WO2012071381A1 - Antibodies to notum pectinacetylesterase - Google Patents
Antibodies to notum pectinacetylesterase Download PDFInfo
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- WO2012071381A1 WO2012071381A1 PCT/US2011/061785 US2011061785W WO2012071381A1 WO 2012071381 A1 WO2012071381 A1 WO 2012071381A1 US 2011061785 W US2011061785 W US 2011061785W WO 2012071381 A1 WO2012071381 A1 WO 2012071381A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
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- 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]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- 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
- This invention relates to antibody inhibitors of Notum Pectinacetylesterase, compositions comprising them, and methods of their use.
- Bone health depends on the coordinated activities of bone forming osteoblasts and bone resorbing osteoclasts. "Bone turnover reflects a balance between these anabolic and catabolic cellular functions and ensures that the mature skeleton can repair itself when damaged and sustain its endocrine function by release of minerals such as calcium and phosphorous into the circulation.” Allen, J.G. et al., J. Med. Chem., 53 (June 10, 2010), pp. 4332 - 4353, 4332. Many disease states alter this balance, resulting in increased or decreased bone mass or changes in bone quality. Gradual loss of bone mineral density is known as osteopenia; severe loss of bone is known as osteoporosis. Id.
- the current standard of care for the treatment and prevention of osteoporosis utilizes the bisphosphonate class of oral, small molecule antiresportives. Id. at 4333. Zoledronic acid, raloxifene, calcium, and vitamin D supplements are also typically used in the osteoporosis treatment. Id. While antiresorptive agents can help prevent bone loss, anabolic agents "are capable of increasing bone mass to a greater degree ... and also have the capacity to improve bone quality and increase bone strength." Guo, H., et al., J. Med. Chem., 53 (February 25, 2010), pp. 1819 - 1829, 1819. In the United States, human PTH is the only FDA-approved anabolic agent.
- WNT pathway Another of these pathways is the WNT pathway, which is implicated in a variety of developmental and regenerative processes. Allen at 4340.
- the pathway is complex, however, and much about it and about how its components affect bone remains unclear. For example, it has been suggested that LRP-5, mutations of which are associated with increased bone mass in humans, and ⁇ -catenin, through which canonical WNT signaling occurs, "may not be linked directly via WNT signaling to the control of bone mass.” Id.
- a monoclonal antibody that binds human notum binds human notum
- the antibody binds to a NOTUM selected from mouse NOTUM, guinea pig NOTUM, cynomolgus monkey NOTUM, and rhesus monkey NOTUM.
- the antibody has at least one activity selected from reducing NOTUM activity in a trisodium 8- octanoyloxypyrene-l,3,6-trisulfonate (OPTS) assay in vitro, and reducing NOTUM activity in a Wnt signaling assay in vitro.
- OPTS trisodium 8- octanoyloxypyrene-l,3,6-trisulfonate
- the antibody has at least one activity selected from increasing serum PINP levels in vivo, increasing bone mineral density in vivo, increasing midshaft femur cortical thickness in vivo, increasing midshaft femur bone area in vivo, increasing midshaft humerus cortical thickness in vivo, increasing endocortical bone formation in vivo, increasing the proportion of cortical bone volume in the LV5 vertebral body in vivo, and increasing the proportion of femoral neck bone volume to femoral neck total volume in vivo.
- an antibody that binds NOTUM binds to a polypeptide having the amino acid sequence of SEQ ID NO: 1 with K D of less than 50 nM, less than 20 nM, or less than 10 nM.
- the antibody has at least one binding characteristic selected from: a) binds to a polypeptide having the amino acid sequence of SEQ ID NO: 83 with a binding affinity that is at least 5-fold stronger than the binding affinity of the antibody for a polypeptide having the amino acid sequence of SEQ ID NO: 84; b) binds to a polypeptide having the amino acid sequence of SEQ ID NO: 85 with a binding affinity that is at least 5-fold stronger than the binding affinity of the antibody for a polypeptide having the amino acid sequence of SEQ ID NO: 86; c) binds to a polypeptide having the amino acid sequence of SEQ ID NO: 1 with a binding affinity that is at least 5-fold stronger than the binding affinity of the antibody for a polypeptide having the amino acid sequence of SEQ ID NO: 94; d) binds to a polypeptide having the amino acid sequence of SEQ ID NO: 1 with a binding affinity that is at least 5-fold stronger than the binding affinity of the antibody for a polypeptide having the amino acid
- the antibody is selected from a mouse antibody, a chimeric antibody, a humanized antibody, and a human antibody.
- an antibody that binds NOTUM comprises a heavy chain and a light chain, wherein the heavy chain comprises at least one CDR selected from: a) a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 9, 17, 25, 33, 41, 49, and 90; b) a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 10, 18, 26, 34, 42, and 50; and c) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, and 91.
- the heavy chain comprises a set comprising a CDR1, a CDR2, and a CDR3, wherein the set is selected from: a) a set comprising a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 10, and a CDR3 having the amino acid sequence of SEQ ID NO: 11; b) a set comprising a CDR1 having the amino acid sequence of SEQ ID NO: 90, a CDR2 having the amino acid sequence of SEQ ID NO: 18, and a CDR3 having the amino acid sequence of SEQ ID NO: 91; c) a set comprising a CDR1 having the amino acid sequence of SEQ ID NO: 17, a CDR2 having the amino acid sequence of SEQ ID NO: 18, and a CDR3 having the amino acid sequence of SEQ ID NO: 19; d) a set comprising a CDR1 having the amino acid sequence of SEQ ID NO: 90, a CDR2 having the amino acid sequence
- an antibody that binds NOTUM comprises a heavy chain and a light chain, wherein the light chain comprises at least one CDR selected from: a) a CDRl comprising an amino acid sequence selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, and 92; b) a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, and 93; and c) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, and 62.
- the light chain comprises at least one CDR selected from: a) a CDRl comprising an amino acid sequence selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, and 92; b) a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, and 93; and c) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 14, 22,
- the light chain comprises a set comprising a CDRl, a CDR2, and a CDR3, wherein the set is selected from: a) a set comprising a CDRl having the amino acid sequence of SEQ ID NO: 12, a CDR2 having the amino acid sequence of SEQ ID NO: 13, and a CDR3 having the amino acid sequence of SEQ ID NO: 14; b) a set comprising a CDRl having the amino acid sequence of SEQ ID NO: 92, a CDR2 having the amino acid sequence of SEQ ID NO: 93, and a CDR3 having the amino acid sequence of SEQ ID NO: 22; c) a set comprising a CDRl having the amino acid sequence of SEQ ID NO: 20, a CDR2 having the amino acid sequence of SEQ ID NO: 21, and a CDR3 having the amino acid sequence of SEQ ID NO: 22; d) a set comprising a CDRl having the amino acid sequence of SEQ ID NO: 92, a
- an antibody that binds NOTUM comprises a heavy chain variable region and a light chain variable region, wherein: a) the heavy chain variable region comprises a CDRl having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 10, and a CDR3 having the amino acid sequence of SEQ ID NO: 11, and wherein the light chain variable region comprises a CDRl having the amino acid sequence of SEQ ID NO: 12, a CDR2 having the amino acid sequence of SEQ ID NO: 13, and a CDR3 having the amino acid sequence of SEQ ID NO: 14; or b) the heavy chain variable region comprises a CDRl having the amino acid sequence of SEQ ID NO: 90, a CDR2 having the amino acid sequence of SEQ ID NO: 18, and a CDR3 having the amino acid sequence of SEQ ID NO: 91, and wherein the light chain variable region comprises a CDRl having the amino acid sequence of SEQ ID NO: 92, a CDR
- an antibody that binds NOTUM comprises a heavy chain variable region and a light chain variable region, wherein a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; or b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16; or c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 73; or d) the heavy chain comprises the amino acid sequence of SEQ ID NO: 72 and the light chain comprises the amino acid sequence of SEQ ID NO: 74; or e) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24; or f) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 75 and the
- a nucleic acid molecule comprises a polynucleotide sequence that encodes a heavy chain or a light chain of an antibody that binds NOTUM and neutralizes at least one activity of NOTUM.
- the nucleic acid molecule comprises a first polynucleotide sequence that encodes the heavy chain, and a second polynucleotide sequence that encodes the light chain.
- the nucleic acid molecule is a vector.
- a host cell comprising a nucleic acid molecule that comprises a polynucleotide sequence that encodes a heavy chain or a light chain of an antibody that binds NOTUM and neutralizes at least one activity of NOTUM is provided.
- a host cell comprising a nucleic acid molecule that comprises a first polynucleotide sequence that encodes a heavy chain, and a second polynucleotide sequence that encodes a light chain, is provided.
- a host cell comprises a first nucleic acid molecule comprising a polynucleotide sequence that encodes a heavy chain, and a second nucleic acid molecule comprising a polynucleotide sequence that encodes a light chain.
- a method of producing an antibody that binds to NOTUM and neutralizes at least one activity of NOTUM comprising incubating a host cell under conditions sufficient to express the antibody.
- a pharmaceutical composition comprising an antibody that binds NOTUM and neutralizes at least one activity of NOTUM is provided.
- a method of stimulating endocortical bone formation in a patient, comprising administering an effective amount of the pharmaceutical composition is provided.
- a method of treating, managing, or preventing a disease or disorder characterized by bone loss in a patient, comprising administering an effective amount of the pharmaceutica l composition is provided.
- the disease or disorder is osteoporosis.
- a single unit dosage form comprising the pharmaceutical composition is provided.
- Figure 1 provides a graphical representation of differences between the cortical thicknesses of various bone sites in NOTUM homozygous knockout mice (“HOM”) and those in the wildtype littermates (“WT").
- Figure 2 provides a graphical representation of an increase in cortical bone thicknesses observed in both NOTUIVI homozygous and heterozygous (“HET”) knockout mice as compared to their wildtype littermates.
- Figure 3 provides a graphical representation of results obtained from femur breaking strength and spine compression tests performed on the bones of male NOTUM homozygous and heterozygous knockout mice and their wildtype littermates.
- Figure 4 provides a graphical representation of results obtained from femur breaking strength and spine compression tests performed on the bones of female NOTUIVI homozygous and heterozygous knockout mice and their wildtype littermates
- Figure 5 provides a graphical representation of certain human/mouse chimeric proteins, and indicates a region that appears to be involved in binding of NOTUM neutralizing antibodies in Bin 1, as described in Example 6.7.
- Figure 6 provides a graphical representation of midshaft femur cortical thickness measurements obtained in mice after eight weeks of administering MAb 2.1029 or MAb 2.78, as described in Example 6.9.1.
- Figure 7 provides a graphical representation of midshaft femur cortical thickness measurements obtained in mice after four weeks of administering various dosages of MAb 2.1029, as described in Example 6.9.2.
- Figure 8 provides a graphical representation of midshaft femur cortical thickness measurements obtained in mice after four weeks of administering various dosages of MAb 2.78b, described in Example 6.9.3.
- Figure 8A shows 3 mg/kg, 10 mg/kg, and 30 mg/kg dosages of MAb 2.78b.
- Figure 8B shows 0.3 mg/kg, 1 mg/kg, and 3 mg/kg dosages of MAb 2.78b.
- Figure 9 provides a graphical representation of midshaft femur cortical thickness measurements (A) and serum PINP levels (B) obtained in mice after 4 weeks of administering MAb 2.78b, with and without pretreatment with zoledronate, as described in Example 6.9.4.
- Figure 10 provides a graphical representation of midshaft femur cortical thickness measurements obtained in mice after 4 weeks of administering MAb 2.78a, as described in Example 6.9.5.
- Figure 11 provides a graphical representation of midshaft femur cortical thickness measurements (A) and midshaft humerus cortical thickness measurements (B) obtained in mice after 12 weeks of administering MAb 2.78a, as described in Example 6.9.6.
- Figure 12 provides a graphical representation of midshaft femur cortical thickness measurements (A), midshaft humerus cortical thickness measurements (B), and ninth rib cortical thickness (C) obtained in mice after 24 weeks of administering MAb 2.78a, as described in Example 6.9.6.
- Figure 13 provides a graphical representation of midshaft femur cortical thickness (A) and midshaft femur mineralized bone area (B) in sham surgery and ovariectomized mice administered NOTUM neutralizing antibody 2.78b or control antibody, as described in Example 6.10.3.
- Figure 14 provides a graphical representation of the proportion in the LV5 vertebral body of bone volume to total volume (A), the proportion in the LV5 vertebral body of cortical bone volume to total volume (B), and the proportion in the LV5 vertebral body of trabecular bone volume to total volume (C) in sham surgery and ovariectomized mice administered NOTUM neutralizing antibody 2.78b or control antibody, as described in Example 6.10.3.
- Figure 15 provides a graphical representation of the proportion of femoral neck bone volume to total volume in sham surgery and ovariectomized mice administered NOTUM neutralizing antibody 2.78b or control antibody, as described in Example 6.10.3.
- Figure 16 provides a graphical representation of the percentage of the endocortical surface of the midshaft femur cross-sections that were labeled with calcein, alizarin, and tetracycline in sham surgery and ovariectomized mice administered NOTUM neutralizing antibody 2.78b or control antibody, as described in Example 6.10.4.
- Figure 17 provides a graphical representation of the mineral apositional rate (A) and the volume-referent bone formation rate (B) in sham surgery and ovariectomized mice
- This invention is based, in part, on the discovery that inhibition of NOTUM can affect endocortical bone formation.
- Particular aspects of the invention are based on studies of mice lacking a functional NOTUM gene ("knockout mice"), on the development of antibodies that inhibit NOTU M, and on the discovery that such antibodies can be used to stimulate cortical bone formation in mice and rats.
- antibody refers to an intact antibody or a fragment of an antibody that com petes with the intact antibody for antigen bind ing.
- Antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134.
- antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies.
- antibody fragments are produced by recombinant DNA techniques.
- antigen-binding site refers to a portion of an antibody capable of specifically binding an antigen.
- an antigen-binding site is provided by one or more antibody variable regions.
- binding affinity refers to a qualitative or quantitative determination of the strength with which an antibody binds to an antigen.
- the binding affinity is the dissociation constant (K D ) of the antibody for the antigen.
- the binding affinity of an antibody for an antigen is determined qualitatively, such as relative to the binding affinity of a different antibody for an a ntigen, or relative to the binding affinity of the same antibody for a different antigen (such as the antigen with one or more changes in its amino acid sequence).
