# Patent

## Title
Sclerostin binding antibodies

## Abstract

Compositions and methods relating to epitopes of sclerostin protein, and sclerostin binding agents, such as antibodies capable of binding to sclerostin, are provided.

## Background
RELATED APPLICATIONS
The present application is a divisional of U.S. patent application Ser. No. 12/950,094, filed Nov. 19, 2010, which is a divisional of U.S. patent application Ser. No. 12/276,889 (now U.S. Pat. No. 7,872,106), filed Nov. 24, 2008, which is a divisional of U.S. patent application Ser. No. 11/411,003 (now U.S. Pat. No. 7,592,429), filed Apr. 25, 2006, which claims the benefit of priority from U.S. Provisional Patent Application No. 60/792,645, filed Apr. 17, 2006, U.S. Provisional Patent Application No. 60/782,244, filed Mar. 13, 2006, U.S. Provisional Patent Application No. 60/776,847, filed Feb. 24, 2006, and U.S. Provisional Patent Application No. 60/677,583, filed May 3, 2005, under 35 U.S.C. §119. The foregoing patent applications are incorporated herein by reference in their entirety.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: ASCII (text) file named “40000C_SubSeqListing.txt,” 507,625 bytes, created Jan. 15, 2013.


TECHNICAL FIELD
The present invention relates generally to epitopes of sclerostin protein, including human sclerostin protein, and binding agents (such as antibodies) capable of binding to sclerostin or fragments thereof.
BACKGROUND OF THE INVENTION
Two or three distinct phases of changes to bone mass occur over the life of an individual (see Riggs, West I Med. 154:63-77 (1991)). The first phase occurs in both men and women and proceeds to attainment of a peak bone mass. This first phase is achieved through linear growth of the endochondral growth plates and radial growth due to a rate of periosteal apposition. The second phase begins around age 30 for trabecular bone (flat bones such as the vertebrae and pelvis) and about age 40 for cortical bone (e.g., long bones found in the limbs) and continues to old age. This phase is characterized by slow bone loss and occurs in both men and women. In women, a third phase of bone loss also occurs, most likely due to postmenopausal estrogen deficiencies. During this phase alone, women may lose an additional bone mass from the cortical bone and from the trabecular compartment (see Riggs, supra).
Loss of bone mineral content can be caused by a wide variety of conditions and may result in significant medical problems. For example, osteoporosis is a debilitating disease in humans and is characterized by marked decreases in skeletal bone mass and mineral density, structural deterioration of bone, including degradation of bone microarchitecture and corresponding increases in bone fragility (i.e., decreases in bone strength), and susceptibility fracture in afflicted individuals. Osteoporosis in humans is generally preceded by clinical osteopenia (bone mineral density that is greater than one standard deviation but less than 2.5 standard deviations below the mean value for young adult bone), a condition found in approximately 25 million people in the United States. Another 7-8 million patients in the United States have been diagnosed with clinical osteoporosis (defined as bone mineral content greater than 2.5 standard deviations below that of mature young adult bone). The frequency of osteoporosis in the human population increases with age. Among Caucasians, osteoporosis is predominant in women who, in the United States, comprise 80% of the osteoporosis patient pool. The increased fragility and susceptibility to fracture of skeletal bone in the aged is aggravated by the greater risk of accidental falls in this population. Fractured hips, wrists, and vertebrae are among the most common injuries associated with osteoporosis. Hip fractures in particular are extremely uncomfortable and expensive for the patient, and for women, correlate with high rates of mortality and morbidity.
Although osteoporosis has been regarded as an increase in the risk of fracture due to decreased bone mass, few of the presently available treatments for skeletal disorders can increase the bone density of adults, and most of the presently available treatments work primarily by inhibiting further bone resorption rather than stimulating new bone formation. Estrogen is now being prescribed to retard bone loss. However, some controversy exists over whether patients gain any long-term benefit and whether estrogen has any effect on patients over 75 years old. Moreover, use of estrogen is believed to increase the risk of breast and endometrial cancer. Calcitonin, osteocalcin with vitamin K, or high doses of dietary calcium, with or without vitamin D, have also been suggested for postmenopausal women. High doses of calcium, however, often have undesired gastrointestinal side effects, and serum and urinary calcium levels must be continuously monitored (e.g., Khosla and Riggs, Mayo Clin. Proc. 70:978982, 1995).
Other current therapeutic approaches to osteoporosis include bisphosphonates (e.g., FosamaxTM, ActonelTM, BonvivaTM, ZometaTM, olpadronate, neridronate, skelid, bonefos), parathyroid hormone, calcilytics, calcimimetics (e.g., cinacalcet), statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. Such therapeutics, however, are often associated with undesirable side effects (see Khosla and Riggs, supra).
Sclerostin, the product of the SOST gene, is absent in sclerosteosis, a skeletal disease characterized by bone overgrowth and strong dense bones (Brunkow et al., Am. J. Hum. Genet., 68:577-589, 2001; Balemans et al., Hum. Mol. Genet., 10:537-543, 2001). The amino acid sequence of human sclerostin is reported by Brunkow et al. ibid and is disclosed herein as SEQ ID NO:1.
BRIEF SUMMARY OF THE INVENTION
Disclosed herein are compositions and methods that can be used to increase at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality and bone strength, and that therefore may be used to treat a wide variety of conditions in which an increase in at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality and bone strength is desirable. The present invention also offers other related advantages described herein.
The invention relates to regions (epitopes) of human sclerostin recognized by the binding agents disclosed herein, methods of using these epitopes, and methods of making such epitopes.
The invention also relates to epitopes specific to the region of sclerostin identified as Loop 2, and binding agents which specifically bind to that region.
The invention also relates to epitopes specific to the cystine-knot region of sclerostin, and binding agents such as antibodies specifically binding to that region.
The invention relates to binding agents, such as antibodies, that specifically bind to sclerostin. The binding agents can be characterized by their ability to cross-block the binding of at least one antibody disclosed herein to sclerostin and/or to be cross-blocked from binding sclerostin by at least one antibody disclosed herein. The antibodies and other binding agents can also be characterized by their binding pattern to human sclerostin peptides in a “human sclerostin peptide epitope competition binding assay” as disclosed herein.
The invention relates to binding agents, such as antibodies, that can increase at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality and bone strength in a mammal.
The invention relates to binding agents, such as antibodies, that can block the inhibitory effect of sclerostin in a cell based mineralization assay.
The invention further relates to polypeptide constructs comprising two, three, or four polypeptide fragments linked by at least one disulfide bond, representing a core region of the cystine-knot of sclerostin, and antibodies capable of specifically binding thereto.
The invention relates to methods of obtaining epitopes suitable for use as immunogens for generating, in mammals, binding agents, such as antibodies capable of binding specifically to sclerostin; in certain embodiments the binding agents generated are capable of neutralizing sclerostin activity in vivo.
The invention relates to a composition for eliciting an antibody specific for sclerostin when the composition is administered to an animal, the composition comprising a polypeptide having the amino acid sequence of SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69.
The invention also relates to a composition for eliciting an antibody specific for sclerostin when the composition is administered to an animal, the composition comprising at least one polypeptide consisting essentially of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; the composition may comprise at least two or at least three of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and the composition may comprise all four of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
The invention further relates to a composition for eliciting an antibody specific for sclerostin when the composition is administered to an animal, the composition comprising a polypeptide having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, wherein SEQ ID NO:2 and 4 are joined by a disulfide bond at amino acid positions 57 and 111 with reference to SEQ ID NO:1, and SEQ ID NO:3 and 5 are joined by at least one of (a) a disulfide bond at amino acid positions 82 and 142 with reference to SEQ ID NO:1, and (b) a disulfide bond at amino acid positions 86 and 144 with reference to SEQ ID NO:1; the polypeptide may retain the tertiary structure of the corresponding polypeptide region of human sclerostin of SEQ ID NO:1.
The invention also relates to polypeptide T20.6 consisting essentially of a multiply truncated human sclerostin protein of SEQ ID NO:1, wherein amino acids 1-50, 65-72, 91-100, 118-137, and 150-190 of SEQ ID NO:1 are absent from the polypeptide; this polypeptide may be obtained by tryptic digestion of human sclerostin, and the protein may be isolated by HPLC fractionation.
The invention further relates to immunogenic portion T20.6 of human sclerostin comprising amino acids 51-64, 73-90, 101-117, and 138-149 of SEQ ID NO:1, wherein the immunogenic portion comprises at least one of:
(a) a disulfide bond between amino acids 57 and 111;
(b) a disulfide bond between amino acids 82 and 142; and
(c) a disulfide bond between amino acids 86 and 144;
the immunogenic portion may have at least two of these disulfide bonds; and the immunogenic portion may have all three disulfide bonds.
The invention further relates to an immunogenic portion T20.6 derivative of human sclerostin comprising amino acids 57-64, 73-86, 111-117, and 138-144 of SEQ ID NO:1, wherein the immunogenic portion comprises at least one of:
(a) a disulfide bond between amino acids 57 and 111;
(b) a disulfide bond between amino acids 82 and 142; and
(c) a disulfide bond between amino acids 86 and 144;
the immunogenic portion may have at least two of these disulfide bonds; and the immunogenic portion may have all three disulfide bonds.
The invention yet further relates to a polypeptide consisting essentially of a human sclerostin protein of SEQ ID NO:1 truncated at the C-terminal and N-terminal ends, wherein amino acids 1-85 and 112-190 of SEQ ID NO:1 are absent from the polypeptide.
The invention also relates to an immunogenic portion of human sclerostin, comprising amino acids 86-111 of SEQ ID NO:1; the immunogenic portion may consist essentially of contiguous amino acids CGPARLLPNAIGRGKWWRPSGPDFRC (SEQ ID NO:6).
The invention further relates to an immunogenic portion of rat sclerostin, comprising amino acids 92-109 of SEQ ID NO:98; the immunogenic portion may consist essentially of contiguous amino acids PNAIGRVKWWRPNGPDFR (SEQ ID NO:96).
The invention still further relates to an immunogenic portion of rat sclerostin, comprising amino acids 99-120 of SEQ ID NO:98; the immunogenic portion may consist essentially of contiguous amino acids KWWRPNGPDFRCIPDRYRAQRV (SEQ ID NO:97).
The invention relates to a method of producing an immunogenic portion of human sclerostin, comprising the steps of:

    
    
        (a) treating human sclerostin to achieve complete tryptic digestion;
        (b) collecting the tryptic digest sample having average molecular weight of 7,122.0 Daltons (theoretical mass 7121.5 Daltons) or retention time of about 20.6 minutes as determined by elution from a reverse-phase HPLC column with linear gradient from 0.05% trifluoroacetic acid to 90% acetonitrile in 0.05% TFA at a flow rate of 0.2 mL/min; and
        (c) purifying the immunogenic portion.
    
    


The invention relates to a method of generating an antibody capable of specifically binding to sclerostin, comprising:

    
    
        (a) immunizing an animal with a composition comprising a polypeptide of SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:96, or SEQ ID NO:97;
        (b) collecting sera from the animal; and
        (c) isolating from the sera an antibody capable of specifically binding to sclerostin.
    
    


The invention also relates to a method of generating an antibody capable of specifically binding to sclerostin, the method comprising:

    
    
        (a) immunizing an animal with a composition comprising polypeptide T20.6 or a derivative of T20.6;
        (b) collecting sera from the animal; and
        (c) isolating from the sera an antibody capable of specifically binding to sclerostin.
    
    


The invention further relates to a method of detecting an anti-sclerostin antibody in a biological sample, comprising the steps of

    
    
        (a) contacting the biological sample with a polypeptide consisting essentially of SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:96, or SEQ ID NO:97 under conditions allowing a complex to form between the antibody and the polypeptide; and
        (b) detecting the presence or absence of the complex,

wherein the presence of the complex indicates that the biological sample contains an anti-sclerostin antibody.

    
    


The invention also relates to a method of detecting an anti-sclerostin antibody in a biological sample, comprising the steps of

    
    
        (a) contacting the biological sample with polypeptide T20.6 or a derivative of T20.6 under conditions allowing a complex to form between the antibody and the polypeptide; and
        (b) detecting the presence or absence of the complex,

wherein the presence of the complex indicates that the biological sample contains an anti-sclerostin antibody.

    
    


The invention further relates to a sclerostin binding agent, such as an antibody, that cross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C, or Ab-D to a sclerostin protein. The sclerostin binding agent may also be cross-blocked from binding to sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, or Ab-D. The isolated antibody, or an antigen-binding fragment thereof, may be a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody or the like.
The invention further relates to a sclerostin binding agent, such as an antibody, that is cross-blocked from binding to sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, or Ab-D. The isolated antibody, or an antigen-binding fragment thereof, may be a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody or the like.
The invention further relates to a sclerostin binding agent, such as an isolated antibody, that cross-blocks the binding of at least one of antibodies 1-24 (Ab-1 to Ab-24) to a sclerostin protein. The sclerostin binding agent may also be cross-blocked from binding to sclerostin by at least one of antibodies 1-24 (Ab-1 to Ab-24). The isolated antibody, or an antigen-binding fragment thereof, may be a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or a chimeric antibody.
The invention further relates to a sclerostin binding agent, such as an isolated antibody, that is cross-blocked from binding to sclerostin by at least one of antibodies 1-24 (Ab-1 to Ab-24); the isolated antibody, or an antigen-binding fragment thereof, may be a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or a chimeric antibody.
The invention further relates to a binding agent, such as an isolated antibody that exhibits a similar binding pattern to human sclerostin peptides in a “human sclerostin peptide epitope competition binding assay” as that exhibited by at least one of the antibodies Ab-A, Ab-B, Ab-C or Ab-D; the isolated antibody, or an antigen-binding fragment thereof, may be a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or a chimeric antibody.
The invention still further relates to a method for treating a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength in a mammalian subject which comprises providing to a subject in need of such treatment an amount of an anti-sclerostin binding agent sufficient to increase at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality and bone strength wherein the anti-sclerostin binding agent comprises an antibody, or sclerostin-binding fragment thereof.
The invention also relates to an isolated sclerostin polypeptide or fragments thereof, wherein the polypeptide contains 6 conserved cysteine residues and the fragments thereof comprise from 7 to 14 amino acids of SEQ ID NO:2; 8 to 17 amino acids of SEQ ID NO:3; 8 to 18 residues of SEQ ID NO:4; and 6 to 12 residues of SEQ ID NO:5, and the polypeptide or fragments thereof are stabilized by disulfide bonds between SEQ ID NO:2 and 4, and between SEQ ID NO:3 and 5; the polypeptide or fragments may comprise 10-14 amino acids of SEQ ID NO:2; 14 to 17 amino acids of SEQ ID NO:3; 13 to 18 amino acids of SEQ ID NO:4; and 8 to 12 residues of SEQ ID NO:5; and the polypeptide or fragments may comprise SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
Provided herein are antibodies that specifically bind to human sclerostin. The antibodies are characterized by their ability to cross-block the binding of at least one antibody disclosed herein to human sclerostin and/or to be cross-blocked from binding human sclerostin by at least one antibody disclosed herein.
Also provided is an isolated antibody, or an antigen-binding fragment thereof, that can increase at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality and bone strength in a mammal.
Also provided in an isolated antibody, or an antigen-binding fragment thereof, that can block the inhibitory effect of sclerostin in a cell based mineralization assay.
Also provided is a binding agent, such as an antibody, that specifically binds to human sclerostin and has at least one CDR sequence selected from SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 78, 79, 80, 81, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 351, 352, 353, 358, 359, and 360, and variants thereof, wherein the antibody or antigen-binding fragment thereof neutralizes sclerostin.
Also provided is a binding agent, such as an antibody, that specifically binds to human sclerostin and has at least one CDR sequence selected from SEQ ID NOs:39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 78, 79, 80, 81, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 351, 352, 353, 358, 359, and 360, and variants thereof.
Also provided are regions of human sclerostin which are important for the in vivo activity of the protein.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entireties as if each was incorporated individually.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the amino acid sequences of the mature form (signal peptides cleaved off) of the light chain (FIG. 1A) (SEQ ID NO:23) and heavy chain (FIG. 1B) (SEQ ID NO:27) for the anti-human sclerostin and anti-mouse sclerostin antibody Ab-A.
FIG. 2 depicts the amino acid sequences of the mature form (signal peptides cleaved off) of the light chain (FIG. 2A) (SEQ ID NO:31) and heavy chain (FIG. 2B) (SEQ ID NO:35) for the anti-human sclerostin and anti-mouse sclerostin antibody Ab-B.
FIG. 3 depicts the amino acid sequences of the mature form (signal peptides cleaved oft) of the light chain (FIG. 3A) (SEQ ID NO:15) and heavy chain (FIG. 3B) (SEQ ID NO:19) for the anti-human sclerostin and anti-mouse sclerostin antibody Ab-C.
FIG. 4 depicts the amino acid sequences of the mature form (signal peptides cleaved off) of the light chain (FIG. 4A) (SEQ ID NO:7) and heavy chain (FIG. 4B) (SEQ ID NO:11) for the anti-human sclerostin and anti-mouse sclerostin antibody Ab-D.
FIG. 5 depicts bone mineral density in mice measured at two skeletal sites (lumbar vertebrae and tibial metaphysis) after 3 weeks of treatment with vehicle, PTH (1-34), Ab-A or Ab-B.
FIG. 6 shows bone mineral density in mice measured at two skeletal sites (lumbar vertebrae and tibial metaphysis) after 2 weeks of treatment with vehicle, PTH (1-34) or Ab-C.
FIG. 7 depicts bone mineral density in mice measured at two skeletal sites (lumbar vertebrae and tibial metaphysis) after 3 weeks of treatment with vehicle or Ab-D.
FIG. 8 depicts the amino acid sequence of the mature form (signal peptide cleaved oft) of human sclerostin (SEQ ID NO:1). Also depicted is the nucleotide sequence of the human sclerostin coding region that encodes the mature form of human sclerostin. The eight cysteines are numbered C1 through C8. The cystine-knot is formed by three disulfide bonds (C1-C5; C3-C7; C4-C8). C2 and C6 also form a disulfide bond, however this disulfide is not part of the cystine-knot.
FIG. 9 depicts a schematic of the basic structure of human sclerostin. There is an N-terminal arm (from the first Q to C1) and a C-terminal arm (from C8 to the terminal Y). In between these arms there is the cystine-knot structure (formed by three disulfides: C1-C5; C3-C7; C4-C8) and three loops which are designated Loop1, Loop 2 and Loop 3. The distal regions of Loop 1 and Loop 3 are linked by the C2-C6 disulfide. Potential trypsin cleavage sites are indicated (arginine=R and lysine=K). Some of the potential AspN cleavage sites are indicated (only aspartic acid (D) residues are shown).
FIG. 10 depicts the HPLC peptide maps of human sclerostin after digestion with either trypsin or AspN. The human sclerostin peptides generated by trypsin digestion are indicated (T19.2, T20, T20.6 and T21-22) as are the human sclerostin peptides generated by AspN digestion (AspN14.6, AspN18.6 and AspN22.7-23.5).
FIG. 11 depicts sequence and mass information for the isolated human sclerostin disulfide linked peptides generated by trypsin digestion. Seq. pos.=sequence position. Obs.=observed. Observed mass was determined by ESI-LC-MS analysis.
FIG. 12 depicts sequence and mass information for the isolated human sclerostin peptides generated by AspN digestion. The AspN22.7-23.5 peptide contains the 4 disulfide bonds. Seq. pos.=sequence position. Obs.=observed. Observed mass was determined by ESI-LC-MS analysis.
FIG. 13 shows a linear schematic of four human sclerostin peptides (T19.2, T20, T20.6 and T21-22) generated by trypsin digestion.
FIG. 14 shows a linear schematic of five human sclerostin peptides (AspN14.6, AspN18.6 and AspN22.7-23.5) generated by AspN digestion. The AspN14.6 HPLC peak is composed of three peptides not linked by any disulfide bonds.
FIG. 15 shows the resonance unit (Ru) signal from the Biacore-based “human sclerostin peptide epitope competition binding assay.” Relative Mab binding to various human sclerostin-peptides (in solution) versus Mab binding to intact mature form human sclerostin (immobilized on Biacore chip) was assessed. Data shown is for Ab-A. Human sclerostin peptides used were T19.2, T20, T20.6, T21-22, AspN14.6, AspN18.6 and AspN22.7-23.5.
FIG. 16 shows the resonance unit (Ru) signal from the Biacore-based “human sclerostin peptide epitope competition binding assay.” Relative Mab binding to various human sclerostin-peptides (in solution) versus Mab binding to intact mature form human sclerostin (immobilized on Biacore chip) was assessed. Data shown is for Ab-B. Human sclerostin peptides used were T19.2, T20, T20.6, T21-22, AspN14.6, AspN18.6 and AspN22.7-23.5.
FIG. 17 shows the resonance unit (Ru) signal from the Biacore-based “human sclerostin peptide epitope competition binding assay.” Relative Mab binding to various human sclerostin-peptides (in solution) versus Mab binding to intact mature form human sclerostin (immobilized on Biacore chip) was assessed. Data shown is for Ab-C. Human sclerostin peptides used were T19.2, T20, T20.6, T21-22, AspN14.6, AspN18.6 and AspN22.7-23.5.
FIG. 18 shows the resonance unit (Ru) signal from Biacore-based “human sclerostin peptide epitope competition binding assay.” Relative Mab binding to various human sclerostin-peptides (in solution) versus Mab binding to intact mature form human sclerostin (immobilized on Biacore chip) was assessed. Data shown is for Ab-D. Human sclerostin peptides used were T19.2, T20, T20.6, T21-22, AspN14.6, AspN18.6 and AspN22.7-23.5.
FIG. 19 shows two Mab binding epitopes of human sclerostin. FIG. 19A shows sequence of the Loop 2 epitope for binding of Ab-A and Ab-B to human sclerostin (SEQ ID NO:6). FIG. 19B shows sequence, disulfide bonding and schematic of the T20.6 epitope for binding of Ab-C and Ab-D to human sclerostin (SEQ ID NO:2-5).
FIG. 20 depicts the HPLC peptide maps of human sclerostin after digestion with trypsin. FIG. 20A shows digestion of the human sclerostin Ab-D complex. FIG. 20B shows digestion of human sclerostin alone. The T19.2, T20, T20.6 and T21-22 peptide peaks are indicated.
FIG. 21 shows the sequence, disulfide bonding and schematic of the “T20.6 derivative 1 (cystine-knot+4 arms)” epitope for binding of Ab-D to human sclerostin. (SEQ ID NO:70-73).
FIG. 22 shows results from the MC3T3-E1-BF osteoblast cell line mineralization assay used for identifying anti-sclerostin neutralizing Mabs. Mouse sclerostin (Scl) was used at 1 μg/ml. Monoclonal antibodies were used at 10 and 5 μg/ml. Extent of mineralization (various types of insoluble calcium phosphate) was quantitated by measuring calcium.
FIG. 23 depicts results from the MC3T3-E1-BF osteoblast cell line mineralization assay used for identifying anti-sclerostin neutralizing Mabs. Human sclerostin (Scl) was used at 1 μg/ml. Monoclonal antibodies were used at 8 and 4 μg/ml. Extent of mineralization (various types of insoluble calcium phosphate) was quantitated by measuring calcium.
FIG. 24 shows results from the MC3T3-E1-BF osteoblast cell line mineralization assay used for identifying anti-sclerostin neutralizing Mabs. Human sclerostin (Scl) was used at 1 μg/ml. Monoclonal antibodies were used at 10 μg/ml. Extent of mineralization (various types of insoluble calcium phosphate) was quantitated by measuring calcium.
FIG. 25 depicts results from an inflammation-induced bone loss SCID mouse model. Ab-A treatment protected mice from inflammation-related bone loss associated with colitis when measured as total bone mineral density (FIG. 25A), vertebral bone density (FIG. 25B), and femur bone density (FIG. 25C).



