 in a TRAP assay for osteoclastogenesis, induced by human RANKL of about 0 3 πM, (ιv) IC50, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL of about 0.6 nM, (v) maximum inhibition, in a TRAP assay for osteoclastogenesis, induced by human RANKL of about 97% (vi) maximum inhibition, in a TRAP assay, for osteoclastogenesis induced by mouse RANKL of about 100%, (VII) maximum inhibition in a RANK receptor inhibition assay, wherein signaling is induced by human RANKL of about 99%, and (vin) maximum inhibition, in a RANK receptor inhib t on assay, wherein signaling is induced by mouse RANKL of about 98% The present invention provides an isolated antibody or antigen-binding fragment thereof comprising one or more members selected from the group consisting of (a) CDR-H1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12 004, (b) CDR-H1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12 020, (c) CDR-H1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12 039 (d) CDR-H1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12 041 (e) CDR-H1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12 042 (f) CDR-L1 CDR-L2 and CDR-L3 of the variable region of antibody XPA12 004, (g) CDR-L1 , CDR-L2 and CDR-L3 of the variable region of antibody XPA12 020, (h) CDR-L1 , CDR-L2 and CDR-L3 of the variable region of antibody XPA12 039, (ι) CDR L1 , CDR L2 and CDR L3 of the variable region of antibody XPA12 041 , and (j) CDR-L1 , CDR-L2 and CDR-L3 of the variable region of antibody XPA12 042 For example, in an embodiment of the invention, any such antibody or fragment is a monoclonal antibody, a polyclonal antibody, a labeled antibody, a bivalent antibody, a bispecific antibody, a chimeric ant body, a humanized antibody a recombinant antibody, an anti-idiotypic antibody, a carnelized 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. In an embodiment of the invention, the antibody or fragment is linked to a constant chain (e g , a K light chain λ light chain, γ1 heavy chain, γ2 heavy chain γ3 heavy chain or γ4 heavy chain) The present invention also provides a
pharmaceutical 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. In addition, 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 004 CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO 70, (b) XPA12 020 CDR-H 1 comprising the am no acid sequence set forth in SEQ ID NO 63 XPA12 020 CDR-H2 comprising the ammo acid sequence set forth in SEQ ID NO 67 and XPA12 020 CDR-H3 comprising the am no acid sequence set forth in SEQ ID NO 71 , (c) XPA12 039 CDR H1 compnsing the amino acid sequence set forth in SEQ ID NO 64 XPA12 039 CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO 68 and XPA12 039 CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO 72 (d) XPA12 041 CDR H1 comprising the amino acid sequence set forth in SEQ ID NO 65 XPA12 041 CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO 67 and XPA12 041 CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO 73, (e) XPA12 042 CDR-H 1 comprising the ammo acid sequence set forth in SEQ ID NO 65 XPA12 042 CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO 69 and XPA12 042 CDR H3 comprising the amino acid sequence set forth in SEQ ID NO 74 (f) XPA12 004 CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO 35 XPA12 004 CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO 39 and XPA12 004 CDR-L3 compr sing the amino acid sequence set forth in SEQ ID NO 44, (g) XPA12 020 CDR L1 comprising the amino acid sequence set forth in SEQ ID NO 36, XPA12 020 CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO 40 and XPA12 020 CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO 45, (h) XPA12 039 CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO 36, XPA12 039 CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO 41 and XPA12 039 CDR L3 comprising the amino ac d sequence set forth in SEQ ID NO 46 (i) XPA12 041 CDR-L1 comprising the ammo acid sequence set forth in SEQ ID NO 37 XPA12 041 CDR L2 comprising the amino acid sequence set forth in SEQ ID NO 42 and XPA12 041 CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO 47, and (j) XPA12 042 CDR-L1 comprising the ammo acid sequence set forth in SEQ ID NO 38, XPA12 042 CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 43 and XPA12 042 CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO 48 In an embodiment of the mt/ention the antibody or fragment is linked to a constant chain (e.g., a K light chain.a λ light chain γ1 heavy chain, γ2 heavy chain γ3 heavy chain or γ4 heavy chain) The present invention further comprises a pharmaceutical composition comprising the antibody or fragment and a pharmaceutically acceptable carrier The present invention also provides a composition comprising the antibody or fragment of the invention in association with one or more further chemotherapeutic agents selected from the group consisting of an anti-inflammatory agent, an immunosuppressive agent, an anti-cancer agent and a bone condition treating agent The present invention also provides an antibody or fragment of the invention wh en is bound to RANKL or a fragment thereof (ι e a complex)
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 SEQ ID NO 17, (c) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 18, (d) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 19, (e) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 20, (f) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 6, (g) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 7 (h) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO B, (ι) a mature light mmunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 9 and (j) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 10 In an embodiment of the nvention, the antibody or fragment is linked to a constant chain (e g a K light chain y1 heavy chain v2 heavy chain, /3 heavy chain or γ4 heavy chain) The present nvention also provides a pharmaceutical composition compr siπg the antibody or fragment and a pharmaceutically acceptable carrier In an embodiment the invention comprises a composition comprising the antibody or fragment in association with one or more further chemotherapeutic agents selected from the group consisting of an anti-inflammatory agent, an immunosuppressive agent, an and cancer agent and a bone condition treating agent The present invention also provides the antibody or fragment which is bound to RANKL or a fragment thereof (/ e , a complex)
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, and (e) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 20 and a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 10
The present invention further provdes an isolated hybπdoma cell producing an antibody of the invention The present invention further includes a method for producing an isolated antibody comprising cultuπng the hybπdoma cell in a culture medium under conditions suitable for expression of said antibody by said hybπdoma 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) Embodiments of the present invention include 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 or RANKL or by an imbalance of bone formation and bone resorption such that bone resorption is favored, comprising administering (e g , by a parenteral route) a therapeutically effective amount of any antibody or fragment of the present invention to said subject (e g , a human) For example, in an embodiment of the invention, said subject is administered said antibody or fragment in a pharmaceutical composition which comprises a pharmaceutically acceptable carrier In an embodiment of the present invention, the antibody or fragment is administered to the subject in association with a therapeutic procedure {e g , application of low level, high frequency stress to bone, radiation therapy, and surgical tumorectomy) and/or one or more further chemotherapeutic agents selected from the group consisting of an anti- inflammatory agent, an immunosuppressive agent, and anti-cancer agent and a bone condition treating agent.
DETAILED DESCRIPTION
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-κB ligand (RANKL), e g , the protein of any one of SEQ ID NOs: 102-104. In certain embodiments, 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-κB (RANK) both in vitro and in vivo
In an embodimant of the invention, 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-κB that is capable of binding to an NF-κB DNA binding site promotion of osteoclastogenesiε [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 resorption factors such as active form Vitamin D3 and parathyroid hormone, induction of bone resorption.
In an embodiment of the invention, 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-κB that is capable of binding to an NF-κB DNA binding site; and/or inhibits osteoclaεtogenesis; and/or inhibits osteoclast- mediated or dependent bone resorption
The term "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 Thθ terms "RANKL" and "receptor activator of NF-κB ligand" are well known in the art. RANKL is also indicated by the terms Tumor Necrosis Factor Related Activation-Induced Cytokine (TRANCE) (Wong.θf a/., J. Exp. Med. 186 2075, 1997), osteoprotegerin ligand (OPGL) (Lacey, et a/., Cell 93:165, 1998) and osteoprotegerin binding protein (OPGbp) (U S. Patent no. 5,843,678) Although 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%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% ammo acid identity with an amino acid sθquence selected from the group consisting of SEQ ID NOS: 102-104 and Genbank Accession Nos NM_003701 and NM__033012 In one embodiment of the invention, said protein has the ability to specifically bind to RANK and/or OPG.
The terms "RANK" and "receptor activator of NF-κB" 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. NM_003839 The term "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. In one embodiment of the invention, said protein has the ability to activate NF-κB or the ability to bind to TNFR-associated factors TRAF1 , TRAF2, TRAF3, TRAF4, TRAF5 or TRAF6.
The term "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
The term 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
The term Osteoclast precursor" includes any cellular entity on the pathway of differentiation between a macrophage and a differentiated and functional osteoclast The term "osteoclast function" as used herein includes bone resorption and the processes required for bone resorption
In an embodiment of the invention, an antibody or antigen-binding fragment thereof binds to an antigen 'specifically" if it binds with a KD of about 10 nM or a lower number
Polypeptides and variants thereof
The amino acid sequences of the variable regions of fully human, monoclonal anti-RANKL antibodies and antigen-binding fragments thereof of the invention (e g , XPA12 004, XPA12 020, XPA12 039 XPA12 041 and XPA12 042), along with the nucleotide sequences of nucleic acids which encode the regions, 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 herein Isolated polypeptides set forth below linked to any immunoglobulin light or heavy chain constant chain also form part of the present invention along with polynucleotides encoding such polypeptides and methods for recombinant expression thereof (e g , wherein the a polynucleotide encoding a polypeptide set forth herein, linked to a constant chain or unlinked, is introduced onto a host cell under conditions wherein the polypeptide is expressed and optionally thereafter, isolated). Moreover, 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 Where the amino acid and nucleotide sequences are identical between two or more antibodies, only a single sequence is shown
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
The invention also provides isolated polypeptides comprising the VL domains (e.g., SEQ ID Noε.: 6-10) and isolatθd 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. In another embodiment, 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,
In an embodiment of the invention, 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. For example, if Aεn-Gly or Asp-Gly is in a CDR of the present invention (e.g., heavy or light chain of XPA12.004; XPA12.020; XPA12.039; XPA12.041 ; and XPA12.042), 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. Also, if a Met is in a CDR in an exposed position, 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. If 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. Furthermore, if 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 sαssile 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.
For example, 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.
Figure imgf000024_0001
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.
Figure imgf000024_0002
Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagine in CDR-H1 (N1) is mutated to GIn or Ala and/or wherein, independently, the Asp-Gly (D2G3) 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 N1 is left unmutated and/or wherein, independently, D2G3 are left unmutated.
For example, 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 , aεpartic acid 2 and glycine 3 {i.e., N1, D2 and G3) 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., N1, N2, N3, N4, or N5) are independently mutated to GIn or Ala or any one is independently left as an unmutated asparagine.
For example, 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., N1, N2, N3, N4 and N5) are as any of the permutations set forth below:
Figure imgf000025_0002
Figure imgf000026_0001
Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagine in CDR-H1 (N3) is mutated to GIn or Ala and/or wherein, independently, the Asp-Gly in CDR-H1 (D1G2) 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 N3 is left unmutated and/or wherein, independently, the D1G2 is left unmutated
For example, 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 , D1, G2 and N3) are as any of the permutations set forth below
Figure imgf000026_0002
DC N
For example 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
Figure imgf000027_0001