- the binding affinity of an antibody for a first antigen is considered “stronger” than its affinity for a second antigen, for example, when the K D of the antibody for the first antigen is lower than the K D of the antibody for the second antigen.
- the binding affinity of an antibody for a first antigen is considered “stronger” when the K D of the antibody for the first antigen is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold lower than the K D of the antibody for the second antigen.
- the binding affinity of an antibody for a first a ntigen is considered “weaker” than its affinity for a second antigen, for example, when the K D of the antibody for the first antigen is higher than the K D of the antibody for the second antigen.
- the binding affinity of an antibody for a first antigen is considered “weaker” when the K D of the antibody for the first antigen is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold higher than the K D of the antibody for the second antigen.
- a "chimeric" antibody refers to an antibody made up of components from at least two different sources.
- a chimeric antibody comprises a portion of an antibody derived from a first species fused to another molecule, e.g., a portion of an antibody derived from a second species.
- a chimeric antibody comprises a portion of an antibody derived from a non-human animal fused to a portion of an antibody derived from a human.
- a chimeric antibody comprises all or a portion of a variable region of an antibody derived from a non-human animal fused to a constant region of an antibody derived from a human.
- epitope refers to any polypeptide determinant capable of specifically binding to an immunoglobulin or a T-cell receptor.
- an epitope is a region of an antigen that is specifically bound by an antibody.
- an epitope may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups.
- an epitope may have specific three dimensional structural characteristics (e.g., a "conformational" epitope) and/or specific charge characteristics.
- An epitope is defined as "the same” as another epitope if a particular antibody specifically binds to both epitopes.
- polypeptides having different primary amino acid sequences may comprise epitopes that are the same. Different antibodies are said to bind to the same epitope if they compete for specific binding to that epitope.
- a "fragment" of a reference polypeptide refers to a contiguous stretch of amino acids from any portion of the reference polypeptide.
- a fragment may be of any length that is less than the length of the reference polypeptide.
- a fragment is a contiguous stretch of amino acids from any portion of the reference polypeptide that has a particular activity or contains a particular epitope.
- human antibody refers to a monoclonal antibody that contains human antibody sequences and does not contain antibody sequences from a non-human animal.
- a human antibody may contain synthetic sequences not found in native antibodies. The term is not limited by the manner in which the antibodies are made.
- a human antibody may be made in a transgenic mouse, by phage display, by human B-lymphocytes, or by recombinant methods.
- a “humanized” antibody refers to a non-human antibody that has been modified so that it more closely matches (in amino acid sequence) a human antibody.
- a humanized antibody is thus a type of chimeric antibody.
- amino acid residues outside of the antigen binding residues of the variable region of the non-human antibody are modified.
- a humanized antibody is constructed by replacing all or a portion of one or more complementarity determining region (CDRs) of a human antibody with all or a portion of one or more CDRs from another antibody, such as a non-human antibody, having the desired antigen binding specificity.
- CDRs complementarity determining region
- a humanized antibody comprises variable regions in which all or substantially all of the CDRs correspond to CDRs of a non-human antibody and all or substantially all of the framework regions (FRs) correspond to FRs of a human antibody.
- one or more amino acids within one or more CDRs of the non-human antibody are changed in the humanized antibody, e.g., through a process of affinity maturation. Exemplary methods of affinity maturation are known in the art.
- a humanized antibody further comprises a constant region (Fc) of a human antibody.
- the term “include” has the same meaning as “include, but are not limited to,” the term “includes” has the same meaning as “includes, but is not limited to,” and the term “including” has the same meaning as “including, but not limited to.” Similarly, the term “such as” has the same meaning as the term “such as, but not limited to.”
- the terms “manage,” “managing” and “management” encompass preventing the recurrence of the specified disease or disorder in a patient who has already suffered from the disease or disorder, and/or lengthening the time that a patient who has suffered from the disease or disorder remains in remission.
- the terms encompass modulating the threshold, development and/or duration of the disease or disorder, or changing the way that a patient responds to the disease or disorder.
- the term "monoclonal antibody” refers to an antibody from a substantially homogeneous population of antibodies that specifically bind to the same epitope.
- a monoclonal antibody is secreted by a hybridoma.
- a hybridoma is produced according to some methods known to those skilled in the art. See, e.g., Kohler and Milstein (1975) Nature 256: 495-499.
- a monoclonal antibody is produced using recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
- a monoclonal antibody refers to an antibody fragment isolated from a phage display library.
- neutralizing antibody or “antibody that neutralizes” refers to an antibody that reduces at least one activity of a polypeptide comprising the epitope to which the antibody specifically binds. In some embodiments, a neutralizing antibody reduces an activity of the polypeptide in vitro and/or in vivo.
- NOTUM refers to notum pectinaceylesterase having an amino acid sequence from any vertebrate or mammalian source, including human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise.
- the term also refers to fragments and variants of native NOTUM that maintain at least one in vivo or in vitro activity of a native NOTUM.
- the term encompasses full-length unprocessed precursor forms of NOTUM as well as mature forms resulting from post-translational cleavage of a signal peptide and other forms of proteolytic processing.
- a full-length, unprocessed human NOTUM has the amino acid sequence set forth in SEQ ID NO: 1.
- a full- length, unprocessed mouse NOTUM has the amino acid sequence set forth in SEQ ID NO: 2.
- polypeptide refers to a polymer of amino acid residues.
- the terms apply to amino acid polymers containing naturally occurring amino acids as well as amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid.
- the amino acid polymers can be of any length.
- native polypeptide refers to a naturally occurring polypeptide.
- the terms “prevent,” “preventing” and “prevention” contemplate an action that occurs before a patient begins to suffer from the specified disease or disorder, which inhibits or reduces the severity of the disease or disorder. In other words, the terms encompass prophylaxis.
- a “prophylactically effective amount” of a compound is an amount sufficient to prevent a disease or condition, or one or more symptoms associated with the disease or condition, or prevent its recurrence.
- a “prophylactically effective amount” of a compound means an amount of therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease.
- the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
- an antibody "specifically binds" an antigen when it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules.
- an antibody comprises an antigen-binding site that specifically binds to a particular epitope.
- the antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope.
- homologous proteins from different species may comprise the same epitope.
- an antibody is said to specifically bind an antigen when the dissociation constant (K D ) is ⁇ 1 ⁇ , in some embodiments, when the dissociation constant is ⁇ 100 nM, and in some embodiments, when the dissociation constant is ⁇ 10 nM.
- a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition.
- a “therapeutically effective amount” of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the disease or condition.
- the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.
- the terms “treat,” “treating” and “treatment” contemplate an action that occurs while a patient is suffering from the specified disease or disorder, which reduces the severity of the disease or disorder, or retards or slows the progression of the disease or disorder.
- a native antibody typically has a tetrameric structure.
- a tetramer typically comprises two identical pairs of polypeptide chains, each pair having one light chain (In some embodiments, about 25 kDa) and one heavy chain (In some embodiments, about 50-70 kDa).
- a heavy chain comprises a variable region, VH, and three constant regions, CHI, CH2, and CH3.
- the VH domain is at the amino-terminus of the heavy chain
- the CH3 domain is at the carboxy-terminus.
- a light chain comprises a variable region, VL, and a constant region, CL.
- the variable region of the light chain is at the amino-terminus of the light chain.
- the variable regions of each light/heavy chain pair typically form the antigen binding site.
- the constant regions are typically responsible for effector function.
- Native human light chains are typically classified as kappa and lambda light chains.
- Native human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
- IgG has subclasses, including IgGl, lgG2, lgG3, and lgG4.
- IgM has subclasses including IgMl and lgM2.
- IgA has subclasses including IgAl and lgA2.
- variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
- J Fundamental Immunology (1989) Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.).
- variable regions typically exhibit the same general structure in which relatively conserved framework regions (FRs) are joined by three hypervariable regions, also called complementarity determining regions (CDRs).
- the CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope.
- both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
- the CDRs on the heavy chain are referred to as HI, H2, and H3, while the CDRs on the light chain are referred to as LI, L2, and L3.
- CDR3 is the greatest source of molecular diversity within the antigen binding site.
- H3 for example, in certain instances, can be as short as two amino acid residues or greater than 26.
- the assignment of amino acids to each domain is typically in accordance with the definitions of Kabat et al. (1991) Sequences of Proteins of Immunological Interest (National Institutes of Health,
- CDR refers to a CDR from either the light or heavy chain, unless otherwise specified.
- a "Fab” fragment comprises one light chain and the CHI and variable region of one heavy chain.
- the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
- a " Fab' " fragment comprises one light chain and one heavy chain that comprises additional constant region, extending between the CHI and CH2 domains.
- An interchain disulfide bond can be formed between two heavy chains of a Fab' fragment to form a "F(ab')2" molecule.
- An "Fv” fragment comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
- a single-chain Fv (scFv) fragment comprises heavy and light chain variable regions connected by a flexible linker to form a single polypeptide chain with an antigen-binding region.
- Exemplary single chain antibodies are discussed in detail in WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.
- a single variable region i.e., a heavy chain variable region or a light chain variable region
- heavy chain refers to a polypeptide comprising sufficient heavy chain variable region sequence to confer antigen specificity either alone or in combination with a light chain.
- light chain refers to a polypeptide comprising sufficient light chain variable region sequence to confer antigen specificity either alone or in combination with a heavy chain.
- monoclonal antibodies that specifically bind to NOTUM are provided.
- the monoclonal antibodies are neutralizing antibodies that reduce at least one activity of NOTUM in vivo and/or in vitro.
- a neutralizing antibody against NOTUM reduces NOTUM activity in a trisodium 8-octanoyloxypyrene-l,3,6-trisulfonate (OPTS) assay in vitro. In some embodiments, a neutralizing antibody against NOTUM reduces NOTUM activity in a Wnt signaling assay in vitro.
- OPTS trisodium 8-octanoyloxypyrene-l,3,6-trisulfonate
- a neutralizing antibody against NOTUM increases serum PINP levels in vivo when administered to a subject in a sufficient amount and for a sufficient duration. Exemplary dosages and dosing schedules for administering a sufficient amount for a sufficient duration are discussed herein.
- a neutralizing antibody against NOTUM increases bone mineral density.
- a neutralizing antibody against NOTUM increases midshaft femur cortical thickness in vivo.
- a neutralizing antibody against NOTUM increases midshaft femur bone area in vivo.
- a neutralizing antibody against NOTUM increases midshaft humerus cortical thickness in vivo.
- a neutralizing antibody against NOTUM increases endocorticai bone formation in vivo. In some embodiments, a neutralizing antibody against NOTUM increases the proportion of cortical bone volume in the LV5 vertebral body in vivo. By “proportion of cortical bone volume in the LV5 vertebral body” is meant the proportion of cortical bone volume to total volume of the LV5 vertebral body. In some embodiments, a neutralizing antibody against NOTUM increases the proportion of femoral neck bone volume to total volume of the femoral neck in vivo.
- neutralizing antibodies that specifically bind to mouse NOTUM are provided.
- neutralizing antibodies that specifically bind to human NOTUM are provided.
- neutralizing antibodies that bind to a region from Q47 to M177 of human NOTUM are provided.
- neutralizing antibodies that depend upon a region from Q47 to M177 of human NOTUM for binding are provided.
- neutralizing antibodies that specifically bind to the same region of NOTUM from different species are provided.
- neutralizing antibodies that bind to human NOTUM and NOTUM from at least one species selected from mouse, rat, guinea pig, cynomolgus monkey, marmoset, and rhesus macaque, are provided.
- the antibodies specifically bind to both non-human primate NOTUM and human NOTUM.
- the antibodies specifically bind to both mouse NOTUM and human NOTUM.
- neutralizing antibodies that bind to a region of human NOTUM from Q47 to M177 are provided.
- neutralizing antibodies that depend upon a region of human NOTUM from Q47 to M177 for binding are provided.
- NOTUM neutralizing antibodies are provided that bind to human-mouse chimeric NOTUM (SEQ ID NO: 83) with an affinity that is at least 5-fold, at least 10-fold, or at least 20-fold stronger than the affinity for mouse-human chimeric NOTUM (SEQ ID NO: 84).
- NOTUM neutralizing antibodies are provided that bind to human-mouse-human chimeric NOTUM (SEQ ID NO: 85) with an affinity that is at least 5-fold, at least 10-fold, or at least 20-fold stronger than the affinity for mouse-human-mouse chimeric NOTUM (SEQ ID NO: 86).
- NOTUM neutralizing antibodies are provided that bind to human NOTUM (SEQ ID NO: 1) with an affinity that is at least 5-fold, at least 10-fold, or at least 20-fold stronger than the affinity for NOTUM D141S (SEQ ID NO: 94).
- NOTUM neutralizing antibodies are provided that bind to mouse NOTUM S148D (SEQ ID NO: 95) with an affinity that is at least 5- fold, at least 10-fold, or at least 20-fold stronger than the affinity for mouse NOTUM (SEQ ID NO: 2).
- NOTUM neutralizing antibodies are provided that bind to human NOTUM (SEQ ID NO: 1) with an affinity that is at least 5-fold, at least 10-fold, or at least 20-fold stronger than the affinity for human NOTUM R144A/R145A (SEQ ID NO: 99).
- a neutralizing antibody against NOTUM binds to human NOTUM (SEQ ID NO: 1) with an affinity (K D ) of less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 3 nM, or less than 2 nM, determined as described in Example 6.8.
- a neutralizing antibody against NOTUM has an IC 50 in an OPTS assay of less than 100 nM, less than 75 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM, determined as described in Example 6.4.1.
- a neutralizing antibody against NOTUM has an IC 50 in a Wnt signaling assay of less than 100 nM, less than 75 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM, determined as described in Example 6.4.2.
- the IC 50 is for human NOTUM. In some embodiments, the IC 50 is for mouse NOTUM.
- neutralizing antibodies are non-human monoclonal antibodies. In some such embodiments, neutralizing antibodies are rodent monoclonal antibodies. In some such embodiments, neutralizing antibodies are mouse monoclonal antibodies. In some embodiments, neutralizing antibodies are chimeric monoclonal antibodies. In some embodiments, neutralizing antibodies are humanized monoclonal antibodies. In some embodiments, neutralizing antibodies are human monoclonal antibodies. In some embodiments, chimeric, humanized, and/or human monoclonal antibodies are useful as therapeutic antibodies in humans.
- neutralizing antibodies are antibody fragments.
- Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, scFv, Fd, diabodies, and the like.