DETAILED DESCRIPTION
The present invention relates to regions of the human sclerostin protein that contain epitopes recognized by antibodies that also bind to full-length sclerostin, and methods of making and using these epitopes. The invention also provides binding agents (such as antibodies) that specifically bind to sclerostin or portions of sclerostin, and methods for using such binding agents. The binding agents are useful to block or impair binding of human sclerostin to one or more ligand.
Recombinant human sclerostin/SOST is commercially available from R&D Systems (Minneapolis, Minn., USA; 2006 cat#1406-ST-025). Additionally, recombinant mouse sclerostin/SOST is commercially available from R&D Systems (Minneapolis, Minn., USA; 2006 cat#1589-ST-025). Research grade sclerostin binding monoclonal antibodies are commercially available from R&D Systems (Minneapolis, Minn., USA; mouse monoclonal: 2006 cat#MAB1406; rat monoclonal: 2006 cat# MAB1589). U.S. Pat. Nos. 6,395,511 and 6,803,453, and U.S. Patent Publications 20040009535 and 20050106683 refer to anti-sclerostin antibodies generally.
As used herein, the term human sclerostin is intended to include the protein of SEQ ID NO:1 and allelic variants thereof. Sclerostin can be purified from 293T host cells that have been transfected by a gene encoding sclerostin by elution of filtered supernatant of host cell culture fluid using a Heparin HP column, using a salt gradient. The preparation and further purification using cation exchange chromatography are described in Examples 1 and 2.
Binding agents of the invention are preferably antibodies, as defined herein. The term “antibody” refers to an intact antibody, or a binding fragment thereof. An antibody may comprise a complete antibody molecule (including polyclonal, monoclonal, chimeric, humanized, or human versions having full length heavy and/or light chains), or comprise an antigen binding fragment thereof. Antibody fragments include F(ab′)2, Fab, Fab′, Fv, Fc, and Fd fragments, and can be incorporated into single domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (See e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). Antibody polypeptides are also disclosed in U.S. Pat. No. 6,703,199, including fibronectin polypeptide monobodies. Other antibody polypeptides are disclosed in U.S. Patent Publication 2005/0238646, which are single-chain polypeptides.
Antigen binding fragments derived from an antibody can be obtained, for example, by proteolytic hydrolysis of the antibody, for example, pepsin or papain digestion of whole antibodies according to conventional methods. By way of example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment termed F(ab′)2. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab′ monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. I 73:119, 1959; Edelman et al., in Methods in Enzymology 1:422 (Academic Press 1967); and by Andrews, S. M. and Titus, J. A. in Current Protocols in Immunology (Coligan J. E., et al., eds), John Wiley & Sons, New York (2003). pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating heavy chains to form monovalent light-heavy chain fragments (Fd), further cleaving of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
An antibody fragment may also be any synthetic or genetically engineered protein. For example, antibody fragments include isolated fragments consisting of the light chain variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (scFv proteins).
Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also termed “minimal recognition units”, or “hypervariable region”) can be obtained by constructing polynucleotides that encode the CDR of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).
Thus, in one embodiment, the binding agent comprises at least one CDR as described herein. The binding agent may comprise at least two, three, four, five or six CDR's as described herein. The binding agent further may comprise at least one variable region domain of an antibody described herein. The variable region domain may be of any size or amino acid composition and will generally comprise at least one CDR sequence responsible for binding to human sclerostin, for example CDR-H1, CDR-H2, CDR-H3 and/or the light chain CDRs specifically described herein and which is adjacent to or in frame with one or more framework sequences. In general terms, the variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (VH) and/or light (VL) chain variable domains. Thus, for example, the V region domain may be monomeric and be a VH or VL domain, which is capable of independently binding human sclerostin with an affinity at least equal to 1×10−7M or less as described below. Alternatively, the V region domain may be dimeric and contain VH-VH, VH-VL, or VL-VL, dimers. The V region dimer comprises at least one VH and at least one VL chain that may be non-covalently associated (hereinafter referred to as Fv). If desired, the chains may be covalently coupled either directly, for example via a disulfide bond between the two variable domains, or through a linker, for example a peptide linker, to form a single chain Fv (scFv).
The variable region domain may be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain that has been created using recombinant DNA engineering techniques. Such engineered versions include those created, for example, from a specific antibody variable region by insertions, deletions, or changes in or to the amino acid sequences of the specific antibody. Particular examples include engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.
The variable region domain may be covalently attached at a C-terminal amino acid to at least one other antibody domain or a fragment thereof. Thus, for example, a VH domain that is present in the variable region domain may be linked to an immunoglobulin CH1 domain, or a fragment thereof. Similarly a VL domain may be linked to a CK domain or a fragment thereof. In this way, for example, the antibody may be a Fab fragment wherein the antigen binding domain contains associated VH and VL domains covalently linked at their C-termini to a CH1 and CK domain, respectively. The CH1 domain may be extended with further amino acids, for example to provide a hinge region or a portion of a hinge region domain as found in a Fab′ fragment, or to provide further domains, such as antibody CH2 and CH3 domains.
As described herein, binding agents comprise at least one of these CDRs. For example, one or more CDR may be incorporated into known antibody framework regions (IgG1, IgG2, etc.), or conjugated to a suitable vehicle to enhance the half-life thereof. Suitable vehicles include, but are not limited to Fc, polyethylene glycol (PEG), albumin, transferrin, and the like. These and other suitable vehicles are known in the art. Such conjugated CDR peptides may be in monomeric, dimeric, tetrameric, or other form. In one embodiment, one or more water-soluble polymer is bonded at one or more specific position, for example at the amino terminus, of a binding agent.
In certain preferred embodiments, a binding agent comprises one or more water soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337. In certain embodiments, a derivative binding agent comprises one or more of monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of such polymers. In certain embodiments, one or more water-soluble polymer is randomly attached to one or more side chains. In certain embodiments, PEG can act to improve the therapeutic capacity for a binding agent, such as an antibody. Certain such methods are discussed, for example, in U.S. Pat. No. 6,133,426, which is hereby incorporated by reference for any purpose.
It will be appreciated that a binding agent of the present invention may have at least one amino acid substitution, providing that the binding agent retains binding specificity. Therefore, modifications to the binding agent structures are encompassed within the scope of the invention. These may include amino acid substitutions, which may be conservative or non-conservative, that do not destroy the sclerostin binding capability of a binding agent. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. A conservative amino acid substitution may also involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid mimetics for a member from another class with different physical properties (e.g. size, polarity, hydrophobicity, charge). Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.
Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.
A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.
One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules.
A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.
Additional methods of predicting secondary structure include “threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and “evolutionary linkage” (See Holm, supra (1999), and Brenner, supra (1997)).
In certain embodiments, variants of binding agents include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide. In certain embodiments, variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants may be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.
Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify important residues of antibodies to sclerostin, or to increase or decrease the affinity of the antibodies to sclerostin described herein.
According to certain embodiments, preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physiochemical or functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991), which are each incorporated herein by reference.
In certain embodiments, binding agents of the invention may be chemically bonded with polymers, lipids, or other moieties.
The binding agents may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In one example, the biocompatible framework structure comprises a polypeptide or portion thereof that is sufficient to form a conformationally stable structural support, or framework, or scaffold, which is able to display one or more sequences of amino acids that bind to an antigen (e.g., CDRs, a variable region, etc.) in a localized surface region. Such structures can be a naturally occurring polypeptide or polypeptide “fold” (a structural motif), or can have one or more modifications, such as additions, deletions or substitutions of amino acids, relative to a naturally occurring polypeptide or fold. These scaffolds can be derived from a polypeptide of any species (or of more than one species), such as a human, other mammal, other vertebrate, invertebrate, plant, bacteria or virus.
Typically the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1, coiled coil, LAC1-D1, Z domain and tendramisat domains may be used (See e.g., Nygren and Uhlen, 1997, Current Opinion in Structural Biology, 7, 463-469).
In preferred embodiments, it will be appreciated that the binding agents of the invention include the humanized antibodies described herein. Humanized antibodies such as those described herein can be produced using techniques known to those skilled in the art (Zhang, W., et al., Molecular Immunology. 42(12):1445-1451, 2005; Hwang W. et al., Methods. 36(1):35-42, 2005; Dall'Acqua W F, et al., Methods 36(1):43-60, 2005; and Clark, M., Immunology Today. 21(8):397-402, 2000).
Additionally, one skilled in the art will recognize that suitable binding agents include portions of these antibodies, such as one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as specifically disclosed herein. At least one of the regions of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 may have at least one amino acid substitution, provided that the binding agent retains the binding specificity of the non-substituted CDR. The non-CDR portion of the binding agent may be a non-protein molecule, wherein the binding agent cross-blocks the binding of an antibody disclosed herein to sclerostin and/or neutralizes sclerostin. The non-CDR portion of the binding agent may be a non-protein molecule in which the binding agent exhibits a similar binding pattern to human sclerostin peptides in a “human sclerostin peptide epitope competition binding assay” as that exhibited by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24, and/or neutralizes sclerostin. The non-CDR portion of the binding agent may be composed of amino acids, wherein the binding agent is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of an antibody disclosed herein to sclerostin and/or neutralizes sclerostin. The non-CDR portion of the binding agent may be composed of amino acids, wherein the binding agent is a recombinant binding protein, and the recombinant binding protein exhibits a similar binding pattern to human sclerostin peptides in the human sclerostin peptide epitope competition binding assay (described hereinbelow) as that exhibited by at least one of the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24, and/or neutralizes sclerostin.
Where an antibody comprises one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as described above, it may be obtained by expression from a host cell containing DNA coding for these sequences. A DNA coding for each CDR sequence may be determined on the basis of the amino acid sequence of the CDR and synthesized together with any desired antibody variable region framework and constant region DNA sequences using oligonucleotide synthesis techniques, site-directed mutagenesis and polymerase chain reaction (PCR) techniques as appropriate. DNA coding for variable region frameworks and constant regions is widely available to those skilled in the art from genetic sequences databases such as GenBank®. Each of the above-mentioned CDRs will be typically located in a variable region framework at positions 31-35 (CDR-H1), 50-65 (CDR-H2) and 95-102 (CDR-H3) of the heavy chain and positions 24-34 (CDR-L1), 50-56 (CDR-L2) and 89-97 (CDR-L3) of the light chain according to the Kabat numbering system (Kabat et al., 1987 in Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA).
Once synthesized, the DNA encoding an antibody of the invention or fragment thereof may be propagated and expressed according to any of a variety of well-known procedures for nucleic acid excision, ligation, transformation, and transfection using any number of known expression vectors. Thus, in certain embodiments expression of an antibody fragment may be preferred in a prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods Enzymol. 178:497-515). In certain other embodiments, expression of the antibody or a fragment thereof may be preferred in a eukaryotic host cell, including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), animal cells (including mammalian cells) or plant cells. Examples of suitable animal cells include, but are not limited to, myeloma (such as a mouse NSO line), COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells.
One or more replicable expression vectors containing DNA encoding an antibody variable and/or constant region may be prepared and used to transform an appropriate cell line, for example, a non-producing myeloma cell line, such as a mouse NSO line or a bacteria, such as E. coli, in which production of the antibody will occur. In order to obtain efficient transcription and translation, the DNA sequence in each vector should include appropriate regulatory sequences, particularly a promoter and leader sequence operatively linked to the variable domain sequence. Particular methods for producing antibodies in this way are generally well-known and routinely used. For example, basic molecular biology procedures are described by Maniatis et al. (Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; see also Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory, New York, (2001)). DNA sequencing can be performed as described in Sanger et al. (PNAS 74:5463, (1977)) and the Amersham International plc sequencing handbook, and site directed mutagenesis can be carried out according to methods known in the art (Kramer et al., Nucleic Acids Res. 12:9441, (1984); Kunkel Proc. Natl. Acad. Sci. USA 82:488-92 (1985); Kunkel et al., Methods in Enzymol. 154:367-82 (1987); the Anglian Biotechnology Ltd handbook). Additionally, numerous publications describe techniques suitable for the preparation of antibodies by manipulation of DNA, creation of expression vectors, and transformation and culture of appropriate cells (Mountain A and Adair, J R in Biotechnology and Genetic Engineering Reviews (ed. Tombs, M P, 10, Chapter 1, 1992, Intercept, Andover, UK); “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed.), Wiley Interscience, New York).
Where it is desired to improve the affinity of antibodies according to the invention containing one or more of the above-mentioned CDRs can be obtained by a number of affinity maturation protocols including maintaining the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutation strains of E. coli. (Low et al., J. Mol. Biol., 250, 350-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 7-88, 1996) and sexual PCR (Crameri, et al., Nature, 391, 288-291, 1998). All of these methods of affinity maturation are discussed by Vaughan et al. (Nature Biotechnology, 16, 535-539, 1998).
Other antibodies according to the invention may be obtained by conventional immunization and cell fusion procedures as described herein and known in the art. Monoclonal antibodies of the invention may be generated using a variety of known techniques. In general, monoclonal antibodies that bind to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256:495, 1975; Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991); U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); Picksley et al., “Production of monoclonal antibodies against proteins expressed in E. coli,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). Antibody fragments may be derived therefrom using any suitable standard technique such as proteolytic digestion, or optionally, by proteolytic digestion (for example, using papain or pepsin) followed by mild reduction of disulfide bonds and alkylation. Alternatively, such fragments may also be generated by recombinant genetic engineering techniques as described herein.
Monoclonal antibodies can be obtained by injecting an animal, for example, a rat, hamster, a rabbit, or preferably a mouse, including for example a transgenic or a knock-out, as known in the art, with an immunogen comprising human sclerostin of SEQ ID NO:1, or a fragment thereof, according to methods known in the art and described herein. The presence of specific antibody production may be monitored after the initial injection and/or after a booster injection by obtaining a serum sample and detecting the presence of an antibody that binds to human sclerostin or peptide using any one of several immunodetection methods known in the art and described herein. From animals producing the desired antibodies, lymphoid cells, most commonly cells from the spleen or lymph node, are removed to obtain B-lymphocytes. The B lymphocytes are then fused with a drug-sensitized myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal and that optionally has other desirable properties (e.g., inability to express endogenous Ig gene products, e.g., P3X63-Ag 8.653 (ATCC No. CRL 1580); NSO, SP20) to produce hybridomas, which are immortal eukaryotic cell lines. The lymphoid (e.g., spleen) cells and the myeloma cells may be combined for a few minutes with a membrane fusion-promoting agent, such as polyethylene glycol or a nonionic detergent, and then plated at low density on a selective medium that supports the growth of hybridoma cells but not unfused myeloma cells. A preferred selection media is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient time, usually about one to two weeks, colonies of cells are observed. Single colonies are isolated, and antibodies produced by the cells may be tested for binding activity to human sclerostin, using any one of a variety of immunoassays known in the art and described herein. The hybridomas are cloned (e.g., by limited dilution cloning or by soft agar plaque isolation) and positive clones that produce an antibody specific to sclerostin are selected and cultured. The monoclonal antibodies from the hybridoma cultures may be isolated from the supernatants of hybridoma cultures. An alternative method for production of a murine monoclonal antibody is to inject the hybridoma cells into the peritoneal cavity of a syngeneic mouse, for example, a mouse that has been treated (e.g., pristane-primed) to promote formation of ascites fluid containing the monoclonal antibody. Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., “Purification of Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). Monoclonal antibodies may be purified by affinity chromatography using an appropriate ligand selected based on particular properties of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of a suitable ligand, immobilized on a solid support, include Protein A, Protein G, an anticonstant region (light chain or heavy chain) antibody, an anti-idiotype antibody, and a TGF-beta binding protein, or fragment or variant thereof.
An antibody of the present invention may also be a human monoclonal antibody. Human monoclonal antibodies may be generated by any number of techniques with which those having ordinary skill in the art will be familiar. Such methods include, but are not limited to, Epstein Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures as known in the art and based on the disclosure herein. For example, human monoclonal antibodies may be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun. 6:579, 1994; U.S. Pat. No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58; Jakobovits et al., 1995 Ann. N.Y. Acad. Sci. 764:525-35. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, human immunoglobulin transgenes may be mini-gene constructs, or transloci on yeast artificial chromosomes, which undergo B cell-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue. Human monoclonal antibodies may be obtained by immunizing the transgenic mice, which may then produce human antibodies specific for sclerostin. Lymphoid cells of the immunized transgenic mice can be used to produce human antibody-secreting hybridomas according to the methods described herein. Polyclonal sera containing human antibodies may also be obtained from the blood of the immunized animals.
Another method for generating human antibodies of the invention includes immortalizing human peripheral blood cells by EBV transformation. See, e.g., U.S. Pat. No. 4,464,456. Such an immortalized B cell line (or lymphoblastoid cell line) producing a monoclonal antibody that specifically binds to sclerostin can be identified by immunodetection methods as provided herein, for example, an ELISA, and then isolated by standard cloning techniques. The stability of the lymphoblastoid cell line producing an anti-sclerostin antibody may be improved by fusing the transformed cell line with a murine myeloma to produce a mouse-human hybrid cell line according to methods known in the art (see, e.g., Glasky et al., Hybridoma 8:377-89 (1989)). Still another method to generate human monoclonal antibodies is in vitro immunization, which includes priming human splenic B cells with human sclerostin, followed by fusion of primed B cells with a heterohybrid fusion partner. See, e.g., Boerner et al., 1991. J. Immunol. 147:86-95.
In certain embodiments, a B cell that is producing an anti-human sclerostin antibody is selected and the light chain and heavy chain variable regions are cloned from the B cell according to molecular biology techniques known in the art (WO 92/02551; U.S. Pat. No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. B cells from an immunized animal may be isolated from the spleen, lymph node, or peripheral blood sample by selecting a cell that is producing an antibody that specifically binds to sclerostin. B cells may also be isolated from humans, for example, from a peripheral blood sample. Methods for detecting single B cells that are producing an antibody with the desired specificity are well known in the art, for example, by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibody, and the like. Methods for selection of specific antibody-producing B cells include, for example, preparing a single cell suspension of B cells in soft agar that contains human sclerostin. Binding of the specific antibody produced by the B cell to the antigen results in the formation of a complex, which may be visible as an immunoprecipitate. After the B cells producing the desired antibody are selected, the specific antibody genes may be cloned by isolating and amplifying DNA or mRNA according to methods known in the art and described herein.
An additional method for obtaining antibodies of the invention is by phage display. See, e.g., Winter et al., 1994 Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280. Human or murine immunoglobulin variable region gene combinatorial libraries may be created in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind specifically to TGF-beta binding protein or variant or fragment thereof. See, e.g., U.S. Pat. No. 5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and references cited therein. For example, a library containing a plurality of polynucleotide sequences encoding Ig variable region fragments may be inserted into the genome of a filamentous bacteriophage, such as M13 or a variant thereof, in frame with the sequence encoding a phage coat protein. A fusion protein may be a fusion of the coat protein with the light chain variable region domain and/or with the heavy chain variable region domain. According to certain embodiments, immunoglobulin Fab fragments may also be displayed on a phage particle (see, e.g., U.S. Pat. No. 5,698,426).
Heavy and light chain immunoglobulin cDNA expression libraries may also be prepared in lambda phage, for example, using λImmunoZapTM(H) and λImmunoZapTM(L) vectors (Stratagene, La Jolla, Calif.). Briefly, mRNA is isolated from a B cell population, and used to create heavy and light chain immunoglobulin cDNA expression libraries in the λImmunoZap(H) and λImmunoZap(L) vectors. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid that allows high level expression of monoclonal antibody fragments from E. coli. 
In one embodiment, in a hybridoma the variable regions of a gene expressing a monoclonal antibody of interest are amplified using nucleotide primers. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. (See, e.g., Stratagene (La Jolla, Calif.), which sells primers for mouse and human variable regions including, among others, primers for VHa, VHb, VHc, VHd, CHl, VL and CL regions.) These primers may be used to amplify heavy or light chain variable regions, which may then be inserted into vectors such as ImmunoZAPTMH or ImmunoZAPTML (Stratagene), respectively. These vectors may then be introduced into E. coli, yeast, or mammalian-based systems for expression. Large amounts of a single-chain protein containing a fusion of the VH and VL domains may be produced using these methods (see Bird et al., Science 242:423-426, 1988).
Once cells producing antibodies according to the invention have been obtained using any of the above-described immunization and other techniques, the specific antibody genes may be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures as described herein. The antibodies produced therefrom may be sequenced and the CDRs identified and the DNA coding for the CDRs may be manipulated as described previously to generate other antibodies according to the invention.
Preferably the binding agents specifically bind to sclerostin. As with all binding agents and binding assays, one of skill in this art recognizes that the various moieties to which a binding agent should not detectably bind in order to be therapeutically effective and suitable would be exhaustive and impractical to list. Therefore, for a binding agent disclosed herein, the term “specifically binds” refers to the ability of a binding agent to bind to sclerostin, preferably human sclerostin, with greater affinity than it binds to an unrelated control protein. Preferably the control protein is hen egg white lysozyme. Preferably the binding agents bind to sclerostin with an affinity that is at least, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for a control protein. A binding agent may have a binding affinity for human sclerostin of less than or equal to 1×10−7 M, less than or equal to 1×10−8 M, less than or equal to 1×10−9 M, less than or equal to 1×10−10 M, less than or equal to 1×10−11 M, or less than or equal to 1×10−12 M.
Affinity may be determined by an affinity ELISA assay. In certain embodiments, affinity may be determined by a BIAcore assay. In certain embodiments, affinity may be determined by a kinetic method. In certain embodiments, affinity may be determined by an equilibrium/solution method. Such methods are described in further detail herein or known in the art.
Sclerostin binding agents of the present invention preferably modulate sclerostin function in the cell-based assay described herein and/or the in vivo assay described herein and/or bind to one or more of the epitopes described herein and/or cross-block the binding of one of the antibodies described in this application and/or are cross-blocked from binding sclerostin by one of the antibodies described in this application. Accordingly such binding agents can be identified using the assays described herein.
In certain embodiments, binding agents are generated by first identifying antibodies that bind to one more of the epitopes provided herein and/or neutralize in the cell-based and/or in vivo assays described herein and/or cross-block the antibodies described in this application and/or are cross-blocked from binding sclerostin by one of the antibodies described in this application. The CDR regions from these antibodies are then used to insert into appropriate biocompatible frameworks to generate sclerostin binding agents. The non-CDR portion of the binding agent may be composed of amino acids, or may be a non-protein molecule. The assays described herein allow the characterization of binding agents. Preferably the binding agents of the present invention are antibodies as defined herein.
It will be understood by one skilled in the art that some proteins, such as antibodies, may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the protein as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperizine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, R J. Journal of Chromatography 705:129-134, 1995).
Antibodies referred to as Ab-A, Ab-B, Ab-C, Ab-D and Ab-1 are described below. “HC” refers to the heavy chain and “LC” refers to the light chain. For some antibodies below, the CDRs are box shaded and the constant (C) regions are shown in bold italics.
Ab-D
Antibody D (also referred to herein as Ab-D and Mab-D) is a mouse antibody which exhibits high affinity binding to sclerostin. The BIAcore binding pattern of Ab-D is shown in FIG. 18.
The amino acid sequence of the mature form (signal peptide removed) of Ab-D light chain:








(SEQ ID NO: 7)














Nucleic acid sequence encoding the mature form (signal peptide removed) of Ab-D LC is as follows:








(SEQ ID NO: 8)










1
GATGTCCAGA TGATTCAGTC TCCATCCTCC CTGTCTGCAT CTTTGGGAGA



 


51
CATAGTCACC ATGACTTGCC AGGCAAGTCA GGGCACTAGC ATTAATTTAA


 


101
ACTGGTTTCA GCAAAAACCA GGGAAGGCTC CTAAGCTCCT GATCTATGGT


 


151
TCAAGCAACT TGGAAGATGG GGTCCCATCA AGGTTCAGTG GCAGTAGATA


 


201
TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTGGAGGAT GAAGATCTGG


 


251
CAACTTATTT CTGTCTACAA CATAGTTATC TCCCGTACAC GTTCGGAGGG


 


301
GGGACCAAGC TGGAAATAAA ACGGGCTGAT GCTGCACCAA CTGTATCCAT


 


351
CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC TCAGTCGTGT


 


401
GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA GTGGAAGATT


 


451
GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA CTGATCAGGA


 


501
CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG


 


551
ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC TCACAAGACA


 


601
TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT GTTAG






The amino acid sequence of Ab-D LC including signal peptide is as follows:








(SEQ ID NO: 9)










1
MNTRAPAEFL GFLLLWFLGA RCDVQMIQSP SSLSASLGDI VTMTCQASQG



 


51
TSINLNWFQQ KPGKAPKLLI YGSSNLEDGV PSRFSGSRYG TDFTLTISSL


 


101
EDEDLATYFC LQHSYLPYTF GGGTKLEIKR ADAAPTVSIF PPSSEQLTSG


 


151
GASVVCFLNN FYPKDINVKW KIDGSERQNG VLNSWTDQDS KDSTYSMSST


 


201
LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRNEC






Nucleic acid sequence of Ab-D LC including signal peptide encoding sequence:








(SEQ ID NO: 10)










1
ATGAACACGA GGGCCCCTGC TGAGTTCCTT GGGTTCCTGT TGCTCTGGTT



 


51
TTTAGGTGCC AGATGTGATG TCCAGATGAT TCAGTCTCCA TCCTCCCTGT


 


101
CTGCATCTTT GGGAGACATA GTCACCATGA CTTGCCAGGC AAGTCAGGGC


 


151
ACTAGCATTA ATTTAAACTG GTTTCAGCAA AAACCAGGGA AGGCTCCTAA


 


201
GCTCCTGATC TATGGTTCAA GCAACTTGGA AGATGGGGTC CCATCAAGGT


 


251
TCAGTGGCAG TAGATATGGG ACAGATTTCA CTCTCACCAT CAGCAGCCTG


 


301
GAGGATGAAG ATCTGGCAAC TTATTTCTGT CTACAACATA GTTATCTCCC


 


351
GTACACGTTC GGAGGGGGGA CCAAGCTGGA AATAAAACGG GCTGATGCTG


 


401
CACCAACTGT ATCCATCTTC CCACCATCCA GTGAGCAGTT AACATCTGGA


 


451
GGTGCCTCAG TCGTGTGCTT CTTGAACAAC TTCTACCCCA AAGACATCAA


 


501
TGTCAAGTGG AAGATTGATG GCAGTGAACG ACAAAATGGC GTCCTGAACA


 


551
GTTGGACTGA TCAGGACAGC AAAGACAGCA CCTACAGCAT GAGCAGCACC


 


601
CTCACGTTGA CCAAGGACGA GTATGAACGA CATAACAGCT ATACCTGTGA


 


651
GGCCACTCAC AAGACATCAA CTTCACCCAT TGTCAAGAGC TTCAACAGGA


 


701
ATGAGTGTTA G






The amino acid sequence of the mature form (signal peptide removed) of Ab-D HC heavy chain is as follows:








(SEQ ID NO: 11)














The nucleic acid sequence encoding the mature form (signal peptide removed) of Ab-D HC is:








(SEQ ID NO: 12)










1
GAGGTCCAGC TGCAACAGTC TGGACCTGAA CTGGTGACGC CTGGGGCTTC



 


51
AGTGAAGATA TCTTGTAAGG CTTCTGGATA CACATTCACT GACCACTACA


 


101
TGAGCTGGGT GAAGCAGAGT CATGGAAAAA GCCITGAGTG GATTGGAGAT


 


151
ATTAATCCCT ATTCTGGTGA AACTACCTAC AACCAGAAGT TCAAGGGCAC


 


201
GGCCACATTG ACTGTAGACA AGTCTTCCAG TATAGCCTAC ATGGAGATCC


 


251
GCGGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC AAGAGATGAT


 


301
TACGACGCCT CTCCGTTTGC TTACTGGGGC CAAGGGACTC TGGTCACTGT


 


351
CTCTGCAGCC AAAACGACAC CCCCATCTGT CTATCCACTG GCCCCTGGAT


 


401
CTGCTGCCCA AACTAACTCC ATGGTGACCC TGGGATGCCT GGTCAAGGGC


 


451
TATTTCCCTG AGCCAGTGAC AGTGACCTGG AACTCTGGAT CCCTGTCCAG


 


501
CGGTGTGCAC ACCTTCCCAG CTGTCCTGCA GTCTGACCTC TACACTCTGA


 


551
GCAGCTCAGT GACTGTCCCC TCCAGCACCT GGCCCAGCGA GACCGTCACC


 


601
TGCAACGTTG CCCACCCGGC CAGCAGCACC AAGGTGGACA AGAAAATTGT


 


651
GCCCAGGGAT TGTGGTTGTA AGCCTTGCAT ATGTACAGTC CCAGAAGTAT


 


701
CATCTGTCTT CATCTTCCCC CCAAAGCCCA AGGATGTGCT CACCATTACT


 


751
CTGACTCCTA AGGTCACGTG TGTTGTGGTA GACATCAGCA AGGATGATCC


 


801
CGAGGTCCAG TTCAGCTGGT TTGTAGATGA TGTGGAGGTG CACACAGCTC


 


851
AGACGCAACC CCGGGAGGAG CAGTTCAACA GCACTTTCCG CTCAGTCAGT


 


901
GAACTTCCCA TCATGCACCA GGACTGGCTC AATGGCAAGG AGTTCAAATG


 


951
CAGGGTCAAC AGTCCAGCTT TCCCTGCCCC CATCGAGAAA ACCATCTCCA


 


1001
AAACCAAAGG CAGACCGAAG GCTCCACAGG TGTACACCAT TCCACCTCCC


 


1051
AAGGAGCAGA TGGCCAAGGA TAAAGTCAGT CTGACCTGCA TGATAACAGA


 


1101
CTTCTTCCCT GAAGACATTA CTGTGGAGTG GCAGTGGAAT GGGCAGCCAG


 


1151
CGGAGAACTA CAAGAACACT CAGCCCATCA TGGACACAGA TGGCTCTTAC


 


1201
TTCATCTACA GCAAGCTCAA TGTGCAGAAG AGCAACTGGG AGGCAGGAAA


 


1251
TACTTTCACC TGCTCTGTGT TACATGAGGG CCTGCACAAC CACCATACTG


 


1301
AGAAGAGCCT CTCCCACTCT CCTGGTAAAT GA






The amino acid sequence of Ab-D HC including signal peptide is:








(SEQ ID NO: 13)










1
MRCRWIFLFL LSGTAGVLSE VQLQQSGPEL VTPGASVKIS CKASGYTFTD



 


51
HYMSWVKQSH GKSLEWIGDI NPYSGETTYN QKFKGTATLT VDKSSSIAYM


 


101
EIRGLTSEDS AVYYCARDDY DASPFAYWGQ GTLVTVSAAK TTPPSVYPLA


 


151
PGSAAQTNSM VTLGCLVKGY FPEPVTVTWN SGSLSSGVHT FPAVLQSDLY


 


201
TLSSSVTVPS STWPSETVTC NVAHPASSTK VDKKIVPRDC GCKPCICTVP


 


251
EVSSVFIFPP KPKDVLTITL TPKVTCVVVD ISKDDPEVQF SWFVDDVEVH


 


301
TAQTQPREEQ FNSTFRSVSE LPIMHQDWLN GKEFKCRVNS PAFPAPIEKT


 


351
ISKTKGRPKA PQVYTIPPPK EQMAKDKVSL TCMITDFFPE DITVEWQWNG


 


401
QPAENYKNTQ PIMDTDGSYF IYSKLNVQKS NWEAGNTFTC SVLHEGLHNH


 


451
HTEKSLSHSP GK






The nucleic acid sequence of Ab-D HC including signal peptide encoding sequence is:








(SEQ ID NO: 14)










1
ATGAGATGCA GGTGGATCTT TCTCTTTCTC CTGTCAGGAA CTGCAGGTGT



 


51
CCTCTCTGAG GTCCAGCTGC AACAGTCTGG ACCTGAACTG GTGACGCCTG


 


101
GGGCTTCAGT GAAGATATCT TGTAAGGCTT CTGGATACAC ATTCACTGAC


 


151
CACTACATGA GCTGGGTGAA GCAGAGTCAT GGAAAAAGCC TTGAGTGGAT


 


201
TGGAGATATT AATCCCTATT CTGGTGAAAC TACCTACAAC CAGAAGTTCA


 


251
AGGGCACGGC CACATTGACT GTAGACAAGT CTTCCAGTAT AGCCTACATG


 


301
GAGATCCGCG GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG


 


351
AGATGATTAC GACGCCTCTC CGTTTGCTTA CTGGGGCCAA GGGACTCTGG


 


401
TCACTGTCTC TGCAGCCAAA ACGACACCCC CATCTGTCTA TCCACTGGCC


 


451
CCTGGATCTG CTGCCCAAAC TAACTCCATG GTGACCCTGG GATGCCTGGT


 


501
CAAGGGCTAT TTCCCTGAGC CAGTGACAGT GACCTGGAAC TCTGGATCCC


 


551
TGTCCAGCGG TGTGCACACC TTCCCAGCTG TCCTGCAGTC TGACCTCTAC


 


601
ACTCTGAGCA GCTCAGTGAC TGTCCCCTCC AGCACCTGGC CCAGCGAGAC


 


651
CGTCACCTGC AACGTTGCCC ACCCGGCCAG CAGCACCAAG GTGGACAAGA


 


701
AAATTGTGCC CAGGGATTGT GGTTGTAAGC CTTGCATATG TACAGTCCCA


 


751
GAAGTATCAT CTGTCTTCAT CTTCCCCCCA AAGCCCAAGG ATGTGCTCAC


 


801
CATTACTCTG ACTCCTAAGG TCACGTGTGT TGTGGTAGAC ATCAGCAAGG


 


851
ATGATCCCGA GGTCCAGTTC AGCTGGTTTG TAGATGATGT GGAGGTGCAC


 


901
ACAGCTCAGA CGCAACCCCG GGAGGAGCAG TTCAACAGCA CTTTCCGCTC


 


951
AGTCAGTGAA CTTCCCATCA TGCACCAGGA CTGGCTCAAT GGCAAGGAGT


 


1001
TCAAATGCAG GGTCAACAGT CCAGCTTTCC CTGCCCCCAT CGAGAAAACC


 


1051
ATCTCCAAAA CCAAAGGCAG ACCGAAGGCT CCACAGGTGT ACACCATTCC


 


1101
ACCTCCCAAG GAGCAGATGG CCAAGGATAA AGTCAGTCTG ACCTGCATGA


 


1151
TAACAGACTT CTTCCCTGAA GACATTACTG TGGAGTGGCA GTGGAATGGG


 


1201
CAGCCAGCGG AGAACTACAA GAACACTCAG CCCATCATGG ACACAGATGG


 


1251
CTCTTACTTC ATCTACAGCA AGCTCAATGT GCAGAAGAGC AACTGGGAGG


 


1301
CAGGAAATAC TTTCACCTGC TCTGTGTTAC ATGAGGGCCT GCACAACCAC


 


1351
CATACTGAGA AGAGCCTCTC CCACTCTCCT GGTAAATGA






The CDR (complementarity determining region) sequences in the variable region of the heavy chain of Ab-D are as follows:









CDR-H1:
DHYMS
(SEQ ID NO: 39)


 


CDR-H2:
DINPYSGETTYNQKFKG
(SEQ ID NO: 40)


 


CDR-H3:
DDYDASPFAY
(SEQ ID NO: 41)






The light chain variable region CDR sequences of Ab-D are:











CDR-L1:
QASQGTSINLN
(SEQ ID NO: 42)



 



CDR-L2:
GSSNLED
(SEQ ID NO: 43)



 



CDR-L3:
LQHSYLPYT
(SEQ ID NO: 44)