Embodiments of the invention comprise polypeptides, antibodies and antigen- bmding fragments thereof wherein the asparagines in CDR-L1 and/or CDR-L2 (N1 and N^) are independently mutated to GIn or Ala or left as unmutated asparagines
For example, embodiments of the invention comprise those wherein the light chain immunoglobulin is as set forth above wherein the residues at the positions αf asparagines 1 and 2 / e N1 and N2 are as an of the ermutations set forth below
Figure imgf000027_0002
Embodiments of the invention comprise polypeptides, antibodies and antigen binding fragments thereof wherein the asparagines in CDR-L1 and/or CDR-L2 (N1 N2 and N3) are independently mutated to GIn or Ala or left as unmutated asparagines
For example, 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 , N1 N2 and N3) are as any of the permutations set forth below
Figure imgf000028_0001
Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagines in CDR-L1 and/or CDR-L2 (N1 and N2) are independently mutated to GIn or Ala or left as unmutated asparagines.
For example, 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. , N1 and N2) are as any of the permutations set forth below:
Figure imgf000028_0002
Embodiments of the invention comprise polypeptides, antibodies and antigen- binding fragments thereof wherein the asparagines in CDR-L1 and/or CDR-L2 (N1, N2, N3 and N4) are independently mutated to GIn or Ala or left as unmutated asparagines.
For example, 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., N1, N2, N3 and N4) are as any of the permutations set forth below.
Figure imgf000029_0001
Figure imgf000030_0001
Embodiments of the invention comprise polypeptides, antibodies and antigen- bmding fragments thereof wherein the asparagines in CDR-L1 (N1, N2 and N3) are independently mutated to GIn or Ala or left as unmutated asparagines
For example, θmbodiments 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 , N1, N2 and N3) are as any of the permutations set forth below
Figure imgf000030_0002
Figure imgf000031_0001
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.
In an embodiment of the invention, 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
Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, Md (1991 ); and/or, Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987) An XPA12 004 antibody or XPA12 020 antibody or XPA12 041 antibody or XPA12.039 antibody or XPA12.042 antibody comprises light and heavy
immunoglobulin chains by these same names
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) The one, two, three, four, five, or six CDRε may be independently selected from the described CDR sequences of the antibodies and antigen-binding fragments of the invention.
Alternatively, 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.
In an embodiment 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.
In an embodiment of the invention 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. 20 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 Alternatively, 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 dθscπbed 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. Molecular biology
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art Such techniques are explained fully in the literature. See, e.g , Sambrook Fπtsch & Maniatis, Molecular Cloning A Laboratory Manual Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sam brook, el a/., 1989"); DNA Cloning: A Practical Approach, Volumes I and Il (D N. Glover ed 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984), Nucleic Acid Hybridization (B. D. Hames & SJ. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & SJ. Higgins, eds (1984)); Animal Cell Culture (R I. Freshney, ed (1986)); Immobilized Cells And Enzymes (I RL Press, (1986)), B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
A polypeptide or protein comprises two or more amino acids.
The term "isolated protein", "isolated polypeptide" or "isolated antibody" 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. Thus, 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.
A "polynucleotide seguence", "nucleic acid sequence" or "nucleotide sequence" is a series of nucleotide bases (also called "nucleotides") in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides.
An amino acid sequence comprises two or more amino acids.
A "coding sequence" or a sequence "encoding" an expression product, such aε an RNA or polypeptide, is a nucleotide sequence that, when expressed, results in production of the product. The term "gene' means a DNA sequence that codes for or corresponds to a particular sequence of ribonucleotides or amino acids which comprise all or part of one or more RNA molecules or proteins, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed Genes may be transcribed from DIMA to RNA which may or may not be translated into an amino acid sequence
"Amplification ' of DNA as used herein includes the use of polymerase chain reaction (PCR) to increase the coπceπtraton of a particular DNA sequence within a rn xture of DNA sequences For a description of PCR see Saiki, et al , Science (1988) 239 487 In a specific embodiment, 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
As used herein the term "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 hybπdizable 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 32P-nucleotιdes, 3H-nucleotιdes 14C-nucleαtιdes, 35S-nucleotιdes or nucleotides to which a label such as biotin has been covalently conjugated Ia ons embodiment a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of the gene, or to detect the presence of nucleic acids Generally, oligonucleotides are prepared synthetically, e g on a nucleic acid synthesizer
The sequence of any nucleic acid (e g , a nucleic acid encoding a RANKL gene or a nucleic acid encoding an anti RANKL antibody or a fragment or portion thereof) 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)
The nucleic acids herein may bθ flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal πbosorne entry sites (IRES) and other πbosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
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 (5r direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found 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. 5,385,839 and 5,168,062), the SV40 early promoter region (Benαst, et al., (1981 ) Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, ef al., (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner, et al., (1981 ) Proc Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., (1982) Nature 296:39-42), prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff, et al , (1978) Proc. Natl. Acad. Sci USA 75 3727-3731 ), or the tac promoter (DeBoer, ef al , (1983) Proc. Natl Acad. Sci. USA 80 21-25); see also "Useful proteins from recombinant bacteria" in Scientific American (1980) 242:74-94; and promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter
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
The terms "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
The terms "vector", "cloning vector" and 'expression vector" mean 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
The term "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
The term "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 Examples of 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)
The term "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. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors In a specific embodiment 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) Other suitable cells include CHO (Chinese hamster ovary) cells, HeLa cells and NIH 3T3 cells and NSO cells (non-lg-producing murine myeloma cell line)
In one embod ment of the invention, the nucleic acid molecules, vectors and host cells may be used to make mutated anti-RANKL antibαdiθs. Specific non exclusive examples of 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 For example, a mutation may be made in one or more of the CDR regions to increase or decrease the K0 of the antibody for RANKL, to increase or decrease koff, or to alter the binding specificity of the antibody. Techniques in site- directed mutagenesis are well-known in the art See, e g , Sambrook et al. and Ausubel ef a/ , supra. 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
In another embodiment of the invention the framework region is mutated so that the resulting framework regιon(s) have the amino acid sequence of the corresponding germline gene Accordingly, 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. See, e g., PCT Publication No. WO 00/09560, incorporated herein by reference 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
In some embodiments, there are from 1 to 8 including any number in between amino acid mutations in either the VH or VL domains of the mutated anti- RANKL antibody compared to the anti-RANKL antibody prior to mutation In any of the above embodiments the mutations may occur in one or more CDR regions Further, any of the mutations can be conservative amino acid substitutions In some embodiments, there are no more than 5 4, 3 2 or 1 amino acid changes in the constant domains
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 In particular the present invention contemplates 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 of the antibodies of the invention are also contemplated by the present invention "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 For example, polar/hydrophilic amino acids which may bθ 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 For example, the present invention contemplates variants and mutants as discussed herein, e g , function-conservative variants of the immunoglobulins set forth in Table 1 (e g VH or VL of XPA12 004, XPA12 020, XPA12 039, XPA12 041 or XPA12 042) as well as immunoglobulins comprising one or more variant or mutant CDRs of the immunoglobulins of Table 1 (e g , VH or VL of XPA12 004, XPA12 020, XPA12 039, XPA12.041 or XPA12 042) that retain 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 υivoy RANK binding; upregulation of the amount of active NF-κB that is capable of binding to an NF-κB DNA binding site; promotion of osteoclastogenesis (Le , 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 resorption factors such as active form Vitamin D3 and parathyroid hormone; induction of bone resorption). For example, in an embodiment of the invention, such a mutant or variant exhibits one or more of the following characteristics"
(i) Ratio, of KD for human RANKL binding to K0 for mouse RANKL binding, of about 1 n to about 10/1 ;
(ii) KD for human RANKL binding of about 0.2 nM to about 0 6 nM;
(iii) KD for mouse RANKL binding of about 0 03 nM to about 0.6 nM;
(iv) Ratio, of IC50, in a TRAP a3say for osteoclastogenesis, induced by human
RANKL, to IC50, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL, of about 0 19 nM to about 1.25 nM;
(v) IC50, in a TRAP assay for osteoclastogenesis, induced by human RANKL, of about
0.1 nM to about 0.5 nM,
(Vi) IC50, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL of about
0 1 nM to about 1.6 nM;
(vii) Ratio, of IC50, in an RANK receptor inhibition assay, wherein signaling is induced by human RANKL to IC50 in an RANK receptor inhibition assay, wherein signaling is induced by mouse RANlKL, of about 0.25 nM to about 3.3 nM;
(viii) IC50, in an RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 1.5 nM to about 2.2 nM,
(ix) IC50, in an RANK receptor inhibition assay, wherein signaling is induced by mouse
RANKL, of about 0.6 nM to about 7.3 nM,
(x) Ratio, of maximum inhibition, in a TRAP assay, for osteoclastogenesis, induced by human RANKL to maximum inhibition, in a TRAP assay, for αsteoclastogenesis, induced by mouse RANKL, of about 0.9 to about 1.0; (xi) maximum inhibition in a TRAP assay, for osteoclastogenesis, induced by human
RANKL, about 89 8% to about 100%,
(XIi) maximum inhibition, in a TRAP assay for osteoclastogenesis induced by mouse
RANKL, of about 100%,
(xiii) 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 RANIKL, of about 0 96 to about 1 1
(xiv) maximum inhibition in a RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 95 7% to about 100%
(xv) maximum inhibition, in a RANK receptor inhibition assay wherein signaling is induced by mouse RANKL of about 90 8% to about 100%,
(xvi) KD for binding to human RANKL of about 0 4 nM,
(XVIi) KD for binding to mouse RANKL of about 0 2 nM,
(xvni) IC50 in a TRAP assay for osteoclastogenesis induced by human RANKL, of about 0 3 nM,
(xix) IC50, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL of about 0 6 nM,
(xx) maximum inhibition, in a TRAP assay for osteoclastogenesis, induced by human RANKL, of about 97%
(xxi) maximum inhibit on in a TRAP assay, for osteoclastogenesis, induced by mouse
RANKL of about 100%
(xxn) maximum inhibition, in a RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 99%, or
(XXiIi) maximum inhibition, in a RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 98%
The present invention includes anii-RANKL antibodies and fragments thereof comprising VL or VH immunoglobulins or CDRs thereof which are encoded by nucleic acids as described in Table 1 as well as nucleic acids which hybridize thereto In an embodiment of the invention, the nucleic aαds hybridize under low stringency conditions, or under moderate stringency conditions or under high stringency conditions A nucleic acid molecule is "hybπdizable1 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). The conditions of temperature and ionic strength determine the "stringency" of the hybridization Stringent hybridization conditions include: prθ~hybrιdιzatιon for 2 hours at 600C in 6X SSC, 0 5% SDS, 5X Denhardt's solution, and 100 μg/ml heat denatured salmon sperm DNA; hybridization for 18 hours at 600C; washing twice in 4X SSC, 0.5% SDS, 0.1 % sodium pyrophosphate for 30 minutes at 6O0C, and twice in 2X SSC, 0 1 % SDS for 30 minutes at 6O0C; or, for example, hybridization in 4X SSC at 650C, followed by a washing in 0.1X SSC at 650C 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 ). 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).