- Nonlimiting exemplary NOTUM neutralizing antibodies include MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78. Each of MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78 neutralizes at least one activity of NOTUM. Further, at least MAbs 1.802, 1.815, 1.846, and 2.78 are dependent for binding to NOTUM on at least a portion of the region of human NOTUM bounded by amino acids Q47 to M177.
- a NOTUIVI neutralizing antibody competes for binding to NOTUM with at least one antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78.
- a NOTU M neutralizing antibody binds to an epitope of NOTUM that at least partially overlaps with the epitope bound by at least one antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78.
- an antibody that competes for binding to NOTUM with at least one antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78 is predicted to be a NOTUM neutralizing antibody.
- the sequences of the CDRs and variable regions of MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78 are shown in Section 7, below.
- NOTUM neutralizing antibodies are provided that bind to the same epitope to which MAb 1.731 binds. In some embodiments, NOTUM neutralizing antibodies are provided that bind to the same epitope to which MAb 1.802 binds. In some embodiments, NOTUM neutralizing antibodies are provided that bind to the same epitope to which MAb 1.815 binds. In some embodiments, NOTUM neutralizing antibodies are provided that bind to the same epitope to which MAb 1.846 binds. In some embodiments, NOTUM neutralizing antibodies are provided that bind to the same epitope to which MAb 2.1029 binds. In some embodiments, NOTUM neutralizing antibodies are provided that bind to the same epitope to which MAb 2.55 binds. In some embodiments, NOTUM neutralizing antibodies are provided that bind to the same epitope to which MAb 2.78 binds.
- a NOTUM neutralizing antibody comprises a heavy chain variable region selected from SEQ ID NOs: 7, 15, 23, 31, 39, and 47. In some embodiments, a NOTUM neutralizing antibody comprises a light chain variable region selected from SEQ ID NOs: 8, 16, 24, 32, 40, and 48. In some embodiments, a NOTUM neutralizing antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7, and a light chain variable region having the amino acid sequence of SEQ ID NO: 8. In some embodiments, a NOTUM neutralizing antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15, and a light chain variable region having the amino acid sequence of SEQ ID NO: 16.
- a NOTUM neutralizing antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23, and a light chain variable region having the amino acid sequence of SEQ ID NO: 24. In some embodiments, a NOTUM neutralizing antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 31, and a light chain variable region having the amino acid sequence of SEQ ID NO: 32. In some
- a NOTUM neutralizing antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 39, and a light chain variable region having the amino acid sequence of SEQ ID NO: 40.
- a NOTUM neutralizing antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47, and a light chain variable region having the amino acid sequence of SEQ ID NO: 48.
- a NOTU neutralizing antibody comprises a heavy chain
- a NOTUM neutralizing antibody comprises a heavy chain CDR2 selected from SEQ ID NOs: 10, 18, 26, 34, 42, and 50.
- a NOTUM neutralizing antibody comprises a heavy chain CDR3 selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, and 91.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 10, and a CDR3 having the amino acid sequence of SEQ ID NO: 11.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 17 and 90, a CDR2 having the amino acid sequence of SEQ ID NO: 18, and a CDR3 having an amino acid sequence selected from SEQ ID NOs: 19 and 91.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 17, a CDR2 having the amino acid sequence of SEQ ID NO: 18, and a CDR3 having the amino acid sequence of SEQ ID NO: 19.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 25 and 90, a CDR2 having the amino acid sequence of SEQ ID NO: 26, and a CDR3 having the amino acid sequence of SEQ ID NO: 27.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 25, a CDR2 having the amino acid sequence of SEQ ID NO: 26, and a CDR3 having the amino acid sequence of SEQ ID NO: 27.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 33 and 90, a CDR2 having the amino acid sequence of SEQ ID NO: 34, and a CDR3 having an amino acid sequence selected from SEQ ID NOs: 35 and 91.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 33, a CDR2 having the amino acid sequence of SEQ ID NO: 34, and a CDR3 having the amino acid sequence of SEQ ID NO: 35.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 41, a CDR2 having the amino acid sequence of SEQ ID NO: 42, and a CDR3 having the amino acid sequence of SEQ ID NO: 43.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 49, a CDR2 having the amino acid sequence of SEQ ID NO: 50, and a CDR3 having the amino acid sequence of SEQ ID NO: 51.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 57, a CDR2 having the amino acid sequence of SEQ ID NO: 58, and a CDR3 having the amino acid sequence of SEQ ID NO: 59.
- X x in SEQ ID NO: 90 is selected from Y and F.
- X 2 in SEQ ID NO: 91 is selected from H and N.
- a NOTUM neutralizing antibody comprises a light chain CDRl selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, and 92.
- a NOTUM neutralizing antibody comprises a light chain CDR2 selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, and 93. In some embodiments, a NOTUM neutralizing antibody comprises a light chain CDR3 selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, and 62. In some embodiments, a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 12, a CDR2 having the amino acid sequence of SEQ ID NO: 13, and a CDR3 having the amino acid sequence of SEQ ID NO: 14.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 20 and 92, a CDR2 having an amino acid sequence selected from SEQ ID NOs: 21 and 93, and a CDR3 having the amino acid sequence of SEQ ID NO: 22.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 20, a CDR2 having the amino acid sequence of SEQ ID NO: 21, and a CDR3 having the amino acid sequence of SEQ ID NO: 22.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 28 and 92, a CDR2 having an amino acid sequence selected from SEQ ID NOs: 29 and 93, and a CDR3 having the amino acid sequence of SEQ ID NO: 30.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 28, a CDR2 having the amino acid sequence of SEQ ID NO: 29, and a CDR3 having the amino acid sequence of SEQ ID NO: 30.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 36 and 92, a CDR2 having an amino acid sequence selected from SEQ ID NOs: 37 and 93, and a CDR3 having the amino acid sequence of SEQ ID NO: 38.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 36, a CDR2 having the amino acid sequence of SEQ ID NO: 37, and a CDR3 having the amino acid sequence of SEQ ID NO: 38.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 44, a CDR2 having the amino acid sequence of SEQ ID NO: 45, and a CDR3 having the amino acid sequence of SEQ ID NO: 46.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 52, a CDR2 having the amino acid sequence of SEQ ID NO: 53, and a CDR3 having the amino acid sequence of SEQ ID NO: 54.
- a NOTUM neutralizing antibody comprises a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 60, a CDR2 having the amino acid sequence of SEQ ID NO: 61, and a CDR3 having the amino acid sequence of SEQ ID NO: 62.
- X 3 in SEQ ID NO: 92 is selected from I and S;
- X 4 in SEQ ID NO: 92 is selected from T and E; and
- X 5 in SEQ ID NO: 92 is selected from and I.
- X 6 in SEQ ID NO: 93 is selected from D and N.
- a NOTU neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 10, and a CDR3 having the amino acid sequence of SEQ ID NO: 11; and a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 12, a CDR2 having the amino acid sequence of SEQ ID NO: 13, and a CDR3 having the amino acid sequence of SEQ ID NO: 14.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 17 and 90, a CDR2 having the amino acid sequence of SEQ ID NO: 18, and a CDR3 having an amino acid sequence selected from SEQ ID NOs: 19 and 91; and a light chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 20 and 92, a CDR2 having an amino acid sequence selected from SEQ ID NOs: 21 and 93, and a CDR3 having the amino acid sequence of SEQ ID NO: 22.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 17, a CDR2 having the amino acid sequence of SEQ ID NO: 18, and a CDR3 having the amino acid sequence of SEQ ID NO: 19; and a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 20, a CDR2 having the amino acid sequence of SEQ ID NO: 21, and a CDR3 having the amino acid sequence of SEQ ID NO: 22.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 25 and 90, a CDR2 having the amino acid sequence of SEQ ID NO: 26, and a CDR3 having the amino acid sequence of SEQ ID NO: 27; and a light chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 28 and 92, a CDR2 having an amino acid sequence selected from SEQ ID NOs: 29 and 93, and a CDR3 having the amino acid sequence of SEQ ID NO: 30.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 25, a CDR2 having the amino acid sequence of SEQ ID NO: 26, and a CDR3 having the amino acid sequence of SEQ ID NO: 27; and a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 28, a CDR2 having the amino acid sequence of SEQ ID NO: 29, and a CDR3 having the amino acid sequence of SEQ ID NO: 30.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 33 and 90, a CDR2 having the amino acid sequence of SEQ ID NO: 34, and a CDR3 having an amino acid sequence selected from SEQ ID NOs: 35 and 91; and a light chain comprising a CDRl having an amino acid sequence selected from SEQ ID NOs: 36 and 92, a CDR2 having an amino acid sequence selected from SEQ ID NOs: 37 and 93, and a CDR3 having the amino acid sequence of SEQ ID NO: 38.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 33, a CDR2 having the amino acid sequence of SEQ ID NO: 34, and a CDR3 having the amino acid sequence of SEQ ID NO: 35; and a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 36, a CDR2 having the amino acid sequence of SEQ ID NO: 37, and a CDR3 having the amino acid sequence of SEQ ID NO: 38.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 41, a CDR2 having the amino acid sequence of SEQ ID NO: 42, and a CDR3 having the amino acid sequence of SEQ ID NO: 43; and a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 44, a CDR2 having the amino acid sequence of SEQ ID NO: 45, and a CDR3 having the amino acid sequence of SEQ ID NO: 46.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 49, a CDR2 having the amino acid sequence of SEQ ID NO: 50, and a CDR3 having the amino acid sequence of SEQ ID NO: 51; and a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 52, a CDR2 having the amino acid sequence of SEQ ID NO: 53, and a CDR3 having the amino acid sequence of SEQ ID NO: 54.
- a NOTUM neutralizing antibody comprises a heavy chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 57, a CDR2 having the amino acid sequence of SEQ ID NO: 58, and a CDR3 having the amino acid sequence of SEQ ID NO: 59; and a light chain comprising a CDRl having the amino acid sequence of SEQ ID NO: 60, a CDR2 having the amino acid sequence of SEQ ID NO: 61, and a CDR3 having the amino acid sequence of SEQ ID NO: 62.
- X x in SEQ ID NO: 90 is selected from Y and F.
- X 2 in SEQ ID NO: 91 is selected from H and N.
- X 3 in SEQ ID NO: 92 is selected from I and S; X 4 in SEQ ID NO: 92 is selected from T and E; and X 5 in SEQ ID NO: 92 is selected from M and I.
- X 6 in SEQ ID NO: 93 is selected from D and N.
- NOTUM neutralizing antibodies that specifically bind human NOTUM are provided.
- NOTUM neutralizing antibodies that specifically bind to the same epitope in NOTUM from different species i.e., antibodies that demonstrate cross- reactivity
- NOTUM neutralizing antibodies that specifically bind human NOTUM and also specifically bind at least one species of NOTUM selected from mouse, rat, guinea pig, cynomolgus monkey, marmoset, and rhesus macaque are provided.
- NOTUM neutralizing antibodies that specifically bind human NOTUM and NOTUM from at least one species of non-human primate are provided.
- NOTUM neutralizing antibodies that specifically bind human NOTUM and mouse NOTUM are provided.
- non-human antibodies are chimerized.
- mouse monoclonal antibodies that specifically bind human NOTUM are chimerized.
- Certain exemplary methods for making chimeric antibodies are provided, for example, in Morrison et a l. (1984) Proc. Nat'l Acad. Sci. USA 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; Ta keda et al. (1985) Nature 314:452-454; and U.S. Patent Nos. 6,075,181 and 5,877,397.
- non-human antibodies are "huma nized .”
- mouse monoclonal antibodies that specifically bind hu man NOTUM are humanized.
- mouse monoclonal antibodies raised against mouse NOTUM, but which specifically bind (i.e., cross react) with human NOTUM are humanized.
- humanized antibodies retain their binding specificity and have reduced imm unogenicity (e.g., reduced human anti-mouse antibody (HAMA) response) when administered to a human.
- HAMA reduced human anti-mouse antibody
- humanization is achieved by methods including CDR grafting and human engineering, as described in detail below.
- one or more complementarity determining regions (CDRs) from the light and heavy chain variable regions of an antibody with the desired binding specificity are grafted onto human framework regions (FRs) in an "acceptor” antibody.
- CDR grafting is described, e.g., in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, a nd 5,530,101; Queen et al. ( 1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033.
- one or more CDRs from the light a nd heavy chain variable regions are grafted onto consensus human FRs in an acceptor antibody.
- consensus human FRs in some embodiments, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid seq uence.
- FR amino acids in the acceptor antibody are replaced with FR amino acids from the donor antibody.
- FR amino acids from the donor antibody are amino acids that contribute to the affinity of the donor antibody for the target antigen. See, e.g., in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.
- computer programs are used for modeling donor and/or acceptor antibodies to identify residues that are likely to be involved in binding antigen and/or to contribute to the structure of the antigen binding site, thus assisting in the selection of residues, such as FR residues, to be replaced in the donor antibody.
- CDRs from a donor antibody are grafted onto an acceptor antibody comprising a human constant region.
- FRs are also grafted onto the acceptor.
- CDRs from a donor antibody are derived from a single chain Fv antibody.
- FRs from a donor antibody are derived from a single chain Fv antibody.
- grafted CDRs in a humanized antibody are further modified (e.g., by amino acid substitutions, deletions, or insertions) to increase the affinity of the huma nized a ntibody for the target antigen.
- grafted FRs in a humanized a ntibody are further modified (e.g., by amino acid substitutions, deletions, or insertions) to increase the affinity of the humanized antibody for the target antigen.
- non-human antibodies may be humanized using a "human engineering" method. See, e.g., U.S. Patent Nos. 5,766,886 and 5,869,619.
- information on the structure of antibody va riable domains e.g., information obtained from crystal structures and/or molecular modeling
- human variable region consensus sequences are generated to identify residues that are conserved among human variable regions. In some embodiments, that information provides guidance as to whether an amino acid residue in the variable region of a non-huma n antibody should be substituted.
- a humanized NOTUM neutra lizing antibody comprises a heavy chain com prising at least one of CDRl, CDR2, and CDR3 of an a ntibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78.
- a NOTUM neutralizing antibody comprises a heavy chain comprising CDRl, CDR2, and CDR3 of an antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78.
- a NOTUM neutralizing a ntibody com prises a light chain comprising at least one of CDRl, CDR2, and CDR3 of an antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78.
- a NOTUM neutralizing antibody comprises a light chain comprising CDRl, CDR2, and CDR3 of an antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78.