Ab-C

Antibody C (also referred to herein as Ab-C and Mab-C) is a mouse antibody which exhibits high affinity binding to sclerostin. The BIAcore binding pattern of Ab-C is shown in FIG. 17. The amino acid sequence of the mature form (signal peptide removed) of Ab-C Light Chain is as follows:








(SEQ ID NO: 15)














The nucleic acid sequence encoding the mature form (signal peptide removed) of Ab-C LC is:







(SEQ ID NO: 16)








  1
GACATTGTGC TGACCCAATC TCCAGCTTCT TTGACTGTGT 



CTCTAGGCCT


 


 51
GAGGGCCACC ATCTCCTGCA AGGCCAGCCA AAGTGTTGAT 



TATGATGGTG


 


101
ATAGTTATAT GAACTGGTAC CAGCAGAAAC CAGGACAGCC 



ACCCAAACTC


 


151
CTCATCTATG CTGCATCCAA TCTAGAATCT GGGATCCCAG 



CCAGGTTTAG


 


201
TGGCAATGGG TCTGGGACAG ACTTCACCCT CAACATCCAT 



CCTGTGGAGG


 


251
AGGAGGATGC TGTAACCTAT TACTGTCAAC AAAGTAATGA 



GGATCCGTGG


 


301
ACGTTCGGTG GAGGCACCAA GCTGGAAATC AAACGGGCTG 



ATGCTGCACC


 


351
AACTGTATCC ATCTTCCCAC CATCCAGTGA GCAGTTAACA 



TCTGGAGGTG


 


401
CCTCAGTCGT GTGCTTCTTG AACAACTTCT ACCCCAAAGA 



CATCAATGTC


 


451
AAGTGGAAGA TTGATGGCAG TGAACGACAA AATGGCGTCC 



TGAACAGTTG


 


501
GACTGATCAG GACAGCAAAG ACAGCACCTA CAGCATGAGC 



AGCACCCTCA


 


551
CGTTGACCAA GGACGAGTAT GAACGACATA ACAGCTATAC 



CTGTGAGGCC


 


601
ACTCACAAGA CATCAACTTC ACCCATTGTC AAGAGCTTCA 



ACAGGAATGA


 


651
GTGTTAG






The amino acid sequence of Ab-C LC including signal peptide is:







(SEQ ID NO: 17)








  1
METDTILLWV LLLWVPGSTG DIVLTQSPAS LTVSLGLRAT



ISCKASQSVD


 


 51
YDGDSYMNWY QQKPGQPPKL LIYAASNLES GIPARFSGNG



SGTDFTLNIH


 


101
PVEEEDAVTY YCQQSNEDPW TFGGGTKLEI KRADAAPTVS



IFPPSSEQLT


 


151
SGGASVVCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ



DSKDSTYSMS


 


201
STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC






The nucleic acid sequence of Ab-C LC including signal peptide encoding sequence is:







(SEQ ID NO: 18)








  1
ATGGAGACAG ACACAATCCT GCTATGGGTG CTGCTGCTCT



GGGTTCCAGG


 


 51
CTCCACTGGT GACATTGTGC TGACCCAATC TCCAGCTTCT



TTGACTGTGT


 


101
CTCTAGGCCT GAGGGCCACC ATCTCCTGCA AGGCCAGCCA



AAGTGTTGAT


 


151
TATGATGGTG ATAGTTATAT GAACTGGTAC CAGCAGAAAC



CAGGACAGCC


 


201
ACCCAAACTC CTCATCTATG CTGCATCCAA TCTAGAATCT



GGGATCCCAG


 


251
CCAGGTTTAG TGGCAATGGG TCTGGGACAG ACTTCACCCT



CAACATCCAT


 


301
CCTGTGGAGG AGGAGGATGC TGTAACCTAT TACTGTCAAC



AAAGTAATGA


 


351
GGATCCGTGG ACGTTCGGTG GAGGCACCAA GCTGGAAATC



AAACGGGCTG


 


401
ATGCTGCACC AACTGTATCC ATCTTCCCAC CATCCAGTGA



GCAGTTAACA


 


451
TCTGGAGGTG CCTCAGTCGT GTGCTTCTTG AACAACTTCT



ACCCCAAAGA


 


501
CATCAATGTC AAGTGGAAGA TTGATGGCAG TGAACGACAA



AATGGCGTCC


 


551
TGAACAGTTG GACTGATCAG GACAGCAAAG ACAGCACCTA



CAGCATGAGC


 


601
AGCACCCTCA CGTTGACCAA GGACGAGTAT GAACGACATA



ACAGCTATAC


 


651
CTGTGAGGCC ACTCACAAGA CATCAACTTC ACCCATTGTC



AAGAGCTTCA


 


701
ACAGGAATGA GTGTTAG






Ab-C Heavy Chain

The amino acid sequence of the mature form (signal peptide removed) of Ab-C HC is:








(SEQ ID NO: 19)














The nucleic acid sequence encoding the mature form (signal peptide removed) of Ab-C HC is as follows:







(SEQ ID NO: 20)








   1
GAGGTCCAGC TGCAACAATC TGGACCTGAG CTGGTGAAGC 



CTGGGACTTC


 


  51
AGTGAAGATG TCCTGTAAGG CTTCTGGATA CACATTCACT 



GACTGCTACA


 


 101
TGAACTGGGT GAAGCAGAGC CATGGGAAGA GCCTTGAATG 



GATTGGAGAT


 


 151
ATTAATCCTT TCAACGGTGG TACTACCTAC AACCAGAAGT 



TCAAGGGCAA


 


 201
GGCCACATTG ACTGTAGACA AATCCTCCAG CACAGCCTAC 



ATGCAGCTCA


 


 251
ACAGCCTGAC ATCTGACGAC TCTGCAGTCT ATTACTGTGC 



AAGATCCCAT


 


 301
TATTACTTCG ATGGTAGAGT CCCTTGGGAT GCTATGGACT 



ACTGGGGTCA


 


 351
AGGAACCTCA GTCACCGTCT CCTCAGCCAA AACGACACCC 



CCATCTGTCT


 


 401
ATCCACTGGC CCCTGGATCT GCTGCCCAAA CTAACTCCAT 



GGTGACCCTG


 


 451
GGATGCCTGG TCAAGGGCTA TTTCCCTGAG CCAGTGACAG 



TGACCTGGAA


 


 501
CTCTGGATCC CTGTCCAGCG GTGTGCACAC CTTCCCAGCT 



GTCCTGCAGT


 


 551
CTGACCTCTA CACTCTGAGC AGCTCAGTGA CTGTCCCCTC 



CAGCACCTGG


 


 601
CCCAGCGAGA CCGTCACCTG CAACGTTGCC CACCCGGCCA 



GCAGCACCAA


 


 651
GGTGGACAAG AAAATTGTGC CCAGGGATTG TGGTTGTAAG 



CCTTGCATAT


 


 701
GTACAGTCCC AGAAGTATCA TCTGTCTTCA TCTTCCCCCC 



AAAGCCCAAG


 


 751
GATGTGCTCA CCATTACTCT GACTCCTAAG GTCACGTGTG 



TTGTGGTAGA


 


 801
CATCAGCAAG GATGATCCCG AGGTCCAGTT CAGCTGGTTT 



GTAGATGATG


 


 851
TGGAGGTGCA CACAGCTCAG ACGCAACCCC GGGAGGAGCA 



GTTCAACAGC


 


 901
ACTTTCCGCT CAGTCAGTGA ACTTCCCATC ATGCACCAGG 



ACTGGCTCAA


 


 951
TGGCAAGGAG TTCAAATGCA GGGTCAACAG TGCAGCTTTC 



CCTGCCCCCA


 


1001
TCGAGAAAAC CATCTCCAAA ACCAAAGGCA GACCGAAGGC 



TCCACAGGTG


 


1051
TACACCATTC CACCTCCCAA GGAGCAGATG GCCAAGGATA 



AAGTCAGTCT


 


1101
GACCTGCATG ATAACAGACT TCTTCCCTGA AGACATTACT 



GTGGAGTGGC


 


1151
AGTGGAATGG GCAGCCAGCG GAGAACTACA AGAACACTCA 



GCCCATCATG


 


1201
GACACAGATG GCTCTTACTT CATCTACAGC AAGCTCAATG 



TGCAGAAGAG


 


1251
CAACTGGGAG GCAGGAAATA CTTTCACCTG CTCTGTGTTA 



CATGAGGGCC


 


1301
TGCACAACCA CCATACTGAG AAGAGCCTCT CCCACTCTCC 



TGGTAAATGA






The amino acid sequence of Ab-C HC including signal peptide is:







(SEQ ID NO: 21)








  1
MGWNWIFLFL LSGTAGVYSE VQLQQSGPEL VKPGTSVKMS



CKASGYTFTD


 


 51
CYMNWVKQSH GKSLEWIGDI NPFNGGTTYN QKFKGKATLT



VDKSSSTAYM


 


101
QLNSLTSDDS AVYYCARSHY YFDGRVPWDA MDYWGQGTSV



TVSSAKTTPP


 


151
SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL



SSGVHTFPAV


 


201
LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK



IVPRDCGCKP


 


251
CICTVPEVSS VFIFPPKPKD VLTITLTPKV TCVVVDISKD



DPEVQFSWFV


 


301
DDVEVHTAQT QPREEQFNST FRSVSELPIM HQDWLNGKEF



KCRVNSAAFP


 


351
APIEKTISKT KGRPKAPQVY TIPPPKEQMA KDKVSLTCMI



TDFFPEDITV


 


401
EWQWNGQPAE NYKNTQPIMD TDGSYFIYSK LNVQKSNWEA



GNTFTCSVLH


 


451
EGLHNHHTEK SLSHSPGK






The nucleic acid sequence of Ab-C HC including signal peptide encoding sequence is:







(SEQ ID NO: 22)








   1
ATGGGATGGA ACTGGATCTT TCTCTTCCTC TTGTCAGGAA



CTGCAGGTGT


 


  51
CTACTCTGAG GTCCAGCTGC AACAATCTGG ACCTGAGCTG



GTGAAGCCTG


 


 101
GGACTTCAGT GAAGATGTCC TGTAAGGCTT CTGGATACAC



ATTCACTGAC


 


 151
TGCTACATGA ACTGGGTGAA GCAGAGCCAT GGGAAGAGCC



TTGAATGGAT


 


 201
TGGAGATATT AATCCTTTCA ACGGTGGTAC TACCTACAAC



CAGAAGTTCA


 


 251
AGGGCAAGGC CACATTGACT GTAGACAAAT CCTCCAGCAC



AGCCTACATG


 


 301
CAGCTCAACA GCCTGACATC TGACGACTCT GCAGTCTATT



ACTGTGCAAG


 


 351
ATCCCATTAT TACTTCGATG GTAGAGTCCC TTGGGATGCT



ATGGACTACT


 


 401
GGGGTCAAGG AACCTCAGTC ACCGTCTCCT CAGCCAAAAC



GACACCCCCA


 


 451
TCTGTCTATC CACTGGCCCC TGGATCTGCT GCCCAAACTA



ACTCCATGGT


 


 501
GACCCTGGGA TGCCTGGTCA AGGGCTATTT CCCTGAGCCA



GTGACAGTGA


 


 551
CCTGGAACTC TGGATCCCTG TCCAGCGGTG TGCACACCTT



CCCAGCTGTC


 


 601
CTGCAGTCTG ACCTCTACAC TCTGAGCAGC TCAGTGACTG



TCCCCTCCAG


 


 651
CACCTGGCCC AGCGAGACCG TCACCTGCAA CGTTGCCCAC



CCGGCCAGCA


 


 701
GCACCAAGGT GGACAAGAAA ATTGTGCCCA GGGATTGTGG



TTGTAAGCCT


 


 751
TGCATATGTA CAGTCCCAGA AGTATCATCT GTCTTCATCT



TCCCCCCAAA


 


 801
GCCCAAGGAT GTGCTCACCA TTACTCTGAC TCCTAAGGTC



ACGTGTGTTG


 


 851
TGGTAGACAT CAGCAAGGAT GATCCCGAGG TCCAGTTCAG



CTGGTTTGTA


 


 901
GATGATGTGG AGGTGCACAC AGCTCAGACG CAACCCCGGG



AGGAGCAGTT


 


 951
CAACAGCACT TTCCGCTCAG TCAGTGAACT TCCCATCATG



CACCAGGACT


 


1001
GGCTCAATGG CAAGGAGTTC AAATGCAGGG TCAACAGTGC



AGCTTTCCCT


 


1051
GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGCAGAC



CGAAGGCTCC


 


1101
ACAGGTGTAC ACCATTCCAC CTCCCAAGGA GCAGATGGCC



AAGGATAAAG


 


1151
TCAGTCTGAC CTGCATGATA ACAGACTTCT TCCCTGAAGA



CATTACTGTG


 


1201
GAGTGGCAGT GGAATGGGCA GCCAGCGGAG AACTACAAGA



ACACTCAGCC


 


1251
CATCATGGAC ACAGATGGCT CTTACTTCAT CTACAGCAAG



CTCAATGTGC


 


1301
AGAAGAGCAA CTGGGAGGCA GGAAATACTT TCACCTGCTC



TGTGTTACAT


 


1351
GAGGGCCTGC ACAACCACCA TACTGAGAAG AGCCTCTCCC



ACTCTCCTGG


 


1401
TAAATGA






The CDR (complementarity determining region) sequences in the variable region of the heavy chain of Ab-C are as follows:









CDR-H1:
DCYMN
(SEQ ID NO: 45)


 


CDR-H2:
DINPFNGGTTYNQKFKG
(SEQ ID NO: 46)


 


CDR-H3:
SHYYFDGRVPWDAMDY
(SEQ ID NO: 47)






The light chain variable region CDR sequences of Ab-C are:











CDR-L1:
KASQSVDYDGDSYMN
(SEQ ID NO: 48)



 



CDR-L2:
AASNLES
(SEQ ID NO: 49)



 



CDR-L3:
QQSNEDPWT
(SEQ ID NO: 50)






Ab-A

Antibody A (also referred to herein as Ab-A and Mab-A) is a rabbit-mouse chimeric antibody which exhibits high affinity binding to sclerostin. The BIAcore binding pattern of Ab-A is shown in FIG. 15.
Ab-A Light Chain
The amino acid sequence of the mature form (signal peptide removed) of Ab-A LC:








(SEQ ID NO: 23)














The nucleic acid sequence encoding the mature form (signal peptide removed) of Ab-A LC:







(SEQ ID NO: 24)








  1
GCGCAAGTGC TGACCCAGAC TCCAGCCTCC GTGTCTGCAG



CTGTGGGAGG


 


 51
CACAGTCACC ATCAATTGCC AGTCCAGTCA GAGTGTTTAT



GATAACAACT


 


101
GGTTAGCCTG GTTTCAGCAG AAACCAGGGC AGCCTCCCAA



GCTCCTGATT


 


151
TATGATGCAT CCGATCTGGC ATCTGGGGTC CCATCGCGGT



TCAGTGGCAG


 


201
TGGATCTGGG ACACAGTTCA CTCTCACCAT CAGCGGCGTG



CAGTGTGCCG


 


251
ATGCTGCCAC TTACTACTGT CAAGGCGCTT ATAATGATGT



TATTTATGCT


 


301
TTCGGCGGAG GGACCGAGGT GGTGGTCAAA CGTACGGATG



CTGCACCAAC


 


351
TGTATCCATC TTCCCACCAT CCAGTGAGCA GTTAACATCT



GGAGGTGCCT


 


401
CAGTCGTGTG CTTCTTGAAC AACTTCTACC CCAAAGACAT



CAATGTCAAG


 


451
TGGAAGATTG ATGGCAGTGA ACGACAAAAT GGCGTCCTGA



ACAGTTGGAC


 


501
TGATCAGGAC AGCAAAGACA GCACCTACAG CATGAGCAGC



ACCCTCACGT


 


551
TGACCAAGGA CGAGTATGAA CGACATAACA GCTATACCTG



TGAGGCCACT


 


601
CACAAGACAT CAACTTCACC CATTGTCAAG AGCTTCAACA



GGAATGAGTG


 


651
TTAG






The amino acid sequence of Ab-A LC including signal peptide is:







(SEQ ID NO: 25)








  1
MDTRAPTQLL GLLLLWLPGA TFAQVLTQTP ASVSAAVGGT



VTINCQSSQS


 


 51
VYDNNWLAWF QQKPGQPPKL LIYDASDLAS GVPSRFSGSG



SGTQFTLTIS


 


101
GVQCADAATY YCQGAYNDVI YAFGGGTEVV VKRTDAAPTV



SIFPPSSEQL


 


151
TSGGASVVCF LNNFYPKDIN VKWKIDGSER QNGVLNSWTD



QDSKDSTYSM


 


201
SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNEC






The nucleic acid sequence of Ab-A LC including signal peptide encoding sequence is:







(SEQ ID NO: 26)








  1
ATGGACACGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC



TGCTCTGGCT


 


 51
CCCAGGTGCC ACATTTGCGC AAGTGCTGAC CCAGACTCCA



GCCTCCGTGT


 


101
CTGCAGCTGT GGGAGGCACA GTCACCATCA ATTGCCAGTC



CAGTCAGAGT


 


151
GTTTATGATA ACAACTGGTT AGCCTGGTTT CAGCAGAAAC



CAGGGCAGCC


 


201
TCCCAAGCTC CTGATTTATG ATGCATCCGA TCTGGCATCT



GGGGTCCCAT


 


251
CGCGGTTCAG TGGCAGTGGA TCTGGGACAC AGTTCACTCT



CACCATCAGC


 


301
GGCGTGCAGT GTGCCGATGC TGCCACTTAC TACTGTCAAG



GCGCTTATAA


 


351
TGATGTTATT TATGCTTTCG GCGGAGGGAC CGAGGTGGTG



GTCAAACGTA


 


401
CGGATGCTGC ACCAACTGTA TCCATCTTCC CACCATCCAG



TGAGCAGTTA


 


451
ACATCTGGAG GTGCCTCAGT CGTGTGCTTC TTGAACAACT



TCTACCCCAA


 


501
AGACATCAAT GTCAAGTGGA AGATTGATGG CAGTGAACGA



CAAAATGGCG


 


551
TCCTGAACAG TTGGACTGAT CAGGACAGCA AAGACAGCAC



CTACAGCATG


 


601
AGCAGCACCC TCACGTTGAC CAAGGACGAG TATGAACGAC



ATAACAGCTA


 


651
TACCTGTGAG GCCACTCACA AGACATCAAC TTCACCCATT



GTCAAGAGCT


 


701
TCAACAGGAA TGAGTGTTAG






The amino acid sequence of the mature form (signal peptide removed) of Ab-A HC is:








(SEQ ID NO: 27)














The nucleic acid sequence encoding the mature form (signal peptide removed) of Ab-A HC:







(SEQ ID NO: 28)








   1
CAGTCGCTGG AGGAGTCCGG GGGTCGCCTG GTCACGCCTG



GGACACCCCT


 


  51
GACACTCACC TGCACAGCCT CTGGATTCTC CCTCAGTAGT



TATTGGATGA


 


 101
ACTGGGTCCG CCAGGCTCCA GGGGAGGGGC TGGAATGGAT



CGGAACCATT


 


 151
GATTCTGGTG GTAGGACGGA CTACGCGAGC TGGGCAAAAG



GCCGATTCAC


 


 201
CATCTCCAGA ACCTCGACTA CGATGGATCT GAAAATGACC



AGTCTGACGA


 


 251
CCGGGGACAC GGCCCGTTAT TTCTGTGCCA GAAATTGGAA



CTTGTGGGGC


 


 301
CAAGGCACCC TCGTCACCGT CTCGAGCGCT TCTACAAAGG



GCCCATCTGT


 


 351
CTATCCACTG GCCCCTGGAT CTGCTGCCCA AACTAACTCC



ATGGTGACCC


 


 401
TGGGATGCCT GGTCAAGGGC TATTTCCCTG AGCCAGTGAC



AGTGACCTGG


 


 451
AACTCTGGAT CCCTGTCCAG CGGTGTGCAC ACCTTCCCAG



CTGTCCTGCA


 


 501
GTCTGACCTC TACACTCTGA GCAGCTCAGT GACTGTCCCC



TCCAGCACCT


 


 551
GGCCCAGCGA GACCGTCACC TGCAACGTTG CCCACCCGGC



CAGCAGCACC


 


 601
AAGGTGGACA AGAAAATTGT GCCCAGGGAT TGTGGTTGTA



AGCCTTGCAT


 


 651
ATGTACAGTC CCAGAAGTAT CATCTGTCTT CATCTTCCCC



CCAAAGCCCA


 


 701
AGGATGTGCT CACCATTACT CTGACTCCTA AGGTCACGTG



TGTTGTGGTA


 


 751
GACATCAGCA AGGATGATCC CGAGGTCCAG TTCAGCTGGT



TTGTAGATGA


 


 801
TGTGGAGGTG CACACAGCTC AGACGCAACC CCGGGAGGAG



CAGTTCAACA


 


 851
GCACTTTCCG CTCAGTCAGT GAACTTCCCA TCATGCACCA



GGACTGGCTC


 


 901
AATGGCAAGG AGTTCAAATG CAGGGTCAAC AGTGCAGCTT



TCCCTGCCCC


 


 951
CATCGAGAAA ACCATCTCCA AAACCAAAGG CAGACCGAAG



GCTCCACAGG


 


1001
TGTACACCAT TCCACCTCCC AAGGAGCAGA TGGCCAAGGA



TAAAGTCAGT


 


1051
CTGACCTGCA TGATAACAGA CTTCTTCCCT GAAGACATTA



CTGTGGAGTG


 


1101
GCAGTGGAAT GGGCAGCCAG CGGAGAACTA CAAGAACACT



CAGCCCATCA


 


1151
TGGACACAGA TGGCTCTTAC TTCGTCTACA GCAAGCTCAA



TGTGCAGAAG


 


1201
AGCAACTGGG AGGCAGGAAA TACTTTCACC TGCTCTGTGT



TACATGAGGG


 


1251
CCTGCACAAC CACCATACTG AGAAGAGCCT CTCCCACTCT



CCTGGTAAAT


 


1301
GA






The amino acid sequence of the Ab-A HC including signal peptide is:







(SEQ ID NO: 29)








  1
METGLRWLLL VAVLKGVHCQ SLEESGGRLV TPGTPLTLTC



TASGFSLSSY


 


 51
WMNWVRQAPG EGLEWIGTID SGGRTDYASW AKGRFTISRT



STTMDLKMTS


 


101
LTTGDTARYF CARNWNLWGQ GTLVTVSSAS TKGPSVYPLA



PGSAAQTNSM


 


151
VTLGCLVKGY FPEPVTVTWN SGSLSSGVHT FPAVLQSDLY



TLSSSVTVPS


 


201
STWPSETVTC NVAHPASSTK VDKKIVPRDC GCKPCICTVP



EVSSVFIFPP


 


251
KPKDVLTITL TPKVTCVVVD ISKDDPEVQF SWFVDDVEVH



TAQTQPREEQ


 


301
FNSTFRSVSE LPIMHQDWLN GKEFKCRVNS AAFPAPIEKT



ISKTKGRPKA


 


351
PQVYTIPPPK EQMAKDKVSL TCMITDFFPE DITVEWQWNG



QPAENYKNTQ


 


401
PIMNTNGSYF VYSKLNVQKS NWEAGNTFTC SVLHEGLHNH



HTEKSLSHSP


 


451
GK






The nucleic acid sequence of Ab-A HC including signal peptide encoding sequence:








(SEQ ID NO: 30)










   1
ATGGAGACTG GGCTGCGCTG GCTTCTCCTG GTCGCTGTGC TCAAAGGTGT



 


  51
CCACTGTCAG TCGCTGGAGG AGTCCGGGGG TCGCCTGGTC ACGCCTGGGA


 


 101
CACCCCTGAC ACTCACCTGC ACAGCCTCTG GATTCTCCCT CAGTAGTTAT


 


 151
TGGATGAACT GGGTCCGCCA GGCTCCAGGG GAGGGGCTGG AATGGATCGG


 


 201
AACCATTGAT TCTGGTGGTA GGACGGACTA CGCGAGCTGG GCAAAAGGCC


 


 251
GATTCACCAT CTCCAGAACC TCGACTACGA TGGATCTGAA AATGACCAGT


 


 301
CTGACGACCG GGGACACGGC CCGTTATTTC TGTGCCAGAA ATTGGAACTT


 


 351
GTGGGGCCAA GGCACCCTCG TCACCGTCTC GAGCGCTTCT ACAAAGGGCC


 


 401
CATCTGTCTA TCCACTGGCC CCTGGATCTG CTGCCCAAAC TAACTCCATG


 


 451
GTGACCCTGG GATGCCTGGT CAAGGGCTAT TTCCCTGAGC CAGTGACAGT


 


 501
GACCTGGAAC TCTGGATCCC TGTCCAGCGG TGTGCACACC TTCCCAGCTG


 


 551
TCCTGCAGTC TGACCTCTAC ACTCTGAGCA GCTCAGTGAC TGTCCCCTCC


 


 601
AGCACCTGGC CCAGCGAGAC CGTCACCTGC AACGTTGCCC ACCCGGCCAG


 


 651
CAGCACCAAG GTGGACAAGA AAATTGTGCC CAGGGATTGT GGTTGTAAGC


 


 701
CTTGCATATG TACAGTCCCA GAAGTATCAT CTGTCTTCAT CTTCCCCCCA


 


 751
AAGCCCAAGG ATGTGCTCAC CATTACTCTG ACTCCTAAGG TCACGTGTGT


 


 801
TGTGGTAGAC ATCAGCAAGG ATGATCCCGA GGTCCAGTTC AGCTGGTTTG


 


 851
TAGATGATGT GGAGGTGCAC ACAGCTCAGA CGCAACCCCG GGAGGAGCAG


 


 901
TTCAACAGCA CTTTCCGCTC AGTCAGTGAA CTTCCCATCA TGCACCAGGA


 


 951
CTGGCTCAAT GGCAAGGAGT TCAAATGCAG GGTCAACAGT GCAGCTTTCC


 


1001
CTGCCCCCAT CGAGAAAACC ATCTCCAAAA CCAAAGGCAG ACCGAAGGCT


 


1051
CCACAGGTGT ACACCATTCC ACCTCCCAAG GAGCAGATGG CCAAGGATAA


 


1101
AGTCAGTCTG ACCTGCATGA TAACAGACTT CTTCCCTGAA GACATTACTG


 


1151
TGGAGTGGCA GTGGAATGGG CAGCCAGCGG AGAACTACAA GAACACTCAG


 


1201
CCCATCATGG ACACAGATGG CTCTTACTTC GTCTACAGCA AGCTCAATGT


 


1251
GCAGAAGAGC AACTGGGAGG CAGGAAATAC TTTCACCTGC TCTGTG1TAC


 


1301
ATGAGGGCCT GCACAACCAC CATACTGAGA AGAGCCTCTC CCACTCTCCT


 


1351
GGTAAATGA






The CDR (complementarity determining region) sequences in the variable region of the heavy chain of Ab-A are as follows:









(SEQ ID NO: 51)



CDR-H1: SYWMN



 



(SEQ ID NO: 52)



CDR-H2: TIDSGGRTDYASWAKG



 



(SEQ ID NO: 53)



CDR-H3: NWNL






The light chain variable region CDR sequences of Ab-A are:









(SEQ ID NO: 54)



CDR-L1: QSSQSVYDNNWLA



 



(SEQ ID NO: 55)



CDR-L2: DASDLAS



 



(SEQ ID NO: 56)



CDR-L3: QGAYNDVIYA






Ab-A was humanized, and is referred to as Antibody 1 (also referred to herein as Ab-1), having the following sequences:
The nucleic acid sequence of the Ab-1 LC variable region including signal peptide encoding sequence is







(SEQ ID NO: 74)


ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT


 


CCCAGGTGCCACATTTGCTCAAGTTCTGACCCAGAGTCCAAGCAGTCTCT


 


CCGCCAGCGTAGGCGATCGTGTGACTATTACCTGTCAATCTAGTCAGAGC


 


GTGTATGATAACAATTGGCTGGCGTGGTACCAGCAAAAACCGGGCAAAGC


 


CCCGAAGCTGCTCATCTATGACGCGTCCGATCTGGCTAGCGGTGTGCCAA


 


GCCGTTTCAGTGGCAGTGGCAGCGGTACTGACTTTACCCTCACAATTTCG


 


TCTCTCCAGCCGGAAGATTTCGCCACTTACTATTGTCAAGGTGCTTACAA


 


CGATGTGATTTATGCCTTCGGTCAGGGCACTAAAGTAGAAATCAAACGT






The amino acid sequence of Ab-1 LC variable region including signal peptide is:







(SEQ ID NO: 75)












The nucleic acid sequence of Ab-1 HC variable region including signal peptide encoding sequence is:







(SEQ ID NO: 76)


ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT


 


CCACTGTGAGGTGCAGCTGTTGGAGTCTGGAGGCGGGCTTGTCCAGCCTG


 


GAGGGAGCCTGCGTCTCTCTTGTGCAGCAAGCGGCTTCAGCTTATCCTCT


 


TACTGGATGAATTGGGTGCGGCAGGCACCTGGGAAGGGCCTGGAGTGGGT


 


GGGCACCATTGATTCCGGAGGCCGTACAGACTACGCGTCTTGGGCAAAGG


 


GCCGTTTCACCATTTCCCGCGACAACTCCAAAAATACCATGTACCTCCAG


 


ATGAACTCTCTCCGCGCAGAGGACACAGCACGTTATTACTGTGCACGCAA


 


CTGGAATCTGTGGGGTCAAGGTACTCTTGTAACAGTCTCGAGC






Amino acid sequence of Ab-1 HC variable region including signal peptide








(SEQ ID NO: 77)












The CDR (complementarity determining region) sequences in the variable region of the heavy chain of Ab-1 are as follows:









(SEQ ID NO: 51)



CDR-H1: SYWMN



 



(SEQ ID NO: 52)



CDR-H2: TIDSGGRTDYASWAKG



 



(SEQ ID NO: 53)



CDR-H3: NWNL






The light chain variable region CDR sequences of Ab-1 are:









(SEQ ID NO: 54)



CDR-L1: QSSQSVYDNNWLA



 



(SEQ ID NO: 55)



CDR-L2: DASDLAS



 



(SEQ ID NO: 56)



CDR-L3: QGAYNDVIYA






Ab-B

Antibody B (also referred to herein as Ab-B and Mab-B) is a mouse antibody which exhibits high affinity binding to sclerostin. The BIAcore binding pattern of Ab-B is shown in FIG. 16.
Ab-B Light Chain
The amino acid sequence of the mature form (signal peptide removed) of the Ab-B LC is:








(SEQ ID NO: 31)














The nucleic acid sequence encoding the mature form (signal peptide removed) of Ab-B LC is:








(SEQ ID NO: 32)










  1
CAAATTGTTC TCACCCAGTC TCCAACAATC GTGTCTGCAT CTCCAGGGGA



 


 51
GAAGGTCACC CTAATCTGCA GTGCCAGTTC AAGTGTAAGT TTCGTGGACT


 


101
GGTTCCAGCA GAAGCCAGGC ACTTCTCCCA AACGCTGGAT TTACAGAACA


 


151
TCCAACCTGG GTTTTGGAGT CCCTGCTCGC TTCAGTGGCG GTGGATCTGG


 


201
GACCTCTCAC TCTCTCACAA TCAGCCGAAT GGAGGCTGAA GATGCTGCCA


 


251
CTTATTACTG CCAGCAAAGG AGTACTTACC CACCCACGTT CGGTGCTGGG


 


301
ACCAAGCTGG AACTGAAACG GGCTGATGCT GCACCAACTG TATCCATCTT


 


351
CCCACCATCC AGTGAGCAGT TAACATCTGG AGGTGCCTCA GTCGTGTGCT


 


401
TCTTGAACAA CTTCTACCCC AAAGACATCA ATGTCAAGTG GAAGATTGAT


 


451
GGCAGTGAAC GACAAAATGG CGTCCTGAAC AGTTGGACTG ATCAGGACAG


 


501
CAAAGACAGC ACCTACAGCA TGAGCAGCAC CCTCACGTTG ACCAAGGACG


 


551
AGTATGAACG ACATAACAGC TATACCTGTG AGGCCACTCA CAAGACATCA


 


601
ACTTCACCCA TTGTCAAGAG CTTCAACAGG AATGAGTGTT AG






The amino acid sequence of Ab-B LC including signal peptide is:








(SEQ ID NO: 33)










  1
MHFQVQIFSF LLISASVIVS RGQIVLTQSP TIVSASPGEK VTLICSASSS



 


 51
VSFVDWFQQK PGTSPKRWIY RTSNLGFGVP ARFSGGGSGT SHSLTISRME


 


101
AEDAATYYCQ QRSTYPPTFG AGTKLELKRA DAAPTVSIFP PSSEQLTSGG


 


151
ASVVCFLNNF YPKDINVKWK IDGSERQNGV LNSWTDQDSK DSTYSMSSTL


 


201
TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC






The nucleic acid sequence of Ab-B LC including signal peptide encoding sequence is:








(SEQ ID NO: 34)










  1
ATGCATTTTC AAGTGCAGAT TTTCAGCTTC CTGCTAATCA GTGCCTCAGT



 


 51
CATAGTGTCC AGAGGGCAAA TTGTTCTCAC CCAGTCTCCA ACAATCGTGT


 


101
CTGCATCTCC AGGGGAGAAG GTCACCCTAA TCTGCAGTGC CAGTTCAAGT


 


151
GTAAGTTTCG TGGACTGGTT CCAGCAGAAG CCAGGCACTT CTCCCAAACG


 


201
CTGGATTTAC AGAACATCCA ACCTGGGTTT TGGAGTCCCT GCTCGCTTCA


 


251
GTGGCGGTGG ATCTGGGACC TCTCACTCTC TCACAATCAG CCGAATGGAG


 


301
GCTGAAGATG CTGCCACTTA TTACTGCCAG CAAAGGAGTA CTTACCCACC


 


351
CACGTTCGGT GCTGGGACCA AGCTGGAACT GAAACGGGCT GATGCTGCAC


 


401
CAACTGTATC CATCTTCCCA CCATCCAGTG AGCAGTTAAC ATCTGGAGGT


 


451
GCCTCAGTCG TGTGCTTCTT GAACAACTTC TACCCCAAAG ACATCAATGT


 


501
CAAGTGGAAG ATTGATGGCA GTGAACGACA AAATGGCGTC CTGAACAGTT


 


551
GGACTGATCA GGACAGCAAA GACAGCACCT ACAGCATGAG CAGCACCCTC


 


601
ACGTTGACCA AGGACGAGTA TGAACGACAT AACAGCTATA CCTGTGAGGC


 


651
CACTCACAAG ACATCAACTT CACCCATTGT CAAGAGCTTC AACAGGAATG


 


701
AGTGTTAG






Ab-B Heavy Chain

The amino acid sequence of the mature form (signal peptide removed) of Ab-B HC:








(SEQ ID NO: 35)














The nucleic acid sequence encoding the mature form (signal peptide removed) of Ab-B HC:








(SEQ ID NO: 36)










   1
CAGGTTACTC TGAAAGAGTC TGGCCCTGGG ATATTGCAGC CCTCCCAGAC



 


  51
CCTCAGTCTG ACTTGTTCTT TCTCTGGGTT TTCACTGAGC ACTTCTGGTA


 


 101
TGGGTGTAGG CTGGATTCGT CACCCATCAG GGAAGAATCT GGAGTGGCTG


 


 151
GCACACATTT GGTGGGATGA TGTCAAGCGC TATAACCCAG TCCTGAAGAG


 


 201
CCGACTGACT ATCTCCAAGG ATACCTCCAA CAGCCAGGTA TTCCTCAAGA


 


 251
TCGCCAATGT GGACACTGCA GATACTGCCA CATACTACTG TGCTCGAATA


 


 301
GAGGACTTTG ATTACGACGA GGAGTATTAT GCTATGGACT ACTGGGGTCA


 


 351
AGGAACCTCA GTCATCGTCT CCTCAGCCAA AACGACACCC CCATCTGTCT


 


 401
ATCCACTGGC CCCTGGATCT GCTGCCCAAA CTAACTCCAT GGTGACCCTG


 


 451
GGATGCCTGG TCAAGGGCTA TTTCCCTGAG CCAGTGACAG TGACCTGGAA


 


 501
CTCTGGATCC CTGTCCAGCG GTGTGCACAC CTTCCCAGCT GTCCTGCAGT


 


 551
CTGACCTCTA CACTCTGAGC AGCTCAGTGA CTGTCCCCTC CAGCACCTGG


 


 601
CCCAGCGAGA CCGTCACCTG CAACGTTGCC CACCCGGCCA GCAGCACCAA


 


 651
GGTGGACAAG AAAATTGTGC CCAGGGATTG TGGTTGTAAG CCTTGCATAT


 


 701
GTACAGTCCC AGAAGTATCA TCTGTCTTCA TCTTCCCCCC AAAGCCCAAG


 


 751
GATGTGCTCA CCATTACTCT GACTCCTAAG GTCACGTGTG TTGTGGTAGA


 


 801
CATCAGCAAG GATGATCCCG AGGTCCAGTT CAGCTGGTTT GTAGATGATG


 


 851
TGGAGGTGCA CACAGCTCAG ACGCAACCCC GGGAGGAGCA GTTCAACAGC


 


 901
ACTTTCCGCT CAGTCAGTGA ACTTCCCATC ATGCACCAGG ACTGGCTCAA


 


 951
TGGCAAGGAG TTCAAATGCA GGGTCAACAG TGCAGCTTTC CCTGCCCCCA


 


1001
TCGAGAAAAC CATCTCCAAA ACCAAAGGCA GACCGAAGGC TCCACAGGTG


 


1051
TACACCATTC CACCTCCCAA GGAGCAGATG GCCAAGGATA AAGTCAGTCT


 


1101
GACCTGCATG ATAACAGACT TCTTCCCTGA AGACATTACT GTGGAGTGGC


 


1151
AGTGGAATGG GCAGCCAGCG GAGAACTACA AGAACACTCA GCCCATCATG


 


1201
GACACAGATG GCTCTTACTT CGTCTACAGC AAGCTCAATG TGCAGAAGAG


 


1251
CAACTGGGAG GCAGGAAATA CTTTCACCTG CTCTGTGTTA CATGAGGGCC


 


1301
TGCACAACCA CCATACTGAG AAGAGCCTCT CCCACTCTCC TGGTAAATGA






The amino acid sequence of Ab-B HC including signal peptide:








(SEQ ID NO: 37)










  1
MGRLTSSFLL LIVPAYVLSQ VTLKESGPGI LQPSQTLSLT CSFSGFSLST



 


 51
SGMGVGWIRH PSGKNLEWLA HIWWDDVKRY NPVLKSRLTI SKDTSNSQVF


 


101
LKIANVDTAD TATYYCARIE DFDYDEEYYA MDYWGQGTSV IVSSAKTTPP


 


151
SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV


 


201
LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRDCGCKP


 


251
CICTVPEVSS VFIFPPKPKD VLTITLTPKV TCVVVDISKD DPEVQFSWFV


 


301
DDVEVHTAQT QPREEQFNST FRSVSELPIM HQDWLNGKEF KCRVNSAAFP


 


351
APIEKTISKT KGRPKAPQVY TIPPPKEQMA KDKVSLTCMI TDFFPEDITV


 


401
EWQWNGQPAE NYKNTQPIMD TDGSYFVYSK LNVQKSNWEA GNTFTCSVLH


 


451
EGLHNHHTEK SLSHSPGK






The nucleic acid sequence of Ab-B HC including signal peptide encoding sequence:








(SEQ ID NO: 38)










   1
ATGGGCAGGC TTACTTCTTC ATTCCTGCTA CTGATTGTCC CTGCATATGT



 


  51
CCTGTCCCAG GTTACTCTGA AAGAGTCTGG CCCTGGGATA TTGCAGCCCT


 


 101
CCCAGACCCT CAGTCTGACT TGTTCTTTCT CTGGGTTTTC ACTGAGCACT


 


 151
TCTGGTATGG GTGTAGGCTG GATTCGTCAC CCATCAGGGA AGAATCTGGA


 


 201
GTGGCTGGCA CACATTTGGT GGGATGATGT CAAGCGCTAT AACCCAGTCC


 


 251
TGAAGAGCCG ACTGACTATC TCCAAGGATA CCTCCAACAG CCAGGTATTC


 


 301
CTCAAGATCG CCAATGTGGA CACTGCAGAT ACTGCCACAT ACTACTGTGC


 


 351
TCGAATAGAG GACTTTGATT ACGACGAGGA GTATTATGCT ATGGACTACT


 


 401
GGGGTCAAGG AACCTCAGTC ATCGTCTCCT CAGCCAAAAC GACACCCCCA


 


 451
TCTGTCTATC CACTGGCCCC TGGATCTGCT GCCCAAACTA ACTCCATGGT


 


 501
GACCCTGGGA TGCCTGGTCA AGGGCTATTT CCCTGAGCCA GTGACAGTGA


 


 551
CCTGGAACTC TGGATCCCTG TCCAGCGGTG TGCACACCTT CCCAGCTGTC


 


 601
CTGCAGTCTG ACCTCTACAC TCTGAGCAGC TCAGTGACTG TCCCCTCCAG


 


 651
CACCTGGCCC AGCGAGACCG TCACCTGCAA CGTTGCCCAC CCGGCCAGCA


 


 701
GCACCAAGGT GGACAAGAAA ATTGTGCCCA GGGATTGTGG TTGTAAGCCT


 


 751
TGCATATGTA CAGTCCCAGA AGTATCATCT GTCTTCATCT TCCCCCCAAA


 


 801
GCCCAAGGAT GTGCTCACCA TTACTCTGAC TCCTAAGGTC ACGTGTGTTG


 


 851
TGGTAGACAT CAGCAAGGAT GATCCCGAGG TCCAGTTCAG CTGGTTTGTA


 


 901
GATGATGTGG AGGTGCACAC AGCTCAGACG CAACCCCGGG AGGAGCAGTT


 


 951
CAACAGCACT TTCCGCTCAG TCAGTGAACT TCCCATCATG CACCAGGACT


 


1001
GGCTCAATGG CAAGGAGTTC AAATGCAGGG TCAACAGTGC AGCTTTCCCT


 


1051
GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGCAGAC CGAAGGCTCC


 


1101
ACAGGTGTAC ACCATTCCAC CTCCCAAGGA GCAGATGGCC AAGGATAAAG


 


1151
TCAGTCTGAC CTGCATGATA ACAGACTTCT TCCCTGAAGA CATTACTGTG


 


1201
GAGTGGCAGT GGAATGGGCA GCCAGCGGAG AACTACAAGA ACACTCAGCC


 


1251
CATCATGGAC ACAGATGGCT CTTACTTCGT CTACAGCAAG CTCAATGTGC


 


1301
AGAAGAGCAA CTGGGAGGCA GGAAATACTT TCACCTGCTC TGTGTTACAT


 


1351
GAGGGCCTGC ACAACCACCA TACTGAGAAG AGCCTCTCCC ACTCTCCTGG


 


1401
TAAATGA






The CDR (complementarity determining region) sequences in the variable region of the heavy chain of Ab-B are as follows:









(SEQ ID NO: 57)



CDR-H1: TSGMGVG



 



(SEQ ID NO: 58)



CDR-H2: HIWWDDVKRYNPVLKS



 



(SEQ ID NO: 59)



CDR-H3: EDFDYDEEYYAMDY






The light chain variable region CDR sequences of Ab-B are:









(SEQ ID NO: 60)



CDR-L1: SASSSVSFVD



 



(SEQ ID NO: 61)



CDR-L2: RTSNLGF



 



(SEQ ID NO: 62)



CDR-L3: QQRSTYPPT






Antibodies disclosed herein bind to regions of human sclerostin which are important for the in vivo activity of the protein. Binding of an antibody to sclerostin can be correlated with increases in, for example, the bone mineral density achieved by use of the antibody in vivo such as described in Examples 5 and 9 (mice) and Example 12 (monkey). Increases in at least one of bone formation, bone mineral content, bone mass, bone quality and bone strength can also be achieved by use of the antibody in vivo such as described in Examples 5 and 9 (mice) and Example 12 (monkey). Since the binding of an antibody to sclerostin is primarily determined by its CDR sequences, an antibody for practicing the invention may be generated with all or some of the disclosed CDR sequences in an appropriate framework, wherein the antibody retains the ability to bind specifically to sclerostin, and can be expected to achieve increases in, for example, bone mineral density. Such antibodies are useful in the treatment of human or animal conditions that are caused by, associated with, or result in at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength. Methods of constructing and expressing antibodies and fragments thereof comprising CDR's of the present invention are known to those of skill in the art.
The present invention therefore relates in one embodiment to an isolated antibody, including Ab-A, or an antigen binding fragment thereof, which specifically binds to sclerostin and wherein the variable domain of the heavy chain comprises at least one CDR having the sequences given in SEQ ID NO:51 for CDR-H1, SEQ ID NO:52 for CDR-H2 and SEQ ID NO:53 for CDR-H3. The antibody or antigen binding fragment thereof may comprise a heavy chain variable domain in which the CDRs consist of at least one of the peptides of SEQ ID NO:51 for CDR-H1, SEQ ID NO:52 for CDR-H2 and SEQ ID NO:53 for CDR-H3.
When in antibodies of the invention a light chain is present the light chain may be any suitable complementary chain and may in particular be selected from a light chain wherein the variable domain comprises at least one CDR having the sequences given in SEQ ID NO:54 for CDR-L1, SEQ ID NO:55 for CDR-L2 and SEQ ID NO:56 for CDR-L3. The antibody or antigen binding fragment thereof may comprise a light chain variable domain in which the CDRs consist of at least one of the peptides of SEQ ID NO:54 for CDR-L1, SEQ ID NO:55 for CDR-L2 and SEQ ID NO:56 for CDR-L3.
The present invention further relates to an isolated antibody, including Ab-B, or an antigen binding fragment hereof, which specifically binds to sclerostin and wherein the variable domain of the heavy chain comprises at least one CDR having the sequences given in SEQ ID NO:57 for CDR-H1, SEQ ID NO:58 for CDR-H2 and SEQ ID NO:59 for CDR-H3. The antibody or antigen binding fragment thereof may comprise a heavy chain variable domain in which the CDRs consist of at least one of the peptides of SEQ ID NO:57 for CDR-H1, SEQ ID NO:58 for CDR-H2 and SEQ ID NO:59 for CDR-H3.
When in antibodies of the invention a light chain is present the light chain may be any suitable complementary chain and may in particular be selected from a light chain wherein the variable domain comprises at least one CDR having the sequences given in SEQ ID NO:60 for CDR-L1, SEQ ID NO:61 for CDR-L2 and SEQ ID NO:62 for CDR-L3. The antibody or antigen binding fragment thereof may comprise a light chain variable domain in which the CDRs consist of at least one of the peptides of SEQ ID NO:60 for CDR-L1, SEQ ID NO:61 for CDR-L2 and SEQ ID NO:62 for CDR-L3.
The present invention still further relates to an isolated antibody, including Ab-C, or an antigen binding fragment hereof, which specifically binds to sclerostin and wherein the variable domain of the heavy chain comprises at least one CDR having the sequences given in SEQ ID NO:45 for CDR-H1, SEQ ID NO:46 for CDR-H2 and SEQ ID NO:47 for CDR-H3. The antibody or antigen binding fragment thereof may comprise a heavy chain variable domain in which the CDRs consist of at least one of the peptides of SEQ ID NO:45 for CDR-H1, SEQ ID NO:46 for CDR-H2 and SEQ ID NO:47 for CDR-H3.
When in antibodies of the invention a light chain is present the light chain may be any suitable complementary chain and may in particular be selected from a light chain wherein the variable domain comprises at least one CDR having the sequences given in SEQ ID NO:48 for CDR-L1, SEQ ID NO:49 for CDR-L2 and SEQ ID NO:50 for CDR-L3. The antibody or antigen binding fragment thereof may comprise a light chain variable domain in which the CDRs consist of at least one of the peptides of SEQ ID NO:48 for CDR-L1, SEQ ID NO:49 for CDR-L2 and SEQ ID NO:50 for CDR-L3.
The present invention also relates to an isolated antibody, including Ab-D, or an antigen binding fragment hereof, which specifically binds to sclerostin and wherein the variable domain of the heavy chain comprises at least one CDR having the sequences given in SEQ ID NO:39 for CDR-H1, SEQ ID NO:40 for CDR-H2 and SEQ ID NO:41 for CDR-H3. The antibody or antigen binding fragment thereof may comprise a heavy chain variable domain in which the CDRs consist of at least one of the peptides of SEQ ID NO:39 for CDR-H1, SEQ ID NO:40 for CDR-H2 and SEQ ID NO:41 for CDR-H3.
When in antibodies of the invention a light chain is present the light chain may be any suitable complementary chain and may in particular be selected from a light chain wherein the variable domain comprises at least one CDR having the sequences given in SEQ ID NO:42 for CDR-L1, SEQ ID NO:43 for CDR-L2 and SEQ ID NO:44 for CDR-L3. The antibody or antigen binding fragment thereof may comprise a light chain variable domain in which the CDRs consist of at least one of the peptides of SEQ ID NO:42 for CDR-L1, SEQ ID NO:43 for CDR-L2 and SEQ ID NO:44 for CDR-L3.
Additional anti-sclerostin antibodies are described below. For some of the amino acid sequences the complementarity-determining regions (CDRs) are boxed-shaded and the constant regions are in bold-italics.
Ab-2
The sequences of the Antibody 2 (also referred to as Ab-2) LC and HC are as follows:
Ab-2 Light Chain:
Amino acid sequence of the mature form (signal peptide removed) of the Ab-2 LC:








(SEQ ID NO: 117)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-2 LC:








(SEQ ID NO: 118)










  1
CAAATTGTTC TCTCCCAGTC TCCAGCAATC CTGTCTACAT CTCCAGGGGA



 


 51
GAAGGTCACA ATGACTTGCA GGGCCAGCTC AAGTGTATAT TACATGCACT


 


101
GGTACCAGCA GAAGCCAGGA TCCTCCCCCA AACCCTGGAT TTATGCCACA


 


151
TCCAACCTGG CTTCTGGAGT CCCTGTTCGC TTCAGTGGCA GTGGGTCTGG


 


201
GACCTCTTAC TCTCTCACAA TCACCAGAGT GGAGGCTGAA GATGCTGCCA


 


251
CTTATTACTG CCAGCAGTGG AGTAGTGACC CACTCACGTT CGGTGCTGGG


 


301
ACCAAGCTGG AGCTGAAACG GGCTGATGCT GCACCAACTG TATCCATCTT


 


351
CCCACCATCC AGTGAGCAGT TAACATCTGG AGGTGCCTCA GTCGTGTGCT


 


401
TCTTGAACAA CTTCTACCCC AAAGACATCA ATGTCAAGTG GAAGATTGAT


 


451
GGCAGTGAAC GACAAAATGG CGTCCTGAAC AGTTGGACTG ATCAGGACAG


 


501
CAAAGACAGC ACCTACAGCA TGAGCAGCAC CCTCACGTTG ACCAAGGACG


 


551
AGTATGAACG ACATAACAGC TATACCTGTG AGGCCACTCA CAAGACATCA


 


601
ACTTCACCCA TTGTCAAGAG CTTCAACAGG AATGAGTGTT AG






Amino acid sequence of the Ab-2 LC including signal peptide:









(SEQ ID NO: 119)










  1
MDFQVQIFSF LLISASVIMS RGQIVLSQSP AILSTSPGEK VTMTCRASSS



 


 51
VYYMHWYQQK PGSSPKPWIY ATSNLASGVP VRFSGSGSGT SYSLTITRVE


 


101
AEDAATYYCQ QWSSDPLTFG AGTKLELKRA DAAPTVSIFP PSSEQLTSGG


 


151
ASVVCFLNNF YPKDINVKWK IDGSERQNGV LNSWTDQDSK DSTYSMSSTL


 


201
TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC






Nucleic acid sequence of the Ab-2 LC including signal peptide encoding sequence:









(SEQ ID NO: 120)










  1
ATGGATTTTC AAGTGCAGAT TTTCAGCTTC CTGCTAATCA GTGCTTCAGT



 


 51
CATTATGTCC AGGGGACAAA TTGTTCTCTC CCAGTCTCCA GCAATCCTGT


 


101
CTACATCTCC AGGGGAGAAG GTCACAATGA CTTGCAGGGC CAGCTCAAGT


 


151
GTATATTACA TGCACTGGTA CCAGCAGAAG CCAGGATCCT CCCCCAAACC


 


201
CTGGATTTAT GCCACATCCA ACCTGGCTTC TGGAGTCCCT GTTCGCTTCA


 


251
GTGGCAGTGG GTCTGGGACC TCTTACTCTC TCACAATCAC CAGAGTGGAG


 


301
GCTGAAGATG CTGCCACTTA TTACTGCCAG CAGTGGAGTA GTGACCCACT


 


351
CACGTTCGGT GCTGGGACCA AGCTGGAGCT GAAACGGGCT GATGCTGCAC


 


401
CAACTGTATC CATCTTCCCA CCATCCAGTG AGCAGTTAAC ATCTGGAGGT


 


451
GCCTCAGTCG TGTGCTTCTT GAACAACTTC TACCCCAAAG ACATCAATGT


 


501
CAAGTGGAAG ATTGATGGCA GTGAACGACA AAATGGCGTC CTGAACAGTT


 


551
GGACTGATCA GGACAGCAAA GACAGCACCT ACAGCATGAG CAGCACCCTC


 


601
ACGTTGACCA AGGACGAGTA TGAACGACAT AACAGCTATA CCTGTGAGGC


 


651
CACTCACAAG ACATCAACTT CACCCATTGT CAAGAGCTTC AACAGGAATG


 


701
AGTGTTAG






Ab-2 Heavy Chain

Amino acid sequence of the mature form (signal peptide removed) of the Ab-2 HC:









(SEQ ID NO: 121)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-2 HC:









(SEQ ID NO: 122)










   1
GAGGTTCAGG TGCAGCAGTC TGGGCCAGAA CTTGTGAAGC CAGGGGCCTC



 


  51
AGTCAAGTTG TCCTGCACAG CTTCTGGCTT CAACATTAAA GACTACTTTA


 


 101
TACACTGGGT GAAGCAGAGG CCTGAACAGG GCCTGGAGTG GATTGGAAGG


 


 151
CTTGATCCTG AGGATGGTGA AAGTGATTAT GCCCCGAAGT TCCAGGACAA


 


 201
GGCCATTATG ACAGCAGACA CATCATCCAA CACAGCCTAT CTTCAGCTCA


 


 251
GAAGCCTGAC ATCTGAGGAC ACTGCCATCT ATTATTGTGA GAGAGAGGAC


 


 301
TACGATGGTA CCTACACCTT TTTTCCTTAC TGGGGCCAAG GGACTCTGGT


 


 351
CACTGTCTCT GCAGCCAAAA CGACACCCCC ATCTGTCTAT CCACTGGCCC


 


 401
CTGGATCTGC TGCCCAAACT AACTCCATGG TGACCCTGGG ATGCCTGGTC


 


 451
AAGGGCTATT TCCCTGAGCC AGTGACAGTG ACCTGGAACT CTGGATCCCT


 


 501
GTCCAGCGGT GTGCACACCT TCCCAGCTGT CCTGCAGTCT GACCTCTACA


 


 551
CTCTGAGCAG CTCAGTGACT GTCCCCTCCA GCACCTGGCC CAGCGAGACC


 


 601
GTCACCTGCA ACGTTGCCCA CCCGGCCAGC AGCACCAAGG TGGACAAGAA


 


 651
AATTGTGCCC AGGGATTGTG GTTGTAAGCC TTGCATATGT ACAGTCCCAG


 


 701
AAGTATCATC TGTCTTCATC TTCCCCCCAA AGCCCAAGGA TGTGCTCACC


 


 751
ATTACTCTGA CTCCTAAGGT CACGTGTGTT GTGGTAGACA TCAGCAAGGA


 


 801
TGATCCCGAG GTCCAGTTCA GCTGGTTTGT AGATGATGTG GAGGTGCACA


 


 851
CAGCTCAGAC GCAACCCCGG GAGGAGCAGT TCAACAGCAC TTTCCGCTCA


 


 901
GTCAGTGAAC TTCCCATCAT GCACCAGGAC TGGCTCAATG GCAAGGAGTT


 


 951
CAAATGCAGG GTCAACAGTG CAGCTTTCCC TGCCCCCATC GAGAAAACCA


 


1001
TCTCCAAAAC CAAAGGCAGA CCGAAGGCTC CACAGGTGTA CACCATTCCA


 


1051
CCTCCCAAGG AGCAGATGGC CAAGGATAAA GTCAGTCTGA CCTGCATGAT


 


1101
AACAGACTTC TTCCCTGAAG ACATTACTGT GGAGTGGCAG TGGAATGGGC


 


1151
AGCCAGCGGA GAACTACAAG AACACTCAGC CCATCATGGA CACAGATGGC


 


1201
TCTTACTTCA TCTACAGCAA GCTCAATGTG CAGAAGAGCA ACTGGGAGGC


 


1251
AGGAAATACT TTCACCTGCT CTGTGTTACA TGAGGGCCTG CACAACCACC


 


1301
ATACTGAGAA GAGCCTCTCC CACTCTCCTG GTAAATGA






Amino acid sequence of the Ab-2 HC including signal peptide:









(SEQ ID NO: 123)










  1
MKCSWVIFFL MAVVTGVNSE VQVQQSGPEL VKPGASVKLS CTASGFNIKD



 


 51
YFIHWVKQRP EQGLEWIGRL DPEDGESDYA PKFQDKAIMT ADTSSNTAYL


 


101
QLRSLTSEDT AIYYCEREDY DGTYTFFPYW GQGTLVTVSA AKTTPPSVYP


 


151
LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD


 


201
LYTLSSSVTV PSSTWPSETV TCNVAHPASS TKVDKKIVPR DCGCKPCICT


 


251
VPEVSSVFIF PPKPKDVLTI TLTPKVTCVV VDISKDDPEV QFSWFVDDVE


 


301
VHTAQTQPRE EQFNSTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE


 


351
KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITDFF PEDITVEWQW


 


401
NGQPAENYKN TQPIMDTDGS YFIYSKLNVQ KSNWEAGNTF TCSVLHEGLH


 


451
NHHTEKSLSH SPGK






Nucleic acid sequence of the Ab-2 HC including signal peptide encoding sequence:









(SEQ ID NO: 124)










   1
ATGAAATGCA GCTGGGTCAT CTTCTTCCTG ATGGCAGTGG TTACAGGGGT



 


  51
CAATTCAGAG GTTCAGGTGC AGCAGTCTGG GCCAGAACTT GTGAAGCCAG


 


 101
GGGCCTCAGT CAAGTTGTCC TGCACAGCTT CTGGCTTCAA CATTAAAGAC


 


 151
TACTTTATAC ACTGGGTGAA GCAGAGGCCT GAACAGGGCC TGGAGTGGAT


 


 201
TGGAAGGCTT GATCCTGAGG ATGGTGAAAG TGATTATGCC CCGAAGTTCC


 


 251
AGGACAAGGC CATTATGACA GCAGACACAT CATCCAACAC AGCCTATCTT


 


 301
CAGCTCAGAA GCCTGACATC TGAGGACACT GCCATCTATT ATTGTGAGAG


 