Also included in the present invention are 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
The following references regarding the BLAST algorithm are herein incorporated by reference- BLAST ALGORITHMS: Altschul, S F , ef al , (1990) J. MoI Biol 215 403-410, Gish, W ef a/ , (1993) Nature Genet 3 266-272 Madden, T.L, er a/ , (1996) Meth. Enzymol 26Θ 131-141 , Altschul, S F , ef a/ , (1997) Nucleic Acids Res 25-3389-3402; Zhang, J , ef a/ , (1997) Genome Res 7 649-656, Wootton, J.C , et al , (1993) Cornput Chem 17 149-163, Hancock, J. M ef a/ , (1994) Comput Appl Biosci 10 67-70, ALIGNMENT SCORING SYSTEMS Dayhoff, M O , ef a/., 'A model of evolutionary change in proteins " in Atlas of Protein Sequence and
Structure, (1978) vol. 5, suppl 3 M O Dayhoff (ed ), pp. 345 352, Natl. Biomed. Res Found , Washington, DC, Schwartz, R.M , et al., "Matrices for detecting distant relationships " in Atlas of Protein Sequence and Structure, (1978) vol 5 suppl 3 " M O Dayhoff (ed ), pp 353-358, Natl Biomed Res Found., Washington, DC Altschul, S F , (1991) J MoI Biol 219 555-565, States, DJ, et al., (1991 ) Methods 3 66-70 Heπikoff S , ef al , (1992) Proc Natl Acad. Sci USA 89:10915-10919; Altschul, S.F., ef al , (1993) J. MoI Evol 36"290-300, ALIGNMENT STATISTICS: Karlm, S , ef a/ , (1990) Proc. Natl. Acad Sci USA 87-2264-2268; Karlin, S , et al, (1993) Proc. Natl Acad. Sci. USA 90 5873-5877, Dembo, A., ef a/ (1994) Ann Prob. 22.2022-2039 and Altschul, S F. "Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (S Suhai, ed.), (1997) pp 1-14, Plenum, New York.
Antibodies and antigen-binding fragments thereof The term "antibody or antigen-binding fragment thereof, and the like, 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., VH 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. Furthermore, antibodies oi the invention may be fully human antibodies or chimeric or humanized antibodies. In some embodiments of the invention, the antibodies are monoclonal, fully human antibodies. In some embodiments of the invention, the antibodies of the invention are XPA12.004, XPA12 020, XPA12.039, XPA12.041 or XPA12.042. In some embodiments of the invention, 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 (θ g , XPA12.004, XPA12.020, XPA12.039, XPA12.041 or XPA12.042 heavy or light chains) 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. For example, 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.
Figure imgf000044_0001
Figure imgf000045_0001
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 For example the scope of thθ present invention includes embodiments wherein the antibody or antigen-binding fragment comprises the following arrangement
(1 ) CDR-L1 with > 85% similar or identical to any of SEQ ID NOs 35-38-CDR-L2- CDRL3, and, optionally
CDR H1 with > 85% similar or identical to any of SEQ ID NOs 62-66-CDR-H2-
CDRH3, or CDRH1-CDRH2 with > 85% similar or identical to any of SEQ ID NOs 67-69- CDRH3, or
CDRH1 -CDRH2 -CDRH3 with > 85% similar or identical to any of SEQ ID NOs 70-74; or
(2) CDRL1-CDRL2 with > 85% similar or identical to any of SEQ ID NOs- 39-43- CDRL3, and, optionally,
CDRH1 with > 85% similar or identical to any of SEQ ID NOs: 62-66-CDRH2- CDRH3, or
CDRH1-CDRH2 with > 85% similar or identical to any of SEQ ID NOs: 67-69- CDRH3; or
CDRH1 -CDRH2 -CDRH3 with > 85% similar or identical to any of SEQ ID NOs 70-74; or
(3) CDRL1-CDRL2 -CDRL3 with > 85% similar or identical to any of SEQ ID NOs 44- 48 and, optionally,
CDRH1 with > 85% similar or identical to any of SEQ ID NOs: 62-66-CDRH2-
CDRH3, or
CDRH1-CDRH2 with > 85% similar or identical to any of SEQ ID NOs 67-69-
CDRH3; or
CDRH1-CDRH2 -CDRH3 with > 85% similar or identical to any of SEQ ID NOs: 70-74, or
(4) CDRH1 with > 85% similar or identical to any of SEQ ID NOs: 62-66-CDRH2- CDRH3; 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-
CDRL3; or
CDRL1-CDRL2 -CDRL3 with > 85% similar or identical to any of SEQ ID NOs- 44-48; or
(5) CDRH 1 -CDRH2 with > 85% similar or identical to any of SEQ ID NOs: 67-69- CDRH3; 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-
CDRL3; or CDRL1-CDRL2 -CDRL3 with > 85% similar or identical to any of SEQ ID NOs
44-48, or
(6) CDRH1 -CDRH2 -CDRH3 with > 85% similar or identical to any of SEQ ID NOs: 70-74, and, optionally
CDRL1 with > 35% 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-
CDRL3, or
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. For example, in an embodiment of the 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-κB 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) 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-Ib 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). For example, in an embodiment of the invention, the antibody or fragment described above (1 -6) exhibits one or more of the following characteristics
(ι) Ratio, of K0 for human RANKL binding to KD for mouse RANKL binding, of about 1/1 to about 10/1 ,
(ii) KD for human RANKL binding of about 0.2 nM to about 0 6 nM;
(ιιi) KD for mouse RANKL binding of about 0.03 nM to about 0.6 nM,
(itf) Ratio, of IC5O, 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; (v) IC50, in a TRAP assay for osteoclastogenesis, induced by human RANKL, of about
0 1 nM to about 0 5 nM,
(v ) IC60, in a TRAP assay for osteoclastogenesis induced by mouse RANKL of about
0 1 nM to about 1 6 nM,
(VII) Ratio, of IC50, in an RANK receptor inhibition assay, wherein signaling is induced by human RANKL to IC50 in an RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 0 25 nM to about 3 3 nM,
(vin) ICso in an RANK receptor inhibition assay wherein signaling is induced by human RANKL of about 1 5 nM to about 2 2 nM,
(ιx) IC50, in an RANK receptor inhibition assay, wherein signaling is induced by mouse
RANKL of about 0 6 nM to about 7.3 nM,
(x) Ratio, of maximum inhibition in a TRAP assay, for osteoclastogenesis induced by human RANKL to maximum inhibition, in a TRAP assay, for osteoclastogenesis, nduced by mouse RANKL of about 0 9 to about 1 0,
(xι) maximum inhibition in a TRAP assay, for osteoclastogenesis, induced by human
RANKL, about 89 8% to about 100%,
(xii) maximum inhibition , in a TRAP assay for osteoclastogenesis, induced by mouse
RANKL, of about 100%,
(xni) Ratio of maximum inhibition, in a RANK receptor inhibition assay wherein s gnaling is induced by human RANKL, to maximum inhibition in a RANK receptor inhibition assay wherein signaling is induced by mouse RANKL of about 0 96 to about 1 1
(xiv) maximum inhibition in a RANK receptor inhibition assay, wherein signaling Is induced by human RANKL, of about 95 7% to about 100%,
(xv) maximum inhibition, in a RANK receptor inhibition assay wherein signaling is induced by mouse RANKL, of about 90 8% to about 100%;
(xvi) KD for binding to human RANKL of about 0 4 nM,
(xvn) KD for binding to mouse RANKL of about 0 2 nM,
(XVIII) IC50, in a TRAP assay for osteoclastogenesis, induced by human RANKL, of about 0 3 nM,
(xix) IC50, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL of about 0 6 nM,
(XX) maximum inhibition, in a TRAP assay, for osteoclastogenesis, induced by human
RANKL, of about 97% (xxi) maximum inhibition, in a TRAP assay for osteoclastogenesis induced by mouse RANKL of about 100%,
(XXII) maximum inhibition in a RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 99%; or
(xxiii) maximum inhibition, in a RANK receptor inhibition assay wherein signaling is induced by mouse RANKL, of about 98%
In an embodiment of the invention, 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 vaπable 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,
or
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;
or
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;
or
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,
or a mature heavy immunoglobulin chain variable region which has at least about 85% (Θ 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
or
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,
or
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,
or
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
or
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;
or
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" 10; optionally, with any of the heavy chain sequences described above In an embodiment of the invention, 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-κB that is capable of binding to an NF-κB DNA binding site, promotion of osteoclastogenesis (/ e , generation and/or maturation of mononudeated and/or multinucleated osteoclasts), support or promotion of osteoclast (e.g. mononudeated 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 resorption factors such as active form Vitamin D3 and parathyroid hormone, induction of bone resorption) For example, in an embodiment of the invention, thθ antibody or fragment described above exhibits one or more of the following characteristics (ι) Ratio, of KD for human RANKL binding to K0 for mouse RANKL binding, of about 1/1 to about 10/1 ,
(ιι) K0 for human RANKL binding of about 0 2 nM to about 0 6 nM,
(HI) KD for mouse RANKL binding of about 0 03 nM to about 0 6 nM
(ιv) Ratio, of IC50, in a TRAP assay for osteoclastogenesis, induced by human RANKL, to IC50, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL, of about 0 19 nM to about 1 25 nM;
(v) IC50, in a TRAP assay for osteoclastogenesis induced by human RANKL, of about 0 1 nM to about 0.5 nM,
(vi) IC50, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL of about 0 1 nM to about 1.6 nM
(VII) Ratio, of IC50, in an RANK receptor inhibition assay, wherein signaling is induced by human RANKL to IC50 in an RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 0 25 nM to about 3 3 nM;
(VIII) IC50, in an RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 1 5 nM to about 2.2 nM;
(ιx) IC50, in an RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 0 6 nM to about 7 3 nM; (x) 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,
(xι) maximum inhibition, in a TRAP assay, for osteoclastogenesis, induced by human RANKL about 89 8% to about 100%,
(xii) maximum inhibition in a TRAP assay, for osteoclastogenesis induced by mouse
RANKL of about 100%,
(xni) Ratio, of maximum inhibition in a RAMK 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 96 to about 1 1 ;
(xiv) maximum inhibition, in a RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 95.7% to about 100%
(xv) maximum inhibition in a RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 90 8% to about 100%,
(xvi) K0 for binding to human RANKL of about 0 4 nM,
(xvn) K0 for binding to mouse RANKL of about 0 2 nM,
(XViIi) IC50, in a TRAP assay for osteoclastogenesis, induced by human RANKL, of about 0 3 nM,
(xix) IC50 in a TRAP assay for osteoclastogenesis, induced by mouse RANKL, of about 0 6 nM,
(xx) maximum inhibition, in a TRAP assay for osteoclastogenesis, induced by human
RANKL of about 97%
(xxi) maximum inhibition, in a TRAP assay, for osteoclastogenesis, induced by mouse RANKL, of about 100%,
(xxii) maximum inhibition, in a RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 99%, or
(xxin) maximum inhibition, in a RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 98%
In an embodiment of the invention any anti-RANKL aniibody or antigen- biπding fragment thereof of the invention binds to an extracellular domain of RANKL
(e.g., amino acids 68-317), and/or binds to RANKL so as to inhibit RANK binding, and/or not to inhibit OPG binding As discussed 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
In one embodiment of the invention the anti-RANKL antibodies and antigen- bmding fragments thereof of the invention recognize human RANKL However, the present invention includes antibody molecules which recognize RANKL from different species, e.g., mammals (e g , mouse, rat, rabbit, sheep or dog). In one embodimBnt, 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 Such complexes may be made by contacting the antibody or antibody fragment with RANKL or a RANKL fragment
In one embodiment of the invention, fully human monoclonal antibodies directed against RANKL are generated using transgenic mice carrying parts of the human immune system rather than the mouse system These transgenic mice, which may be referred to, herein, as "HuMAb" mice, contain a human immunoglobulin gene miπiloci that encodes unrearranged human heavy (μ and γ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and K chain loci (Lonberg, N., et al , (1994) Nature 368(6474) 856- 859). Accordingly, the 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 lgGκ monoclonal antibodies (Lonberg, N , et at. , (1994), supra; reviewed in Lonberg, N.
(1994) Handbook of Experimental Pharmacology 1 13:49-101 , Lonberg, N., et al ,
(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, Tuailloπ, Θt al. , (1993) Proc Natl. Acad Sci USA 90 3720-3724, Choi, et al , (1993) Nature Genetics 4.117-123 Chen, J , et al., (1993)EMBO J. 12: 821 - 830; Tuaillon, et al.
(1994) J Immunol 152"2912-292O1 Lonberg, et al., (1994) Nature 368(6474)- 856- 859; Lonberg, N (1994) Handbook of Experimental Pharmacology 113 49-101 ;
Taylor, L , et al., (1994) International Immunology 6: 579-591 ; Lonberg, N , et al.
(1995) Intern Rev Immunol VoI 13 65-93, Harding, F , ef a/ , (1995) Ann N. Y Acad. So 764 536-546 Fishwild D , et al., (1996) Nature Biotechnology 14 845-851 and Harding, et al , (1995) Annals NY Acad Sci. 764 536-546, the contents of all of which are hereby incorporated by reference in their entirety. See further, U.S Patent Nos 5,545,806, 5, 569 825; 5,625,126, 5,533,425; 5,789,650; 5,877,397, 5,661,016; 5,β14,318, 5,874, 299 5,770,429 and 5,545,807, and International Patent Application Publication Nos. WO 98/24884, WO 94/25585; WO 93/12227; WO 92/22645 and WO 92/03918 the disclosures of all of which are hereby incorporated by reference in their entity.