- a NOTU M neutralizing antibody comprises heavy chain CDRl, CDR2, and CDR3, and light chain CDRl, CDR2, and CDR3 from an antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, and 2.78.
- a NOTUM neutralizing antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 63, 67, 71, 75, and 79.
- a NOTU M neutralizing antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ I D NOs: 64, 68, 72, 76, and 80.
- a NOTUM neutralizing antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 65, 69, 73, 77, and 81.
- a NOTUM neutralizing antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 66, 70, 74, 78, and 82.
- a NOTUM neutralizing antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a light chain comprising the amino acid sequence of SEQ I D NO: 65.
- a NOTUM neutralizing antibody comprises a heavy chain comprising the a mino acid sequence of SEQ ID NO: 67 a nd a light chain comprising the a mino acid seq uence of SEQ I D NO: 69.
- a NOTUM neutralizing antibody com prises a heavy chain comprising the amino acid sequence of SEQ ID NO: 71 and a light chain comprising the amino acid sequence of SEQ ID NO: 73.
- a NOTU M neutralizing antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a light chain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, a NOTU M neutralizing antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 79 and a light chain comprising the amino acid sequence of SEQ ID NO: 81. In some embodiments, a NOTUM neutra lizing antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 66.
- a NOTUM neutralizing antibody comprises a heavy chain comprising the amino acid seq uence of SEQ I D NO: 68 and a light chain com prising the amino acid sequence of SEQ ID NO: 70.
- a NOTUM neutralizing antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 74.
- a NOTU M neutralizing antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76 and a light chain comprising the amino acid sequence of SEQ I D NO: 78.
- a NOTUM neutralizing antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 82.
- an antibody against NOTUM is of any isotype selected from IgM, IgD, IgG, IgA, and IgE.
- an antibody against NOTUM is of the IgG isotype.
- an antibody is of the subclass IgGl, lgG2, lgG3, or lgG4.
- an antibody against NOTUM is of the IgM isotype.
- an antibody is of the subclass IgM l or lgM2.
- an antibody against NOTUM is of the IgA isotype. In certain such embodiments, an antibody is of the subclass IgAl or lgA2.
- An antibody against NOTUM may comprise a lambda or kappa light chain constant region of, e.g., either human or mouse origin.
- an antibody against NOTUM comprises a human kappa light chain constantnt region and a human IgGl, lgG2, or lgG4 heavy chain constant region.
- an antibody against NOTU M comprises a mouse kappa light chain and a mouse IgGl or lgG2 heavy chain.
- an antibody is modified to alter one or more of its properties.
- a modified antibody may possess advantages over an unmodified antibody, such as increased stability, increased time in circulation, or decreased immunogenicity ⁇ see, e.g., U.S. Patent No. 4,179,337).
- an antibody is modified by linking it to a nonproteinaceous moiety.
- an antibody is modified by altering the glycosylation state of the antibody, e.g., by altering the number, type, linkage, and/or position of carbohydrate chains on the antibody.
- an antibody is altered so that it is not glycosylated.
- one or more chemical moieties are linked to the amino acid backbone and/or carbohydrate residues of the antibody.
- Certain exemplary methods for linking a chemical moiety to an antibody include, but are not limited to, acylation reactions or alkylation reactions. See, e.g, EP 0 401 384; Malik et al. (1992), Exp. Hematol..
- any of these reactions are used to generate an antibody that is chemically modified at its amino-terminus.
- an antibody is linked to a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label.
- a detectable label allows for the detection or isolation of the antibody.
- a detectable label allows for the detection of an antigen bound by the antibody.
- an antibody is modified by linking it to one or more polymers.
- an antibody is linked to one or more water-soluble polymers.
- linkage to a water-soluble polymer reduces the likelihood that the antibody will precipitate in an aqueous environment, such as a physiological environment.
- a therapeutic antibody is linked to a water-soluble polymer.
- one skilled in the art can select a suitable water-soluble polymer based on considerations including whether the polymer/antibody conjugate will be used in the treatment of a patient and, if so, the pharmacological profile of the antibody (e.g., half-life, dosage, activity, antigenicity, and/or other factors).
- Certain exemplary clinically acceptable, water-soluble polymers include, but are not limited to, polyethylene glycol (PEG); polyethylene glycol propionaldehyde; copolymers of ethylene glycol/propylene glycol; monomethoxy-polyethylene glycol; carboxymethylcellulose; dextran; polyvinyl alcohol (PVA); polyvinyl pyrrolidone, poly-1, 3-dioxolane; poly-l,3,6-trioxane;
- ethylene/maleic anhydride copolymer ethylene/maleic anhydride copolymer; poly-p-amino acids (either homopolymers or random copolymers); poly(n-vinyl pyrrolidone)polyethylene glycol; polypropylene glycol homopolymers (PPG) and other polyalkylene oxides; polypropylene oxide/ethylene oxide copolymers;
- PEG polyoxyethylated polyols
- glycerol e.g., glycerol
- other polyoxyethylated polyols e.g., glycerol
- PEGs include, but are not limited to, certain forms known in the art to be useful in antibody modification, such as mono-(Ci-C 10 ) alkoxy- or aryloxy-PEG.
- PEG propionaldehyde may have advantages in
- a water-soluble polymer is of any molecular weight. In some embodiments, a water-soluble polymer is branched or unbranched. In some embodiments, a water-soluble polymer has an average molecular weight of about 2 kDa to about 100 kDa, including all points between the end points of the range. In some embodiments, a water-soluble polymer has an average molecular weight of about 5 kDa to about 40 kDa. In some embodiments, a water- soluble polymer has an average molecular weight of about 10 kDa to about 35 kDa. In some embodiments, a water-soluble polymer has an average molecular weight of about 15 kDa to about 30 kDa.
- an antibody is linked to polyethylene glycol (PEG; i.e., an antibody is "pegylated”).
- PEG polyethylene glycol
- PEG has low toxicity in mammals. See Carpenter et al. (1971) Toxicol. Appl. Pharmacol., 18:35-40.
- a PEG adduct of adenosine deaminase was approved in the United States for use in humans for the treatment of severe combined immunodeficiency syndrome.
- PEG may reduce the immunogenicity of antibodies.
- linkage of PEG to an antibody having non-huma n sequences may red uce the antigenicity of that antibody when administered to a human.
- a polymer is linked to one or more reactive amino acid residues in an antibody.
- Certain exemplary reactive amino acid residues include, but are not limited to, the alpha-amino group of the amino-terminal amino acid, the epsilon amino groups of lysine side chains, the sulfhydryl groups of cysteine side chains, the carboxyl groups of aspartyl and glutamyl side chains, the alpha-carboxyl group of the carboxy-terminal amino acid, tyrosine side chains, a nd activated glycosyl chains linked to certain asparagine, serine or threonine residues.
- PEG reagents suitable for direct reaction with proteins are known to those skilled in the art.
- PEG reagents suitable for linkage to amino groups include, but are not limited to, active esters of carboxylic acid or carbonate derivatives of PEG, for example, those in which the leaving groups are N-hydroxysuccinimide, p- nitrophenol, imidazole or l-hydroxy-2-nitrobenzene-4-sulfonate.
- PEG reagents containing maleimido or haloacetyl groups are used to modify sulfhydryl groups.
- PEG reagents containing amino, hydrazine and/or hydrazide groups may be used in reactions with aldehydes generated by periodate oxidation of carbohydrate groups in proteins.
- a water-soluble polymer has at least one reactive group.
- an activated derivative of a water-soluble polymer such as PEG, is created by reacting the water-soluble polymer with a n activating group.
- an activating group may be monofunctional, bifunctional, or multifunctional.
- Certain exemplary activating groups that can be used to link a water-soluble polymer to two or more antibodies include, but are not limited to, the following groups: sulfone (e.g., chlorosulfone, vi nylsulfone and divinylsulfone), maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and 5-pyridyl.
- sulfone e.g., chlorosulfone, vi nylsulfone and divinylsulfone
- maleimide e.g., sulfhydryl
- thiol e.g., triflate
- tresylate e.g., azidirine, oxirane and 5-pyridyl.
- a PEG derivative is typically stable against hydrolysis for extended periods in aqueous environments at pHs of about 11 or less.
- a PEG derivative linked to another molecule, such as an antibody confers stability from hydrolysis on that molecule.
- Certain exemplary homobifunctional PEG derivatives include, but are not limited to, PEG-bis-chlorosulfone and PEG-bis-vinylsulfone (see WO 95/13312).
- monoclonal antibodies are produced by standard techniques.
- monoclonal antibodies are produced by hybridoma-based methods. Certain such methods are known to those skilled in the art. See, e.g., Kohler et al. (1975) Nature 256:495-497; Harlow and Lane (1988) Antibodies: A Laboratory Manual Ch. 6 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
- a suitable animal such as a mouse, rat, hamster, monkey, or other mammal, is immunized with an immunogen to produce antibody-secreting cells.
- the antibody-secreting cells are B-cells, such as lymphocytes or splenocytes.
- lymphocytes e.g., human lymphocytes
- lymphocytes are immunized in vitro to generate antibody-secreting cells. See, e.g., Borreback et al. (1988) Proc. Nat'l Acad. Sci. USA 85:3995-3999.
- antibody secreting cells are fused with an "immortalized" cell line, such as a myeloid-type cell line, to produce hybridoma cells.
- hybridoma cells that produce the desired antibodies are identified, for example, by ELISA.
- such cells can then be subcloned and cultured using standard methods.
- such cells can also be grown in vivo as ascites tumors in a suitable animal host.
- monoclonal antibodies are isolated from hybridoma culture medium, serum, or ascites fluid using standard separation procedures, such as affinity chromatography. Guidance for the production of hybridomas and the purification of monoclonal antibodies according to certain embodiments is provided, for example, in Harlow and Lane (1988) Antibodies: A Laboratory Manual Ch. 8 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
- mouse monoclonal antibodies are produced by immunizing genetically altered mice with an immunogen.
- the mice are NOTUM- deficient mice, which partially or completely lack NOTUM function.
- the mice are "knockout" mice that lack all or part of a gene encoding NOTUM.
- such knockout mice are immunized with mouse NOTU M.
- such knockout mice are immunized with human NOTUM .
- human monoclonal antibodies are raised in transgenic anima ls (e.g., mice) that are capable of producing human antibodies. See, e.g., U.S. Patent Nos. 6,075,181 A and 6,114,598 A; and WO 98/24893 A2.
- human immunoglobulin genes are introduced ⁇ e.g., using yeast artificial chromosomes, huma n
- mice in which the endogenous Ig genes have been inactivated.
- Ig genes e.g., Jakobovits et al. (1993) Nature 362:255-258; Tomizuka et al. (2000) Proc. Nat'l Acad. Sci. USA 97:722-727; and Mendez et al. (1997) Nat. Genet. 15:146-156 (describing the XenoMouse I I ® line of transgenic mice).
- such transgenic mice are immunized with an imm unogen.
- lymphatic cells such as B-cells
- recovered cells are fused with an "immortalized" cell line, such as a myeloid-type cell line, to produce hybridoma cells.
- hybridoma cells are screened and selected to identify those that produce antibodies specific to the antigen of interest.
- human monoclonal antibodies are produced using a display- based method, such as, for example, any of those described below.
- a monoclonal antibody is produced using phage display techniques.
- phage display techniques are known to those skilled in the art and are described, for example, in Hoogenboom, Overview of Antibody Phage-Display Technology and Its Applications, from Methods in Molecular Biology: Antibody Phage Display: Methods and Protocols (2002) 178: 1-37 (O'Brien and Aitken, eds., Human Press, Totowa, NJ).
- a library of antibodies are displayed on the surface of a filamentous phage, such as the nonlytic filamentous phage fd or M 13.
- the antibodies are antibody fragments, such as scFvs, Fabs, Fvs with an engineered intermoiecular disulfide bond to stabilize the V H -V L pair, and diabodies.
- antibodies with the desired binding specificity can then be selected.
- Nonlimiting exemplary embodiments of antibody phage display methods are described in further detail below.
- an antibody phage-display library can be prepared using certain methods known to those skilled in the art. See, e.g., Hoogenboom, Overview of Antibody Phage-Display Technology and Its Applications, from Methods in Molecular Biology: Antibody Phage Display: Methods and Protocols (2002) 178:1-37 (O'Brien and Aitken, eds., Human Press, Totowa, NJ).
- variable gene repertoires are prepared by PCR amplification of genomic DNA or cDNA derived from the mRNA of antibody-secreting cells.
- cDNA is prepared from mRNA of B-cells.
- cDNA encoding the variable regions of heavy and light chains is amplified, for example, by PCR.
- heavy chain cDNA and light chain cDNA are cloned into a suitable vector.
- heavy chain cDNA and light chain cDNA are randomly combined during the cloning process, thereby resulting in the assembly of a cDNA library encoding diverse scFvs or Fabs.
- heavy chain cDNA and light chain cDNA are ligated before being cloned into a suitable vector.
- heavy chain cDNA and light chain cDNA are ligated by stepwise cloning into a suitable vector.
- cDNA is cloned into a phage display vector, such as a phagemid vector.
- a phagemid vector such as a phagemid vector.
- Certain exemplary phagemid vectors, such as pCESl, are known to those skilled in the art.
- cDNA encoding both heavy and light chains is present on the same vector.
- cDNA encoding scFvs are cloned in frame with all or a portion of gene III, which encodes the minor phage coat protein pill.
- the phagemid directs the expression of the scFv-plll fusion on the phage surface.
- cDNA encoding heavy chain is cloned in frame with all or a portion of gene III, and cDNA encoding light chain (or heavy chain) is cloned downstream of a signal sequence in the same vector.
- the signal sequence directs expression of the light chain (or heavy chain) into the periplasm of the host cell, where the heavy and light chains assemble into Fab fragments.
- cDNA encoding heavy chain and cDNA encoding light chain are present on separate vectors.
- heavy chain and light chain cDNA is cloned separately, one into a phagemid and the other into a phage vector, which both contain signals for in vivo recombination in the host cell.
- recombinant phagemid or phage vectors are introduced into a suitable bacterial host, such as E. coli.
- a suitable bacterial host such as E. coli.
- the host is infected with helper phage to supply phage structural proteins, thereby allowing expression of phage particles carrying the antibody-pill fusion protein on the phage surface.