 351
AGAGGACTAC GATGGTACCT ACACCTTTTT TCCTTACTGG GGCCAAGGGA


 


 401
CTCTGGTCAC TGTCTCTGCA GCCAAAACGA CACCCCCATC TGTCTATCCA


 


 451
CTGGCCCCTG GATCTGCTGC CCAAACTAAC TCCATGGTGA CCCTGGGATG


 


 501
CCTGGTCAAG GGCTATTTCC CTGAGCCAGT GACAGTGACC TGGAACTCTG


 


 551
GATCCCTGTC CAGCGGTGTG CACACCTTCC CAGCTGTCCT GCAGTCTGAC


 


 601
CTCTACACTC TGAGCAGCTC AGTGACTGTC CCCTCCAGCA CCTGGCCCAG


 


 651
CGAGACCGTC ACCTGCAACG TTGCCCACCC GGCCAGCAGC ACCAAGGTGG


 


 701
ACAAGAAAAT TGTGCCCAGG GATTGTGGTT GTAAGCCTTG CATATGTACA


 


 751
GTCCCAGAAG TATCATCTGT CTTCATCTTC CCCCCAAAGC CCAAGGATGT


 


 801
GCTCACCATT ACTCTGACTC CTAAGGTCAC GTGTGTTGTG GTAGACATCA


 


 851
GCAAGGATGA TCCCGAGGTC CAGTTCAGCT GGTTTGTAGA TGATGTGGAG


 


 901
GTGCACACAG CTCAGACGCA ACCCCGGGAG GAGCAGTTCA ACAGCACTTT


 


 951
CCGCTCAGTC AGTGAACTTC CCATCATGCA CCAGGACTGG CTCAATGGCA


 


1001
AGGAGTTCAA ATGCAGGGTC AACAGTGCAG CTTTCCCTGC CCCCATCGAG


 


1051
AAAACCATCT CCAAAACCAA AGGCAGACCG AAGGCTCCAC AGGTGTACAC


 


1101
CATTCCACCT CCCAAGGAGC AGATGGCCAA GGATAAAGTC AGTCTGACCT


 


1151
GCATGATAAC AGACTTCTTC CCTGAAGACA TTACTGTGGA GTGGCAGTGG


 


1201
AATGGGCAGC CAGCGGAGAA CTACAAGAAC ACTCAGCCCA TCATGGACAC


 


1251
AGATGGCTCT TACTTCATCT ACAGCAAGCT CAATGTGCAG AAGAGCAACT


 


1301
GGGAGGCAGG AAATACTTTC ACCTGCTCTG TGTTACATGA GGGCCTGCAC


 


1351
AACCACCATA CTGAGAAGAG CCTCTCCCAC TCTCCTGGTA AATGA






Ab-3

The sequences of the Antibody 3 (also referred to herein as Ab-3) LC and HC are as follows:

Ab-3 Light Chain

Amino acid sequence of the mature form (signal peptide removed) of the Ab-3 LC:









(SEQ ID NO: 125)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-3 LC:









(SEQ ID NO: 126)










  1
GAAATTGTGC TCACCCAGTC TCCAGCACTC ATGGCTGCAT CTCCGGGGGA



 


 51
GAAGGTCACC ATCACCTGCA GTGTCAGTTC AACTATAAGT TCCAACCACT


 


101
TGCACTGGTT CCAGCAGAAG TCAGACACCT CCCCCAAACC CTGGATTTAT


 


151
GGCACATCCA ACCTGGCTTC TGGAGTCCCT GTTCGCTTCA GTGGCAGTGG


 


201
ATCTGGGACC TCTTATTCTC TCACAATCAG CAGCATGGAG GCTGAGGATG


 


251
CTGCCACTTA TTACTGTCAA CAGTGGAGTA GTTACCCACT CACGTTCGGC


 


301
GCTGGGACCA AGCTGGAGCT GAGACGGGCT GATGCTGCAC CAACTGTATC


 


351
CATCTTCCCA CCATCCAGTG AGCAGTTAAC ATCTGGAGGT GCCTCAGTCG


 


401
TGTGCTTCTT GAACAACTTC TACCCCAAAG ACATCAATGT CAAGTGGAAG


 


451
ATTGATGGCA GTGAACGACA AAATGGCGTC CTGAACAGTT GGACTGATCA


 


501
GGACAGCAAA GACAGCACCT ACAGCATGAG CAGCACCCTC ACGTTGACCA


 


551
AGGACGAGTA TGAACGACAT AACAGCTATA CCTGTGAGGC CACTCACAAG


 


601
ACATCAACTT CACCCATTGT CAAGAGCTTC AACAGGAATG AGTGTTAG






Amino acid sequence of the Ab-3 LC including signal peptide:









(SEQ ID NO: 127)










  1
MDFHVQIFSF MLISVTVILS SGEIVLTQSP ALMAASPGEK VTITCSVSST



 


 51
ISSNHLHWFQ QKSDTSPKPW IYGTSNLASG VPVRFSGSGS GTSYSLTISS


 


101
MEAEDAATYY CQQWSSYPLT FGAGTKLELR RADAAPTVSI FPPSSEQLTS


 


151
GGASVVCFLN NFYPKDINVK WKIDGSERQN GVLNSWTDQD SKDSTYSMSS


 


201
TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNEC






Nucleic acid sequence of the Ab-3 LC including signal peptide encoding sequence:









(SEQ ID NO: 128)










  1
ATGGATTTTC ATGTGCAGAT TTTCAGCTTC ATGCTAATCA GTGTCACAGT



 


 51
CATTTTGTCC AGTGGAGAAA TTGTGCTCAC CCAGTCTCCA GCACTCATGG


 


101
CTGCATCTCC GGGGGAGAAG GTCACCATCA CCTGCAGTGT CAGTTCAACT


 


151
ATAAGTTCCA ACCACTTGCA CTGGTTCCAG CAGAAGTCAG ACACCTCCCC


 


201
CAAACCCTGG ATTTATGGCA CATCCAACCT GGCTTCTGGA GTCCCTGTTC


 


251
GCTTCAGTGG CAGTGGATCT GGGACCTCTT ATTCTCTCAC AATCAGCAGC


 


301
ATGGAGGCTG AGGATGCTGC CACTTATTAC TGTCAACAGT GGAGTAGTTA


 


351
CCCACTCACG TTCGGCGCTG GGACCAAGCT GGAGCTGAGA CGGGCTGATG


 


401
CTGCACCAAC TGTATCCATC TGTCCACCAT CCAGTGAGCA GTTAACATCT


 


451
GGAGGTGCCT CAGTCGTGTG CTTCTTGAAC AACTTCTACC CCAAAGACAT


 


501
CAATGTCAAG TGGAAGATTG ATGGCAGTGA ACGACAAAAT GGCGTCCTGA


 


551
ACAGTTGGAC TGATCAGGAC AGCAAAGACA GCACCTACAG CATGAGCAGC


 


601
ACCCTCACGT TGACCAAGGA CGAGTATGAA CGACATAACA GCTATACCTG


 


651
TGAGGCCACT CACAAGACAT CAACTTCACC CATTGTCAAG AGCTTCAACA


 


701
GGAATGAGTG TTAG






Ab-3 Heavy Chain

Amino acid sequence of the mature form (signal peptide removed) of the Ab-3 HC:









(SEQ ID NO: 129)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-3 HC:









(SEQ ID NO: 130)










   1
GAGGTTCAGC TGCAGCAGTC TGGGGCTGAA CTTGTGAGGC CAGGGGCCTT



 


  51
AGTCAAGTTG TCCTGCACAG CTTCTGACTT CAACATTAAA GACTTCTATC


 


 101
TACACTGGAT GAGGCAGCGG CCTGAACAGG GCCTGGACTG GATTGGAAGG


 


 151
ATTGATCCTG AGAATGGTGA TACTTTATAT GACCCGAAGT TCCAGGACAA


 


 201
GGCCACTCTT ACAACAGACA CATCCTCCAA CACAGCCTAC CTGCAGCTCA


 


 251
GCGGCCTGAC ATCTGAGACC ACTGCCGTCT ATTACTGTTC TAGAGAGGCG


 


 301
GATTATTTCC ACGATGGTAC CTCCTACTGG TACTTCGATG TCTGGGGCGC


 


 351
AGGGACCACA ATCACCGTCT CCTCAGCCAA AACGACACCC CCATCTGTCT


 


 401
ATCCACTGGC CCCTGGATCT GCTGCCCAAA CTAACTCCAT GGTGACCCTG


 


 451
GGATGCCTGG TCAAGGGCTA TTTCCCTGAG CCAGTGACAG TGACCTGGAA


 


 501
CTCTGGATCC CTGTCCAGCG GTGTGCACAC CTTCCCAGCT GTCCTGCAGT


 


 551
CTGACCTCTA CACTCTGAGC AGCTCAGTGA CTGTCCCCTC CAGCACCTGG


 


 601
CCCAGCGAGA CCGTCACCTG CAACGTTGCC CACCCGGCCA GCAGCACCAA


 


 651
GGTGGACAAG AAAATTGTGC CCAGGGATTG TGGTTGTAAG CCTTGCATAT


 


 701
GTACAGTCCC AGAAGTATCA TCTGTCTTCA TCTTCCCCCC AAAGCCCAAG


 


 751
GATGTGCTCA CCATTACTCT GACTCCTAAG GTCACGTGTG TTGTGGTAGA


 


 801
CATCAGCAAG GATGATCCCG AGGTCCAGTT CAGCTGGTTT GTAGATGATG


 


 851
TGGAGGTGCA CACAGCTCAG ACGCAACCCC GGGAGGAGCA GTTCAACAGC


 


 901
ACTTTCCGCT CAGTCAGTGA ACTTCCCATC ATGCACCAGG ACTGGCTCAA


 


 951
TGGCAAGGAG TTCAAATGCA GGGTCAACAG TGCAGCTTTC CCTGCCCCCA


 


1001
TCGAGAAAAC CATCTCCAAA ACCAAAGGCA GACCGAAGGC TCCACAGGTG


 


1051
TACACCATTC CACCTCCCAA GGAGCAGATG GCCAAGGATA AAGTCAGTCT


 


1101
GACCTGCATG ATAACAGACT TCTTCCCTGA AGACATTACT GTGGAGTGGC


 


1151
AGTGGAATGG GCAGCCAGCG GAGAACTACA AGAACACTCA GCCCATCATG


 


1201
GACACAGATG GCTCTTACTT CATCTACAGC AAGCTCAATG TGCAGAAGAG


 


1251
CAACTGGGAG GCAGGAAATA CTTTCACCTG CTCTGTGTTA CATGAGGGCC


 


1301
TGCACAACCA CCATACTGAG AAGAGCCTCT CCCACTCTCC TGGTAAATGA






Amino acid sequence of the Ab-3 HC including signal peptide:









(SEQ ID NO: 131)










  1
MKCSWVIFFL MAVVTGVNSE VQLQQSGAEL VRPGALVKLS CTASDFNIKD



 


 51
FYLHWMRQRP EQGLDWIGRI DPENGDTLYD PKFQDKATLT TDTSSNTAYL


 


101
QLSGLTSETT AVYYCSREAD YFHDGTSYWY FDVWGAGTTI TVSSAKTTPP


 


151
SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV


 


201
LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRDCGCKP


 


251
CICTVPEVSS VFIFPPKPKD VLTITLTPKV TCVVVDISKD DPEVQFSWFV


 


301
DDVEVHTAQT QPREEQFNST FRSVSELPIM HQDWLNGKEF KCRVNSAAFP


 


351
APIEKTISKT KGRPKAPQVY TIPPPKEQMA KDKVSLTCMI TDFFPEDITV


 


401
EWQWNGQPAE NYKNTQPIMD TDGSYFIYSK LNVQKSNWEA GNTFTCSVLH


 


451
EGLHNHHTEK SLSHSPGK






Nucleic acid sequence of the Ab-3 HC including signal peptide encoding sequence:









(SEQ ID NO: 132)










   1
ATGAAATGCA GCTGGGTCAT CTTCTTCCTG ATGGCAGTGG TTACAGGGGT



 


  51
CAATTCAGAG GTTCAGCTGC AGCAGTCTGG GGCTGAACTT GTGAGGCCAG


 


 101
GGGCCTTAGT CAAGTTGTCC TGCACAGCTT CTGACTTCAA CATTAAAGAC


 


 151
TTCTATCTAC ACTGGATGAG GCAGCGGCCT GAACAGGGCC TGGACTGGAT


 


 201
TGGAAGGATT GATCCTGAGA ATGGTGATAC TTTATATGAC CCGAAGTTCC


 


 251
AGGACAAGGC CACTCTTACA ACAGACACAT CCTCCAACAC AGCCTACCTG


 


 301
CAGCTCAGCG GCCTGACATC TGAGACCACT GCCGTCTATT ACTGTTCTAG


 


 351
AGAGGCGGAT TATTTCCACG ATGGTACCTC CTACTGGTAC TTCGATGTCT


 


 401
GGGGCGCAGG GACCACAATC ACCGTCTCCT CAGCCAAAAC GACACCCCCA


 


 451
TCTGTCTATC CACTGGCCCC TGGATCTGCT GCCCAAACTA ACTCCATGGT


 


 501
GACCCTGGGA TGCCTGGTCA AGGGCTATTT CCCTGAGCCA GTGACAGTGA


 


 551
CCTGGAACTC TGGATCCCTG TCCAGCGGTG TGCACACCTT CCCAGCTGTC


 


 601
CTGCAGTCTG ACCTCTACAC TCTGAGCAGC TCAGTGACTG TCCCCTCCAG


 


 651
CACCTGGCCC AGCGAGACCG TCACCTGCAA CGTTGCCCAC CCGGCCAGCA


 


 701
GCACCAAGGT GGACAAGAAA ATTGTGCCCA GGGATTGTGG TTGTAAGCCT


 


 751
TGCATATGTA CAGTCCCAGA AGTATCATCT GTCTTCATCT TCCCCCCAAA


 


 801
GCCCAAGGAT GTGCTCACCA TTACTCTGAC TCCTAAGGTC ACGTGTGTTG


 


 851
TGGTAGACAT CAGCAAGGAT GATCCCGAGG TCCAGTTCAG CTGGTTTGTA


 


 901
GATGATGTGG AGGTGCACAC AGCTCAGACG CAACCCCGGG AGGAGCAGTT


 


 951
CAACAGCACT TTCCGCTCAG TCAGTGAACT TCCCATCATG CACCAGGACT


 


1001
GGCTCAATGG CAAGGAGTTC AAATGCAGGG TCAACAGTGC AGCTTTCCCT


 


1051
GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGCAGAC CGAAGGCTCC


 


1101
ACAGGTGTAC ACCATTCCAC CTCCCAAGGA GCAGATGGCC AAGGATAAAG


 


1151
TCAGTCTGAC CTGCATGATA ACAGACTTCT TCCCTGAAGA CATTACTGTG


 


1201
GAGTGGCAGT GGAATGGGCA GCCAGCGGAG AACTACAAGA ACACTCAGCC


 


1251
CATCATGGAC ACAGATGGCT CTTACTTCAT CTACAGCAAG CTCAATGTGC


 


1301
AGAAGAGCAA CTGGGAGGCA GGAAATACTT TCACCTGCTC TGTGTTACAT


 


1351
GAGGGCCTGC ACAACCACCA TACTGAGAAG AGCCTCTCCC ACTCTCCTGG


 


1401
TAAATGA






Ab-4

The sequences of the Antibody 4 (also referred to herein as Ab-4) LC and HC are as follows:

Ab-4 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-4 LC:









(SEQ ID NO: 133)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-4 LC:









(SEQ ID NO: 134)










  1
GATATCCAGA TGACACAGAT TACATCCTCC CTGTCTGCCT CTCTGGGAGA



 


 51
CAGGGTCTCC ATCAGTTGCA GGGCAAGTCA AGACATTAGC AATTATTTAA


 


101
ACTGGTATCA GCAGAAACCA GATGGAACTT TTAAACTCCT TATCTTCTAC


 


151
ACATCAAGAT TACTCTCAGG AGTCCCATCA AGGTTCAGTG GCAGTGGGTC


 


201
TGGAACAGAT TATTCTCTCA CCATTTACAA CCTGGAGCAA GAAGATTTTG


 


251
CCACTTACTT TTGCCAACAG GGAGATACGC TTCCGTACAC TTTCGGAGGG


 


301
GGGACCAAGC TGGAAATAAA ACGGGCTGAT GCTGCACCAA CTGTATCCAT


 


351
CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC TCAGTCGTGT


 


401
GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA GTGGAAGATT


 


451
GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA CTGATCAGGA


 


501
CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG


 


551
ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC TCACAAGACA


 


601
TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT GTTAG






Amino acid sequence of the Ab-4 LC including signal peptide:









(SEQ ID NO: 135)










  1
MMSSAQFLGL LLLCFQGTRC DIQMTQITSS LSASLGDRVS ISCRASQDIS



 


 51
NYLNWYQQKP DGTFKLLIFY TSRLLSGVPS RFSGSGSGTD YSLTIYNLEQ


 


101
EDFATYFCQQ GDTLPYTFGG GTKLEIKRAD AAPTVSIFPP SSEQLTSGGA


 


151
SVVCFLNNFY PKDINVKWKI DGSERQNGVL NSWTDQDSKD STYSMSSTLT


 


201
LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC






Nucleic acid sequence of the Ab-4 LC including signal peptide encoding sequence:









(SEQ ID NO: 136)










  1
ATGATGTCCT CTGCTCAGTT CCTTGGTCTC CTGTTGCTCT GTTTTCAAGG



 


 51
TACCAGATGT GATATCCAGA TGACACAGAT TACATCCTCC CTGTCTGCCT


 


101
CTCTGGGAGA CAGGGTCTCC ATCAGTTGCA GGGCAAGTCA AGACATTAGC


 


151
AATTATTTAA ACTGGTATCA GCAGAAACCA GATGGAACTT TTAAACTCCT


 


201
TATCTTCTAC ACATCAAGAT TACTCTCAGG AGTCCCATCA AGGTTCAGTG


 


251
GCAGTGGGTC TGGAACAGAT TATTCTCTCA CCATTTACAA CCTGGAGCAA


 


301
GAAGATTTTG CCACTTACTT TTGCCAACAG GGAGATACGC TTCCGTACAC


 


351
TTTCGGAGGG GGGACCAAGC TGGAAATAAA ACGGGCTGAT GCTGCACCAA


 


401
CTGTATCCAT CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC


 


451
TCAGTCGTGT GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA


 


501
GTGGAAGATT GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA


 


551
CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG


 


601
TTGACCAAGG ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC


 


651
TCACAAGACA TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT


 


701
GTTAG






Ab-4 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-4 HC:









(SEQ ID NO: 137)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-4 HC:









(SEQ ID NO: 138)










   1
GAGGTCCAAC TGCAACAGTC TGGACCTGAA CTAATGAAGC CTGGGGCTTC



 


  51
AGTGAAGATG TCCTGCAAGG CTTCTGGATA TACATTCACT GACTACAACA


 


 101
TGCACTGGGT GAAGCAGAAC CAAGGAAAGA CCCTAGAGTG GATAGGAGAA


 


 151
ATTAATCCTA ACAGTGGTGG TGCTGGCTAC AACCAGAAGT TCAAGGGCAA


 


 201
GGCCACATTG ACTGTAGACA AGTCCTCCAC CACAGCCTAC ATGGAGCTCC


 


 251
GCAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC AAGATTGGGC


 


 301
TACGATGATA TCTACGACGA CTGGTACTTC GATGTCTGGG GCGCAGGGAC


 


 351
CACGGTCACC GTCTCCTCAG CCAAAACGAC ACCCCCATCT GTCTATCCAC


 


 401
TGGCCCCTGG ATCTGCTGCC CAAACTAACT CCATGGTGAC CCTGGGATGC


 


 451
CTGGTCAAGG GCTATTTCCC TGAGCCAGTG ACAGTGACCT GGAACTCTGG


 


 501
ATCCCTGTCC AGCGGTGTGC ACACCTTCCC AGCTGTCCTG CAGTCTGACC


 


 551
TCTACACTCT GAGCAGCTCA GTGACTGTCC CCTCCAGCAC CTGGCCCAGC


 


 601
GAGACCGTCA CCTGCAACGT TGCCCACCCG GCCAGCAGCA CCAAGGTGGA


 


 651
CAAGAAAATT GTGCCCAGGG ATTGTGGTTG TAAGCCTTGC ATATGTACAG


 


 701
TCCCAGAAGT ATCATCTGTC TTCATCTTCC CCCCAAAGCC CAAGGATGTG


 


 751
CTCACCATTA CTCTGACTCC TAAGGTCACG TGTGTTGTGG TAGACATCAG


 


 801
CAAGGATGAT CCCGAGGTCC AGTTCAGCTG GTTTGTAGAT GATGTGGAGG


 


 851
TGCACACAGC TCAGACGCAA CCCCGGGAGG AGCAGTTCAA CAGCACTTTC


 


 901
CGCTCAGTCA GTGAACTTCC CATCATGCAC CAGGACTGGC TCAATGGCAA


 


 951
GGAGTTCAAA TGCAGGGTCA ACAGTGCAGC TTTCCCTGCC CCCATCGAGA


 


1001
AAACCATCTC CAAAACCAAA GGCAGACCGA AGGCTCCACA GGTGTACACC


 


1051
ATTCCACCTC CCAAGGAGCA GATGGCCAAG GATAAAGTCA GTCTGACCTG


 


1101
CATGATAACA GACTTCTTCC CTGAAGACAT TACTGTGGAG TGGCAGTGGA


 


1151
ATGGGCAGCC AGCGGAGAAC TACAAGAACA CTCAGCCCAT CATGGACACA


 


1201
GATGGCTCTT ACTTCATCTA CAGCAAGCTC AATGTGCAGA AGAGCAACTG


 


1251
GGAGGCAGGA AATACTTTCA CCTGCTCTGT GTTACATGAG GGCCTGCACA


 


1301
ACCACCATAC TGAGAAGAGC CTCTCCCACT CTCCTGGTAA ATGA






Amino acid sequence of the Ab-4 HC including signal peptide:









(SEQ ID NO: 139)










  1
MGWSWTFLFL LSGTAGVLSE VQLQQSGPEL MKPGASVKMS CKASGYIFTD



 


 51
YNMHWVKQNQ GKTLEWIGEI NPNSGGAGYN QKFKGKATLT VDKSSTTAYM


 


101
ELRSLTSEDS AVYYCARLGY DDIYDDWYFD VWGAGTTVTV SSAKTTPPSV


 


151
YPLAPGSAAQ TNSMVTLGCL VKGYFPEPVT VTWNSGSLSS GVHTFPAVLQ


 


201
SDLYTLSSSV TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV PRDCGCKPCI


 


251
CTVPEVSSVF IFPPKPKDVL TITLTPKVTC VVVDISKDDP EVQFSWFVDD


 


301
VEVHTAQTQP REEQFNSTFR SVSELPIMHQ DWLNGKEFKC RVNSAAFPAP


 


351
IEKTISKTKG RPKAPQVYTI PPPKEQMAKD KVSLTCMITD FFPEDITVEW


 


401
QWNGQPAENY KNTQPIMDTD GSYFIYSKLN VQKSNWEAGN TFTCSVLHEG


 


451
LHNHHTEKSL SHSPGK






Nucleic acid sequence of the Ab-4 HC including signal peptide encoding sequence:









(SEQ ID NO: 140)










   1
ATGGGATGGA GCTGGACCTT TCTCTTCCTC CTGTCAGGAA CTGCAGGTGT



 


  51
CCTCTCTGAG GTCCAACTGC AACAGTCTGG ACCTGAACTA ATGAAGCCTG


 


 101
GGGCTTCAGT GAAGATGTCC TGCAAGGCTT CTGGATATAC ATTCACTGAC


 


 151
TACAACATGC ACTGGGTGAA GCAGAACCAA GGAAAGACCC TAGAGTGGAT


 


 201
AGGAGAAATT AATCCTAACA GTGGTGGTGC TGGCTACAAC CAGAAGTTCA


 


 251
AGGGCAAGGC CACATTGACT GTAGACAAGT CCTCCACCAC AGCCTACATG


 


 301
GAGCTCCGCA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG


 


 351
ATTGGGCTAC GATGATATCT ACGACGACTG GTACTTCGAT GTCTGGGGCG


 


 401
CAGGGACCAC GGTCACCGTC TCCTCAGCCA AAACGACACC CCCATCTGTC


 


 451
TATCCACTGG CCCCTGGATC TGCTGCCCAA ACTAACTCCA TGGTGACCCT


 


 501
GGGATGCCTG GTCAAGGGCT ATTTCCCTGA GCCAGTGACA GTGACCTGGA


 


 551
ACTCTGGATC CCTGTCCAGC GGTGTGCACA CCTTCCCAGC TGTCCTGCAG


 


 601
TCTGACCTCT ACACTCTGAG CAGCTCAGTG ACTGTCCCCT CCAGCACCTG


 


 651
GCCCAGCGAG ACCGTCACCT GCAACGTTGC CCACCCGGCC AGCAGCACCA


 


 701
AGGTGGACAA GAAAATTGTG CCCAGGGATT GTGGTTGTAA GCCTTGCATA


 


 751
TGTACAGTCC CAGAAGTATC ATCTGTCTTC ATCTTCCCCC CAAAGCCCAA


 


 801
GGATGTGCTC ACCATTACTC TGACTCCTAA GGTCACGTGT GTTGTGGTAG


 


 851
ACATCAGCAA GGATGATCCC GAGGTCCAGT TCAGCTGGTT TGTAGATGAT


 


 901
GTGGAGGTGC ACACAGCTCA GACGCAACCC CGGGAGGAGC AGTTCAACAG


 


 951
CACTTTCCGC TCAGTCAGTG AACTTCCCAT CATGCACCAG GACTGGCTCA


 


1001
ATGGCAAGGA GTTCAAATGC AGGGTCAACA GTGCAGCTTT CCCTGCCCCC


 


1051
ATCGAGAAAA CCATCTCCAA AACCAAAGGC AGACCGAAGG CTCCACAGGT


 


1101
GTACACCATT CCACCTCCCA AGGAGCAGAT GGCCAAGGAT AAAGTCAGTC


 


1151
TGACCTGCAT GATAACAGAC TTCTTCCCTG AAGACATTAC TGTGGAGTGG


 


1201
CAGTGGAATG GGCAGCCAGC GGAGAACTAC AAGAACACTC AGCCCATCAT


 


1251
GGACACAGAT GGCTCTTACT TCATCTACAG CAAGCTCAAT GTGCAGAAGA


 


1301
GCAACTGGGA GGCAGGAAAT ACTTTCACCT GCTCTGTGTT ACATGAGGGC


 


1351
CTGCACAACC ACCATACTGA GAAGAGCCTC TCCCACTCTC CTGGTAAATG


 


1401
A






Ab-4 was humanized to generate Ab-5.