To generate fully human, monoclonal antibodies to RANKL, 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
Biotechnology 14 845-851 and WO 98/24884.
Hybπdoma 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 hybπdoma technique originally developed by Kohler, ef a/ , (1975) (Nature 256 495-497), as well as the trioma technique (Heπng, et al , (1988) Biomed. Biochim. Acta 47:211-216 and Hagiwara, et a/., (1993) Hum. Antibod. Hybπdomas 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-hybπdoma technique (Cole, et a/., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). In an embodiment of the invention, mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols The resulting hybπdomas 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;
Rosenberg, A. H., et al., (1987) Gene 56: 125-135; and Dunn, J. J., et aL, (1988) Gene 68; 259; which are herein incorporated by reference. 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; culturing said diploid cell under conditions wherein said first and said second immunoglobulin chains are expressed and secreted as said multimeric protein followed by optional isolation of the antibody or antigen-binding fragment.
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). In this embodiment, nucleic acids encoding the antibodies and antigen-binding fragments thereof of the invention (e.g., VH or VL) may be inserted into a pET-based plasm id and expressed in the E.coWll system. There are several methods by which to produce recombinant antibodies which are known in the art. One example of a method for recombinant production of antibodies is disclosed in U.S. Patent No. 4,816,567 which is herein incorporated by reference. For example, 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 are well known in the art and include dextran-mediated transfectiαn, calcium phosphate precipitation, poiybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotιde(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, 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. When recombinant expression vectors encoding the heavy chain and/or antigen-binding portion thereof, and/or the light chain and/or antigen-binding portion thereof are introduced into mammalian host 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.
It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, 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. As mentioned above, 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 In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B- lyrnphocytes which produced non-identical antibodies Often, 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 hybπdomas or linking of Fab1 fragments See e g , Songsivilai, et a/ , (1990) Clin Exp Immunol 79 315-321 , Kostelny, et al , (1992) J Immunol. 148 1547- 1553 In addition, bispecific antibodies may be formed as "diabodies" (Holliger, θi 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)
The term "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
immunoglobulin sequences For example 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 chimeπzed with an antibody region (e g , constant chain) from another non-human species (e g , mouse, horse, rabbit, dog, cow or chicken).
The present invention also includes "antigen-binding fragments ' An antigen- biπding 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 VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain Generally, the sFv polypeptide further comprises a polypeptide linker between the VH and Vt domains which enables the sFv to form the desired structure for antigen binding Techniques described for the production of single chain antibodies (U.S. Patent Nos. 5,476,786; 5,132,405 and 4,946,778) can be adapted to produce anti- RANKL-spβcific single chain antibodies For a review of 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 (VH) and a variable light chain (VL) 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 VL chain appended to a VH 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 VH domain.
Depending on the amino acid sequences of the constant chain of their heavy chains, 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.
Camelidae (e.g. camels and llamas) possess fully functional antibodies that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). The cloned and isolated VHH domain is a stable polypeptide with the antigen-binding capacity of the original heavy- chain antibody. The present invention includes VHH domains that comprise one or more (e g , 3) CDRs of the present invention as well as humanized versions thereof (wherθin the framework regions between the CDRs have been replace with human framework sequences) These 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 In an embodiment of the invention, 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 radiometal chelator
(d ethylenetriaminpentaacetic acid (DTPA))
The antibodies and antigen-binding fragments thereof of the invention may also be conjugated with labels such as saTc, 90Y, 111In, 32P, 14C, 125I 3H, 131I, 11C 15O, nN, 18F, 35S, 51Cr, 67To, 22Va, 60Co, 59Fe, 67Se, 152Eu, 67CU, 217Ci, 211At, 212Pb, 47Sc, 109Pd, 234Th1 and 40K, 157Gd 55Mn, 52Tr and 56Fe
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 isothiocyaπate phycαerythπn, phycocyanin, allophycocyanin, o- phthaladehyde, fluorescamine, 152Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label an aromatic acridinium ester label, an imidazole label, an acπdimium salt label, an oxalate ester label, an aequoπn label, 2,3-dιhydrophthalazιnedιones, biotin/avidin, spin labels and stable free radicals
The antibodies and antigen-binding fragments thereof of the invention may be conjugated to a cytotoxic factor such as diptheπa toxin, Pseudomonas aeruginosa exotoxin A chain , πcin A chain, abπn 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, restπctocin, phenomycin, and enomyαn 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, θt al. , (1962) Nature 144:945; David, et al , (1974) Biochemistry 13:1014; Pain, et al , (1981 ) J. Immunol. Meth. 40:219, and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating antibodies are conventional and very well known in the art.
"KOfl" refers to the off-rate constant for dissociation of the antibody from an antibody/antigen complex.
"K0n" refers to the rate at which the antibody associates with the antigen . "K0" refers to the dissociation constant of a particular antibody/antigen interaction. K0 = K0nZK0n.
Dispecific antibodies
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. For example, 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. In an embodiment of the invention, 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). 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.
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. , 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
(programmed deatb-1), DEC-205 and EGFR (epidermal growth factor receptor) In addition, the present invention encompasses a bispecific antibody including an immunoglobulin that binds specifically to RANKL associated with CTLA4-lg (e g , abatacept)
In an embodiment of the invention, 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
(programmed death-1 ) DEC205 and EGFR (epidermal growth factor receptor), DEC205/EGFR, e g , comprising an immunoglobulin sequence set forth herein.
The term "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
A "bispecific antibody" refers to a full antibody wherein the VH and VL on one arm binds one epitope and the VH and VL 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 VHi-VLZ and
VL1-VH2 VHI and VLi associate to form one binding site and VH2 and VL2 associate to create another binding site, each binding site binding a distinct epitope In an embodiment of the invention, a bispecific diabody comprises two chains wherein each chain comprises a VH 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-JΛ/LZ and V1 ΓVH? chains together (e.g., disulfide bridge between VHi and VL1)
A dsFv-dsFv' comprises a VHi-VL2 molecule wherein the variable regions are linked, e.g , by a peptide linker, and wherein the Vm disulfide bridges and pairs with an independent Vu to form one binding site and wherein the VL2 disulfide bridges and pairs with a VH2 to form a distinct binding site.
A bispecific F(ab')2 refers to a F(ab')2 wherein the VH and VL on one arm binds one epitope and the VH and VL on the other arm bind a distinct epitope.
In an embodiment of the invention, 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 (TNFα) antibody adalimumab, infliximab or gαlimumab
For example, in an embodiment of the invention, 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):
Light chain:
Figure imgf000062_0001
Heavy chain:
Figure imgf000063_0002
For example, in an embodiment of the invention, 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):
Anti-1L23R Li ht Chain Se uences and Domains
Figure imgf000063_0001
Figure imgf000063_0003
Figure imgf000064_0001
Figure imgf000064_0002
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
In an embodiment of the invention, the antι-IL-23 portion of the bispecific antibody or antigen-binding fragment comprises a 3 light or 3 heavy chain CDRs selected from:
Figure imgf000073_0002
In an embodiment of the invention, 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:
Figure imgf000073_0003
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
See U.S. Patent no. 7,375, 193. 1
In an embodiment of the invention, 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:
Figure imgf000076_0002
In an embodiment of the invention, the aπti-IL23 p19 portion comprises the following CDRs:
Figure imgf000076_0003
Figure imgf000077_0001
In an embodiment of the invention, the antι-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
For example, in an embodiment of the invention the antι-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
Figure imgf000077_0002
TVSS
(SEQ ID NO 171 )
For example, in an embodiment of the invention the antι-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)
Figure imgf000078_0001
For example, in an embodiment oF the invention, 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)
Figure imgf000078_0002
In an embodiment of the invention, 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). For example, in an embodiment of the invention, the CDR sequences are as set forth below:
Figure imgf000079_0001
For example, in an embodiment of the invention, 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 mαre CDRs thereof (e.g., all 3 light or heavy chain CDRs):
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Pharmaceutical compositions
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. 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
For general information concerning formulations, see e g Gilman, et al , (eds ) (1990) The Pharmacological Bases of Therapeutics, 8th Ed Pergamon Press A Gennaro (ed ), Remington s Pharmaceutical Sciences 18th Edition, (1990), Mack Publishing Co , Easton Pennsylvania Avis, er a/ (eds ) (1993) Pharmaceutical Dosage Forms Parenteral Medications Dekksr New York, Lieberman et a/ (eds ) (1990) Pharmaceutical Dosage Forms Tablets Dekker New York, and Lieberman ef a/ , (eds ) (1990), Pharmaceutical Dosage Forms Disperse Systems Dekker New York, Kenneth A Walters (ed ) (2002) Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), VoI 119, Marcel Dekker
Pharmaceutically acceptable carriers 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 In an embodiment of the invention, the carrier is suitable for injection into a subject s body
Examples of suitable aqueouε and nonaqueous carriers which may be employed in the pharmaceutical composrtions of the invention 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 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 α α-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
Examples of pharmaceutically-acceptable antioxidants 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 Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antimicrobial agents such as EDTA, EGTA paraben chlorobutanol, phenol εorbic acid, and the like
Suitable buffers which may be included in the pharmaceutical compositions of the invention include L-histidine based buffers, phosphate based buffers (Θ 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 manπitol or sorbitol) sodium citrate or sodium chloride (e.g., buffered saline) may also be included in the pharmaceutical compositions of the invention
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 The use of such media and agents for pharmaceutically active substances is well known in the art
Sterile injectable solutions can be prepared by incorporating the antibody or antigen-binding fragment of the invention in the required amount in an appropnatθ solvent, optionally with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration Generally, 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 In the case of sterile powders for the preparation of sterile injectable solutions, 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
In yet another embodiment of the present invention, the pharmaceutical composition can be administered by combination therapy. Accordingly, 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. For example 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
chemotherapeutic agents include anti-inflammatory agents, immunosuppressive agents, anti-cancer agents, and/or bone condition treating agents In one aspect of the invention, 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
Bone condition treating agents are known in the art, and include, but are not limitθd to, anti-rθεorptivθ 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. For example, bone condition treating agents may include bisphosphonates (e g , alendronate, risedronate, ibandroπate 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
morphogenetic protein, osteogenin, NaF, PGE2 agonists, statins (e g , simvastatin, rosuvastatin, lovastatn atorvastatin), vitamin D or a derivative or mimic thereof, raloxifene, bazedoxifene and lasofoxifene.