- "synthetic" antibody libraries are constructed using repertoires of variable genes that are rearranged in vitro. For example, in some embodiments, individual gene segments encoding heavy or light chains (V-D-J or V-J, respectively) are randomly combined using PCR. In some embodiments, additional sequence diversity can be introduced into the CDRs, and possibly FRs, e.g., by error prone PCR. In some such embodiments, additional sequence diversity is introduced into CDR3, e.g., H3 of the heavy chain.
- "naive" or “universal” phage display libraries are constructed as described above using nucleic acid from an unimmunized animal.
- the unimmunized animal is a human.
- "immunized” phage display libraries are constructed as described above using nucleic acid from an immunized animal.
- the immunized animal is a human, rat, mouse, hamster, or monkey. In certain such embodiments, the animals are immunized with any of the immunogens described below.
- Certain exemplary universal human antibody phage display libraries are available from commercial sources.
- Certain exemplary libraries include, but are not limited to, the HuCAL * series of libraries from MorphoSys AG ( artinstreid/Munich, Germany); libraries from Crucell (Leiden, the Netherlands) using MAbstract' technology; the n-CoDeRTM Fab library from Biolnvent (Lund, Sweden); and libraries available from Cambridge Antibody Technology (Cambridge, UK).
- the selection of antibodies having the desired binding specificity from a phage display library is achieved by successive panning steps.
- library phage preparations are exposed to antigen.
- the phage-antigen complexes are washed, and unbound phage are discarded.
- bound phage are recovered and subsequently amplified by infecting £ coli.
- monoclonal antibody-producing phage may be cloned by picking single plaques. In some embodiments, the above process is repeated.
- the antigen used in panning is any of the immunogens described below.
- the antigen is immobilized on a solid support to allow purification of antigen-binding phage by affinity chromatography.
- the antigen is biotinylated, thereby allowing the separation of bound phage from unbound phage using streptavidin-coated magnetic beads.
- the antigen may be immobilized on cells (for direct panning), in tissue cryosections, or on membranes (e.g., nylon or nitrocellulose membranes). Other variations of certain panning procedures may be routinely determined by one skilled in the art.
- a yeast display system is used to produce monoclonal antibodies.
- an antibody is expressed as a fusion protein with all or a portion of the yeast AGA2 protein, which becomes displayed on the surface of the yeast cell wall.
- yeast cells expressing antibodies with the desired binding specificity can then be identified by exposing the cells to fluorescently labeled antigen.
- yeast cells that bind the antigen can then be isolated by flow cytometry. See, e.g., Boder et al. (1997) Nat. Biotechnol. 15:553-557.
- the affinity of an antibody for a particular antigen is increased by subjecting the antibody to affinity maturation (or "directed evolution") in vitro, in vivo, native antibodies undergo affinity maturation through somatic hypermutation followed by selection.
- affinity maturation or "directed evolution"
- affinity maturation or "directed evolution”
- in vitro methods mimic that in vivo process, thereby allowing the production of antibodies having affinities that equal or surpass that of native antibodies.
- mutations are introduced into a nucleic acid sequence encoding the variable region of an antibody having the desired binding specificity. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; Brekke et al. (2002) Nat. Reviews 2:52-62.
- mutations are introduced into the variable region of the heavy chain, light chain, or both.
- mutations are introduced into one or more CDRs. In certain such embodiments, mutations are introduced into H3, L3, or both.
- mutations are introduced into one or more FRs.
- a library of mutations is created, for example, in a phage, ribosome, or yeast display library, so that antibodies with increased affinity may be identified by standard screening methods. See, e.g., Boder et al. (2000) Proc. Nat'l Acad. Sci. USA 97:10701-10705; Foote et al. (2000) Proc. Nat'l Acad. Sci. USA
- mutations are introduced by site-specific mutagenesis based on information on the antibody's structure, e.g., the antigen binding site.
- mutations are introduced using combinatorial mutagenesis of CDRs.
- all or a portion of the variable region coding sequence is randomly mutagenized, e.g., using E. coli mutator cells, homologous gene rearrangement, or error prone PCR.
- mutations are introduced using "DNA shuffling.” See, e.g., Crameri et al. (1996) Nat. Med. 2:100-102; Fermer et al. (2004) Tumor Biol. 25:7-13.
- chain shuffling is used to generate antibodies with increased affinity.
- one of the chains e.g., the light chain
- the other chain e.g., the heavy chain
- a library of chain shuffled antibodies is created, wherein the unchanged heavy chain is expressed in combination with each light chain from the repertoire of light chains.
- such libraries may then be screened for antibodies with increased affinity.
- both the heavy and light chains are sequentially replaced. In some embodiments, only the varia ble regions of the heavy and/or light chains are replaced.
- variable regions e.g., CDRs
- CDRs variable regions
- mouse monoclonal antibodies that specifically bind human NOTU M are subject to sequential chain shuffling.
- the heavy chain of a given mouse monoclonal antibody is combined with a new repertoire of human light chains, and antibodies with the desired affinity are selected.
- the light chains of the selected antibodies are then combined with a new repertoire of human heavy chains, and antibodies with the desired affinity are selected.
- human antibodies having the desired antigen binding specificity and affinity are selected.
- the heavy chain of a given mouse monoclonal antibody is combined with a new repertoire of human light chains, and antibodies with the desired affinity are selected from this first round of shuffling.
- the light chain of the original mouse monoclonal antibody is combined with a new repertoire of human heavy chains, and antibodies with the desired affinity are selected from this second round of shuffling.
- human light chains from the antibodies selected in the first round of shuffling are then combined with human heavy chains from the antibodies selected in the second round of shuffling.
- huma n antibodies having the desired a ntigen binding specificity and affinity are selected.
- a "ribosome display” method is used that alternates antibody selection with affinity maturation.
- antibody-encoding nucleic acid is amplified by RT-PCR between the selection steps.
- error prone polymerases may be used to introduce mutations into the nucleic acid. A nonlimiting example of such a method is described in detail in Ha nes et al. (1998) Proc. Natl Acad. Sci. USA 95:14130-14135.
- a monoclonal antibody is produced by recombinant techniques. See, e.g., U.S. Patent No. 4,816,567.
- nucleic acid encoding monoclonal antibody chains are cloned and expressed in a suitable host cell.
- RNA can be prepared from cells expressing the desired antibody, such as mature B-cells or hybridoma cells, using standard methods.
- the RNA can then be used to make cDNA using standard methods.
- cDNA encoding a heavy or light chain polypeptide is amplified, for example, by PCR, using specific oligonucleotide primers.
- the cDNA is cloned into a suitable expression vector.
- the expression vector is then transformed or transfected into a suitable host cell, such as a host cell that does not endogenously produce antibody.
- suitable host cells include, but are not limited to, f. coll, COS cells, Chinese hamster ovary (CHO) cells, and myeloma cells.
- reconstituted antibody may be isolated.
- cDNA encoding a heavy or light chain can be modified.
- the constant region of a mouse heavy or light chain can be replaced with the constant region of a human heavy or light chain.
- a chimeric antibody can be produced which possesses human antibody constant regions but retains the binding specificity of a mouse antibody.
- a nucleic acid molecule comprises a polynucleotide sequence that encodes the heavy chain or the light chain of a NOTUM neutralizing antibody.
- a single nucleic acid molecule comprises a first polynucleotide sequence that encodes the heavy chain of a NOTUM neutralizing antibody and a second polynucleotide sequence that encodes the light chain of a NOTUM neutralizing antibody.
- the coding sequence for the heavy chain and the coding sequence for the light chain are part of a continuous coding sequence such that a single polypeptide is expressed, which comprises both the heavy chain and the light chain of the antibody.
- a single nucleic acid molecule that encodes both a heavy chain and a light chain is capable of expressing the two chains as separate polypeptides.
- each chain is under the control of a separate promoter.
- the two chains are under the control of the same promoter.
- One skilled in the art can select a suitable configuration and suitable control elements for the heavy and light chain of the NOTUM neutralizing antibody according to the intended application.
- the nucleic acid is a vector, such as an expression vector suitable for expressing the heavy chain and/or light chain in a particular host cell.
- a suitable expression vector, or expression vectors can select a suitable expression vector, or expression vectors, according to the host cell to be used for expression. Many exemplary such vectors are known in the art.
- a nucleic acid molecule comprises a polynucleotide sequence that encodes a heavy chain of a NOTUM neutralizing antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, 2.78, and humanized versions of such MAbs.
- a nucleic acid molecule comprises a polynucleotide sequence selected from SEQ ID NOs: 101, 103, 105, 107, 109, 111, 112, 115, 116, 119, 120, 123, 124, 127, and 128.
- a nucleic acid molecule comprises a polynucleotide sequence that encodes a light chain of a NOTUM neutralizing antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, 2.78, and humanized versions of such MAbs.
- a nucleic acid molecule comprises a polynucleotide sequence selected from SEQ ID NOs: 102, 104, 106, 108, 110, 113, 114, 117, 118, 121, 122, 125, 126, 129, and 130.
- a nucleic acid molecule comprises a first polynucleotide sequence that encodes the heavy chain and a second polynucleotide sequence that encodes the light chain, of a NOTUM neutralizing antibody selected from MAbs 1.731, 1.802, 1.815, 1.846, 2.1029, 2.55, 2.78, and humanized versions of such MAbs.
- recombinant antibodies can be expressed in certain cell lines.
- sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell.
- transformation can be by any known method for introducing polynucleotides into a host cell.
- Certain exemplary methods include, but are not limited to, packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) and using certain transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455.
- the transformation procedure used may depend upon the host to be transformed.
- Certain exemplary methods for introduction of heterologous polynucleotides into mammalian cells include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
- Certain exemplary mammalian cell lines available as hosts for expression are known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), including Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
- ATCC American Type Culture Collection
- CHO Chinese hamster ovary
- HeLa cells HeLa cells
- BHK baby hamster kidney
- COS monkey kidney cells
- human hepatocellular carcinoma cells e.g., Hep G2
- cell lines may be selected by determining which cell lines produce high levels of antibodies that specifically bind NOTUM.
- This invention encompasses a method of stimulating endocortical bone formation in a patient, which comprises administering to a patient in need thereof an effective amount of an antibody of the invention. It also encompasses a method of increasing cortical bone thickness, comprising administering to a patient in need thereof an effective amount of an antibody of the invention. [0129] This invention encompasses a method of treating, managing, or preventing a disease or disorder associated with bone loss, which comprises adm i nostiring to a patient in need thereof a therapeutically or prophylactically effective amount of an a ntibody of the invention.
- osteoporosis e.g., postmenopausal osteoporosis, steroid- or glucocorticoid-induced osteoporosis, male osteoporosis, a nd idiopathic osteoporosis), osteopenia, and Paget's disease.
- Also encom passed by the invention is a method of treating, managing, or preventing bone fractures, which comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of an antibody of the invention.
- Particular bone fractures are associated with metastatic bone disease, i.e., ca ncer that has metastasized to bone.
- metastatic bone disease i.e., ca ncer that has metastasized to bone.
- cancers that can metastasize to bone include prostate, breast, lung, thyroid, and kidney cancer.
- This invention also encompasses a method of treating, managing, or preventing bone loss associated with, or caused by, a disease or disorder, which comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of an antibody of the invention.
- diseases and disorders include celiac disease, Crohn's Disease, Cushing's syndrome, hyperparathyroidism, inflammatory bowel disease, and ulcerative colitis.
- Nonlimiting exemplary patients that may benefit from methods of this invention include men and women aged 55 years or older, post-menopausal women, and patients suffering from renal insufficiency.
- Antibodies of the invention can be administered in combination (e.g., at the same or at different times) with other drugs known to be useful in the treatment, management, or prevention of diseases or conditions affecting the bone.
- drugs known to be useful in the treatment, management, or prevention of diseases or conditions affecting the bone.
- examples include: androgen receptor modulators; bisphosphonates; calcitonin; calcium sensing receptor antagonists; RANKL antibodies, cathepsin K inhibitors; estrogen and estrogen receptor modulators; integrin binders, antibodies, and receptor antagonists; parathyroid hormone (PTH) and analogues and mimics thereof; and vitamin D and synthetic vita min D analogues.
- PTH parathyroid hormone
- Examples of and rogen receptor modulators include finasteride and other 5a- reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.
- bisphosphonates include alendronate, cimadronate, clodronate, etidronate, ibandronate, incadronate, minodronate, neridronate, olpadronate, pamidronate, piridronate, risedronate, tiludronate, and zolendronate, and pharmaceutically acceptable salts and esters thereof.
- cathepsin K inhibitors examples include VEL-0230, AAE581 (balicatib),
- estrogen and estrogen receptor modulators include naturally occurring estrogens (e.g., 7-estradiol, estrone, and estriol), conjugated estrogens [e.g., conjugated equine estrogens), oral contraceptives, sulfated estrogens, progestogen, estradiol, droloxifene, raloxifene, lasofoxifene, TSE-424, ta moxifen, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2- dimethyl-l-oxopropoxy-4-methyl-2-[4-[2-(l-piperidinyl)ethoxy]phenyl]-2H-l-benzopyran-3-yl]- phenyl-2,2-dimethylpropanoate, 4,
- integrin binders examples include vitaxin (M EDI-522), cilengitide a nd L-000845704.
- This invention encompasses pharmaceutica l compositions comprising one or more a ntibodies of the invention, and optionally one or more other drugs, such as those described above.
- a NOTUM neutralizing antibody may be used as a therapeutic antibody.
- Exemplary NOTUM neutralizing antibodies to be used as thera Chamberic antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, and human antibodies. Those skilled in the art are familiar with the use of antibodies as therapeutic agents.
- a pharmaceutical composition that comprises an effective amount of an antibody to NOTUM and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
- a pharmaceutical composition is provided that comprises an effective amount of an antibody to NOTUM and an effective amount of at least one additional therapeutic agent, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
- at least one additional therapeutic agent is selected from those described above.
- formulation materials for pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed.