Ab-5

The sequences of the Antibody 5 (also referred to herein as Ab-5) LC and HC are as follows:

Ab-5 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-5 LC:









(SEQ ID NO: 141)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-5 LC:









(SEQ ID NO: 142)










  1
GACATCCAGA TGACCCAGTC TCCATCCTCC CTCTCCGCAT CCGTAGGCGA



 


 51
CCGCGTAACC ATAACATGTA GAGCATCTCA AGATATTTCC AACTATTTGA


 


101
ATTGGTACCA ACAAAAACCC GGCAAAGCAC CTAAACTCCT CATTTACTAT


 


151
ACATCAAGAC TCCTCTCCGG CGTTCCATCA CGATTCTCAG GCTCCGGCTC


 


201
CGGCACAGAT TTCACACTCA CTATTTCCTC CCTCCAACCA GAAGATTTTG


 


251
CAACCTATTA CTGTCAACAA GGCGATACAC TCCCATACAC ATTCGGCGGC


 


301
GGCACAAAAG TTGAAATTAA ACGTACGGTG GCTGCACCAT CTGTCTTCAT


 


351
CTTCCCGCCA TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT


 


401
GCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACA GTGGAAGGTG


 


451
GATAACGCCC TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA


 


501
CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG


 


551
CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC


 


601
CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GT






Amino acid sequence of the Ab-5 LC including signal peptide:









(SEQ ID NO: 143)










  1
MDMRVPAQLL GLLLLWLRGA RCDIQMTQSP SSLSASVGDR VTITCRASQD



 


 51
ISNYLNWYQQ KPGKAPKLLI YYTSRLLSGV PSRFSGSGSG TDFTLTISSL


 


101
QPEDFATYYC QQGDTLPYTF GGGTKVEIKR TVAAPSVFIF PPSDEQLKSG


 


151
TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST


 


201
LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC






Nucleic acid sequence of the Ab-5 LC including signal peptide encoding sequence:









(SEQ ID NO: 144)










  1
ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTCCTGC TACTCTGGCT



 


 51
CCGAGGTGCC AGATGTGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTCT


 


101
CCGCATCCGT AGGCGACCGC GTAACCATAA CATGTAGAGC ATCTCAAGAT


 


151
ATTTCCAACT ATTTGAATTG GTACCAACAA AAACCCGGCA AAGCACCTAA


 


201
ACTCCTCATT TACTATACAT CAAGACTCCT CTCCGGCGTT CCATCACGAT


 


251
TCTCAGGCTC CGGCTCCGGC ACAGATTTCA CACTCACTAT TTCCTCCCTC


 


301
CAACCAGAAG ATTTTGCAAC CTATTACTGT CAACAAGGCG ATACACTCCC


 


351
ATACACATTC GGCGGCGGCA CAAAAGTTGA AATTAAACGT ACGGTGGCTG


 


401
CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA


 


451
ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA


 


501
AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA


 


551
GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC


 


601
CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA


 


651
AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG


 


701
GAGAGTGT






Ab-5 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-5 HC:









(SEQ ID NO: 145)














Amino acid sequence of the mature form (signal peptide removed) of the Ab-5 HC without carboxy-terminal lysine:









(SEQ ID NO: 392)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-5 HC:









(SEQ ID NO: 146)










   1
GAGGTGCAGC TGGTGCAGAG CGGCGCCGAG GTAAAAAAAC CAGGAGCAAG



 


  51
CGTTAAAGTT TCTTGTAAAG CAAGCGGATA TACATTTACA GATTACAACA


 


 101
TGCATTGGGT AAGACAAGCG CCAGGACAAG GATTGGAATG GATGGGCGAA


 


 151
ATTAACCCTA ATAGTGGAGG AGCAGGCTAC AATCAAAAAT TCAAAGGGAG


 


 201
AGTTACAATG ACAACAGACA CAAGCACTTC AACAGCATAT ATGGAACTGC


 


 251
GATCACTTAG AAGCGACGAT ACAGCTGTAT ACTATTGCGC ACGACTTGGG


 


 301
TATGATGATA TATATGATGA CTGGTATTTC GATGTTTGGG GCCAGGGAAC


 


 351
AACAGTTACC GTCTCTAGTG CCTCCACCAA GGGCCCATCG GTCTTCCCCC


 


 401
TGGCGCCCTG CTCCAGGAGC ACCTCCGAGA GCACAGCGGC CCTGGGCTGC


 


 451
CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT GGAACTCAGG


 


 501
CGCTCTGACC AGCGGCGTGC ACACCTTCCC AGCTGTCCTA CAGTCCTCAG


 


 551
GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG CAACTTCGGC


 


 601
ACCCAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA ACACCAAGGT


 


 651
GGACAAGACA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA CCGTGCCCAG


 


 701
CACCACCTGT GGCAGGACCG TCAGTCTTCC TCTTCCCCCC AAAACCCAAG


 


 751
GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG TGGTGGTGGA


 


 801
CGTGAGCCAC GAAGACCCCG AGGTCCAGTT CAACTGGTAC GTGGACGGCG


 


 851
TGGAGGTGCA TAATGCCAAG ACAAAGCCAC GGGAGGAGCA GTTCAACAGC


 


 901
ACGTTCCGTG TGGTCAGCGT CCTCACCGTT GTGCACCAGG ACTGGCTGAA


 


 951
CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC CCAGCCCCCA


 


1001
TCGAGAAAAC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA ACCACAGGTG


 


1051
TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC AGGTCAGCCT


 


1101
GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC GTGGAGTGGG


 


1151
AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACACC TCCCATGCTG


 


1201
GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG


 


1251
CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC


 


1301
TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC GGGTAAA






Amino acid sequence of the Ab-5 HC including signal peptide:









(SEQ ID NO: 147)










  1
MDWTWRILFL VAAATGAHSE VQLVQSGAEV KKPGASVKVS CKASGYTFTD



 


 51
YNMHWVRQAP GQGLEWMGEI NPNSGGAGYN QKFKGRVTMT TDTSTSTAYM


 


101
ELRSLRSDDT AVYYCARLGY DDIYDDWYFD VWGQGTTVTV SSASTKGPSV


 


151
FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ


 


201
SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP


 


251
CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV


 


301
DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP


 


351
APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV


 


401
EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH


 


451
EALHNHYTQK SLSLSPGK






Nucleic acid sequence of the Ab-5 HC including signal peptide encoding sequence:









(SEQ ID NO: 148)










   1
ATGGACTGGA CCTGGAGGAT CCTCTTCTTG GTGGCAGCAG CCACAGGAGC



 


  51
CCACTCCGAG GTGCAGCTGG TGCAGAGCGG CGCCGAGGTA AAAAAACCAG


 


 101
GAGCAAGCGT TAAAGTTTCT TGTAAAGCAA GCGGATATAC ATTTACAGAT


 


 151
TACAACATGC ATTGGGTAAG ACAAGCGCCA GGACAAGGAT TGGAATGGAT


 


 201
GGGCGAAATT AACCCTAATA GTGGAGGAGC AGGCTACAAT CAAAAATTCA


 


 251
AAGGGAGAGT TACAATGACA ACAGACACAA GCACTTCAAC AGCATATATG


 


 301
GAACTGCGAT CACTTAGAAG CGACGATACA GCTGTATACT ATTGCGCACG


 


 351
ACTTGGGTAT GATGATATAT ATGATGACTG GTATTTCGAT GTTTGGGGCC


 


 401
AGGGAACAAC AGTTACCGTC TCTAGTGCCT CCACCAAGGG CCCATCGGTC


 


 451
TTCCCCCTGG CGCCCTGCTC CAGGAGCACC TCCGAGAGCA CAGCGGCCCT


 


 501
GGGCTGCCTG GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA


 


 551
ACTCAGGCGC TCTGACCAGC GGCGTGCACA CCTTCCCAGC TGTCCTACAG


 


 601
TCCTCAGGAC TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAA


 


 651
CTTCGGCACC CAGACCTACA CCTGCAACGT AGATCACAAG CCCAGCAACA


 


 701
CCAAGGTGGA CAAGACAGTT GAGCGCAAAT GTTGTGTCGA GTGCCCACCG


 


 751
TGCCCAGCAC CACCTGTGGC AGGACCGTCA GTCTTCCTCT TCCCCCCAAA


 


 801
ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACGTGCGTGG


 


 851
TGGTGGACGT GAGCCACGAA GACCCCGAGG TCCAGTTCAA CTGGTACGTG


 


 901
GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCACGGG AGGAGCAGTT


 


 951
CAACAGCACG TTCCGTGTGG TCAGCGTCCT CACCGTTGTG CACCAGGACT


 


1001
GGCTGAACGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGGCCTCCCA


 


1051
GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGGCAGC CCCGAGAACC


 


1101
ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG


 


1151
TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ACCCCAGCGA CATCGCCGTG


 


1201
GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACACCTCC


 


1251
CATGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG


 


1301
ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT


 


1351
GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG


 


1401
TAAA






Ab-5 Variable domains:

Ab-5 light chain variable domain amino acid sequence (without signal sequence):









(SEQ ID NO: 376)














Ab-5 light chain variable domain DNA sequence (without signal sequence):









(SEQ ID NO: 377)










  1
GACATCCAGA TGACCCAGTC TCCATCCTCC CTCTCCGCAT CCGTAGGCGA



 


 51
CCGCGTAACC ATAACATGTA GAGCATCTCA AGATATTTCC AACTATTTGA


 


101
ATTGGTACCA ACAAAAACCC GGCAAAGCAC CTAAACTCCT CATTTACTAT


 


151
ACATCAAGAC TCCTCTCCGG CGTTCCATCA CGATTCTCAG GCTCCGGCTC


 


201
CGGCACAGAT TTCACACTCA CTATTTCCTC CCTCCAACCA GAAGATTTTG


 


251
CAACCTATTA CTGTCAACAA GGCGATACAC TCCCATACAC ATTCGGCGGC


 


301
GGCACAAAAG TTGAAATTAA A






Ab-5 heavy chain variable domain amino acid sequence (without signal sequence):









(SEQ ID NO: 378)














Ab-5 heavy chain variable domain DNA sequence (without signal sequence):









(SEQ ID NO: 379)










  1
GAGGTGCAGC TGGTGCAGAG CGGCGCCGAG GTAAAAAAAC CAGGAGCAAG



 


 51
CGTTAAAGTT TCTTGTAAAG CAAGCGGATA TACATTTACA GATTACAACA


 


101
TGCATTGGGT AAGACAAGCG CCAGGACAAG GATTGGAATG GATGGGCGAA


 


151
ATTAACCCTA ATAGTGGAGG AGCAGGCTAC AATCAAAAAT TCAAAGGGAG


 


201
AGTTACAATG ACAACAGACA CAAGCACTTC AACAGCATAT ATGGAACTGC


 


251
GATCACTTAG AAGCGACGAT ACAGCTGTAT ACTATTGCGC ACGACTTGGG


 


301
TATGATGATA TATATGATGA CTGGTATTTC GATGTTTGGG GCCAGGGAAC


 


351
AACAGTTACC GTCTCTAGT






The CDR (complementarity determining region) sequences in the variable region of the heavy chain of Ab-5 are as follows:









(SEQ ID NO: 245)



CDR-H1: DYNMH



 



(SEQ ID NO: 246)



CDR-H2: EINPNSGGAGYNQKFKG



 



(SEQ ID NO: 247)



CDR-H3: LGYDDIYDDWYFDV






The light chain variable region CDR sequences of Ab-5 are:









(SEQ ID NO: 78)



CDR-L1: RASQDISNYLN



 



(SEQ ID NO: 79)



CDR-L2: YTSRLLS



 



(SEQ ID NO: 80)



CDR-L3: QQGDTLPYT






Ab-6

The sequences of the Antibody 6 (also referred to herein as Ab-6) LC and HC are as follows:

Ab-6 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-6 LC:









(SEQ ID NO: 149)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-6 LC:









(SEQ ID NO: 150)










  1
GATATCCAGA TGACACAGAC TACATCCTCC CTGTCTGCCT CTCTGGGAGA



 


 51
CAGAGTCACC ATCAGTTGCA GGGCAAGTCA GGACATTAGC AATTATTTAA


 


101
ACTGGTTTCA GCAGAAACCA GATGGAACTC TTAAACTCCT GATCTTCTAC


 


151
ACATCAAGAT TACACTCAGG AGTTCCATCA AGGTTCAGTG GCAGTGGGTC


 


201
TGGAACAGAT TATTCTCTCA CCATTAGCAA CCTGGAGCAA GAAGATATTG


 


251
CCACTTACTT TTGCCAACAG GGTGATACGC TTCCGTACAC GTTCGGGGGG


 


301
GGGACCAAGC TGGAAATAAG ACGGGCTGAT GCTGCACCAA CTGTATCCAT


 


351
CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC TCAGTCGTGT


 


401
GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA GTGGAAGATT


 


451
GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA CTGATCAGGA


 


501
CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG


 


551
ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC TCACAAGACA


 


601
TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT GTTAG






Amino acid sequence of the Ab-6 LC including signal peptide:









(SEQ ID NO: 151)










  1
MMSSAQFLGL LLLCFQGTRC DIQMTQTTSS LSASLGDRVT ISCRASQDIS



 


 51
NYLNWFQQKP DGTLKLLIFY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ


 


101
EDIATYFCQQ GDTLPYTFGG GTKLEIRRAD AAPTVSIFPP SSEQLTSGGA


 


151
SVVCFLNNFY PKDINVKWKI DGSERQNGVL NSWTDQDSKD STYSMSSTLT


 


201
LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC






Nucleic acid sequence of the Ab-6 LC including signal peptide encoding sequence:








(SEQ ID NO: 152)








1
ATGATGTCCT CTGCTCAGTT CCTTGGTCTC CTGTTGCTCT 



GTTTTCAAGG


 


51
TACCAGATGT GATATCCAGA TGACACAGAC TACATCCTCC 



CTGTCTGCCT


 


101
CTCTGGGAGA CAGAGTCACC ATCAGTTGCA GGGCAAGTCA 



GGACATTAGC


 


151
AATTATTTAA ACTGGTTTCA GCAGAAACCA GATGGAACTC 



TTAAACTCCT


 


201
GATCTTCTAC ACATCAAGAT TACACTCAGG AGTTCCATCA 



AGGTTCAGTG


 


251
GCAGTGGGTC TGGAACAGAT TATTCTCTCA CCATTAGCAA 



CCTGGAGCAA


 


301
GAAGATATTG CCACTTACTT TTGCCAACAG GGTGATACGC 



TTCCGTACAC


 


351
GTTCGGGGGG GGGACCAAGC TGGAAATAAG ACGGGCTGAT 



GCTGCACCAA


 


401
CTGTATCCAT CTTCCCACCA TCCAGTGAGC AGTTAACATC 



TGGAGGTGCC


 


451
TCAGTCGTGT GCTTCTTGAA CAACTTCTAC CCCAAAGACA 



TCAATGTCAA


 


501
GTGGAAGATT GATGGCAGTG AACGACAAAA TGGCGTCCTG 



AACAGTTGGA


 


551
CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG 



CACCCTCACG


 


601
TTGACCAAGG ACGAGTATGA ACGACATAAC AGCTATACCT



GTGAGGCCAC


 


651
TCACAAGACA TCAACTTCAC CCATTGTCAA GAGCTTCAAC



AGGAATGAGT


 


701
GTTAG






Ab-6 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-6 HC:









(SEQ ID NO: 153)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-6 HC:








(SEQ ID NO: 154)








1
GAGGTCCAGC TGCAACAGTC TGGACCTGAA CTAATGAAGC 



CTGGGGCTTC


 


51
AGTGAAGATG TCCTGCAAGG CTTCTGGATA CACATTCACT 



GACTACAACA


 


101
TGCACTGGGT GAAACAGAAC CAAGGAAAGA GCCTAGAGTG 



GATAGGAGAA


 


151
ATTAATCCTA ACAGTGGTGG TAGTGGCTAC AACCAAAAGT 



TCAAAGGCAA


 


201
GGCCACATTG ACTGTAGACA AGTCTTCCAG CACAGCCTAC 



ATGGAGCTCC


 


251
GCAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC 



AAGATTGGTC


 


301
TACGATGGCA GCTACGAGGA CTGGTACTTC GATGTCTGGG 



GCGCAGGGAC


 


351
CACGGTCACC GTCTCCTCAG CCAAAACGAC ACCCCCATCT 



GTCTATCCAC


 


401
TGGCCCCTGG ATCTGCTGCC CAAACTAACT CCATGGTGAC 



CCTGGGATGC


 


451
CTGGTCAAGG GCTATTTCCC TGAGCCAGTG ACAGTGACCT 



GGAACTCTGG


 


501
ATCCCTGTCC AGCGGTGTGC ACACCTTCCC AGCTGTCCTG 



CAGTCTGACC


 


551
TCTACACTCT GAGCAGCTCA GTGACTGTCC CCTCCAGCAC 



CTGGCCCAGC


 


601
GAGACCGTCA CCTGCAACGT TGCCCACCCG GCCAGCAGCA 



CCAAGGTGGA


 


651
CAAGAAAATT GTGCCCAGGG ATTGTGGTTG TAAGCCTTGC 



ATATGTACAG


 


701
TCCCAGAAGT ATCATCTGTC TTCATCTTCC CCCCAAAGCC 



CAAGGATGTG


 


751
CTCACCATTA CTCTGACTCC TAAGGTCACG TGTGTTGTGG 



TAGACATCAG


 


801
CAAGGATGAT CCCGAGGTCC AGTTCAGCTG GTTTGTAGAT 



GATGTGGAGG


 


851
TGCACACAGC TCAGACGCAA CCCCGGGAGG AGCAGTTCAA 



CAGCACTTTC


 


901
CGCTCAGTCA GTGAACTTCC CATCATGCAC CAGGACTGGC 



TCAATGGCAA


 


951
GGAGTTCAAA TGCAGGGTCA ACAGTGCAGC TTTCCCTGCC 



CCCATCGAGA


 


1001
AAACCATCTC CAAAACCAAA GGCAGACCGA AGGCTCCACA 



GGTGTACACC


 


1051
ATTCCACCTC CCAAGGAGCA GATGGCCAAG GATAAAGTCA 



GTCTGACCTG


 


1101
CATGATAACA GACTTCTTCC CTGAAGACAT TACTGTGGAG 



TGGCAGTGGA


 


1151
ATGGGCAGCC AGCGGAGAAC TACAAGAACA CTCAGCCCAT 



CATGGACACA


 


1201
GATGGCTCTT ACTTCATCTA CAGCAAGCTC AATGTGCAGA 



AGAGCAACTG


 


1251
GGAGGCAGGA AATACTTTCA CCTGCTCTGT GTTACATGAG 



GGCCTGCACA


 


1301
ACCACCATAC TGAGAAGAGC CTCTCCCACT CTCCTGGTAA 



ATGA






Amino acid sequence of the Ab-6 HC including signal peptide:








(SEQ ID NO: 155)








1
MGWSWTFLFL LSGTAGVLSE VQLQQSGPEL MKPGASVKMS 



CKASGYTFTD


 


51
YNMHWVKQNQ GKSLEWIGEI NPNSGGSGYN QKFKGKATLT 



VDKSSSTAYM


 


101
ELRSLTSEDS AVYYCARLVY DGSYEDWYFD VWGAGTTVTV 



SSAKTTPPSV


 


151
YPLAPGSAAQ TNSMVTLGCL VKGYFPEPVT VTWNSGSLSS 



GVHTFPAVLQ


 


201
SDLYTLSSSV TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV 



PRDCGCKPCI


 


251
CTVPEVSSVF IFPPKPKDVL TITLTPKVTC VVVDISKDDP 



EVQFSWFVDD


 


301
VEVHTAQTQP REEQFNSTFR SVSELPIMHQ DWLNGKEFKC 



RVNSAAFPAP


 


351
IEKTISKTKG RPKAPQVYTI PPPKEQMAKD KVSLTCMITD 



FFPEDITVEW


 


401
QWNGQPAENY KNTQPIMDTD GSYFIYSKLN VQKSNWEAGN 



TFTCSVLHEG


 


451
LHNHHTEKSL SHSPGK






Nucleic acid sequence of the Ab-6 HC including signal peptide encoding sequence:








(SEQ ID NO: 156)








1
ATGGGATGGA GCTGGACCTT TCTCTTCCTC CTGTCAGGAA 



CTGCAGGTGT


 


51
CCTCTCTGAG GTCCAGCTGC AACAGTCTGG ACCTGAACTA 



ATGAAGCCTG


 


101
GGGCTTCAGT GAAGATGTCC TGCAAGGCTT CTGGATACAC 



ATTCACTGAC


 


151
TACAACATGC ACTGGGTGAA ACAGAACCAA GGAAAGAGCC 



TAGAGTGGAT


 


201
AGGAGAAATT AATCCTAACA GTGGTGGTAG TGGCTACAAC 



CAAAAGTTCA


 


251
AAGGCAAGGC CACATTGACT GTAGACAAGT CTTCCAGCAC 



AGCCTACATG


 


301
GAGCTCCGCA GCCTGACATC TGAGGACTCT GCAGTCTATT 



ACTGTGCAAG


 


351
ATTGGTCTAC GATGGCAGCT ACGAGGACTG GTACTTCGAT 



GTCTGGGGCG


 


401
CAGGGACCAC GGTCACCGTC TCCTCAGCCA AAACGACACC 



CCCATCTGTC


 


451
TATCCACTGG CCCCTGGATC TGCTGCCCAA ACTAACTCCA 



TGGTGACCCT


 


501
GGGATGCCTG GTCAAGGGCT ATTTCCCTGA GCCAGTGACA 



GTGACCTGGA


 


551
ACTCTGGATC CCTGTCCAGC GGTGTGCACA CCTTCCCAGC 



TGTCCTGCAG


 


601
TCTGACCTCT ACACTCTGAG CAGCTCAGTG ACTGTCCCCT 



CCAGCACCTG


 


651
GCCCAGCGAG ACCGTCACCT GCAACGTTGC CCACCCGGCC 



AGCAGCACCA


 


701
AGGTGGACAA GAAAATTGTG CCCAGGGATT GTGGTTGTAA 



GCCTTGCATA


 


751
TGTACAGTCC CAGAAGTATC ATCTGTCTTC ATCTTCCCCC 



CAAAGCCCAA


 


801
GGATGTGCTC ACCATTACTC TGACTCCTAA GGTCACGTGT 



GTTGTGGTAG


 


851
ACATCAGCAA GGATGATCCC GAGGTCCAGT TCAGCTGGTT 



TGTAGATGAT


 


901
GTGGAGGTGC ACACAGCTCA GACGCAACCC CGGGAGGAGC 



AGTTCAACAG


 


951
CACTTTCCGC TCAGTCAGTG AACTTCCCAT CATGCACCAG 



GACTGGCTCA


 


1001
ATGGCAAGGA GTTCAAATGC AGGGTCAACA GTGCAGCTTT 



CCCTGCCCCC


 


1051
ATCGAGAAAA CCATCTCCAA AACCAAAGGC AGACCGAAGG 



CTCCACAGGT


 


1101
GTACACCATT CCACCTCCCA AGGAGCAGAT GGCCAAGGAT 



AAAGTCAGTC


 


1151
TGACCTGCAT GATAACAGAC TTCTTCCCTG AAGACATTAC 



TGTGGAGTGG


 


1201
CAGTGGAATG GGCAGCCAGC GGAGAACTAC AAGAACACTC 



AGCCCATCAT


 


1251
GGACACAGAT GGCTCTTACT TCATCTACAG CAAGCTCAAT 



GTGCAGAAGA


 


1301
GCAACTGGGA GGCAGGAAAT ACTTTCACCT GCTCTGTGTT 



ACATGAGGGC


 


1351
CTGCACAACC ACCATACTGA GAAGAGCCTC TCCCACTCTC 



CTGGTAAATG


 


1401
A






Ab-7

The sequences of the Antibody 7 (also referred to herein as Ab-7) LC and HC are as follows:

Ab-7 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-7 LC:









(SEQ ID NO: 157)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-7 LC:








(SEQ ID NO: 158)








1
GATATCCAGA TGACACAGAC TACATCCTCC CTGTCTGCCT 



CTCTGGGAGA


 


51
CAGAGTCACC ATCTGTTGCA GGGCAAGTCA GGTCATTACC 



AATTATTTAT


 


101
ACTGGTATCA GCAGAAACCA GATGGAACTT TTAAACTCCT 



GATCTACTAC


 


151
ACATCAAGAT TACACTCAGG AGTCCCATCA AGGTTCAGTG 



GCAGTGGGTC


 


201
TGGAACAGAT TATTCTCTCA CCATTAGCAA CCTGGAACAG 



GAAGATATTG


 


251
CCACTTACTT TTGCCAACAG GGTGATACGC TTCCGTACAC 



GTTCGGAGGG


 


301
GGGACCAAGC TGGAAATAAA ACGGGCTGAT GCTGCACCAA 



CTGTATCCAT


 


351
CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC 



TCAGTCGTGT


 


401
GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA 



GTGGAAGATT


 


451
GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA 



CTGATCAGGA


 


501
CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG 



TTGACCAAGG


 


551
ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC 



TCACAAGACA


 


601
TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT 



GT






Amino acid sequence of the Ab-7 LC including signal peptide:








(SEQ ID NO: 159)








1
MMSSAQFLGL LLLCFQGTRC DIQMTQTTSS LSASLGDRVT 



ICCRASQVIT


 


51
NYLYWYQQKP DGTFKLLIYY TSRLHSGVPS RFSGSGSGTD 



YSLTISNLEQ


 


101
EDIATYFCQQ GDTLPYTFGG GTKLEIKRAD AAPTVSIFPP 



SSEQLTSGGA


 


151
SVVCFLNNFY PKDINVKWKI DGSERQNGVL NSWTDQDSKD 



STYSMSSTLT


 


201
LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC






Nucleic acid sequence of the Ab-7 LC including signal peptide encoding sequence:








(SEQ ID NO: 160)








1
ATGATGTCCT CTGCTCAGTT CCTTGGTCTC CTGTTGCTCT 



GTTTTCAAGG


 


51
TACCAGATGT GATATCCAGA TGACACAGAC TACATCCTCC 



CTGTCTGCCT


 


101
CTCTGGGAGA CAGAGTCACC ATCTGTTGCA GGGCAAGTCA 



GGTCATTACC


 


151
AATTATTTAT ACTGGTATCA GCAGAAACCA GATGGAACTT 



TTAAACTCCT


 


201
GATCTACTAC ACATCAAGAT TACACTCAGG AGTCCCATCA 



AGGTTCAGTG


 


251
GCAGTGGGTC TGGAACAGAT TATTCTCTCA CCATTAGCAA 



CCTGGAACAG


 


301
GAAGATATTG CCACTTACTT TTGCCAACAG GGTGATACGC 



TTCCGTACAC


 


351
GTTCGGAGGG GGGACCAAGC TGGAAATAAA ACGGGCTGAT 



GCTGCACCAA


 


401
CTGTATCCAT CTTCCCACCA TCCAGTGAGC AGTTAACATC 



TGGAGGTGCC


 


451
TCAGTCGTGT GCTTCTTGAA CAACTTCTAC CCCAAAGACA 



TCAATGTCAA


 


501
GTGGAAGATT GATGGCAGTG AACGACAAAA TGGCGTCCTG 



AACAGTTGGA


 


551
CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG 



CACCCTCACG


 


601
TTGACCAAGG ACGAGTATGA ACGACATAAC AGCTATACCT 



GTGAGGCCAC


 


651
TCACAAGACA TCAACTTCAC CCATTGTCAA GAGCTTCAAC 



AGGAATGAGT


 


701
GT






Ab-7 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-7 HC:









(SEQ ID NO: 161)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-7 HC:








(SEQ ID NO: 162)








1
GAGGTCCAGC TGCAACAGTC TGGACCTGAA CTAATGAAGC 



CTGGGGCTTC


 


51
AGTGAAGATG TCCTGCAAGG CTTCTGGATA CACATTCACT 



GACTACAACA


 


101
TGCACTGGAT GAAGCAGAAC CAAGGAAAGA GCCTAGAATG 



GATAGGAGAA


 


151
ATTAATCCTA ACAGTGGTGG TGCTGGCTAC AACCAGCAGT 



TCAAAGGCAA


 


201
GGCCACATTG ACTGTAGACA AGTCCTCCAG GACAGCCTAC 



ATGGAGCTCC


 


251
GCAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC 



AAGATTGGGC


 


301
TACGTTGGTA ATTACGAGGA CTGGTACTTC GATGTCTGGG 



GCGCAGGGAC


 


351
CACGGTCACC GTCTCCTCAG CCAAAACGAC ACCCCCATCT 



GTCTATCCAC


 


401
TGGCCCCTGG ATCTGCTGCC CAAACTAACT CCATGGTGAC 



CCTGGGATGC


 


451
CTGGTCAAGG GCTATTTCCC TGAGCCAGTG ACAGTGACCT 



GGAACTCTGG


 


501
ATCCCTGTCC AGCGGTGTGC ACACCTTCCC AGCTGTCCTG 



CAGTCTGACC


 


551
TCTACACTCT GAGCAGCTCA GTGACTGTCC CCTCCAGCAC 



CTGGCCCAGC


 


601
GAGACCGTCA CCTGCAACGT TGCCCACCCG GCCAGCAGCA 



CCAAGGTGGA


 


651
CAAGAAAATT GTGCCCAGGG ATTGTGGTTG TAAGCCTTGC 



ATATGTACAG


 


701
TCCCAGAAGT ATCATCTGTC TTCATCTTCC CCCCAAAGCC 



CAAGGATGTG


 


751
CTCACCATTA CTCTGACTCC TAAGGTCACG TGTGTTGTGG 



TAGACATCAG


 


801
CAAGGATGAT CCCGAGGTCC AGTTCAGCTG GTTTGTAGAT 



GATGTGGAGG


 


851
TGCACACAGC TCAGACGCAA CCCCGGGAGG AGCAGTTCAA 



CAGCACTTTC


 


901
CGCTCAGTCA GTGAACTTCC CATCATGCAC CAGGACTGGC 



TCAATGGCAA


 


951
GGAGTTCAAA TGCAGGGTCA ACAGTGCAGC TTTCCCTGCC 



CCCATCGAGA


 


1001
AAACCATCTC CAAAACCAAA GGCAGACCGA AGGCTCCACA 



GGTGTACACC


 


1051
ATTCCACCTC CCAAGGAGCA GATGGCCAAG GATAAAGTCA 



GTCTGACCTG


 


1101
CATGATAACA GACTTCTTCC CTGAAGACAT TACTGTGGAG 



TGGCAGTGGA


 


1151
ATGGGCAGCC AGCGGAGAAC TACAAGAACA CTCAGCCCAT 



CATGGACACA


 


1201
GATGGCTCTT ACTTCATCTA CAGCAAGCTC AATGTGCAGA 



AGAGCAACTG


 


1251
GGAGGCAGGA AATACTTTCA CCTGCTCTGT GTTACATGAG 



GGCCTGCACA


 


1301
ACCACCATAC TGAGAAGAGC CTCTCCCACT CTCCTGGTAA 



A






Amino acid sequence of the Ab-7 HC including signal peptide:








(SEQ ID NO: 163)








1
MGWSWTFLFL LSGTAGVLSE VQLQQSGPEL MKPGASVKMS 



CKASGYTFTD


 


51
YNMHWMKQNQ GKSLEWIGEI NPNSGGAGYN QQFKGKATLT 



VDKSSRTAYM


 


101
ELRSLTSEDS AVYYCARLGY VGNYEDWYFD VWGAGTTVTV 



SSAKTTPPSV


 


151
YPLAPGSAAQ TNSMVTLGCL VKGYFPEPVT VTWNSGSLSS 



GVHTFPAVLQ


 


201
SDLYTLSSSV TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV 



PRDCGCKPCI


 


251
CTVPEVSSVF IFPPKPKDVL TITLTPKVTC VVVDISKDDP 



EVQFSWFVDD


 


301
VEVHTAQTQP REEQFNSTFR SVSELPIMHQ DWLNGKEFKC 



RVNSAAFPAP


 


351
IEKTISKTKG RPKAPQVYTI PPPKEQMAKD KVSLTCMITD 



FFPEDITVEW


 


401
QWNGQPAENY KNTQPIMDTD GSYFIYSKLN VQKSNWEAGN 



TFTCSVLHEG


 


451
LHNHHTEKSL SHSPGK






Nucleic acid sequence of the Ab-7 HC including signal peptide encoding sequence:








(SEQ ID NO: 164)








1
ATGGGATGGA GCTGGACCTT TCTCTTCCTC CTGTCAGGAA 



CTGCAGGTGT


 