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 are known in the art, and 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, algestoπe, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortoloπe, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort flucloronide, flumethasone, fluπisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortoloπe, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetaεol propionate, halometasone, haiopredone acetate, hydrocortamate, hydrocortisone loteprednol etabonate, mazipredone, madryeone, meprednisone, methylpredntsolone, mometasone furoate,
paramethasone, prednicarbate, prednisolone, prednisolone 25-dιethylamιno-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, etodαlac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, progiumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyπc acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorolac, tinoπdine), arylpropionic acid derivatives (eg., alminoprofen, benoxaprofen. bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epiπzole), pyrazolones (e.g., apazone, benzpiperylon, fepra2one, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propypheπazone, ramifenazone, suxibuzone, thiazolinobutazone) salicylic acid derivatives (e g , acetaminosalol, aspirin benorylate, bromosaligenin, calcium acefylsalicylate, diflunisal, etersalate, fendosal, gentiεic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (e g , ampiroxicam, droxicam, isoxicam, lornoxicam, piroxicam, tenoxicam), epsilon - acetamidocaproic acid, s-adenosylmethionine, 3-amιno-4-h/droxybutyπc acid, amixetπne, bendazac, benzydamine, alpha -bisabolol, bucolome, difenpirarnide, ditazol, emorfazone fepradinol, guaiazulene nabunπetone, mmesulide, oxaceprol, paranyline, perisoxal proquazone, superoxide dismutase, teπidap, and zileuton
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-glycyrrhetιc acιd, stearyl glycyrrhetinate, and 3- stearyloxy-glycyrrhetmic acid, and disodium 3-sucαnyloxy-beta-glycyrrhetιnate
Immunosuppressive agents include, but are not limited to, interferon, ιnterleukιn-2, cyclosporin A, diftitox, demleukiπ, levamisole, azathiopnne, brequmar guspeπmus, 6-mercaptopuπne, mizoπbine, rapamycin tacrolimus (FK-506), folic acid analogs (e g , denopteπn, edatrexate, methotrexate, piπtrexirn, pteropterin, Tomudex, and trimetrexate), purine analogs (e g , cladribine, fludarabine 6-mercaptopurιne, thiamipπne and thiaguanine), pyrimidine analogs (e g , ancitabine, azacitidine, 6- azaundine, carmofur, cytarabine, doxifluridine, emitefur, enocitabme, floxuπdine fluorouracil, gemcitabine, and tegafur) fluocinolone, tπaminolone, anecortave acetate, fluorometholone, medrysone, and prednislone An "immunosuppressive agent" is a substance which, when administered to a subject, reduces or prevents an immune response in the subject's body.
Antibody therapies which may be administered in conjunction with the antibodies or antigen-binding fragments of the invention include denosumab (Bekker θt at , J Bone Miner Res 16.348-360 (2001)), trastuzumab (e g , herceptin) (see, for example, Sliwkowski, ef a/ , (1999) Semιn. 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
The term ' in association with indicates that the components of a composition of the invention (e g , anti-RANKL antibody or antigen-binding fragment thereof along with denosumab) can be formulated into a single composition for simultaneous delivery or formulated separately into two or more compositions (e g a kit)
Furthermore 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 subjθct by the same or by a different route (e g , wherein an anti- RANKL antibody is administered parenterally and rofecoxib is administered orally)
Dosage
In an embodiment of the invention, 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 For example 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 For example, 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 For example, in an embodiment of the invention, a
therapeutically effective dosage of an antibody or antigen-binding fragment thereof is a dosage which rβducQS 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).
Typically, the administration and dosage of 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
2002), as well as therapeutic protocols well known in the art, e.g., in accordance with dosage and administration instructions on a FDA approved label associated with a particular agent.
For example, in one embodiment of the invention, 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.
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- θnergy 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- telopeptide of type I collagen (uNTX) or low vitamin D levels. Based on the results of these tests, a physician or clinician can adjust dosage of an antibody or antigen- binding fragment thereof of the invention accordingly
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), uπnanalyεis {e.g., to detect hematuria or proteinuria), blood testθ 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 fragment thereof of the invention accordingly.
Therapeutic methods and administration
The antibodies or antigen-binding fragments of the invention and
pharmaceutical 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. 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 osteoclastogeneεis is favored In one embodiment of the invention, 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.
Other diseases or conditions include Lyme-associated arthritis, psoriatic arthritis, ankylosing spondylitis, and osteoarthritis. Though these indications may not be inflammatory in all cases, they may respond to anti-RANKL therapy
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,
hypercalcemia, lytic bone 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, and other conditions where facilitation of bone repair or replacement is desired such as bone fractures, bone defects, plastic surgery, dental and other implantations. Conditions characterized by bone loss also include certain cancers that are known to increase osteoclast activity and induce bone resorption as well as other cancers that result in hypercalcemia and bone loss, such as squamous cell carcinomas. 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. For example, the following four general diagnostic categories for women have been proposed by a World Health Organization Study Group based on measurements by dual-energy X-ray absorptiometry (DXA): 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 of Osteoporosis- Report of a WHO Scientific Group, WHO Technical Report Series (921 ), Geneva, Switzerland (2003)) The mean volumetric mineral density of bony tissue (in g of hydroxyapatite per cm3) can be determined, in an embodiment of the invention, non- invasively by quantitative computed tomography (QCT) The so-called 'areal' or 'surface" bone mineral density (BMD in g of hydroxyapatite per cm2) can be determined by single- or dual-energy X-ray absorptiometry (SXA and DXA) The values generated by these techniques are directly dependent on both the size and integrated mineral density of the scanned skeletal tissue The integrated mineral density is determined by cortical thickness, the number and thickness of the trabeculae, and the '1rue' mineral density corresponding to Ihe amount of hydroxyapatite per unit volume of the bone organic matrix
In single-energy absorptiometry, 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 125I Use of SPA, however, to determine BMD is within the scope of the present invention
Dual-energy absorptiometry (dual-photon absorptiometry (DPA) or DXA) 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
Other bone disorders that may be treated with an aπti-RANKL based therapy of the invention include bone cancer (e g , osteosarcoma, Ewing's sarcoma chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma or chordoma) and bone metastases
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
Accordingly, 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 ,ιn 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. In an embodiment of the invention, a subject is any mammal possessing a RANKL gene
In embodiments of the invention, 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 noπ-mvasive route is also within the scope of the present invention.
Compositions can be administered with medical devices known in the art For example, in an embodiment of the invention a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle Accordingly, 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. 4,487 603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate, U S Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate, 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.
Assays
The 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. In one embodiment of the invention, the anti-RANKL antibody or antigen-binding fragment thereof is directly labeled with a detectable label and may be detected directly. In another embodiment of the invention, 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. As is well known to one of ordinary skill in the art, a secondary antibody is chosen that is able to specifically bind the specific species and class of the primary antibody. For example, if the primary anti-RANKL antibody or antigen-binding fragment thereof is a human IgG, then 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. For example, after washing away unbound secondary/labeled antibody from a well comprising the primary antibody/antigen complex or from a membrane (e.g., a nitrocellulose or nylon membrane) comprising the complex, 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, β- galactosidase, or acetylcholinesterase, examples of prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of fluorescent materials include urnbelliferone, fluorescein, fluorescein isoihiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; an example of a magnetic agent includes gadolinium, and examples of suitable radioactive material include 125I, 1311, 35S or 3H In an embodiment of the invention, 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. In this assay, 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 One may use the immunoassays disclosed above for a number of purposes. In one embodiment of the invention, 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. Advantageously, 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). In another embodiment of the method, 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. As stated above, in an embodiment of the invention, the anti- RANKL antibodies or antigen-binding fragments thereof are labeled with a detectable agent that can be imaged in a patient. For example, 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 99Tc; or other labels discussed herein. These methods may be used, e.g., to diagnose RANKL-mediated disorders (e g., disorders associated with abnormal osteoclast or T-cell activity) or track the progress of treatment for such disorders.
EXAMPLES
The following examples are provided to further describe the present invention and should not be construed to limit the scope of the invention in any way. Any composition or method disclosed in the Examples section talis within the scope of the present invention.
EXAMPLE 1: Determination of Affinity Constants (KD) of Anti-RANKL Antibodies
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
(PBS+Tween) and captured onto the Protein A/G surface, establishing a fairly uniform antibody capture level across all six spots of the flow cell.
The antigen (human or murine RANKL) 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).
The affinity constants of the described antibodies are shown in Table 2
EXAMPLE 2: Inhibition of Osteoclastogenesis with Anti-RANKL
Antibodies
The ability of aπti-RANKL antibodies XPA.12.004, XPA.12 020, XPA.12 039, XPA 12 041 and XPA 12 042 to inhibit osteoclastogenesis was evaluated by TRAP (tartrate-resistant acid phosphatase) assay TRAP is a histochemical marker of oεteoclast-like cells.
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 10s cells/ml Dy dilution into pre-warmβd 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 πM) were diluted to 4X final concentration in pre-warmed growth medium and combined in an assay plate by addition of 25 μl of each to appropriate wells in a 96-well tissue culture dish 50 μl 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% CO2 for 4 days. At day 4, the cells were rinsed once with PBS and then solubilized by the addition of 50 μl/well of TRAP assay buffer (59 mM sodium citrate, 41 mM citric acid, 0.1% Triton X-100, pH 5.2) 100 μl of TRAP assay buffer containing 1 mg/ml p-nιtrophenolphosρhate (pNPP) and 10% (33 5 mM) tartrate salt were added to each assay well. After a 25 minute incubation at 37°C, 50 μl 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.
These experiments demonstrated that each of the described anti-RANKL antibodies inhibit osteoclastogenesis (see Table 2) EXAMPLE 3. Inhibition of RANIKL/RANK Binding by Anti-RANKL Antibodies
The ability of anti-RANKL antibodies XPA 12 004, XPA 12 020 XPA 12 039, XPA 12 041 and XPA 12 042 to inhibit osteoclastogenesis was evaluated by Ba/F3 assay This 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% CO2 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 πM) Subsequently cells were incubated with Alamar Blue, a redox sensitive dye For determining cell viability for 6 hours at 37°C and 5% CO2 Absorbance was read at 570 nm and 600 nm
The results of the assay demonstrated that the described anti-RANKL antibodies inhibit RANKL binding to RANK (see Table 2)
Figure imgf000098_0001
The present invention is not to be limited in scope by the specific embodiments described herein Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Patents, patent applications, Genbank Accession Numbers and publications are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties.