- the pharmaceutical composition comprises formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
- suitable formulation materials include, but are not limited to, amino acids (for example, glycine, glutamine, asparagine, arginine and lysine); antimicrobials; antioxidants (for example, ascorbic acid, sodium sulfite and sodium hydrogen- sulfite); buffers (for example, borate, bicarbonate, Tris-HCI, citrates, phosphates and other organic acids); bulking agents (for example, mannitol and glycine); chelating agents (for example, ethylenediamine tetraacetic acid (EDTA)); complexing agents (for example, caffeine,
- amino acids for example, glycine, glutamine, asparagine, arginine and lysine
- antimicrobials for example, ascorbic acid, sodium sulfite and sodium hydrogen- sulfite
- buffers for example, borate, bicarbonate, Tris-HCI, citrates, phosphates and other organic acids
- bulking agents for example,
- polyvinylpyrrolidone beta-cyclodextrin, and hydroxypropyl-beta-cyclodextrin
- fillers ; monosaccharides, disaccharides, and other carbohydrates (for exam ple, glucose, mannose and dextrins); proteins (for example, serum albumin, gelatin and immunoglobulins); coloring, flavoring, and diluting agents; emulsifying agents; hydrophilic polymers (for example, polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (for example, sodium); preservatives (for example, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and hydrogen peroxide); solvents (for example, glycerin, propylene glycol and polyethylene glycol); sugar alcohols (for example, mannitol and sorbi
- tromethamine lecithin, cholesterol, and tyloxapal
- stability enhancing agents for example, sucrose and sorbitol
- tonicity enhancing agents for example, alkali metal halides (for example, sodium or potassium chloride), mannitol, and sorbitol
- delivery vehicles diluents; excipients; and
- an antibody to NOTUM or other therapeutic molecule is linked to a half-life extending vehicle.
- exemplary half-life extending vehicles include those known in the art. Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Exemplary such vehicles are described, e.g., in published PCT Application No. WO 99/25044.
- an optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions may influence the physical state, stability, rate of in vivo release, or rate of in vivo clearance of a neutralizing antibody.
- a primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
- a suitable vehicle or carrier may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
- Exemplary vehicles include, but are not limited to, neutral buffered saline and saline mixed with serum albumin.
- pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.
- a composition comprising an antibody to NOTUM, with or without at least one additional therapeutic agents may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution.
- a composition comprising an antibody to NOTUM, with or without at least one additional therapeutic agent may be formulated as a lyophilizate using appropriate excipients such as sucrose.
- a pharmaceutical composition is selected for parenteral delivery. In some embodiments, a pharmaceutical composition is selected for inhalation or for delivery through the digestive tract, such as orally.
- compositions are within the skill of one skilled in the art.
- formulation components are present in concentrations that are acceptable to the site of administration.
- buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
- a pharmaceutical composition when parenteral administration is contemplated, may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired antibody to NOTUM, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle.
- a vehicle for parenteral injection is sterile distilled water in which the antibody to NOTUM, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved.
- the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide for the controlled or sustained release of the product which may then be delivered via a depot injection.
- an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide for the controlled or sustained release of the product which may then be delivered via a depot injection.
- hyaluronic acid may also be used, and may have the effect of promoting sustained duration in the circulation.
- implantable drug delivery devices may be used to introduce the desired molecule.
- a pharmaceutical composition may be formulated for inhalation.
- an antibody to NOTUM, with or without at least one additional therapeutic agent may be formulated as a dry powder for inhalation.
- an inhalation solution comprising an antibody to NOTUM, with or without at least one additional therapeutic agent, may be formulated with a propellant for aerosol delivery.
- solutions may be nebulized.
- a formulation may be administered orally.
- an antibody to NOTUM, with or without at least one additional therapeutic agent, that is administered in this fashion may be formulated with or without carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
- a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
- at least one additional agent can be included to facilitate absorption of the antibody to NOTUM with or without any additional therapeutic agents.
- diluents, flavorings, low melting point waxes, vegetable oils, lubrica nts, suspending agents, tablet disintegrating agents, and/or binders may also be employed.
- a pharmaceutical composition comprises an effective amount of an antibody to NOTUM, with or without at least one additional therapeutic agent, in a mixture with non-toxic excipients which are suitable for the manufacture of tablets.
- excipients include, but are not limited to, inert diluents (for example, calcium carbonate, sodium carbonate, sodium bicarbonate, lactose, and calcium phosphate); binding agents (for example, starch, gelatin, and acacia); and lubricating agents (for example, magnesium stearate, stearic acid, and talc).
- sustained- or controlled-delivery formulations include, but are not limited to, liposome carriers, bio-erodible microparticles, porous beads, and depot injections.
- sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g. films or microcapsules.
- sustained release matrices include, but are not limited to, polyesters, hydrogels, polylactides (see, e.g., U.S.
- Patent No. 3,773,919 and EP 058,481 copolymers of L-glutamic acid and gamma ethyl-L-glutamate (see, e.g., Sidman et al. (1983) Biopolvmers 22:547-556), poly (2- hydroxyethyl-methacrylate) (see, e.g., Langer et al. (1981) J. Biomed. Mater. Res. 15:167-277 and Langer (1982) Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer er al., supra), and poly-D(-)-3- hydroxybutyric acid (EP 133,988).
- L-glutamic acid and gamma ethyl-L-glutamate see, e.g., Sidman et al. (1983) Biopolvmers 22:547-556
- poly (2- hydroxyethyl-methacrylate) see, e.g
- sustained release compositions may include liposomes, which can be prepared, in some embodiments, by any of several methods known in the art. See e.g., Eppstein et al. (1985) Proc. Natl. Acad. Sci. USA. 82:3688-3692; EP 036,676; EP 088,046; and EP 143,949.
- a pharmaceutical composition to be used for in vivo administration typically is sterile. In some embodiments, this may be accomplished by filtration through sterile filtration membranes. In some embodiments, where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. In some embodiments, the composition for parenteral administration may be stored in lyophilized form or in a solution. In some embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- the pharmaceutical com position may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
- such formulations may be stored either in a ready-to- use form or in a form ⁇ e.g., lyophilized) that is reconstituted prior to administration.
- kits for producing a single-dose administration unit are provided.
- the kits may each contain both a first container having a dried protein and a second container having an aqueous formulation.
- kits containing single or multi-chambered pre-filled syringes e.g., liquid syringes and lyosyringes are included.
- the effective amount of a pharmaceutical composition comprising an antibody to NOTUM, with or without at least one additional therapeutic agent, to be employed therapeutically will depend, for example, upon the context and objectives of treatment.
- the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the antibody to NOTU M, with or without at least one additional therapeutic agent, is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient.
- the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
- a typical dosage may range from about 0.1 ⁇ g/kg of patient body weight, up to about 100 mg/kg or more, depending on the factors mentioned above. I n some embodiments, the dosage may range from 0.1 ⁇ g/kg up to about 100 mg/kg; 1 ⁇ g/kg up to about 100 mg/kg; or 5 ⁇ g/kg up to a bout 100 mg/kg, including all points (including fractions) between any of the foregoing endpoints. In some embodiments, the dosage is between about 1 mg/kg body weight and about 60 mg/kg body weight.
- the dosage is about 1 mg/kg body weight, about 3 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 20 mg/kg body weight, about 30 mg/kg body weight, about 40 mg/kg body weight, about 50 mg/kg body weight, or about 60 mg/kg body weight.
- a human dose of a neutralizing antibody against NOTUM is determined based on the efficacious dose of the same antibody in another species, such as mice, dogs, monkeys, etc.
- a human dose of a neutralizing antibody against NOTU M is determined using "Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Hea lthy Volunteers," U.S. Department of Health and Human Services, Food and Drug Administration, and Center for Drug Evaluation and Research (CDER), July 2005 (Pharmacology and Toxicology).
- a suitable dosage may be determined by one skilled in the art, for exa m ple, based on animal studies.
- a neutralizing antibody against NOTU is administered to a patient twice per week, once per week, once every two weeks, once per month, once every other month, or even less frequently.
- the frequency of dosing will take into account the pharmacokinetic parameters of an antibody to NOTUM and, if applicable, any additional therapeutic agents in the formulation used.
- a clinician will administer the composition until a dosage is reached that achieves the desired effect.
- the composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter.
- further refinement of the appropriate dosage is routinely made by those skilled in the art and is within the ambit of tasks routinely performed by them.
- appropriate dosages may be ascertained through use of appropriate dose-response data.
- a patient receives one dose of a pharmaceutical composition comprising an antibody to NOTUM. In some embodiments, a patient receives one, two, three, or four doses per day of a pharmaceutical composition comprising an antibody to NOTUM. In some embodiments, a patient receives one, two, three, four, five, or six doses per week of a pharmaceutical composition comprising an antibody to NOTUM. In some embodiments, a patient receives one, two, three, or four doses per month of a pharmaceutical composition comprising an antibody to NOTUM.
- the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by subcutaneous, intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices.
- the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
- the composition may be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated.
- the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
- an antibody to NOTUM, with or without at least one additional therapeutic agent is delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the
- such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic.
- the cells may be immortalized.
- the cells in order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues.
- the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
- mice homozygous for a genetically engineered mutation in the murine ortholog of the human NOTUM gene were generated using corresponding mutated embryonic stem (ES) cell clones from the OMNIBANK collection of mutated murine ES cell clones (see generally, U.S. Patent No. 6,080,576).
- ES cell clones containing a mutagenic viral insertion into the murine NOTUM locus were microinjected into blastocysts which were in turn implanted into
- mice homozygous (-/-) for the disruption of the NOTUM gene were studied in conjunction with mice heterozygous (+/-) for the disruption of the NOTUM gene and wild-type (+/+) litter mates.
- the mice were subject to a medical work-up using an integrated suite of medical diagnostic procedures designed to assess the function of the major organ systems in a mammalian subject.
- studying the homozygous (-/-) "knockout" mice in the described numbers and in conjunction with heterozygous (+/-) and wild-type (+/+) litter mates more reliable and repeatable data were obtained.
- N number of mice
- HEK293F cells were transfected using Lipofectamine2000 (Invitrogen) and grown in suspension culture in Freestyle 293 Expression Medium (Invitrogen) in shaker flasks. For transient transfections, conditioned medium was harvested four days after transfection, sterile filtered and stored at 4°C. For the generation of cell lines stably expressing NOTUM protein, genomic integration of the expression plasmid was selected for in the presence of puromycin.
- NOTUM protein Expression and secretion of NOTUM protein was confirmed by Western blot of cell lysates and/or conditioned medium, using an anti-His antibody. Subcloning of NOTUM-producing bulk stable transfectants by limiting dilution enabled the identification by anti-His Western blot of individual clones expressing NOTUM at relatively high levels.
- clonal HEK293F cell lines expressing either mouse or human NOTUM were expanded in suspension culture to a volume of 3L.
- the cell density at this volume reached 1 ⁇ 10 ⁇ 6 viable cells per ml, the cells were pelleted by centrifugation and resuspended in fresh Freestyle 293 Expression Medium and maintained in culture for a further 96 hours without additional medium changes. After 96 hours, cultures were harvested, cells were pelleted by centrifugation, and the conditioned medium was sterile filtered and stored at 4°C for subsequent processing.
- NOTUM-containing conditioned medium was concentrated from 3L to 1L and then buffer exchanged into nickel immobilized metal affinity chromatography (IMAC) buffer (20 mM Tris-HCI, 10 mM imidazole, 0.5 M NaCI, pH 7.4) by tangential flow filtration using a membrane with a lOkDa nominal molecular weight cut off.
- IMAC nickel immobilized metal affinity chromatography
- Antibodies were raised against purified recombinant human and mouse NOTUM proteins in two separate immunization campaigns.
- mice homozygous for a gene trap insertion in the NOTUM gene and therefore lacking endogenous NOTUM protein were immunized with human NOTUM protein as follows. Mice were primed with 20 g human NOTUM protein in complete Freund's adjuvant injected intraperitoneal ⁇ . Mice were boosted with 20 g human NOTUM protein in incomplete Freund's adjuvant injected intraperitoneal ⁇ every two to three weeks. Mice exhibiting a robust serum titer against human NOTUM as determined by ELISA received a final boost of 10 ⁇ g human NOTUM protein in PBS injected intravenously (i.v.).
- mice homozygous for a gene trap insertion in the NOTUM gene were immunized via the hind footpads with a priming immunization of 10 ⁇ g mouse NOTUM protein in TiterMax adjuvant with CpG DNA followed by ten boosts of 10 ⁇ g mouse NOTUM protein in Alum adjuvant with CpG DNA at three or four day intervals.
- Inguinal and popliteal lymph nodes were harvested from high titer mice after a final footpad boost with 10 ⁇ g mouse NOTUM protein in PBS.
- Spleens from i.v. boosted mice or lymph nodes from footpad immunized mice were collected four days after the final boost and were minced and strained to yield a cell suspension.
- Red blood cells were lysed and the cell suspension was enriched for B-cells by negative selection using magnetic beads coated with antibodies specific for non-B-cell populations.
- Hybridomas were generated by electro-cell fusion of enriched B-cells with mouse NSl myeloma cells and were seeded onto 96-well plates in hybridoma medium containing hypoxanthine and aminopterin to select for viable B-cell/myeloma cell hybridomas.
- Hybridomas were screened for the production of NOTUM-specific antibodies by assaying hybridoma conditioned medium for immunoreactivity with passively adsorbed NOTUM protein in an ELISA format. Hundreds of hybridomas secreting antibody specific for mouse and/or human NOTUM were found from both immunization campaigns.
- OPTS trisodium 8-octanoyloxypyrene-l,3,6-trisulfonate
- hybridoma conditioned medium in general interfered in the OPTS assay perhaps due to the release from dying cells of hydrolases that could also cleave the OPTS. For this reason, additional hybridoma conditioned medium was generated for those lines originally showing the highest level of binding activity by ELISA and antibody was purified in a 96-well format by affinity chromatography using protein A beads. These purified antibodies were then tested in the OPTS assay at a four-fold dilution without prior quantitation.
- Antibodies were tested in quadruplicate in 384-well plates. 12.5 ⁇ containing 125 ng of purified NOTUM in 4X reaction buffer (20 mM CaCI2, 2mM MgCI2, 50mM Tris-HCI, pH7.4) was added to 12.5 ⁇ of purified antibody. After mixing, antibody and NOTUM were incubated at room temperature for 20 minutes followed by addition of 25 ⁇ of 1.25 ⁇ OPTS (Sigma, catalog # 74875) in 50 mM Tric-HCI, pH7.4. After mixing, the enzyme reaction was allowed to proceed at room temperature for 10 minutes before being stopped by addition of 25 ⁇ of 3% SDS. Plates were read on an Envision plate reader with an excitation wavelength of 485nm and emission wavelength of 535 nm to quantify the amount of cleavage product.
- OPTS assay screening of 1,056 mouse NOTUM immunoreactive hybridomas identified from Campaign 2 yielded six antibodies that showed greater than 50% inhibition of mouse NOTUM. These six together with an additional six hybridomas exhibiting some degree of neutralization in the OPTS assay were selected for subcloning by limiting dilution and small scale purified antibody production by protein A affinity chromatography using 50ml conditioned medium from clonal hybridomas.