51
CCTCTCTGAG GTCCAGCTGC AACAGTCTGG ACCTGAACTA 



ATGAAGCCTG


 


101
GGGCTTCAGT GAAGATGTCC TGCAAGGCTT CTGGATACAC 



ATTCACTGAC


 


151
TACAACATGC ACTGGATGAA GCAGAACCAA GGAAAGAGCC 



TAGAATGGAT


 


201
AGGAGAAATT AATCCTAACA GTGGTGGTGC TGGCTACAAC 



CAGCAGTTCA


 


251
AAGGCAAGGC CACATTGACT GTAGACAAGT CCTCCAGGAC 



AGCCTACATG


 


301
GAGCTCCGCA GCCTGACATC TGAGGACTCT GCAGTCTATT 



ACTGTGCAAG


 


351
ATTGGGCTAC GTTGGTAATT ACGAGGACTG GTACTTCGAT 



GTCTGGGGCG


 


401
CAGGGACCAC GGTCACCGTC TCCTCAGCCA AAACGACACC 



CCCATCTGTC


 


451
TATCCACTGG CCCCTGGATC TGCTGCCCAA ACTAACTCCA 



TGGTGACCCT


 


501
GGGATGCCTG GTCAAGGGCT ATTTCCCTGA GCCAGTGACA 



GTGACCTGGA


 


551
ACTCTGGATC CCTGTCCAGC GGTGTGCACA CCTTCCCAGC 



TGTCCTGCAG


 


601
TCTGACCTCT ACACTCTGAG CAGCTCAGTG ACTGTCCCCT 



CCAGCACCTG


 


651
GCCCAGCGAG ACCGTCACCT GCAACGTTGC CCACCCGGCC 



AGCAGCACCA


 


701
AGGTGGACAA GAAAATTGTG CCCAGGGATT GTGGTTGTAA 



GCCTTGCATA


 


751
TGTACAGTCC CAGAAGTATC ATCTGTCTTC ATCTTCCCCC 



CAAAGCCCAA


 


801
GGATGTGCTC ACCATTACTC TGACTCCTAA GGTCACGTGT 



GTTGTGGTAG


 


851
ACATCAGCAA GGATGATCCC GAGGTCCAGT TCAGCTGGTT 



TGTAGATGAT


 


901
GTGGAGGTGC ACACAGCTCA GACGCAACCC CGGGAGGAGC 



AGTTCAACAG


 


951
CACTTTCCGC TCAGTCAGTG AACTTCCCAT CATGCACCAG 



GACTGGCTCA


 


1001
ATGGCAAGGA GTTCAAATGC AGGGTCAACA GTGCAGCTTT 



CCCTGCCCCC


 


1051
ATCGAGAAAA CCATCTCCAA AACCAAAGGC AGACCGAAGG 



CTCCACAGGT


 


1101
GTACACCATT CCACCTCCCA AGGAGCAGAT GGCCAAGGAT 



AAAGTCAGTC


 


1151
TGACCTGCAT GATAACAGAC TTCTTCCCTG AAGACATTAC 



TGTGGAGTGG


 


1201
CAGTGGAATG GGCAGCCAGC GGAGAACTAC AAGAACACTC 



AGCCCATCAT


 


1251
GGACACAGAT GGCTCTTACT TCATCTACAG CAAGCTCAAT 



GTGCAGAAGA


 


1301
GCAACTGGGA GGCAGGAAAT ACTTTCACCT GCTCTGTGTT 



ACATGAGGGC


 


1351
CTGCACAACC ACCATACTGA GAAGAGCCTC TCCCACTCTC 



CTGGTAAA






Ab-8

The sequences of the Antibody 8 (also referred to herein as Ab-8) LC and HC are as follows:

Ab-8 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-8 LC:









(SEQ ID NO: 165)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-8 LC:








(SEQ ID NO: 166)








1
GATATCCAGA TGACACAGAC TACATCCTCC CTGTCTGCCT 



CTCTGGGAGA


 


51
CAGGGTCTCC ATCAGTTGCA GGGCAAGTCA AGACATTAGC 



AATTATTTAA


 


101
ACTGGTATCA GCAGAAACCA GATGGAACTT TTAAACTCCT 



TATCTTCTAC


 


151
ACATCAAGAT TACTCTCAGG AGTCCCATCA AGGTTCAGTG 



GCAGTGGGTC


 


201
TGGAACAGAT TATTCTCTCA CCATTTACAA CCTGGAGCAA 



GAAGATTTTG


 


251
CCACTTACTT TTGCCAACAG GGAGATACGC TTCCGTACAC 



TTTCGGAGGG


 


301
GGGACCAAAC TGGAAATAAA ACGGGCTGAT GCTGCACCAA 



CTGTATCCAT


 


351
CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC 



TCAGTCGTGT


 


401
GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA 



GTGGAAGATT


 


451
GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA 



CTGATCAGGA


 


501
CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG 



TTGACCAAGG


 


551
ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC 



TCACAAGACA


 


601
TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT  



GTTAG






Amino acid sequence of the Ab-8 LC including signal peptide:








(SEQ ID NO: 167)








1
MMSSAQFLGL LLLCFQGTRC DIQMTQTTSS LSASLGDRVS 



ISCRASQDIS


 


51
NYLNWYQQKP DGTFKLLIFY TSRLLSGVPS RFSGSGSGTD 



YSLTIYNLEQ


 


101
EDFATYFCQQ GDTLPYTFGG GTKLEIKRAD AAPTVSIFPP 



SSEQLTSGGA


 


151
SVVCFLNNFY PKDINVKWKI DGSERQNGVL NSWTDQDSKD 



STYSMSSTLT


 


201
LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC






Nucleic acid sequence of the Ab-8 LC including signal peptide encoding sequence:








(SEQ ID NO: 168)








1
ATGATGTCCT CTGCTCAGTT CCTTGGTCTC CTGTTGCTCT 



GTTTTCAAGG


 


51
TACCAGATGT GATATCCAGA TGACACAGAC TACATCCTCC 



CTGTCTGCCT


 


101
CTCTGGGAGA CAGGGTCTCC ATCAGTTGCA GGGCAAGTCA 



AGACATTAGC


 


151
AATTATTTAA ACTGGTATCA GCAGAAACCA GATGGAACTT 



TTAAACTCCT


 


201
TATCTTCTAC ACATCAAGAT TACTCTCAGG AGTCCCATCA 



AGGTTCAGTG


 


251
GCAGTGGGTC TGGAACAGAT TATTCTCTCA CCATTTACAA 



CCTGGAGCAA


 


301
GAAGATTTTG CCACTTACTT TTGCCAACAG GGAGATACGC 



TTCCGTACAC


 


351
TTTCGGAGGG GGGACCAAAC TGGAAATAAA ACGGGCTGAT 



GCTGCACCAA


 


401
CTGTATCCAT CTTCCCACCA TCCAGTGAGC AGTTAACATC 



TGGAGGTGCC


 


451
TCAGTCGTGT GCTTCTTGAA CAACTTCTAC CCCAAAGACA 



TCAATGTCAA


 


501
GTGGAAGATT GATGGCAGTG AACGACAAAA TGGCGTCCTG 



AACAGTTGGA


 


551
CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG 



CACCCTCACG


 


601
TTGACCAAGG ACGAGTATGA ACGACATAAC AGCTATACCT 



GTGAGGCCAC


 


651
TCACAAGACA TCAACTTCAC CCATTGTCAA GAGCTTCAAC 



AGGAATGAGT


 


701
GTTAG






Ab-8 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-8 HC:















(SEQ ID NO: 169)


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-8 HC:








(SEQ ID NO: 170)








1
GAGGTCCAAC TGCAACAGTC TGGACCTGAA CTAATGAAGC 



CTGGGGCTTC


 


51
AGTGAAGATG TCCTGCAAGG CTTCTGGATA TACATTCACT 



GACTACAACA


 


101
TGCACTGGGT GAAGCAGAAC CAAGGAAAGA CCCTAGACTG 



GATAGGAGAA


 


151
ATTAATCCTA ACAGTGGTGG TGCTGGCTAC AACCAGAAGT 



TCAAGGGCAA


 


201
GGCCACATTG ACTGTAGACA AGTCCTCCAC CACAGCCTAC 



ATGGAGCTCC






Amino acid sequence of the Ab-8 HC including signal peptide:








(SEQ ID NO: 171)








1
MGWSWTFLFL LSGTAGVLSE VQLQQSGPEL MKPGASVKMS 



CKASGYTFTD


 


51
YNMHWVKQNQ GKTLDWIGEI NPNSGGAGYN QKFKGKATLT 



VDKSSTTAYM


 


101
ELRSLTSEDS AVYYCARLGY DDIYDDWYFD VWGAGTTVTV 



SSAKTTPPSV


 


151
YPLAPGSAAQ TNSMVTLGCL VKGYFPEPVT VTWNSGSLSS 



GVHTFPAVLQ


 


201
SDLYTLSSSV TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV 



PRDCGCKPCI


 


251
CTVPEVSSVF IFPPKPKDVL TITLTPKVTC VVVDISKDDP 



EVQFSWFVDD


 


301
VEVHTAQTQP REEQFNSTFR SVSELPIMHQ DWLNGKEFKC 



RVNSAAFPAP


 


351
IEKTISKTKG RPKAPQVYTI PPPKEQMAKD KVSLTCMITD 



FFPEDITVEW


 


401
QWNGQPAENY KNTQPIMDTD GSYFIYSKLN VQKSNWEAGN 



TFTCSVLHEG


 


451
LHNHHTEKSL SHSPGK






Nucleic acid sequence of the Ab-8 HC including signal peptide encoding sequence:









(SEQ ID NO: 172)



   1 ATGGGATGGA GCTGGACCTT TCTCTTCCTC CTGTCAGGAA CTGCAGGTGT



 


  51 CCTCTCTGAG GTCCAACTGC AACAGTCTGG ACCTGAACTA ATGAAGCCTG


 


 101 GGGCTTCAGT GAAGATGTCC TGCAAGGCTT CTGGATATAC ATTCACTGAC


 


 151 TACAACATGC ACTGGGTGAA GCAGAACCAA GGAAAGACCC TAGACTGGAT


 


 201 AGGAGAAATT AATCCTAACA GTGGTGGTGC TGGCTACAAC CAGAAGTTCA


 


 251 AGGGCAAGGC CACATTGACT GTAGACAAGT CCTCCACCAC AGCCTACATG


 


 301 GAGCTCCGCA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG


 


 351 ATTGGGCTAC GATGATATCT ACGACGACTG GTACTTCGAT GTCTGGGGCG


 


 401 CAGGGACCAC GGTCACCGTC TCCTCAGCCA AAACGACACC CCCATCTGTC


 


 451 TATCCACTGG CCCCTGGATC TGCTGCCCAA ACTAACTCCA TGGTGACCCT


 


 501 GGGATGCCTG GTCAAGGGCT ATTTCCCTGA GCCAGTGACA GTGACCTGGA


 


 551 ACTCTGGATC CCTGTCCAGC GGTGTGCACA CCTTCCCAGC TGTCCTGCAG


 


 601 TCTGACCTCT ACACTCTGAG CAGCTCAGTG ACTGTCCCCT CCAGCACCTG


 


 651 GCCCAGCGAG ACCGTCACCT GCAACGTTGC CCACCCGGCC AGCAGCACCA


 


 701 AGGTGGACAA GAAAATTGTG CCCAGGGATT GTGGTTGTAA GCCTTGCATA


 


 751 TGTACAGTCC CAGAAGTATC ATCTGTCTTC ATCTTCCCCC CAAAGCCCAA


 


 801 GGATGTGCTC ACCATTACTC TGACTCCTAA GGTCACGTGT GTTGTGGTAG


 


 851 ACATCAGCAA GGATGATCCC GAGGTCCAGT TCAGCTGGTT TGTAGATGAT


 


 901 GTGGAGGTGC ACACAGCTCA GACGCAACCC CGGGAGGAGC AGTTCAACAG


 


 951 CACTTTCCGC TCAGTCAGTG AACTTCCCAT CATGCACCAG GACTGGCTCA


 


1001 ATGGCAAGGA GTTCAAATGC AGGGTCAACA GTGCAGCTTT CCCTGCCCCC


 


1051 ATCGAGAAAA CCATCTCCAA AACCAAAGGC AGACCGAAGG CTCCACAGGT


 


1101 GTACACCATT CCACCTCCCA AGGAGCAGAT GGCCAAGGAT AAAGTCAGTC


 


1151 TGACCTGCAT GATAACAGAC TTCTTCCCTG AAGACATTAC TGTGGAGTGG


 


1201 CAGTGGAATG GGCAGCCAGC GGAGAACTAC AAGAACACTC AGCCCATCAT


 


1251 GGACACAGAT GGCTCTTACT TCATCTACAG CAAGCTCAAT GTGCAGAAGA


 


1301 GCAACTGGGA GGCAGGAAAT ACTTTCACCT GCTCTGTGTT ACATGAGGGC


 


1351 CTGCACAACC ACCATACTGA GAAGAGCCTC TCCCACTCTC CTGGTAAATG


 


1401 A






Ab-9

The sequences of the Antibody 9 (also referred to herein as Ab-9) LC and HC are as follows:

Ab-9 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-9 LC:















(SEQ ID NO: 173)


 










Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-9 LC:









(SEQ ID NO: 174)



  1 GATATCCAGA TGACACAGAT TACATCCTCC CTGTCTGCCT CTCTGGGAGA



 


 51 CAGGGTCTCC ATCAGTTGCA GGGCAAGTCA AGACATTAGC AATTATTTAA


 


101 ATTGGTATCA GCAGAAACCA GATGGAACTT TTAAACTCCT TATCTTCTAC


 


151 ACATCAAGAT TATTTTCAGG AGTCCCATCA AGGTTCAGTG GCAGTGGGTC


 


201 TGGAACAGAT TATTCTCTCA CCATTTACAA CCTGGAGCAA GAAGATTTTG


 


251 CCACTTACTT TTGCCAACAG GGAGATACGC TTCCGTACAC TTTCGGAGGG


 


301 GGGACCAAGG TGGAAATAAA ACGGGCTGAT GCTGCACCAA CTGTATCCAT


 


351 CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC TCAGTCGTGT


 


401 GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA GTGGAAGATT


 


451 GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA CTGATCAGGA


 


501 CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG


 


551 ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC TCACAAGACA


 


601 TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT GT






Amino acid sequence of the Ab-9 LC including signal peptide:









(SEQ ID NO: 175)



  1 MMSSAQFLGL LLLCFQGTRC DIQMTQITSS LSASLGDRVS ISCRASQDIS



 


 51 NYLNWYQQKP DGTFKLLIFY TSRLFSGVPS RFSGSGSGTD YSLTIYNLEQ


 


101 EDFATYFCQQ GDTLPYTFGG GTKVEIKRAD AAPTVSIFPP SSEQLTSGGA


 


151 SVVCFLNNFY PKDINVKWKI DGSERQNGVL NSWTDQDSKD STYSMSSTLT


 


201 LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC






Nucleic acid sequence of the Ab-9 LC including signal peptide encoding sequence:









(SEQ ID NO: 176)



  1 ATGATGTCCT CTGCTCAGTT CCTTGGTCTC CTGTTGCTCT GTTTTCAAGG



 


 51 TACCAGATGT GATATCCAGA TGACACAGAT TACATCCTCC CTGTCTGCCT


 


101 CTCTGGGAGA CAGGGTCTCC ATCAGTTGCA GGGCAAGTCA AGACATTAGC


 


151 AATTATTTAA ATTGGTATCA GCAGAAACCA GATGGAACTT TTAAACTCCT


 


201 TATCTTCTAC ACATCAAGAT TATTTTCAGG AGTCCCATCA AGGTTCAGTG


 


251 GCAGTGGGTC TGGAACAGAT TATTCTCTCA CCATTTACAA CCTGGAGCAA


 


301 GAAGATTTTG CCACTTACTT TTGCCAACAG GGAGATACGC TTCCGTACAC


 


351 TTTCGGAGGG GGGACCAAGG TGGAAATAAA ACGGGCTGAT GCTGCACCAA


 


401 CTGTATCCAT CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC


 


451 TCAGTCGTGT GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA


 


501 GTGGAAGATT GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA


 


551 CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG


 


601 TTGACCAAGG ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC


 


651 TCACAAGACA TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT


 


701 GT






Ab-9 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-9 HC:















(SEQ ID NO: 177)


 










Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-9 HC:









(SEQ ID NO: 178)



   1 GAGGTCCAAC TGCAACAGTC TGGACCTGAA CTAATGAAGC CTGGGACTTC



 


  51 AGTGAAGATG TCCTGCAAGG CTTCTGGATA TACATTCACT GACTACAACA


 


 101 TGCACTGGGT GAAGCAGACC CAAGGAAAGA CCCTAGAGTG GATAGGAGAA


 


 151 ATTAATCCTA ACAGTGGTGG TGCTGGCTAC AACCAGAAGT TCAAGGGCAA


 


 201 GGCCACATTG ACTGTAGACA AGTCCTCCAC CACAGCCTAC ATGGAGCTCC


 


 251 GCAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC AAAATTGGGC


 


 301 TACGATGATA TCTACGACGA CTGGTATTTC GATGTCTGGG GCGCAGGGAC


 


 351 CACGGTCACC GTCTCCTCAG CCAAAACAAC AGCCCCATCG GTCTATCCAC


 


 401 TGGCCCCTGT GTGTGGAGAT ACAACTGGCT CCTCGGTGAC TCTAGGATGC


 


 451 CTGGTCAAGG GTTATTTCCC TGAGCCAGTG ACCTTGACCT GGAACTCTGG


 


 501 ATCCCTGTCC AGTGATGTGC ACACCTTCCC AGCTCTCCTG CAGTCTGGCC


 


 551 TCTACACCCT CAGCAGCTCA GTGACTGTAA CCACCTGGCC CAGCCAGACC


 


 601 ATCACCTGCA ATGTGGCCCA CCCGGCAAGC AGCACCAAAG TGGACAAGAA


 


 651 AATTGAGCCC AGAGGGTCCC CAACACATAA ACCCTGTCCT CCATGCCCAG


 


 701 CTCCTAACCT CTTGGGTGGA CCATCCGTCT TCATCTTCCC TCCAAAGATC


 


 751 AAGGATGTAC TCATGATCTC CCTGAGCCCC ATGGTCACGT GTGTGGTGGT


 


 801 GGATGTGAGC GAGGATGACC CAGATGTCCA TGTCAGCTGG TTCGTGAACA


 


 851 ACGTGGAAGT ACACACAGCT CAGACACAAA CCCATAGAGA GGATTACAAC


 


 901 AGTACTATCC GGGTGGTCAG TGCCCTCCCC ATCCAGCACC AGGACTGGAT


 


 951 GAGTGGCAAG GAGTTCAAAT GCAAGGTCAA CAACAAAGCC CTCCCAGCGC


 


1001 CCATCGAGAG AACCATCTCA AAACCCAAAG GGCCAGTAAG AGCTCCACAG


 


1051 GTATATGTCT TGCCTCCACC AGAAGAAGAG ATGACTAAGA AACAGGTCAC


 


1101 TCTGACCTGC ATGATCACAG ACTTCATGCC TGAAGACATT TACGTGGAGT


 


1151 GGACCAACAA CGGGCAAACA GAGCTAAACT ACAAGAACAC TGAACCAGTC


 


1201 CTGGACTCTG ATGGTTCTTA CTTCATGTAC AGCAAGCTGA GAGTGGAAAA


 


1251 GAAGAACTGG GTGGAAAGAA ATAGCTACTC CTGTTCAGTG GTCCACGAGG


 


1301 GTCTGCACAA TCACCACACG ACTAAGAGCT TCTCCCGGAC TCCGGGTAAA






Amino acid sequence of the Ab-9 HC including signal peptide:









(SEQ ID NO: 179)



  1 MGWSWTFLFL LSGTAGVLSE VQLQQSGPEL MKPGTSVKMS CKASGYTFTD



 


 51 YNMHWVKQTQ GKTLEWIGEI NPNSGGAGYN QKFKGKATLT VDKSSTTAYM


 


101 ELRSLTSEDS AVYYCAKLGY DDIYDDWYFD VWGAGTTVTV SSAKTTAPSV


 


151 YPLAPVCGDT TGSSVTLGCL VKGYFPEPVT LTWNSGSLSS DVHTFPALLQ


 


201 SGLYTLSSSV TVTTWPSQTI TCNVAHPASS TKVDKKIEPR GSPTHKPCPP


 


251 CPAPNLLGGP SVFIFPPKIK DVLMISLSPM VTCVVVDVSE DDPDVHVSWF


 


301 VNNVEVHTAQ TQTHREDYNS TIRVVSALPI QHQDWMSGKE FKCKVNNKAL


 


351 PAPIERTISK PKGPVRAPQV YVLPPPEEEM TKKQVTLTCM ITDFMPEDIY


 


401 VEWTNNGQTE LNYKNTEPVL DSDGSYFMYS KLRVEKKNWV ERNSYSCSVV


 


451 HEGLHNHHTT KSFSRTPGK






Nucleic acid sequence of the Ab-9 HC including signal peptide encoding sequence:









(SEQ ID NO: 180)



   1 ATGGGATGGA GCTGGACCTT TCTCTTCCTC CTGTCAGGAA CTGCAGGTGT



 


  51 CCTCTCTGAG GTCCAACTGC AACAGTCTGG ACCTGAACTA ATGAAGCCTG


 


 101 GGACTTCAGT GAAGATGTCC TGCAAGGCTT CTGGATATAC ATTCACTGAC


 


 151 TACAACATGC ACTGGGTGAA GCAGACCCAA GGAAAGACCC TAGAGTGGAT


 


 201 AGGAGAAATT AATCCTAACA GTGGTGGTGC TGGCTACAAC CAGAAGTTCA


 


 251 AGGGCAAGGC CACATTGACT GTAGACAAGT CCTCCACCAC AGCCTACATG


 


 301 GAGCTCCGCA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAA


 


 351 ATTGGGCTAC GATGATATCT ACGACGACTG GTATTTCGAT GTCTGGGGCG


 


 401 CAGGGACCAC GGTCACCGTC TCCTCAGCCA AAACAACAGC CCCATCGGTC


 


 451 TATCCACTGG CCCCTGTGTG TGGAGATACA ACTGGCTCCT CGGTGACTCT


 


 501 AGGATGCCTG GTCAAGGGTT ATTTCCCTGA GCCAGTGACC TTGACCTGGA


 


 551 ACTCTGGATC CCTGTCCAGT GATGTGCACA CCTTCCCAGC TCTCCTGCAG


 


 601 TCTGGCCTCT ACACCCTCAG CAGCTCAGTG ACTGTAACCA CCTGGCCCAG


 


 651 CCAGACCATC ACCTGCAATG TGGCCCACCC GGCAAGCAGC ACCAAAGTGG


 


 701 ACAAGAAAAT TGAGCCCAGA GGGTCCCCAA CACATAAACC CTGTCCTCCA


 


 751 TGCCCAGCTC CTAACCTCTT GGGTGGACCA TCCGTCTTCA TCTTCCCTCC


 


 801 AAAGATCAAG GATGTACTCA TGATCTCCCT GAGCCCCATG GTCACGTGTG


 


 851 TGGTGGTGGA TGTGAGCGAG GATGACCCAG ATGTCCATGT CAGCTGGTTC


 


 901 GTGAACAACG TGGAAGTACA CACAGCTCAG ACACAAACCC ATAGAGAGGA


 


 951 TTACAACAGT ACTATCCGGG TGGTCAGTGC CCTCCCCATC CAGCACCAGG


 


1001 ACTGGATGAG TGGCAAGGAG TTCAAATGCA AGGTCAACAA CAAAGCCCTC


 


1051 CCAGCGCCCA TCGAGAGAAC CATCTCAAAA CCCAAAGGGC CAGTAAGAGC


 


1101 TCCACAGGTA TATGTCTTGC CTCCACCAGA AGAAGAGATG ACTAAGAAAC


 


1151 AGGTCACTCT GACCTGCATG ATCACAGACT TCATGCCTGA AGACATTTAC


 


1201 GTGGAGTGGA CCAACAACGG GCAAACAGAG CTAAACTACA AGAACACTGA


 


1251 ACCAGTCCTG GACTCTGATG GTTCTTACTT CATGTACAGC AAGCTGAGAG


 


1301 TGGAAAAGAA GAACTGGGTG GAAAGAAATA GCTACTCCTG TTCAGTGGTC


 


1351 CACGAGGGTC TGCACAATCA CCACACGACT AAGAGCTTCT CCCGGACTCC


 


1401 GGGTAAA






Ab-10

The sequences of the Antibody 10 (also referred to herein as Ab-10) LC and HC are as follows:

Ab-10 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-10 LC:















(SEQ ID NO: 181)


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-10 LC:









(SEQ ID NO: 182)



  1 GATATCCAGA TGACACAGAC TACATCCTCC CTGTCTGCCT CTCTGGGAGA



 


 51 CAGGGTCTCC ATCAGTTGCA GGGCAAGTCA AGACATTAGC AATTATTTAA


 


101 ACTGGTATCA GCAGAAACCA GATGGAACTT TTAAACTCCT TATCTTCTAC


 


151 ACATCAAGAT TACTCTCAGG AGTCCCATCA AGGTTCAGTG GCAGTGGGTC


 


201 TGGAACAGAT TATTCTCTCA CCATTTACAA CCTGGAGCAA GAAGATTTTG


 


251 CCACTTACTT TTGCCAACAG GGAGATACGC TTCCGTACAC TTTCGGAGGG


 


301 GGGACCAAAC TGGAAATAAA ACGGGCTGAT GCTGCACCAA CTGTATCCAT


 


351 CTTCCCACTA TCCAGTGAGC AGTTAACATC TGGAGGTGCC TCAGTCGTGT


 


401 GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA GTGGAAGATT


 


451 GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA CTGATCAGGA


 


501 CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG


 


551 ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC TCACAAGACA


 


601 TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT GTTAG






Amino acid sequence of the Ab-10 LC including signal peptide:









(SEQ ID NO: 183)



  1 MMSSAQFLGL LLLCFQGTRC DIQMTQTTSS LSASLGDRVS ISCRASQDIS



 


 51 NYLNWYQQKP DGTFKLLIFY TSRLLSGVPS RFSGSGSGTD YSLTIYNLEQ


 


101 EDFATYFCQQ GDTLPYTFGG GTKLEIKRAD AAPTVSIFPL SSEQLTSGGA


 


151 SVVCFLNNFY PKDINVKWKI DGSERQNGVL NSWTDQDSKD STYSMSSTLT


 


201 LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC 






Nucleic acid sequence of the Ab-10 LC including signal peptide encoding sequence:









(SEQ ID NO: 184)



  1 ATGATGTCCT CTGCTCAGTT CCTTGGTCTC CTGTTGCTCT GTTTTCAAGG



 


 51 TACCAGATGT GATATCCAGA TGACACAGAC TACATCCTCC CTGTCTGCCT


 


101 CTCTGGGAGA CAGGGTCTCC ATCAGTTGCA GGGCAAGTCA AGACATTAGC


 


151 AATTATTTAA ACTGGTATCA GCAGAAACCA GATGGAACTT TTAAACTCCT


 


201 TATCTTCTAC ACATCAAGAT TACTCTCAGG AGTCCCATCA AGGTTCAGTG


 


251 GCAGTGGGTC TGGAACAGAT TATTCTCTCA CCATTTACAA CCTGGAGCAA


 


301 GAAGATTTTG CCACTTACTT TTGCCAACAG GGAGATACGC TTCCGTACAC


 


351 TTTCGGAGGG GGGACCAAAC TGGAAATAAA ACGGGCTGAT GCTGCACCAA


 


401 CTGTATCCAT CTTCCCACTA TCCAGTGAGC AGTTAACATC TGGAGGTGCC


 


451 TCAGTCGTGT GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA


 


501 GTGGAAGATT GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA


 


551 CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG


 


601 TTGACCAAGG ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC


 


651 TCACAAGACA TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT


 


701 GTTAG






Ab-10 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-10 HC:















(SEQ ID NO: 185)


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-10 HC:









(SEQ ID NO: 186)



   1 GAGGTCCAAC TGCAACAGTC TGGACCTGAA CTAATGAAGC CTGGGGCTTC



 


  51 AGTGAAGATG TCCTGCAAGG CTTCTGGATA TACATTCACT GACTACAACA


 


 101 TGCACTGGGT GAAGCAGAAC CAAGGAAAGA CCCTAGAATG GATAGGAGAA


 


 151 ATTAATCCTA ACAGTGGTGG TGCTGGCTAC AACCAGAAGT TCAAGGGCAA


 


 201 GGCCACATTG ACTGTAGACA AGTCCTCCAC CACAGCCTAC ATGGAGCTCC


 


 251 GCAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC AAGATTGGGC


 


 301 TACGATGATA TCTACGACGA CTGGTACTTC GATGTCTGGG GCGCAGGGAC


 


 351 CACGGTCACC GTCTCCTCAG CCAAAACGAC ACCCCCATCT GTCTATCCAC


 


 401 TGGCCCCTGG ATCTGCTGCC CAAACTAACT CCATGGTGAC CCTGGGATGC


 


 451 CTGGTCAAGG GCTATTTCCC TGAGCCAGTG ACAGTGACCT GGAACTCTGG


 


 501 ATCCCTGTCC AGCGGTGTGC ACACCTTCCC AGCTGTCCTG CAGTCTGACC


 


 551 TCTACACTCT GAGCAGCTCA GTGACTGTCC CCTCCAGCAC CTGGCCCAGC


 


 601 GAGACCGTCA CCTGCAACGT TGCCCACCCG GCCAGCAGCA CCAAGGTGGA


 


 651 CAAGAAAATT GTGCCCAGGG ATTGTGGTTG TAAGCCTTGC ATATGTACAG


 