Claims

WE CLAIM:
1 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
(ι) Ratio, of KD for human RANKL binding to K0 for mouse RANKL binding, of about
1/1 to about 10/1 ,
(n) K0 for human RANKL binding of about 0 2 nM to about 0 6 nM
(HI) K0 for mouse RANKL binding of about 0 03 nM to about 0 6 nM,
(ιv) Ratio, of IC50, in a TRAP assay for osteoclastogenesis, induced by human
RANKL to IC50 in a TRAP assay for osteoclastogenesis, induced by mouse RANKL, of about 0 19 nM to about 1 25 nM
(v) IC50, in a TRAP assay for osteoclastogenesis, induced by human RANKL, of about
0 1 nM to about 0 5 nM;
(vι) IC50 in a TRAP assay for osteoclastogenesis, induced by mouse RANKL of about
0 1 nM to about 1 6 nM,
(VII) Ratio, of IC50, in a Ba/F3 RANK receptor inhibition assay, wherein signaling is induced by human RANKL to IC50 in an RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 0.25 nM to about 3 3 nM,
( Yin) IC50, in a Ba/F3 RANK receptor inhibition assay wherein signaling is induced by human RANKL, of about 1 5 nM to about 2 2 nM
(ιx) IC50 in a Ba/F3 RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL of about 0 6 nM to about 7 3 nM;
(x) 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
(xι) maximum inhibition in a TRAP assay, for osteoclastogenesis, induced by human
RANKL, about 89.8% to about 100%,
(xii) maximum inhibition, in a TRAP assay, for osteoclastogenesis, induced by mouse RANKL, of about 100%,
(xiii) Ratio, of maximum inhibition, in a Ba/F3 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 0.96 to about 1 1 ; (xiv) maximum inhibition, in a Ba/F3 RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 95 7% to about 100%, and
(xv) maximum inhibition, in a Ba/F3 RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL of about 90 8% to about 100%.
2. 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
(ι) KD for binding to human RANKL of about 0 4 nM,
[ii) KD for binding to mouse RANKL of about 0 2 nM,
(in) IC50, in a TRAP assay for osteoclastogenesis, induced by human RANKL, of about 0 3 nM,
(ιv) IC50, in a TRAP assay for osteoclastogenesis, induced by mouse RANKL, of about 0 6 nM,
(v) maximum inhibition, in a TRAP assay For osteoclastogenesis, induced by human
RANKL, of about 97%,
(vi) maximum inhibition, in a TRAP assay, for osteoclastogenesis, induced by mouse
RANKL, of about 100%;
(VII) maximum inhibition, in a Ba/F3 RANK receptor inhibition assay, wherein signaling is induced by human RANKL, of about 99%, and
(vni) maximum inhibition, in a Ba/F3 RANK receptor inhibition assay, wherein signaling is induced by mouse RANKL, of about 98%
3 An isolated antibody or antigen-binding fragment thereof comprising one or more members selected from the group consisting of
(a) CDR-H1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12.004,
(b) CDR-H 1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12.020,
(c) CDR-HI , CDR-H2 and CDR-H3 of the variable region of antibody
XPA12.039,
(d) CDR-H 1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12.041 ; (e) CDR-H1 , CDR-H2 and CDR-H3 of the variable region of antibody XPA12.042;
(f) CDR L1 , CDR L2 and CDR-L3 of the variable region of antibody
XPA12.004;
(g) CDR-L1 , CDR-L2 and CDR-L3 of the variable region of antibody
XPA12.020;
(h) CDR-L1 , CDR-L2 and CDR-L3 of the variable region of antibody
XPA12 039;
(i) CDR-L1 , CDR-L2 and CDR-L3 of the variable region of antibody XPA12 041 ; and
(j) CDR-L1 , CDR-L2 and CDR-L3 of the variable region of antibody XPA12 042
4. The antibody or fragment of claim 1 which is a monoclonal antibody.
5. The antibody or fragment of claim 1 which is a biεpecific antibody or antigen-biding fragment thereof, polyclonal antibody, labeled antibody, bivalent antibody, chimeric antibody, humanized antibody, recombinant antibody, anti-idiotypic antibody, camehzed single domain antibody, diabody, scfv, scfv dimer, dsfv, (dsfv)2, ds diabody, Fv, an Fab or F(ab')2-
6. The antibody or fragment of claim 1 which is a bispecific antibody or antigen- binding fragment thereof which binds specifically to an epitope on RANKL and to a distinct epitope on a polypeptide selected from the group consisting of: RANKL, VEGF, HGF, CD40, IL-10, TNF, IL17, APRIL, IL-23p19, IL-23p40, IL-23R, IL-
12Rbeta1 , IGF1 R, MDL-1 , IL-1 beta, IL-1 RAcP, IL-1 RA, PD-1 , DEC205, EGFR and DEC205/EGFR
7 The antibody or fragment of claim 1 which is linked to an immunoglobulin constant chain.
8. The antibody or fragment of claim 7 wherein the constant chain is a K or λ light chain, and/or a γ1 heavy chain, γ2 heavy chain, γ3 heavy chain or γ4 heavy chain 9 A pharmaceutical composition comprising the antibody or fragment of claim 1 and a pharmaceutically acceptable carrier
10. A composition comprising the antibody or fragment of claim 1 in association with one or more further chemotherapeutic agents
11. The antibody or fragment of claim 1 which is bound to RANKL or an antigenic fragment thereof 12 An isolated antibody or antigen-binding fragment thereof comprising one or more members selected from the group consisting of:
(a) XPA12.004 CDR-H1 comprising the amino 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
XPA12.004 CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO 70,
(b) XPA12.020 CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO' 63,
XPA12 020 CDR-H2 comprising the amino acid sequence set forth in
SEQ ID NO" 67, and
XPA12.020 CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 71 ;
(c) XPA12 039 CDR-H1 comprising the ammo acid sequence set forth in SEQ ID NO 64,
XPA12 039 CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO 68, and
XPA12 039 CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 72;
(d) XPA12.041 CDR-H1 comprising the amino acid sequence set forth in
SEQ ID NO: 65,
XPA12.041 CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO. 67, and
Figure imgf000104_0001
XPA12.042 CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO. 43, and
XPA12.042 CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 48
13 The antibody or fragment of claim 12 which is a monoclonal antibody.
14. The antibody or fragment of claim 12 which is a bispecific antibody or antigen- bmding fragment thereof, polyclonal antibody, labeled antibody, bivalent antibody, chimeric antibody, humanized antibody, recombinant antibody, anti-idiotypic antibody, camelized single domain antibody, diabody, scfv, scfv dimer, dsfv, (dsfv)2, ds diabody, Fv, an Fab or F(ab')2.
15. The antibody or fragment of claim 12 which is a bispecific antibody which binds specifically to an epitope on RANKL and to a distinct epitope on a polypeptide selected from the group consisting of. RANKL, VEGF, HGF, CD40, IL-10, TNF, IL17, APRIL, IL-23p19, IL-23p40, IL-23R, IL-12Rbeta1 , IGF1 R, MDL-1 , IL-1 beta, IL- 1 RAcP, IL-1 RA, PD-1 , DEC205, EGFR and DEC205/EGFR . 16. The antibody or fragment of claim 12 which is linked to an immunoglobulin constant chain.
17. The antibody or fragment of claim 16 wherein the constant chain is a K or λ light chain, γ1 heavy chain, γ2 heavy chain, γ3 heavy chain or γ4 heavy chain.
18. A pharmaceutical composition comprising the antibody or fragment of claim 12 and a pharmaceutically acceptable carrier
19. A composition comprising the antibody or fragment of claim 12 in association with one or more further chemotherapeutic agents.
20. The antibody or fragment of claim 12 which is bound to RANKL or an antigenic fragment thereof.
21. An isolated antibody or antigen-binding fragment thereof 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 SEQ ID NO' 17,
(c) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18,
(d) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19;
(e) a mature heavy immunoglobulin chain variable region comprising the ammo acid sequence set forth in SEQ ID NO: 20,
(f) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 6;
(g) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7;
(h) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8;
(i) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 9; and
(j) a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 10.
22. The antibody or fragment of claim 21 which is a monoclonal antibody.
23. The antibody or fragment of claim 21 which is a bispecific antibody or antigen- binding fragment thereof, polyclonal antibody, labeled antibody, bivalent antibody, chimeric antibody, humanized antibody, recombinant antibody, anti-idiotypic antibody, camelized single domain antibody, diabody, scfv, scfv dimer, dsfv, (dsfv)2, ds diabody, Fv, an Fab or F(ab')2.
24. The antibody or fragment of claim 21 which is a bispecific antibody which binds specifically to an epitope on RANKL and to a distinct epitope on a polypeptide selected from the group consisting of: RANKL, VEGF, HGF, CD40, IL-10, TNF, IL17, APRIL, IL-23p19, IL-23p40, IL-23R, IL-12Rbeta1 , IGF1 R, MDL-1 , IL-1 beta, IL-1 RAcP, IL-1 RA, PD-1 , DEC205, EGFR and DEC205/EGFR .
25. The antibody or fragment of claim 21 which is linked to an immunoglobulin constant chain
26. The antibody or fragment of claim 25 wherein the constant chain is a K or λ light chain and/or a γ1 heavy chain, γ2 heavy chain, γ3 heavy chain or γ4 heavy chain 27 A pharmaceutical composition compnsing the antibody or fragment of claim 21 and a pharmaceutically acceptable carrier
28. A composition comprising the antibody or fragment of claim 21 in association with one or more further chemotherapeutic agents.
29. The antibody or fragment of claim 21 which is bound to RANKL or an antigenic fragment thereof
30. 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 16, 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 chain variable region comprising the ammo acid sequence set forth in SEQ ID NO' 17; and a mature light immunoglobulin chain variable region comprising the ammo acid sequence set forth in SEQ ID NO: 7;
(c) a mature heavy immunoglobulin chain variable region comprising the amino 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 sequence set forth in SEQ ID NO 9, and
(e) a mature heavy immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 20 and a mature light immunoglobulin chain variable region comprising the amino acid sequence set forth in SEQ ID NO 10
31 An isolated hybπdoma cell producing the antibody of claim 1 32. A method for producing an isolated antibody comprising culturing the hybπdoma cell of claim 31 in a culture medium under conditions suitable for expression of said antibody by said hybndoma and optionally purifying the antibody from the culture medium 33 An isolated antibody produced by the method of claim 32
34 An isolated polypeptide comprising
(I) CDR-HI , CDR-H2 and CDR-H3 of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO 16, 17, 18, 19 or 20,
(n) CDR-L1 , CDR-L2 and CDR-L3 of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO 6, 7, 8, 9 or 10, or
(in) an amino acid sequence selected from the group consisting of SEQ ID NOs 6-10 and 16-20 or a fragment thereof 35. An isolated polynucleotide encoding the polypeptide of claim 34 36 An isolated vector comprising the polynucleotide of claim 35
37. An isolated host cell comprising the vector of claim 36.
38. The host cell of claim 37 which is bacterial, yeast or mammalian.
39. The host cell of claim 38 wherein the host cell is E.coli, a Pichia cell or a Chinese hamster ovary cell 40 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 or a fragment thereof
41. An isolated vector comprising the polynucleotide of claim 40
42 An isolated host cell comprising the vector of claim 41 43 The host cell of claim 42 which is bacterial, yeast or mammalian.
44 The host cell of claim 43 wherein the host cell is E.coli, a Pichia cell or a Chinese hamster ovary cell. 45. A method for treating or preventing a medical condition in a subject, wherein said medical condition is mediated by elevated expression or activity of RANK or RANKL or by an imbalance of bone formation and bone resorption such that bone resorption is favored, comprising administering a therapeutically effective amount of an antibody or fragment of claim 1 to said subject
46 The method of claim 45 wherein said subject is administered said antibody or fragment in a pharmaceutical composition which comprises a pharmaceutically acceptable carrier 47. The method of claim 45 wherein the subject is a human.
48. A method for treating or preventing a medical condition in a subject, wherein said medical condition is mediated by elevated expression or activity of RANK or RANKL or by an imbalance of bone formation and bone resorption such that bone resorption is favored, comprising administering a therapeutically effective amount of an antibody □r fragment of claim 2 to said subject.
49. A method for treating or preventing a medical condition in a subject, wherein said medical condition is mediated by elevated expression or activity of RANK or RANKL or by an imbalance of bone formation and bone resorption such that bone resorption is favored, comprising administering a therapeutically effective amount of an antibody or fragment of claim 3 to said subject 50. A method for treating or preventing a medical condition in a subject, wherein said medical condition is mediated by elevated expression or activity of RANK or RANKL or by an imbalance of bone formation and bone resorption such that bone resorption is favored comprising administering a therapeutically effective amount of an antibody or fragment of claim 12 to said subject
51 A method for treating or preventing a medical condition in a subject, wherein said medical condition is mediated by elevated expression or activity of RANK or RANKL or by an imbalance of bone formation and bone resorption such that bone resorption is favored comprising administering a therapeutically effective amount of an antibody or fragment of claim 21 to said subject
52 A method for treating or preventing a medical condition in a subject wherein said medical condition is mediated by elevated expression or activity of RANK or RANKL or by an imbalance of bone formation and bone resorption such that bone resorption is favored, comprising administering a therapeutically effective amount of a monoclonal antibody of claim 30 to said subject
53 The method of claim 45 wherein the medical condition is a member selected from the group consisting of an inflammatory disorder an osteopenic disorder, psoriasis 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 bone 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 54 The method of claim 53 wherein the medical condition is osteoporosis
55 The method of claim 45 wherein said antibody or fragment is an antibody which is a monoclonal antibody
56 The method of claim 45 wherein the antibody or fragment is administered to the subject by a parenteral route
57 The method of claim 45 wherein the antibody or fragment is administered to the subject in association with one or more therapeutic procedures and/or one or more further chemotherapeutic agents
58 The method of claim 57 wherein the therapeutic procedure is selected from the group consisting of application of low level, high frequency stress to bone, radiation therapy and surgical tumorectomy
59 The method of claim 45 wherein the antibody or fragment administered to the subject is a monoclonal antibody
PCT/US2010/044206 2009-08-07 2010-08-03 Human anti-rankl antibodies WO2011017294A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US23222109P 2009-08-07 2009-08-07
US61/232,221 2009-08-07

Publications (1)

Publication Number Publication Date
WO2011017294A1 true WO2011017294A1 (en) 2011-02-10

Family

ID=42752017

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/044206 WO2011017294A1 (en) 2009-08-07 2010-08-03 Human anti-rankl antibodies

Country Status (2)