- NOTUM can act as a negative regulator of Wnt signaling.
- Antibody neutralizing activity determined through the effect on Wnt signaling, was determined in a Wnt signaling assay, which uses CellSensor ® technology and conditioned media prepared as follows. Plasmid containing human NOTUM in pcDNA3.1(+) vector was transfected into HEK293 cells and clones were selecting by growing in presence of 400 ⁇ g/mL of G418. Condition media from these cells was used for the assay. L cells overexpressing and secreting Wnt3a into the conditioned media were purchased from ATCC.
- the assay protocol was as follows. CellSensor ® LEF/TCF-bla FreestyleTM 293F cells (Invitrogen) were grown to near confluency in 15-cm plates in DMEM with 10% Dialyzed FBS, 5 ⁇ Blasticidin (Invitrogen, R210-01), 0.1 mM NEAA, 25 mM HEPES and lxGPS. Cells were trypsinized by first rinsing with PBS, followed by addition of 5 mL trypsin and incubation of plates at room temperature for two minutes.
- a total of 10 mL of assay media (Opti-MEM, plus 0.5% dialyzed FBS, 0.1 mM NEAA, ImM sodium pyruvate, 10 mM HEPES, lx GPS) was then added per 15 cm plate. Cells were counted and suspended at 50,000 cells per mL. Cells were seeded into Biocoat 384-well plates (Fisher, Catalogue #356663) at a density of 10000 cells per 20 ⁇ per well. After incubation of cells at 37°C for 3 hours, 10 ⁇ of 30 mM LiCI in assay medium was added per well, followed by incubation at 37°C overnight.
- assay media Opti-MEM, plus 0.5% dialyzed FBS, 0.1 mM NEAA, ImM sodium pyruvate, 10 mM HEPES, lx GPS
- the assay plate was incubated for 5 hours at 37°C to enable Wnt-mediated beta-lactamase upregulation, and then 8 ⁇ LiveBLAzerTM-FRET B/G Substrate (CCF4- AM, Invitrogen) was added to each well and the plate incubated in the dark at room temperature for 3 hours. Plates were then read on an Envision plate reader using an excitation wavelength of 400 nm and emission wavelengths of 460 nm and 535 nm.
- Antibodies purified from clonal hybridomas were characterized with respect to their species cross-reactivity by ELISA, their ability to recognize reduced, denatured NOTUM protein by Western blot, and their neutralizing potency in the cell-free OPTS assay and the cell-based Wnt signaling assay, both of which are described above in Example 6.4.
- Table 1 shows the results of various characterization experiments for certain antibodies from Campaign 1. The data in the "Bin” column was generated using the method described in Example 6.6, below.
- Table 2 shows the results of various characterization experiments for certain antibodies from Campaign 2. The data in the "Bin” column was generated using the method described in Example 6.6, below. Table 2: Characterization of certain antibodies raised against mouse NOTUM
- Antibodies from both immunization campaigns were assessed for their ability to interfere with each other's binding to NOTUM protein in an epitope binning assay.
- This assay was performed in an ELISA format using anti-His captured NOTUM protein.
- the captured NOTUM protein was incubated with an excess of an unlabelled NOTUM-specific antibody (the 'blocking' antibody) followed by addition of a biotinylated NOTUM-specific antibody (the 'probe' antibody). Binding of the probe antibody was measured using HRP conjugated to streptavidin. If the two antibodies compete for binding in the same epitope space or if the blocking antibody otherwise affects the ability of the probe antibody to bind, e.g., by allosteric interference, no signal is generated.
- Antibodies are tested in a reciprocal matrix format. Typically, a pair of antibodies will show the same level of interference regardless of which of the two is the blocking antibody and which is the probe antibody. Antibodies exhibiting similar profiles are assigned to the same epitope 'bin'.
- MAbs 1.802, 1.815, 1.846, 2.78, and 2.1029 all interfere with each other's binding to human NOTUM while they do not interfere with the binding of several other less potent neutralizers or non-neutralizers.
- MAbs 1.802, 1.815, and 1.846 depend on human NOTUM amino acids between Q47 and M177 for binding. See Figure 5. Within this region, mouse and human NOTUM differ at five positions (R115K, D141S. R150K, R154H, and Y171H, based on the human sequence numbering). Human NOTUM point mutants were generated by transient transfection of constructs expressing human NOTUM with the mouse amino acid at each of these five positions and the point mutants were all shown to be functional in the OPTS assay.
- MAbs 1.802, 1.815, and 1.846 bound all point mutants except human NOTUM D141S, indicating that this amino acid is important for their binding to human NOTUM.
- Mouse NOTUM with the reciprocal point mutation, mouse NOTUM S148D was generated by transient transfection, shown to be active in the OPTS assay, and was shown to support binding of the human NOTUM-specific MAbs. Therefore, the species specificity of MAbs 1.802, 1.815, and 1.846 appears to be dependent upon the amino acid at position 141 in human NOTUM, which is aspartic acid in the native human NOTUM protein.
- human NOTUM mutants Five human NOTUM mutants were constructed, each with a pair of charged residues mutated to alanines: human NOTUM N132A/R133A (SEQ ID NO: 96); human NOTUM E134A/N135A (SEQ ID NO: 97); human NOTUM D137A/R139A (SEQ ID NO: 98); human NOTUM R144A/R145A (SEQ ID NO: 99); and human NOTUM R150A/D151A (SEQ ID NO: 100). All five human mutants were effectively expressed and secreted after transient transfection. Four of the five mutants exhibited significant activity in the OPTS assay while the fifth (human NOTUM D137A/R139A) showed little to no activity.
- Binding affinities of certain anti-NOTUM MAbs was determined using a Biacore 3000.
- antibody FAb fragments were generated by digestion of whole IgG with the protease Ficin, followed by removal of undigested IgG and Fc fragments by protein A affinity chromatography. Affinity values for binding of FAbs and whole IgG to human NOTUM corresponded, and their affinity values were in the single to low double digit nM range, as shown in Table 3.
- mice Eight week old male Fl hybrid (129 x C57) mice were administered NOTUM neutralizing antibody 2.1029 or 2.78b, or a control antibody, by intraperitoneal injection at 30 mg/kg once per week for eight weeks. There were 12 mice per group. At the end of the study, the mice were sacrificed. Bone mass and architecture were determined by microCT following necropsy, using a Scanco ⁇ " 40 with a threshold value of 240, an integration time of 200 milliseconds, and an X-ray tube voltage of 55 keV.
- midshaft femur cortical thickness increased by 12% (P ⁇ 0.001) with administration of NOTUM neutralizing antibody 2.1029, and 16% (P ⁇ 0.001) with administration of NOTUM neutralizing antibody 2.78b, as compared to the control antibody.
- mice Eight week old male Fl hybrid (129 x C57) mice were administered NOTUM neutralizing antibody 2.1029 by intraperitoneal injection at 3 mg/kg, 10 mg/kg, or 30 mg/kg once per week for four weeks. There were 10 mice per group. At the end of the study, the mice were sacrificed. Bone mass and architecture were determined by microCT following necropsy, using a Scanco ⁇ ( ⁇ 40 with a threshold value of 240, an integration time of 200 milliseconds, and an X-ray tube voltage of 55 keV.
- mice Eight week old male Fl hybrid (129 x C57) mice were administered NOTUM neutralizing antibody 2.78b by intraperitoneal injection at 3 mg/kg, 10 mg/kg, or 30 mg/kg once per week for four weeks. There were 10 mice per group in the first experiment. In a second experiment, NOTUM neutralizing antibody 2.78b was administered by intraperitoneal injection at 0.3 mg/kg, 1 mg/kg, or 3 mg/kg once per week for four weeks. There were 12 mice per group in the second experiment. At the end of each study, the mice were sacrificed. Bone mass and architecture were determined by microCT following necropsy, using a Scanco ⁇ 40 with a threshold value of 240, an integration time of 200 milliseconds, and an X-ray tube voltage of 55 keV.
- midshaft femur cortical thickness increased by 13% (P ⁇ 0.001), 17% (P ⁇ 0.001), and 16% (P ⁇ 0.001) with administration of 3 mg/kg, 10 mg/kg, and 30 mg/kg, respectively, of NOTUM neutralizing antibody 2.78b, relative to administration of control antibody, in the first experiment.
- PINP levels increased by 14 ng/mL, or 47% (P ⁇ 0.001) in mice administered NOTUM neutralizing antibody 2.78b without zoledronate pretreatment, relative to mice administered saline and control antibody, and increased by 12 ng/mL, or 79% (P ⁇ 0.001) in mice administered NOTUM neutralizing antibody 2.78b with zoledronate pretreatment, relative to mice administered zeledronate and control antibody.
- Mab 2.78 (also referred to as "2.78b”), which is an lgG2b antibody, was reformatted as an lgG2a antibody (lgG2a antibodies often have longer half-lives than lgG2b antibodies). Reformatted Mab 2.78 is referred to as "2.78a.”
- 13-week old male Fl hybrid mice (129 x C57) were administered NOTUM neutralizing antibody 2.78a by intraperitoneal injection at 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg once per week for four weeks. There were 10 or 12 mice per group. At the end of each study, the mice were sacrificed. Bone mass and architecture were determined by microCT following necropsy, using a Scanco ⁇ 40 with a threshold value of 240, an integration time of 200 milliseconds, and an X-ray tube voltage of 55 keV.
- mice Ten week old male Fl hybrid mice (129 x C57) were administered a control antibody, 0.3 mg/kg NOTUM neutralizing antibody 2.78a by i.p. injection weekly for 12 weeks, or 1 mg/kg NOTUM neutralizing antibody 2.78a by i.p. injection every other week (biweekly) for 12 weeks or 24 weeks. There were twelve mice per administration group. At the end of each study, the mice were sacrificed. Bone mass and architecture were determined by microCT following necropsy, using a Scanco ⁇ ( ⁇ 40 with a threshold value of 240, an integration time of 200 milliseconds, and an X-ray tube voltage of 55 keV.
- the midshaft femur cortical thickness increased by 6% (P ⁇ 0.001) and 9% (P ⁇ 0.001) in mice administered 0.3 mg/kg weekly and 1 mg/kg biweekly, respectively, of NOTUM neutralizing antibody 2.78a for 12 weeks.
- mice Following surgery and prior to the start of treatment, ovariectomized mice showed increased bone remodeling relative to sham surgery mice, as shown in Table 4. Since trabecular bone contains many more bone cells than cortical bone, these data likely reflect primarily increased trabecular bone remodeling.
- NOTUM neutralizing antibody 2.78b or a control antibody was administered at 10 mg/kg by intraperitoneal injection once per week for 4 weeks, starting 8 weeks after surgery.
- fluorochrome bone labels were administered on treatment days 7, 14, and 21 (i.e., with the 2 nd , 3 rd , and 4 th treatments). Calcein, which fluoresces green, was administered on day 7; alizarin, which fluoresces red, was administered on day 14; and tetracycline, which fluoresces yellow, was administered on day 21. The mice were sacrificed at the end of the 4 week treatment. Uterine weight at necropsy confirmed that the ovariectomy surgery was successful. (Data not shown.)
- Bone mass and architecture were determined by microCT following necropsy, using a Scanco ⁇ " 40 with a threshold value of 240, an integration time of 200 milliseconds, and an X-ray tube voltage of 55 keV.
- the midshaft femur, LV5 vertebral body, and the femoral neck were scanned.
- the midshaft femur cortical thickness increased by 22 ⁇ , or 9%, in sham surgery mice administered NOTUM neutralizing antibody 2.78b, relative to sham surgery mice administered control antibody, and increased by 26 ⁇ , or 12%, in ovariectomized mice administered NOTUM neutralizing antibody 2.78b, relative to ovariectomized mice administered control antibody.
- the midshaft femur mineralized bone area increased by 0.1 mm 2 , or 11%, in sham surgery mice administered NOTUM neutralizing antibody 2.78b, relative to sham surgery mice administered control antibody, and increased by 0.08 mm 2 , or 10%, in ovariectomized mice administered NOTUM neutralizing antibody 2.78b, relative to ovariectomized mice administered control antibody.
- the proportion in the LV5 vertebral body of cortical bone volume to total volume increased by 13% in sham surgery mice administered NOTUM neutralizing antibody 2.78b, relative to sham surgery mice administered control antibody, and increased by 9% in ovariectomized mice administered NOTUM neutralizing antibody 2.78b, relative to ovariectomized mice administered control antibody.
- the proportion in the LV5 vertebral body of trabecular bone volume to total volume was not significantly affected by administration of NOTUM neutralizing antibody 2.78b in either the sham surgery mice or the ovariectomized mice.
- Femur shafts were embedded in methylmethacrylate using a rapid embedding protocol. See Brlow and Vafai, Calcified Tissue Int'l 67: 479 (2000). Midshaft cross-sections with a thickness of about 80 ⁇ were prepared using a Leica SP160O bone saw. Sections were then examined with an Olympus BX60 fluorescent microscope. Various bone histomorphometric parameters were determined using OsteoMeasureTM software (OsteoMetrics, Decatur, GA). Both static parameters (such as bone area and thickness) and dynamic parameters (such as single label surface (SLS), mineral aposition rater (MAR), and bone formation rate (BFR)) were measured at lOOx magnification.
- SLS single label surface
- MAR mineral aposition rater
- BFR bone formation rate
- Figure 16 shows the percentage of the endocortical surface of the midshaft femur cross-sections that were labeled with calcein, which was administered on day 7, with alizarin, which was administered on day 14, and with tetracycline, which was administered on day 21.
- Table 6 shows the statistical analysis of the data in Figure 16.
- Mice administered NOTUM neutralizing antibody 2.78b showed a significantly higher percentage of endocortical labeling at days 7 and 14 compared to mice administered control antibody.
- Figure 17 shows the mineral appositional rate (A) and the volume-referent bone formation rate (B) of sham surgery and ovariectomized mice that were administered control antibody or NOTUM neutralizing antibody 2.78b.
- the mineral appositional rate ( Figure 17A) was determined by measuring the distance between the calcein label (day 7) and the alizarin label (day 14) and dividing by 7 to obtain the "days 7 to 14 rate,” and measuring the distance between the alizarin label (day 14) and the tetracycline label (day 21) and dividing by 7 to obtain the "days 14 to 21 rate.”