Country Link
AR (1) AR077709A1 (en)
WO (1) WO2011017294A1 (en)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012133914A1 (en) * 2011-03-31 2012-10-04 オリエンタル酵母工業株式会社 Potentiator of cancer immunity containing rankl antagonist
EP2546268A1 (en) 2011-07-13 2013-01-16 F-Star Biotechnologische Forschungs - und Entwicklungsges. M.B.H. Internalising immunoglobulin
WO2013176469A1 (en) * 2012-05-21 2013-11-28 이화여자대학교 산학협력단 Antibody able to suppress osteoclast differentiation
WO2014159430A1 (en) * 2013-03-14 2014-10-02 Apexigen, Inc. Anti-rankl antibodies and methods of use
WO2015030701A1 (en) * 2013-08-24 2015-03-05 R-Pharm Overseas, Inc. Fully human antibodies against human rankl
WO2015033144A1 (en) * 2013-09-05 2015-03-12 Queen Mary University Of London Bispecific antibody against tnf-alpha and synovial microvasculature of arthritis patients
WO2014210545A3 (en) * 2013-06-27 2015-06-25 The University Of Chicago Compositions and methods related to recombinant antibodies to histone posttranslational modifications
EP2878670A4 (en) * 2012-06-08 2016-03-02 Univ Kinki ANTIBODY DIRECTED AGAINST A CARRIER AND USE THEREOF
EP2914292A4 (en) * 2012-10-30 2016-05-04 Univ Emory STIMULATION OF BONE FORMATION BY INHIBITING CD28 COSTIMULATION
EP3091030A4 (en) * 2013-12-31 2017-11-29 Genor Biopharma Co., Ltd Anti-human rankl antibody, humanized antibody, pharmaceutical compositions and uses thereof
CN108064249A (en) * 2014-11-05 2018-05-22 伊莱利利公司 Anti- TNF/ anti-il-23s bispecific antibody
WO2018223182A1 (en) * 2017-06-05 2018-12-13 The Council Of The Queensland Institute Of Medical Research A combination of, or a bispecific binding molecule to, an immune checkpoint molecule antagonist and a rank-l (nf- kb ligand) antagonist for cancer therapy or prophylaxis and uses therefor.
CN109072303A (en) * 2016-02-05 2018-12-21 詹森生物科技公司 Anti-TNF antibodies, compositions, methods and uses for treating or preventing type 1 diabetes
WO2020183270A1 (en) * 2019-03-14 2020-09-17 Janssen Biotech, Inc. Methods for producing anti-tnf antibody compositions
WO2020183271A1 (en) * 2019-03-14 2020-09-17 Janssen Biotech, Inc. Methods for producing anti-tnf antibody compositions
WO2020183269A1 (en) * 2019-03-14 2020-09-17 Janssen Biotech, Inc. Manufacturing methods for producing anti-tnf antibody compositions
EP3576782A4 (en) * 2017-02-02 2020-12-30 Silverback Therapeutics, Inc. CONSTRUCTIVE PEPTIDE COMPOSITIONS AND METHOD OF USING THEREOF
US11248054B2 (en) 2017-06-12 2022-02-15 Bluefin Biomedicine, Inc. Anti-IL1RAP antibodies and antibody drug conjugates
CN116041513A (en) * 2017-10-27 2023-05-02 河北药明生物技术有限公司 Therapeutic antibodies targeting RANKL
WO2023104172A1 (en) * 2021-12-10 2023-06-15 深圳先进技术研究院 Nanobody and preparation method therefor and application thereof
CN117264071A (en) * 2023-11-22 2023-12-22 江苏迈威康新药研发有限公司 Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof
CN118271446A (en) * 2024-05-31 2024-07-02 深圳真实生物医药科技有限公司 Antibodies and antigen-binding fragments of RANKL and their uses
WO2025011639A1 (en) * 2023-07-13 2025-01-16 深圳泽安生物医药有限公司 Multispecific antibody and use thereof

Citations (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4740461A (en) 1983-12-27 1988-04-26 Genetics Institute, Inc. Vectors and methods for transformation of eucaryotic cells
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4912040A (en) 1986-11-14 1990-03-27 Genetics Institute, Inc. Eucaryotic expression system
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US4952496A (en) 1984-03-30 1990-08-28 Associated Universities, Inc. Cloning and expression of the gene for bacteriophage T7 RNA polymerase
US4959455A (en) 1986-07-14 1990-09-25 Genetics Institute, Inc. Primate hematopoietic growth factors IL-3 and pharmaceutical compositions
WO1992003918A1 (en) 1990-08-29 1992-03-19 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5132405A (en) 1987-05-21 1992-07-21 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
WO1992022645A1 (en) 1991-06-14 1992-12-23 Genpharm International, Inc. Transgenic immunodeficient non-human animals
WO1993012227A1 (en) 1991-12-17 1993-06-24 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1994025585A1 (en) 1993-04-26 1994-11-10 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5476786A (en) 1987-05-21 1995-12-19 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5693489A (en) 1984-03-30 1997-12-02 Associated Universities, Inc. Cloning and expression of the gene for bacteriophage T7 RNA polymerase
WO1998024884A1 (en) 1996-12-02 1998-06-11 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5843678A (en) 1997-04-16 1998-12-01 Amgen Inc. Osteoprotegerin binding proteins
US5874299A (en) 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO2000009560A2 (en) 1998-08-17 2000-02-24 Abgenix, Inc. Generation of modified molecules with increased serum half-lives
WO2002015846A2 (en) * 2000-08-21 2002-02-28 Smithkline Beecham Corporation Anti-rank ligand monoclonal antibodies useful in treatment of rank ligand mediated disorders
WO2003086289A2 (en) * 2002-04-05 2003-10-23 Amgen Inc. Human anti-opgl neutralizing antibodies as selective opgl pathway inhibitors
US20050003457A1 (en) * 1997-04-15 2005-01-06 Kyoji Yamaguchi Antibodies to OCIF-binding molecules
US7094874B2 (en) 2000-05-26 2006-08-22 Bristol-Myers Squibb Co. Soluble CTLA4 mutant molecules
WO2007024846A2 (en) 2005-08-25 2007-03-01 Eli Lilly And Company Anit-il-23 antibiodies
US7247711B2 (en) 2003-05-09 2007-07-24 Centocor, Inc. IL-23p40 specific antibody
US20070218064A1 (en) 2005-12-29 2007-09-20 Jacqueline Benson Human anti-il-23 antibodies, compositions, methods and uses
US20080095775A1 (en) 2006-06-13 2008-04-24 Lewis Katherine E Il-17 and il-23 antagonists and methods of using the same
WO2008088594A2 (en) * 2007-01-12 2008-07-24 Anaptysbio, Inc. Isolation of antibodies that cross-react and neutralize rankl orginating from multiple species
WO2008142164A2 (en) * 2007-05-24 2008-11-27 Ablynx N.V. Amino acid sequences directed against rank-l and polypeptides comprising the same for the treatment of bone diseases and disorders
US7491391B2 (en) 2005-06-30 2009-02-17 Centocor, Inc. Anti-IL-23 antibodies, compositions, methods and uses
WO2010022120A1 (en) * 2008-08-19 2010-02-25 Regeneron Pharmaceuticals, Inc. Human antibodies to human rankl

Patent Citations (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4740461A (en) 1983-12-27 1988-04-26 Genetics Institute, Inc. Vectors and methods for transformation of eucaryotic cells
US5693489A (en) 1984-03-30 1997-12-02 Associated Universities, Inc. Cloning and expression of the gene for bacteriophage T7 RNA polymerase
US4952496A (en) 1984-03-30 1990-08-28 Associated Universities, Inc. Cloning and expression of the gene for bacteriophage T7 RNA polymerase
US5869320A (en) 1984-03-30 1999-02-09 Brookhaven Science Associates Llc Cloning and expression of the gene for bacteriophage T7 RNA polymerase
US5385839A (en) 1985-01-30 1995-01-31 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter regulatory DNA sequence
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US4959455A (en) 1986-07-14 1990-09-25 Genetics Institute, Inc. Primate hematopoietic growth factors IL-3 and pharmaceutical compositions
US4912040A (en) 1986-11-14 1990-03-27 Genetics Institute, Inc. Eucaryotic expression system
US5132405A (en) 1987-05-21 1992-07-21 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US5476786A (en) 1987-05-21 1995-12-19 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
WO1992003918A1 (en) 1990-08-29 1992-03-19 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5874299A (en) 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
WO1992022645A1 (en) 1991-06-14 1992-12-23 Genpharm International, Inc. Transgenic immunodeficient non-human animals
WO1993012227A1 (en) 1991-12-17 1993-06-24 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1994025585A1 (en) 1993-04-26 1994-11-10 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1998024884A1 (en) 1996-12-02 1998-06-11 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies
US20050003457A1 (en) * 1997-04-15 2005-01-06 Kyoji Yamaguchi Antibodies to OCIF-binding molecules
US5843678A (en) 1997-04-16 1998-12-01 Amgen Inc. Osteoprotegerin binding proteins
WO2000009560A2 (en) 1998-08-17 2000-02-24 Abgenix, Inc. Generation of modified molecules with increased serum half-lives
US7094874B2 (en) 2000-05-26 2006-08-22 Bristol-Myers Squibb Co. Soluble CTLA4 mutant molecules
WO2002015846A2 (en) * 2000-08-21 2002-02-28 Smithkline Beecham Corporation Anti-rank ligand monoclonal antibodies useful in treatment of rank ligand mediated disorders
WO2003086289A2 (en) * 2002-04-05 2003-10-23 Amgen Inc. Human anti-opgl neutralizing antibodies as selective opgl pathway inhibitors
US7247711B2 (en) 2003-05-09 2007-07-24 Centocor, Inc. IL-23p40 specific antibody
US7491391B2 (en) 2005-06-30 2009-02-17 Centocor, Inc. Anti-IL-23 antibodies, compositions, methods and uses
WO2007024846A2 (en) 2005-08-25 2007-03-01 Eli Lilly And Company Anit-il-23 antibiodies
US20070218064A1 (en) 2005-12-29 2007-09-20 Jacqueline Benson Human anti-il-23 antibodies, compositions, methods and uses
US20080095775A1 (en) 2006-06-13 2008-04-24 Lewis Katherine E Il-17 and il-23 antagonists and methods of using the same
WO2008088594A2 (en) * 2007-01-12 2008-07-24 Anaptysbio, Inc. Isolation of antibodies that cross-react and neutralize rankl orginating from multiple species
WO2008142164A2 (en) * 2007-05-24 2008-11-27 Ablynx N.V. Amino acid sequences directed against rank-l and polypeptides comprising the same for the treatment of bone diseases and disorders
WO2010022120A1 (en) * 2008-08-19 2010-02-25 Regeneron Pharmaceuticals, Inc. Human antibodies to human rankl

Non-Patent Citations (68)