- Table 7 shows the statistical analysis of the data in Figure 17A.
- Mice administered NOTUM neutralizing antibody 2.78b showed a greater rate of mineral apposition than mice administered control antibody during the time period from days 7 to 14.
- the volume-referent bone formation rate (Figure 17B) was determined by standard calculations involving multiplying the endocortical mineralization surface (percentage of double- labeled surface plus one-half of the single labeled surface, derived from Figure 16) by the mineral apposition rate (see Figure 17A). The result is the bone formation rate divided by the bone volume, expressed as a percentage per 7 days.
- Table 8 shows the statistical a nalysis of the data in Figure 17B. As evident in Figure 17B, the bone formation rate per bone volume is significantly higher in mice administered NOTUIVI neutralizing antibody 2.78b than in mice administered control antibody. Table 8: Two-factor ANOVA of Volume-Referent Bone Formation Rate
- MAb 2.78 bound guinea pig NOTUM with lower affinity than MAb 1.802, and had correspondingly lower inhibiting activity in the OPTS assay.
- MAb 2.1029 bound guinea pig NOTUM only weakly, and did not significantly inhibit it in the OPTS assay.
- Cynomoigus and rhesus monkey NOTUM were cloned from cDNA preparations from those species. Analysis of the sequences revealed that the amino acid at the position equivalent to human NOTUM D141 is an asparagine, which is different from the amino acid at that position in both mouse and human NOTUM . Active (as determined by OPTS assay) cynomoigus and rhesus NOTUM proteins were generated by transient transfection, and it was found that MAb 1.802 neither binds nor inhibits either protein. An active human NOTUM point mutant, human NOTUM D141N, was generated by transient transfection, and it was found that MAb 1.802 does not bind to that human NOTUM point mutant.
- MAb 2.78 bound both cynomoigus and rhesus NOTU M weakly by ELISA, but did not inhibit either protein significantly in the OPTS assay.
- MAb 2.1029 bound both cynomoigus and rhesus monkey NOTUM by ELISA as well as it binds human NOTUM, and also inhibited both proteins in the OPTS assay as well as it inhibited human NOTUM . 6.12. Antibody Sequencing and Humanization
- Heavy and light chain variable regions were sequenced by specific RT-PCR using total RNA from the relevant hybridoma cell line followed by sequencing of the PCR product.
- the variable region sequences, without signal sequences, for each of those antibodies are shown in Section 7 (Table of Sequences), below. Section 7 also shows the sequences for the heavy and light chain CDRl, CDR2, and CDR3 for each of those antibodies.
- the following table shows the SEQ ID NOs corresponding to the heavy and light chain variable regions, and to CDRl, CDR2, and CDR3, for each of those antibodies.
- MAbs 1.802 and 1.846 share an identical heavy chain CDRl (GFTFSDYGMH; SEQ ID NOs: 17 and 33), while heavy chain CDRl of MAb 1.815 (GFTFSDFGMH; SEQ ID NO: 25) differs from MAbs 1.802 and 1.846 by only one conservative amino acid substitution (Phenylalanine (F) in place of Tyrosine (Y)).
- the consensus sequence for the heavy chain CDRl for those antibodies is therefore GFTFSDXiGMH (SEQ ID NO: 90), wherein X x is F or Y.
- Heavy chain CDR3 of MAbs 1.802 and 1.846 differ by only one conservative amino acid substitution (histidine (H) versus asparagine (N)).
- the consensus sequence for the heavy chain CDR3 for those antibodies is therefore KX 2 YNGGYFDV (SEQ ID NO: 91), wherein X 2 is H or N.
- MAbs 1.802 and 1.846 share an identical light chain CDR2 (LASNLES; SEQ ID NOs: 21 and 37), while light chain CDR2 of MAb 1.815 (LASDLES; SEQ ID NO: 29) differs from MAbs 1.802 and 1.846 by only one conservative amino acid substitution (aspartic acid (D) in place of asparagine (N)).
- LASX 6 LES SEQ ID NO: 93
- X 6 is D or N
- a consensus sequence for the light chain CDRl for the three antibodies from Campaign 1, 1.802, 1.846, and 1.815 is RASKX 3 VSX 4 SGYSYX 5 H (SEQ ID NO: 92), wherein X 3 is I or S, X 4 is T or E, and X 5 is M or I.
- BLAST searching was performed against public databases to identify the human germline variable region sequences with greatest similarity to each of the mouse heavy and light chain variable regions.
- CDRs from the mouse variable regions were then grafted in silico into these human germline variable sequences in place of the human germline CDRs.
- the resulting humanized variable regions for five of the mouse antibodies (2.78, 2.1029, 1.802, 1.815, and 1.846) were synthesized with a 5' leader sequence encoding an in-frame signal peptide and cloned upstream of sequence encoding human lgG2 constant regions in the case of the heavy chain variable sequences or human kappa constant region in the case of the light chain variable sequences.
- the sequences for each of the humanized variable regions are shown in Section 7 (Table of Sequences), below, along with the sequences for the full-length humanized heavy and light chains (without the signal peptide).
- Coding sequences for full length humanized heavy and light chains were subcloned into mammalian expression vectors and corresponding heavy and light chain constructs were cotransfected into CHO-S cells. The resulting conditioned media were checked by Western blotting with an anti-human secondary antibody to confirm expression and secretion of intact humanized antibody. The conditioned media were then tested in ELISA format to determine whether the humanized antibodies retained the capacity to bind human NOTUM protein.
- Humanized MAbs 1.802, 1.815, 1.846, and 2.1029 bound human NOTUM while humanized MAb 2.78 exhibited little to no binding to either human or mouse NOTUM.
- HumAb 2.1029 1 ght DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY chain variable TSRLHSGVPS RFSGSGTD FTFTISSLQP EDIATYYCQQ GKTLPRTFGG region GTKVE I
- HumAb 2.1029 1 ght DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY chain TSRLHSGVPS RFSGSGTD FTFTI SSLQP EDIATYYCQQ GKTLPRTFGG
- HumAb 1.846 light DIVMTQSPDS LAVSLGERAT INCRASKSVS ESGYSYMHWY QQKPGQPPKL chain variable LIYLASNLES GVPDRFSGSG SGTDFTLTI S SLQAEDVAVY YCQHSRVLPP region TFGQGTKLEI
- CTCTGGATTC ACTTTCAGTA GCTTTGGCAT GCACTGGGTT CGTCAGGCTC CAGAGAAGGG ACTGGAGTGG GTCGCATACA TTACTAGTGG CAGTGGTGCC ATCTACTATG CAGACACAGT GAGGGGCCGA TTCACCATCT CCAGAGACAC T C C CAAGAAC ACCCTGTTCC TGCAGATGAC CAGTCTAAGG TCTGAGGACA CGGCCATGTA TTACTGTGCA AGATCGGCTG ATGGTTTGGA CTACTGGGGT CAAGGAACCT CAGTCACCGT CTCCTCAGCC AAAACAACAC CCCCATCAGT CTATCCACTG GCCCCTGGGT GTGGAGATAC AACTG
- AAACAGGGAA AATCTCCTCA GCTCCTGGTC TATGGTGCAA CAAACTTAGC AGATGGTGTG CCATCAAGGT TCAGTGGCAG TGGATCAGGC ACACAGTATT CCCTCAAGAT CAACAGCCTG AAGTCTGAAG ATTTTGGGAG TTATTACTGT CAACATTTTT GGGGTACTCC ATTCACGTTC GGCTCGGGGA CAAAGTTGGA AATAAAACGG GCTGATGCTG CACCAACTGT ATCCATCTTC CCACCATCCA GTGAGCAGTT AACATCTGGA GGTGCCTCAG TCGTGTGC
- GAGCTACATC ACCAGCGGCA GCGGCCAT CTACTACGCC GACAGCGTGA AGGGCAGATT CACCATCAGC AGAGACAACG CCAAGAACAG CCTGTACCTG CAGATGAACA GCCTGAGAGC CGAGGACACC
- GCCGTGTACT ACTGCGCCAG AAGCGCCGAC GGCCTGGACT ACTGGGGCCA GGGCACCACC GTGACCGTGA GCAGCGATGT GTGGGGCCAG GGCACCACCG TGACCGTGAG CAGCGCGTCG ACCAAGGGCC CATCGGTCTT CCCCCTGGCG CCCTGCTCCA
Abstract
Description
Claims
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CN201180056452.6A CN103298490B (en) | 2010-11-24 | 2011-11-22 | Antibodies to notum pectinacetylesterase |
DK11843652.6T DK2643019T3 (en) | 2010-11-24 | 2011-11-22 | ANTIBODIES AGAINST NOTE PECTINACETYL ESTERASE |
KR1020187016214A KR20180069083A (en) | 2010-11-24 | 2011-11-22 | Antibodies to notum pectinacetylesterase |
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ES11843652T ES2718849T3 (en) | 2010-11-24 | 2011-11-22 | Antibodies against notum pectin acetylesterase |
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SG2013031083A SG189982A1 (en) | 2010-11-24 | 2011-11-22 | Antibodies to notum pectinacetylesterase |
JP2013541012A JP6033229B2 (en) | 2010-11-24 | 2011-11-22 | Antibody binding to NOTUM pectin acetylesterase |
CA2817415A CA2817415C (en) | 2010-11-24 | 2011-11-22 | Antibodies that bind notum pectinacetylesterase |
AU2011332007A AU2011332007C1 (en) | 2010-11-24 | 2011-11-22 | Antibodies to Notum Pectinacetylesterase |
BR112013012858-5A BR112013012858B1 (en) | 2010-11-24 | 2011-11-22 | MONOCLONAL ANTIBODY BINDING PECTINACETYLESTERASE FROM NOTUM, PHARMACEUTICAL COMPOSITION, NUCLEIC ACID MOLECULE, HOST CELL AND METHOD FOR PRODUCING SUCH ANTIBODY |
US13/885,815 US20130302346A1 (en) | 2010-11-24 | 2011-11-22 | Antibodies that bind notum pectinacetylesterase |
MX2013005906A MX357166B (en) | 2010-11-24 | 2011-11-22 | Antibodies to notum pectinacetylesterase. |
RU2013123793/10A RU2013123793A (en) | 2010-11-24 | 2011-11-22 | ANTIBODIES RELATING TO NOTUM PECTINACETHYLESTERASE |
IL225876A IL225876A0 (en) | 2010-11-24 | 2013-04-22 | Antibodies to notum pectinacetylesterase |
ZA2013/02983A ZA201302983B (en) | 2010-11-24 | 2013-04-24 | Antibodies to notum pectinacetylesterase |
US14/952,264 US20160152731A1 (en) | 2010-11-24 | 2015-11-25 | Antibodies that bind notum pectinacetylesterase |
US16/277,466 US11059907B2 (en) | 2010-11-24 | 2019-02-15 | Antibodies that bind Notum Pectinacetylesterase |
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US14/952,264 Continuation US20160152731A1 (en) | 2010-11-24 | 2015-11-25 | Antibodies that bind notum pectinacetylesterase |
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Cited By (4)
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US20120269798A1 (en) * | 2011-03-22 | 2012-10-25 | Peter Reddien | Enhancement of regeneration by modulation of notum activity |
EP2608807A1 (en) * | 2010-08-27 | 2013-07-03 | Stem Centrx, Inc. | Notum protein modulators and methods of use |
JP2015530087A (en) * | 2012-08-31 | 2015-10-15 | アルゲン−エックス エヌ.ブイ. | Method for producing an antibody molecule having interspecific intra-target cross-reactivity |
US11059907B2 (en) | 2010-11-24 | 2021-07-13 | Lexicon Pharmaceuticals, Inc. | Antibodies that bind Notum Pectinacetylesterase |
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EP3604339B1 (en) | 2011-01-14 | 2021-03-10 | The Regents Of The University Of California | Therapeutic antibodies against ror-1 protein and methods for use of same |
WO2020150282A1 (en) * | 2019-01-18 | 2020-07-23 | Askgene Pharma Inc. | Pd-l1 specific monoclonal antibodies for disease treatment and diagnosis |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2608807A1 (en) * | 2010-08-27 | 2013-07-03 | Stem Centrx, Inc. | Notum protein modulators and methods of use |
US11059907B2 (en) | 2010-11-24 | 2021-07-13 | Lexicon Pharmaceuticals, Inc. | Antibodies that bind Notum Pectinacetylesterase |
US20120269798A1 (en) * | 2011-03-22 | 2012-10-25 | Peter Reddien | Enhancement of regeneration by modulation of notum activity |
US8802365B2 (en) * | 2011-03-22 | 2014-08-12 | Whitehead Institute For Biomedical Research | Methods for identifying candidate modulators of NOTUM activity |
JP2015530087A (en) * | 2012-08-31 | 2015-10-15 | アルゲン−エックス エヌ.ブイ. | Method for producing an antibody molecule having interspecific intra-target cross-reactivity |
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HUE043576T2 (en) | 2019-08-28 |
JP6033229B2 (en) | 2016-11-30 |
EP2643019B1 (en) | 2019-01-02 |
CN103298490B (en) | 2017-04-26 |
ES2718849T3 (en) | 2019-07-04 |
MX357166B (en) | 2018-06-28 |
PT2643019T (en) | 2019-04-23 |
MX2013005906A (en) | 2013-06-28 |
NZ609501A (en) | 2014-12-24 |
JP2014501513A (en) | 2014-01-23 |
CA2817415C (en) | 2020-05-12 |
AU2011332007A1 (en) | 2013-05-09 |
EP2643019A1 (en) | 2013-10-02 |
PL2643019T3 (en) | 2019-07-31 |
KR20180069083A (en) | 2018-06-22 |
EP2643019A4 (en) | 2014-12-31 |
CA2817415A1 (en) | 2012-05-31 |
BR112013012858A2 (en) | 2018-09-04 |
CN103298490A (en) | 2013-09-11 |
ZA201302983B (en) | 2017-06-28 |
IL225876A0 (en) | 2013-06-27 |
AU2011332007B2 (en) | 2016-11-10 |
AU2011332007C1 (en) | 2017-03-02 |
CO6721048A2 (en) | 2013-07-31 |
KR20140026334A (en) | 2014-03-05 |
US11059907B2 (en) | 2021-07-13 |
US20160152731A1 (en) | 2016-06-02 |
DK2643019T3 (en) | 2019-04-15 |
US20190241677A1 (en) | 2019-08-08 |
RU2013123793A (en) | 2014-12-27 |
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US20130302346A1 (en) | 2013-11-14 |
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