* Cited by examiner, † Cited by third party
Title
"Useful proteins from recombinant bacteria", SCIENTIFIC AMERICAN, vol. 242, 1980, pages 74 - 94
ALTSCHUL, S.F. ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL, S.F. ET AL., J. MOL. EVOL., vol. 36, 1993, pages 290 - 300
ALTSCHUL, S.F. ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
ALTSCHUL, S.F., J. MOL. BIOL., vol. 219, 1991, pages 555 - 565
BEKKER ET AL., J BONE MINER RES, vol. 16, 2001, pages 348 - 360
BEKKER, J BONE MINER RES, vol. 16, 2001, pages 348 - 360
BENOIST ET AL., NATURE, vol. 290, 1981, pages 304 - 310
BRINSTER ET AL., NATURE, vol. 296, 1982, pages 39 - 42
CHEN, J. ET AL., EMBO J., vol. 12, 1993, pages 821 - 830
CHEN, J. ET AL., INTERNATIONAL IMMUNOLOGY, vol. 5, 1993, pages 647 - 656
CHOI ET AL., NATURE GENETICS, vol. 4, 1993, pages 117 - 123
CHOTHIA; LESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
COTE, PROC. NATL. ACAD. SCI. U.S.A, vol. 80, 1983, pages 2026 - 2030
DAVANLOO, P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 2035 - 2039
DAVID ET AL., BIOCHEMISTRY, vol. 13, 1974, pages 1014
DAVIS ET AL., J. RHEUMATOL., vol. 34, 2007, pages 2204 - 2210
DEBOER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 80, 1983, pages 21 - 25
DEMBO, A. ET AL., ANN. PROB., vol. 22, 1994, pages 2022 - 2039
DUNN, J. J. ET AL., GENE, vol. 68, 1988, pages 259
FISHWILD, D. ET AL., NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 845 - 851
GISH, W. ET AL., NATURE GENET., vol. 3, 1993, pages 266 - 272
HAGIWARA ET AL., HUM. ANTIBOD. HYBRIDOMAS, vol. 4, 1993, pages 15
HANCOCK, J.M. ET AL., COMPUT. APPL. BIOSCI., vol. 10, 1994, pages 67 - 70
HARDING, ANNALS NY ACAD. SCI., vol. 764, 1995, pages 536 - 546
HARDING, F. ET AL., ANN. N. Y ACAD. SCI, vol. 764, 1995, pages 536 - 546
HARDING, F. ET AL., ANN. N.Y ACAD. SCI, vol. 764, 1995, pages 536 - 546
HENIKOFF, S. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 10915 - 10919
HERING, BIOMED. BIOCHIM. ACTA., vol. 47, 1988, pages 211 - 216
HOLLIGER ET AL., PNAS USA, vol. 90, 1993, pages 6444 - 6448
HUNTER ET AL., NATURE, vol. 144, 1962, pages 945
KARLIN, S. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 2264 - 2268
KARLIN, S. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5877
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495 - 497
KOSTELNY ET AL., J IMMUNOL., vol. 148, 1992, pages 1547 - 1553
KOSTENUIK PAUL J ET AL: "Denosumab, a Fully Human Monoclonal Antibody to RANKL, Inhibits Bone Resorption and Increases BMD in Knock-In Mice That Express Chimeric (Murine/Human) RANKL", JOURNAL OF BONE AND MINERAL RESEARCH, vol. 24, no. 2, February 2009 (2009-02-01), pages 182 - 195, XP007915105, ISSN: 0884-0431 *
KOZBOR ET AL., IMMUNOLOGY TODAY, vol. 4, 1983, pages 72
LEE ET AL., BIOCONJ. CHEM., vol. 10, 1999, pages 973 - 981
LONBERG ET AL., NATURE, vol. 368, no. 6474, 1994, pages 856 - 859
LONBERG, N. ET AL., INTERN. REV. IMMUNOL., vol. 13, 1995, pages 65 - 93
LONBERG, N. ET AL., NATURE, vol. 368, no. 6474, 1994, pages 856 - 859
LONBERG, N., HANDBOOK OF EXPERIMENTAL PHARMACOLOGY, vol. 113, 1994, pages 49 - 101
MADDEN, T.L. ET AL., METH. ENZYMOL., vol. 266, 1996, pages 131 - 141
MAXAM; GILBERT, PROC. NATL. ACAD. SCI. USA, vol. 74, 1977, pages 560
NYGREN, J., HISTOCHEM. AND CYTOCHEM., vol. 30, 1982, pages 407
PAIN ET AL., J. IMMUNOL. METH., vol. 40, 1981, pages 219
PREVENTION AND MANAGEMENT OF OSTEOPOROSIS-REPORT OF A WHO SCIENTIFIC GROUP, 2003
ROSENBERG, A. H. ET AL., GENE, vol. 56, 1987, pages 125 - 135
SAIKI ET AL., SCIENCE, vol. 239, 1988, pages 487
SANGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 74, 1977, pages 5463
SLIWKOWSKI ET AL., SEMIN. ONCOL., vol. 26, no. 12, 1999, pages 60 - 70
SONGSIVILAI ET AL., CLIN. EXP. IMMUNOL., vol. 79, 1990, pages 315 - 321
STATES, D.J. ET AL., METHODS, vol. 3, 1991, pages 66 - 70
STUDIER, F. W. ET AL., J. MOL. BIOL., vol. 189, 1986, pages 113 - 130
TAYLOR, L. ET AL., INTERNATIONAL IMMUNOLOGY, vol. 6, 1994, pages 579 - 591
TAYLOR, L. ET AL., NUCLEIC ACIDS RESEARCH, vol. 20, 1992, pages 6287 - 6295
TRAUNECKER ET AL., EMBO J., vol. 10, 1991, pages 3655 - 3659
TRAUNECKER ET AL., INT. J. CANCER SUPPL., vol. 7, 1992, pages 51 - 52
TUAILLON ET AL., J IMMUNOL., vol. 152, 1994, pages 2912 - 2920
TUAILLON ET AL., PROC. NATL. ACAD. SCI USA, vol. 90, 1993, pages 3720 - 3724
VILLA-KOMAROFF ET AL., PROC. NATL. ACAD. SCI. USA, vol. 75, 1978, pages 3727 - 3731
WAGNER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 78, 1981, pages 1441 - 1445
WEN ET AL., BIOCONJ. CHEM., vol. 12, 2001, pages 545 - 553
WHO DRUG INFORMATION, vol. 18, no. 2, 2004
WOOTTON, J.C. ET AL., COMPUT. CHEM., vol. 17, 1993, pages 149 - 163
YAMAMOTO ET AL., CELL, vol. 22, 1980, pages 787 - 797
ZHANG, J. ET AL., GENOME RES., vol. 7, 1997, pages 649 - 656

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2012133914A1 (en) * 2011-03-31 2014-07-28 オリエンタル酵母工業株式会社 Cancer immunity enhancer containing RANKL antagonist
WO2012133914A1 (en) * 2011-03-31 2012-10-04 オリエンタル酵母工業株式会社 Potentiator of cancer immunity containing rankl antagonist
EP2546268A1 (en) 2011-07-13 2013-01-16 F-Star Biotechnologische Forschungs - und Entwicklungsges. M.B.H. Internalising immunoglobulin
WO2013176469A1 (en) * 2012-05-21 2013-11-28 이화여자대학교 산학협력단 Antibody able to suppress osteoclast differentiation
EP2878670A4 (en) * 2012-06-08 2016-03-02 Univ Kinki ANTIBODY DIRECTED AGAINST A CARRIER AND USE THEREOF
US9725519B2 (en) 2012-06-08 2017-08-08 Kinki University Antibody against transporter and use thereof
EP2914292A4 (en) * 2012-10-30 2016-05-04 Univ Emory STIMULATION OF BONE FORMATION BY INHIBITING CD28 COSTIMULATION
WO2014159430A1 (en) * 2013-03-14 2014-10-02 Apexigen, Inc. Anti-rankl antibodies and methods of use
US10259878B2 (en) 2013-03-14 2019-04-16 Apexigen, Inc. Anti-RANKL antibodies and methods of use
WO2014210545A3 (en) * 2013-06-27 2015-06-25 The University Of Chicago Compositions and methods related to recombinant antibodies to histone posttranslational modifications
US10208110B2 (en) 2013-06-27 2019-02-19 The University Of Chicago Compositions and methods related to recombinant antibodies to histone posttranslational modifications
WO2015030701A1 (en) * 2013-08-24 2015-03-05 R-Pharm Overseas, Inc. Fully human antibodies against human rankl
WO2015033144A1 (en) * 2013-09-05 2015-03-12 Queen Mary University Of London Bispecific antibody against tnf-alpha and synovial microvasculature of arthritis patients
EP3091030A4 (en) * 2013-12-31 2017-11-29 Genor Biopharma Co., Ltd Anti-human rankl antibody, humanized antibody, pharmaceutical compositions and uses thereof
CN108064249A (en) * 2014-11-05 2018-05-22 伊莱利利公司 Anti- TNF/ anti-il-23s bispecific antibody
CN109072303A (en) * 2016-02-05 2018-12-21 詹森生物科技公司 Anti-TNF antibodies, compositions, methods and uses for treating or preventing type 1 diabetes
EP3576782A4 (en) * 2017-02-02 2020-12-30 Silverback Therapeutics, Inc. CONSTRUCTIVE PEPTIDE COMPOSITIONS AND METHOD OF USING THEREOF
JP2020522529A (en) * 2017-06-05 2020-07-30 ザ カウンシル オブ ザ クイーンズランド インスティテュート オブ メディカル リサーチ Drugs for the treatment or prevention of cancer and their use
WO2018223182A1 (en) * 2017-06-05 2018-12-13 The Council Of The Queensland Institute Of Medical Research A combination of, or a bispecific binding molecule to, an immune checkpoint molecule antagonist and a rank-l (nf- kb ligand) antagonist for cancer therapy or prophylaxis and uses therefor.
CN111032085A (en) * 2017-06-05 2020-04-17 昆士兰医学研究所理事会 Combination of an immune checkpoint antagonist and a RANK-L (NF-KB ligand) antagonist or bispecific binding molecules thereof for use in cancer treatment or prevention and uses thereof
US12350347B2 (en) 2017-06-12 2025-07-08 Bluefin Biomedicine, Inc. Nucleic acids encoding anti-IL1RAP antibodies and their uses
US11248054B2 (en) 2017-06-12 2022-02-15 Bluefin Biomedicine, Inc. Anti-IL1RAP antibodies and antibody drug conjugates
CN116375866A (en) * 2017-10-27 2023-07-04 河北药明生物技术有限公司 Therapeutic antibodies targeting RANKL
CN116041513A (en) * 2017-10-27 2023-05-02 河北药明生物技术有限公司 Therapeutic antibodies targeting RANKL
CN116514970A (en) * 2017-10-27 2023-08-01 河北药明生物技术有限公司 Therapeutic antibodies targeting RANKL
US12129292B2 (en) 2019-03-14 2024-10-29 Janssen Biotech, Inc. Anti-tumor necrosis factor (TNF) antibodies and compositions thereof
WO2020183271A1 (en) * 2019-03-14 2020-09-17 Janssen Biotech, Inc. Methods for producing anti-tnf antibody compositions
WO2020183270A1 (en) * 2019-03-14 2020-09-17 Janssen Biotech, Inc. Methods for producing anti-tnf antibody compositions
WO2020183269A1 (en) * 2019-03-14 2020-09-17 Janssen Biotech, Inc. Manufacturing methods for producing anti-tnf antibody compositions
US12122825B2 (en) 2019-03-14 2024-10-22 Janssen Biotech, Inc. Nucleic acid molecule encoding, and method of producing, a recombinant anti-tumor necrosis factor (TNF) antibody
US12180271B2 (en) 2019-03-14 2024-12-31 Janssen Biotech, Inc. Manufacturing methods for producing anti-TNF antibody compositions
WO2023104172A1 (en) * 2021-12-10 2023-06-15 深圳先进技术研究院 Nanobody and preparation method therefor and application thereof
WO2025011639A1 (en) * 2023-07-13 2025-01-16 深圳泽安生物医药有限公司 Multispecific antibody and use thereof
CN117264071B (en) * 2023-11-22 2024-03-22 江苏迈威康新药研发有限公司 A binding agent for anti-RANKL monoclonal antibody or its derivatives and its application
CN117264071A (en) * 2023-11-22 2023-12-22 江苏迈威康新药研发有限公司 Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof
CN118271446B (en) * 2024-05-31 2024-10-22 深圳真实生物医药科技有限公司 Antibodies and antigen-binding fragments of RANKL and their uses
CN118271446A (en) * 2024-05-31 2024-07-02 深圳真实生物医药科技有限公司 Antibodies and antigen-binding fragments of RANKL and their uses

Also Published As

Publication number Publication date
AR077709A1 (en) 2011-09-14

Similar Documents

Publication Publication Date Title
WO2011017294A1 (en) Human anti-rankl antibodies
KR102121678B1 (en) HUMAN ANTIGEN BINDING PROTEINS THAT BIND β-KLOTHO, FGF RECEPTORS AND COMPLEXES THEREOF
KR102440044B1 (en) Anti-activin A antibodies and uses thereof
JP5457671B2 (en) M-CSF specific monoclonal antibody and use thereof
EP2176294B1 (en) Uses of mdl-1 antagonists
JP4563171B2 (en) Neutralizing human anti-IGFR antibody
KR102230620B1 (en) Antibody constructs for cdh19 and cd3
KR101098109B1 (en) ANTI αVβ6 ANTIBODIES
ES2753756T3 (en) Antibody polytherapy against human CSF-1R and uses thereof
US8715941B2 (en) Antibodies to LRP6
BRPI0714893A2 (en) isolated monoclonal antibody or antigen-binding portion thereof, antibody fragment, mimetic antibody, immunoconjugate, isolated nucleic acid molecule composition, expression vector, host cell, method for preparing an anti-bmp2 or anti-bmp4 antibody, Method for treating or preventing a disease associated with normal bone formation and ossification, hybridoma and method for preparing the antibody
JP6033229B2 (en) Antibody binding to NOTUM pectin acetylesterase
JP2023526317A (en) Anti-Hemojuvelin (HJV) Antibodies for Treating Anemia in Chronic Disease
US20140056918A1 (en) Oscar antagonists
US20250297019A1 (en) Methods of treating cancer with anti-c-c motif chemokine receptor 8 (ccr8) antibodies
AU2019210504B2 (en) Human antigen binding proteins that bind beta-Klotho, FGF receptors and complexes thereof
HK40074936A (en) Antibodies, uses &amp; methods
HK40075257A (en) Antibodies, uses &amp; methods
AU2007200876A1 (en) Neutralizing human anti-IGFR antibody

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10742382

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10742382

Country of ref document: EP

Kind code of ref document: A1