 701 TCCCAGAAGT ATCATCTGTC TTCATCTTCC CCCCAAAGCC CAAGGATGTG


 


 751 CTCACCATTA CTCTGACTCC TAAGGTCACG TGTGTTGTGG TAGACATCAG


 


 801 CAAGGATGAT CCCGAGGTCC AGTTCAGCTG GTTTGTAGAT GATGTGGAGG


 


 851 TGCACACAGC TCAGACGCAA CCCCGGGAGG AGCAGTTCAA CAGCACTTTC


 


 901 CGCTCAGTCA GTGAACTTCC CATCATGCAC CAGGACTGGC TCAATGGCAA


 


 951 GGAGTTCAAA TGCAGGGTCA ACAGTGCAGC TTTCCCTGCC CCCATCGAGA


 


1001 AAACCATCTC CAAAACCAAA GGCAGACCGA AGGCTCCACA GGTGTACACC


 


1051 ATTCCACCTC CCAAGGAGCA GATGGCCAAG GATAAAGTCA GTCTGACCTG


 


1101 CATGATAACA GACTTCTTCC CTGAAGACAT TACTGTGGAG TGGCAGTGGA


 


1151 ATGGGCAGCC AGCGGAGAAC TACAAGAACA CTCAGCCCAT CATGGACACA


 


1201 GATGGCTCTT ACTTCATCTA CAGCAAGCTC AATGTGCAGA AGAGCAACTG


 


1251 GGAGGCAGGA AATACTTTCA CCTGCTCTGT GTTACATGAG GGCCTGCACA


 


1301 ACCACCATAC TGAGAAGAGC CTCTCCCACT CTCCTGGTAA ATGA






Amino acid sequence of the Ab-10 HC including signal peptide:









(SEQ ID NO: 187)



  1 MGWSWTFLFL LSGTAGVLSE VQLQQSGPEL MKPGASVKMS CKASGYTFTD



 


 51 YNMHWVKQNQ GKTLEWIGEI NPNSGGAGYN QKFKGKATLT VDKSSTTAYM


 


101 ELRSLTSEDS AVYYCARLGY DDIYDDWYFD VWGAGTTVTV SSAKTTPPSV


 


151 YPLAPGSAAQ TNSMVTLGCL VKGYFPEPVT VTWNSGSLSS GVHTFPAVLQ


 


201 SDLYTLSSSV TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV PRDCGCKPCI


 


251 CTVPEVSSVF IFPPKPKDVL TITLTPKVTC VVVDISKDDP EVQFSWFVDD


 


301 VEVHTAQTQP REEQFNSTFR SVSELPIMHQ DWLNGKEFKC RVNSAAFPAP


 


351 IEKTISKTKG RPKAPQVYTI PPPKEQMAKD KVSLTCMITD FFPEDITVEW


 


401 QWNGQPAENY KNTQPIMDTD GSYFIYSKLN VQKSNWEAGN TFTCSVLHEG


 


451 LHNHHTEKSL SHSPGK






Nucleic acid sequence of the Ab-10 HC including signal peptide encoding sequence:









(SEQ ID NO: 188)



   1 ATGGGATGGA GCTGGACCTT TCTCTTCCTC CTGTCAGGAA CTGCAGGTGT



 


  51 CCTCTCTGAG GTCCAACTGC AACAGTCTGG ACCTGAACTA ATGAAGCCTG


 


 101 GGGCTTCAGT GAAGATGTCC TGCAAGGCTT CTGGATATAC ATTCACTGAC


 


 151 TACAACATGC ACTGGGTGAA GCAGAACCAA GGAAAGACCC TAGAATGGAT


 


 201 AGGAGAAATT AATCCTAACA GTGGTGGTGC TGGCTACAAC CAGAAGTTCA


 


 251 AGGGCAAGGC CACATTGACT GTAGACAAGT CCTCCACCAC AGCCTACATG


 


 301 GAGCTCCGCA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG


 


 351 ATTGGGCTAC GATGATATCT ACGACGACTG GTACTTCGAT GTCTGGGGCG


 


 401 CAGGGACCAC GGTCACCGTC TCCTCAGCCA AAACGACACC CCCATCTGTC


 


 451 TATCCACTGG CCCCTGGATC TGCTGCCCAA ACTAACTCCA TGGTGACCCT


 


 501 GGGATGCCTG GTCAAGGGCT ATTTCCCTGA GCCAGTGACA GTGACCTGGA


 


 551 ACTCTGGATC CCTGTCCAGC GGTGTGCACA CCTTCCCAGC TGTCCTGCAG


 


 601 TCTGACCTCT ACACTCTGAG CAGCTCAGTG ACTGTCCCCT CCAGCACCTG


 


 651 GCCCAGCGAG ACCGTCACCT GCAACGTTGC CCACCCGGCC AGCAGCACCA


 


 701 AGGTGGACAA GAAAATTGTG CCCAGGGATT GTGGTTGTAA GCCTTGCATA


 


 751 TGTACAGTCC CAGAAGTATC ATCTGTCTTC ATCTTCCCCC CAAAGCCCAA


 


 801 GGATGTGCTC ACCATTACTC TGACTCCTAA GGTCACGTGT GTTGTGGTAG


 


 851 ACATCAGCAA GGATGATCCC GAGGTCCAGT TCAGCTGGTT TGTAGATGAT


 


 901 GTGGAGGTGC ACACAGCTCA GACGCAACCC CGGGAGGAGC AGTTCAACAG


 


 951 CACTTTCCGC TCAGTCAGTG AACTTCCCAT CATGCACCAG GACTGGCTCA


 


1001 ATGGCAAGGA GTTCAAATGC AGGGTCAACA GTGCAGCTTT CCCTGCCCCC


 


1051 ATCGAGAAAA CCATCTCCAA AACCAAAGGC AGACCGAAGG CTCCACAGGT


 


1101 GTACACCATT CCACCTCCCA AGGAGCAGAT GGCCAAGGAT AAAGTCAGTC


 


1151 TGACCTGCAT GATAACAGAC TTCTTCCCTG AAGACATTAC TGTGGAGTGG


 


1201 CAGTGGAATG GGCAGCCAGC GGAGAACTAC AAGAACACTC AGCCCATCAT


 


1251 GGACACAGAT GGCTCTTACT TCATCTACAG CAAGCTCAAT GTGCAGAAGA


 


1301 GCAACTGGGA GGCAGGAAAT ACTTTCACCT GCTCTGTGTT ACATGAGGGC


 


1351 CTGCACAACC ACCATACTGA GAAGAGCCTC TCCCACTCTC CTGGTAAATG


 


1401 A 






Ab-11

The sequences of the Antibody 11 (also referred to herein as Ab-11) LC and HC are as follows:

Ab-11 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-11 LC:















(SEQ ID NO: 189)


 


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-11 LC:









(SEQ ID NO: 190)



  1 CAAATTGTTC TCTCCCAGTC TCCAGCATTC CTGTCTGTAT CTCCAGGGGA



 


 51 TAAGGTCACA ATGACTTGCA GGGCCAGCTC AAGTATAAGT TACATACACT


 


101 GGTTTCAGCA GAAGCCAGGA TCCTCCCCCA GATCCTGGAT TTATGCCACA


 


151 TCCAACCTGG CTTCTGGAGT CCCTGGTCGC TTCAGTGGCA GTGGGTCTGG


 


201 GACCTCTTAC TCTCTCACAA TCAGCAGAGT GGAGGCTGAG GATGCTGCCA


 


251 CTTATTACTG CCAGCAGTGG AGTAGTGACC CACTCACGTT CGGTGCTGGG


 


301 ACCAAGCTGG AGCTGAAACG GGCTGATGCT GCACCAACTG TATCCATCTT


 


351 CCCACCATCC AGTGAGCAGT TAACATCTGG AGGTGCCTCA GTCGTGTGCT


 


401 TCTTGAACAA CTTCTACCCC AAAGACATCA ATGTCAAGTG GAAGATTGAT


 


451 GGCAGTGAAC GACAAAATGG CGTCCTGAAC AGTTGGACTG ATCAGGACAG


 


501 CAAAGACAGC ACCTACAGCA TGAGCAGCAC CCTCACGTTG ACCAAGGACG


 


551 AGTATGAACG ACATAACAGC TATACCTGTG AGGCCACTCA CAAGACATCA


 


601 ACTTCACCCA TTGTCAAGAG CTTCAACAGG AATGAGTGTT AG






Amino acid sequence of the Ab-11 LC including signal peptide:









(SEQ ID NO: 191)



  1 MDFQVQIFSF LLISASVIMS RGQIVLSQSP AFLSVSPGDK VTMTCRASSS



 


 51 ISYIHWFQQK PGSSPRSWIY ATSNLASGVP GRFSGSGSGT SYSLTISRVE


 


101 AEDAATYYCQ QWSSDPLTFG AGTKLELKRA DAAPTVSIFP PSSEQLTSGG


 


151 ASVVCFLNNF YPKDINVKWK IDGSERQNGV LNSWTDQDSK DSTYSMSSTL


 


201 TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC






Nucleic acid sequence of the Ab-11 LC including signal peptide encoding sequence:









(SEQ ID NO: 192)










1
ATGGATTTTC AAGTGCAGAT TTTCAGCTTC CTGCTAATCA GTGCTTCAGT



 


51
CATAATGTCC AGAGGACAAA TTGTTCTCTC CCAGTCTCCA GCATTCCTGT


 


101
CTGTATCTCC AGGGGATAAG GTCACAATGA CTTGCAGGGC CAGCTCAAGT


 


151
ATAAGTTACA TACACTGGTT TCAGCAGAAG CCAGGATCCT CCCCCAGATC


 


201
CTGGATTTAT GCCACATCCA ACCTGGCTTC TGGAGTCCCT GGTCGCTTCA


 


251
GTGGCAGTGG GTCTGGGACC TCTTACTCTC TCACAATCAG CAGAGTGGAG


 


301
GCTGAGGATG CTGCCACTTA TTACTGCCAG CAGTGGAGTA GTGACCCACT


 


351
CACGTTCGGT GCTGGGACCA AGCTGGAGCT GAAACGGGCT GATGCTGCAC


 


401
CAACTGTATC CATCTTCCCA CCATCCAGTG AGCAGTTAAC ATCTGGAGGT


 


451
GCCTCAGTCG TGTGCTTCTT GAACAACTTC TACCCCAAAG ACATCAATGT


 


501
CAAGTGGAAG ATTGATGGCA GTGAACGACA AAATGGCGTC CTGAACAGTT


 


551
GGACTGATCA GGACAGCAAA GACAGCACCT ACAGCATGAG CAGCACCCTC


 


601
ACGTTGACCA AGGACGAGTA TGAACGACAT AACAGCTATA CCTGTGAGGC


 


651
CACTCACAAG ACATCAACTT CACCCATTGT CAAGAGCTTC AACAGGAATG


 


701
AGTGTTAG






Ab-11 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-11 HC:















(SEQ ID NO: 193)


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-11 HC:









(SEQ ID NO: 194)










1
GAAGTTCAGC TGCAACAGTC TGGGGCAGAC CTTGTGCAGC CAGGGGCCTC



 


51
AGTCAAGGTG TCCTGCACAG CTTCTGGCTT CGACATTAAG GACTACTATA


 


101
TACACTGGAT GAAACAGAGG CCTGACCAGG GCCTGGAGTG GATTGGAAGG


 


151
GTTGATCCTG ACAATGGTGA GACTGAATTT GCCCCGAAGT TCCCGGGCAA


 


201
GGCCACTTTT ACAACAGACA CATCCTCCAA CACAGCCTAC CTACAACTCA


 


251
GAGGCCTGAC ATCTGAGGAC ACTGCCATCT ATTACTGTGG GAGAGAAGAC


 


301
TACGATGGTA CCTACACCTG GTTTCCTTAT TGGGGCCAAG GGACTCTGGT


 


351
CACTGTCTCT GCAGCCAAAA CGACACCCCC ATCTGTCTAT CCACTGGCCC


 


401
CTGGATCTGC TGCCCAAACT AACTCCATGG TGACCCTGGG ATGCCTGGTC


 


451
AAGGGCTATT TCCCTGAGCC AGTGACAGTG ACCTGGAACT CTGGATCCCT


 


501
GTCCAGCGGT GTGCACACCT TCCCAGCTGT CCTGCAGTCT GACCTCTACA


 


551
CTCTGAGCAG CTCAGTGACT GTCCCCTCCA GCACCTGGCC CAGCGAGACC


 


601
GTCACCTGCA ACGTTGCCCA CCCGGCCAGC AGCACCAAGG TGGACAAGAA


 


651
AATTGTGCCC AGGGATTGTG GTTGTAAGCC TTGCATATGT ACAGTCCCAG


 


701
AAGTATCATC TGTCTTCATC TTCCCCCCAA AGCCCAAGGA TGTGCTCACC


 


751
ATTACTCTGA CTCCTAAGGT CACGTGTGTT GTGGTAGACA TCAGCAAGGA


 


801
TGATCCCGAG GTCCAGTTCA GCTGGTTTGT AGATGATGTG GAGGTGCACA


 


851
CAGCTCAGAC GCAACCCCGG GAGGAGCAGT TCAACAGCAC TTTCCGCTCA


 


901
GTCAGTGAAC TTCCCATCAT GCACCAGGAC TGGCTCAATG GCAAGGAGTT


 


951
CAAATGCAGG GTCAACAGTG CAGCTTTCCC TGCCCCCATC GAGAAAACCA


 


1001
TCTCCAAAAC CAAAGGCAGA CCGAAGGCTC CACAGGTGTA CACCATTCCA


 


1051
CCTCCCAAGG AGCAGATGGC CAAGGATAAA GTCAGTCTGA CCTGCATGAT


 


1101
AACAGACTTC TTCCCTGAAG ACATTACTGT GGAGTGGCAG TGGAATGGGC


 


1151
AGCCAGCGGA GAACTACAAG AACACTCAGC CCATCATGGA CACAGATGGC


 


1201
TCTTACTTCA TCTACAGCAA GCTCAATGTG CAGAAGAGCA ACTGGGAGGC


 


1251
AGGAAATACT TTCACCTGCT CTGTGTTACA TGAGGGCCTG CACAACCACC


 


1301
ATACTGAGAA GAGCCTCTCC CACTCTCCTG GTAAATGA






Amino acid sequence of the Ab-11 HC including signal peptide:









(SEQ ID NO: 195)










1
MKCSWVIFFL MAVVTGVNSE VQLQQSGADL VQPGASVKVS CTASGFDIKD



 


51
YYIHWMKQRP DQGLEWIGRV DPDNGETEFA PKFPGKATFT TDTSSNTAYL


 


101
QLRGLTSEDT AIYYCGREDY DGTYTWFPYW GQGTLVTVSA AKTTPPSVYP


 


151
LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD


 


201
LYTLSSSVTV PSSTWPSETV TCNVAHPASS TKVDKKIVPR DCGCKPCICT


 


251
VPEVSSVFIF PPKPKDVLTI TLTPKVTCVV VDISKDDPEV QFSWFVDDVE


 


301
VHTAQTQPRE EQFNSTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE


 


351
KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITDFF PEDITVEWQW


 


401
NGQPAENYKN TQPIMDTDGS YFIYSKLNVQ KSNWEAGNTF TCSVLHEGLH


 


451
NHHTEKSLSH SPGK






Nucleic acid sequence of the Ab-11 HC including signal peptide encoding sequence:









(SEQ ID NO: 196)










1
ATGAAATGCA GCTGGGTCAT CTTCTTCCTG ATGGCAGTGG TTACAGGGGT



 


51
CAATTCAGAA GTTCAGCTGC AACAGTCTGG GGCAGACCTT GTGCAGCCAG


 


101
GGGCCTCAGT CAAGGTGTCC TGCACAGCTT CTGGCTTCGA CATTAAGGAC


 


151
TACTATATAC ACTGGATGAA ACAGAGGCCT GACCAGGGCC TGGAGTGGAT


 


201
TGGAAGGGTT GATCCTGACA ATGGTGAGAC TGAATTTGCC CCGAAGTTCC


 


251
CGGGCAAGGC CACTTTTACA ACAGACACAT CCTCCAACAC AGCCTACCTA


 


301
CAACTCAGAG GCCTGACATC TGAGGACACT GCCATCTATT ACTGTGGGAG


 


351
AGAAGACTAC GATGGTACCT ACACCTGGTT TCCTTATTGG GGCCAAGGGA


 


401
CTCTGGTCAC TGTCTCTGCA GCCAAAACGA CACCCCCATC TGTCTATCCA


 


451
CTGGCCCCTG GATCTGCTGC CCAAACTAAC TCCATGGTGA CCCTGGGATG


 


501
CCTGGTCAAG GGCTATTTCC CTGAGCCAGT GACAGTGACC TGGAACTCTG


 


551
GATCCCTGTC CAGCGGTGTG CACACCTTCC CAGCTGTCCT GCAGTCTGAC


 


601
CTCTACACTC TGAGCAGCTC AGTGACTGTC CCCTCCAGCA CCTGGCCCAG


 


651
CGAGACCGTC ACCTGCAACG TTGCCCACCC GGCCAGCAGC ACCAAGGTGG


 


701
ACAAGAAAAT TGTGCCCAGG GATTGTGGTT GTAAGCCTTG CATATGTACA


 


751
GTCCCAGAAG TATCATCTGT CTTCATCTTC CCCCCAAAGC CCAAGGATGT


 


801
GCTCACCATT ACTCTGACTC CTAAGGTCAC GTGTGTTGTG GTAGACATCA


 


851
GCAAGGATGA TCCCGAGGTC CAGTTCAGCT GGTTTGTAGA TGATGTGGAG


 


901
GTGCACACAG CTCAGACGCA ACCCCGGGAG GAGCAGTTCA ACAGCACTTT


 


951
CCGCTCAGTC AGTGAACTTC CCATCATGCA CCAGGACTGG CTCAATGGCA


 


1001
AGGAGTTCAA ATGCAGGGTC AACAGTGCAG CTTTCCCTGC CCCCATCGAG


 


1051
AAAACCATCT CCAAAACCAA AGGCAGACCG AAGGCTCCAC AGGTGTACAC


 


1101
CATTCCACCT CCCAAGGAGC AGATGGCCAA GGATAAAGTC AGTCTGACCT


 


1151
GCATGATAAC AGACTTCTTC CCTGAAGACA TTACTGTGGA GTGGCAGTGG


 


1201
AATGGGCAGC CAGCGGAGAA CTACAAGAAC ACTCAGCCCA TCATGGACAC


 


1251
AGATGGCTCT TACTTCATCT ACAGCAAGCT CAATGTGCAG AAGAGCAACT


 


1301
GGGAGGCAGG AAATACTTTC ACCTGCTCTG TGTTACATGA GGGCCTGCAC


 


1351
AACCACCATA CTGAGAAGAG CCTCTCCCAC TCTCCTGGTA AATGA






Ab-12

The sequences of the Antibody 12 (also referred to herein as Ab-12) LC and HC are as follows:

Ab-12 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-12 LC:















(SEQ ID NO: 197)


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-12 LC:









(SEQ ID NO: 198)










1
GATCTCCAGA TGACACAGAC TACTTCCTCC CTGTCTGCCT CTCTGGGAGA



 


51
CAGAGTCACC ATCAGTTGCA GGGCAAGTCA GGACATTAGC AATTATTTAA


 


101
ACTGGTATCA GCAGAAACCA GATGGAACTG TTAAGCTCCT GATCTTCTAC


 


151
ACATCAACAT TACAGTCAGG AGTCCCATCG AGGTTCAGTG GCAGTGGGTC


 


201
TGGAACAAAT TATTCTCTCA CCATTACCAA CCTGGAGCAA GATGATGCTG


 


251
CCACTTACTT TTGCCAACAG GGTGATACGC TTCCGTACAC GTTCGGAGGG


 


301
GGGACCAAGC TGGAAATAAA ACGGGCTGAT GCTGCACCAA CTGTATCCAT


 


351
CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC TCAGTCGTGT


 


401
GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA GTGGAAGATT


 


451
GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA CTGATCAGGA


 


501
CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG


 


551
ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC TCACAAGACA


 


601
TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT GTTAG






Amino acid sequence of the Ab-12 LC including signal peptide:









(SEQ ID NO: 199)










1
MMSSAQFLGL LLLCFQGSRC DLQMTQTTSS LSASLGDRVT ISCRASQDIS



 


51
NYLNWYQQKP DGTVKLLIFY TSTLQSGVPS RFSGSGSGTN YSLTITNLEQ


 


101
DDAATYFCQQ GDTLPYTFGG GTKLEIKRAD AAPTVSIFPP SSEQLTSGGA


 


151
SVVCFLNNFY PKDINVKWKI DGSERQNGVL NSWTDQDSKD STYSMSSTLT


 


201
LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC






Nucleic acid sequence of the Ab-12 LC including signal peptide encoding sequence:









(SEQ ID NO: 200)










1
ATGATGTCCT CTGCTCAGTT CCTTGGTCTC CTGTTGCTCT GTTTTCAAGG



 


51
TTCCAGATGT GATCTCCAGA TGACACAGAC TACTTCCTCC CTGTCTGCCT


 


101
CTCTGGGAGA CAGAGTCACC ATCAGTTGCA GGGCAAGTCA GGACATTAGC


 


151
AATTATTTAA ACTGGTATCA GCAGAAACCA GATGGAACTG TTAAGCTCCT


 


201
GATCTTCTAC ACATCAACAT TACAGTCAGG AGTCCCATCG AGGTTCAGTG


 


251
GCAGTGGGTC TGGAACAAAT TATTCTCTCA CCATTACCAA CCTGGAGCAA


 


301
GATGATGCTG CCACTTACTT TTGCCAACAG GGTGATACGC TTCCGTACAC


 


351
GTTCGGAGGG GGGACCAAGC TGGAAATAAA ACGGGCTGAT GCTGCACCAA


 


401
CTGTATCCAT CTTCCCACCA TCCAGTGAGC AGTTAACATC TGGAGGTGCC


 


451
TCAGTCGTGT GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA


 


501
GTGGAAGATT GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA


 


551
CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG


 


601
TTGACCAAGG ACGAGTATGA ACGACATAAC AGCTATACCT GTGAGGCCAC


 


651
TCACAAGACA TCAACTTCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT


 


701
GTTAG






Ab-12 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-12 HC:















(SEQ ID NO: 201)


 


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-12 HC:









(SEQ ID NO: 202)










1
GAGGTCCAGT TGCAACAGTC TGGACCTGAA CTAATGAAGC CTGGGGCTTC



 


51
AGTGAAGATG TCCTGCAAGG CTTCTGGATA CACATTCACT GACTACAACA


 


101
TGCACTGGAT GAAGCAGAAC CAAGGAAAGA GCCTAGAGTG GATAGGAGAG


 


151
ATTAATCCTA ACAGTGGTGG TTCTGGTTAC AACCAGAAGT TCAAAGGCAA


 


201
GGCCACATTG ACTGTAGACA AGTCCTCCAG CACAGCCTAC ATGGAGCTCC


 


251
GCAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC AAGATTGGGC


 


301
TACTATGGTA ACTACGAGGA CTGGTATTTC GATGTCTGGG GCGCAGGGAC


 


351
CACGGTCACC GTCTCCTCTG CCAAAACGAC ACCCCCATCT GTCTATCCAC


 


401
TGGCCCCTGG ATCTGCTGCC CAAACTAACT CCATGGTGAC CCTGGGATGC


 


451
CTGGTCAAGG GCTATTTCCC TGAGCCAGTG ACAGTGACCT GGAACTCTGG


 


501
ATCCCTGTCC AGCGGTGTGC ACACCTTCCC AGCTGTCCTG CAGTCTGACC


 


551
TCTACACTCT GAGCAGCTCA GTGACTGTCC CCTCCAGCAC CTGGCCCAGC


 


601
GAGACCGTCA CCTGCAACGT TGCCCACCCG GCCAGCAGCA CCAAGGTGGA


 


651
CAAGAAAATT GTGCCCAGGG ATTGTGGTTG TAAGCCTTGC ATATGTACAG


 


701
TCCCAGAAGT ATCATCTGTC TTCATCTTCC CCCCAAAGCC CAAGGATGTG


 


751
CTCACCATTA CTCTGACTCC TAAGGTCACG TGTGTTGTGG TAGACATCAG


 


801
CAAGGATGAT CCCGAGGTCC AGTTCAGCTG GTTTGTAGAT GATGTGGAGG


 


851
TGCACACAGC TCAGACGCAA CCCCGGGAGG AGCAGTTCAA CAGCACTTTC


 


901
CGCTCAGTCA GTGAACTTCC CATCATGCAC CAGGACTGGC TCAATGGCAA


 


951
GGAGTTCAAA TGCAGGGTCA ACAGTGCAGC TTTCCCTGCC CCCATCGAGA


 


1001
AAACCATCTC CAAAACCAAA GGCAGACCGA AGGCTCCACA GGTGTACACC


 


1051
ATTCCACCTC CCAAGGAGCA GATGGCCAAG GATAAAGTCA GTCTGACCTG


 


1101
CATGATAACA GACTTCTTCC CTGAAGACAT TACTGTGGAG TGGCAGTGGA


 


1151
ATGGGCAGCC AGCGGAGAAC TACAAGAACA CTCAGCCCAT CATGGACACA


 


1201
GATGGCTCTT ACTTCATCTA CAGCAAGCTC AATGTGCAGA AGAGCAACTG


 


1251
GGAGGCAGGA AATACTTTCA CCTGCTCTGT GTTACATGAG GGCCTGCACA


 


1301
ACCACCATAC TGAGAAGAGC CTCTCCCACT CTCCTGGTAA ATGA






Amino acid sequence of the Ab-12 HC including signal peptide:









(SEQ ID NO: 203)










1
MGWSWTFLFL LSGTSGVLSE VQLQQSGPEL MKPGASVKMS CKASGYTFTD



 


51
YNMHWMKQNQ GKSLEWIGEI NPNSGGSGYN QKFKGKATLT VDKSSSTAYM


 


101
ELRSLTSEDS AVYYCARLGY YGNYEDWYFD VWGAGTTVTV SSAKTTPPSV


 


151
YPLAPGSAAQ TNSMVTLGCL VKGYFPEPVT VTWNSGSLSS GVHTFPAVLQ


 


201
SDLYTLSSSV TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV PRDCGCKPCI


 


251
CTVPEVSSVF IFPPKPKDVL TITLTPKVTC VVVDISKDDP EVQFSWFVDD


 


301
VEVHTAQTQP REEQFNSTFR SVSELPIMHQ DWLNGKEFKC RVNSAAFPAP


 


351
IEKTISKTKG RPKAPQVYTI PPPKEQMAKD KVSLTCMITD FFPEDITVEW


 


401
QWNGQPAENY KNTQPIMDTD GSYFIYSKLN VQKSNWEAGN TFTCSVLHEG


 


451
LHNHHTEKSL SHSPGK






Nucleic acid sequence of the Ab-12 HC including signal peptide encoding sequence:









(SEQ ID NO: 204)










1
ATGGGATGGA GCTGGACCTT TCTCTTCCTC CTGTCAGGAA CTTCGGGTGT



 


51
CCTCTCTGAG GTCCAGTTGC AACAGTCTGG ACCTGAACTA ATGAAGCCTG


 


101
GGGCTTCAGT GAAGATGTCC TGCAAGGCTT CTGGATACAC ATTCACTGAC


 


151
TACAACATGC ACTGGATGAA GCAGAACCAA GGAAAGAGCC TAGAGTGGAT


 


201
AGGAGAGATT AATCCTAACA GTGGTGGTTC TGGTTACAAC CAGAAGTTCA


 


251
AAGGCAAGGC CACATTGACT GTAGACAAGT CCTCCAGCAC AGCCTACATG


 


301
GAGCTCCGCA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG


 


351
ATTGGGCTAC TATGGTAACT ACGAGGACTG GTATTTCGAT GTCTGGGGCG


 


401
CAGGGACCAC GGTCACCGTC TCCTCTGCCA AAACGACACC CCCATCTGTC


 


451
TATCCACTGG CCCCTGGATC TGCTGCCCAA ACTAACTCCA TGGTGACCCT


 


501
GGGATGCCTG GTCAAGGGCT ATTTCCCTGA GCCAGTGACA GTGACCTGGA


 


551
ACTCTGGATC CCTGTCCAGC GGTGTGCACA CCTTCCCAGC TGTCCTGCAG


 


601
TCTGACCTCT ACACTCTGAG CAGCTCAGTG ACTGTCCCCT CCAGCACCTG


 


651
GCCCAGCGAG ACCGTCACCT GCAACGTTGC CCACCCGGCC AGCAGCACCA


 


701
AGGTGGACAA GAAAATTGTG CCCAGGGATT GTGGTTGTAA GCCTTGCATA


 


751
TGTACAGTCC CAGAAGTATC ATCTGTCTTC ATCTTCCCCC CAAAGCCCAA


 


801
GGATGTGCTC ACCATTACTC TGACTCCTAA GGTCACGTGT GTTGTGGTAG


 


851
ACATCAGCAA GGATGATCCC GAGGTCCAGT TCAGCTGGTT TGTAGATGAT


 


901
GTGGAGGTGC ACACAGCTCA GACGCAACCC CGGGAGGAGC AGTTCAACAG


 


951
CACTTTCCGC TCAGTCAGTG AACTTCCCAT CATGCACCAG GACTGGCTCA


 


1001
ATGGCAAGGA GTTCAAATGC AGGGTCAACA GTGCAGCTTT CCCTGCCCCC


 


1051
ATCGAGAAAA CCATCTCCAA AACCAAAGGC AGACCGAAGG CTCCACAGGT


 


1101
GTACACCATT CCACCTCCCA AGGAGCAGAT GGCCAAGGAT AAAGTCAGTC


 


1151
TGACCTGCAT GATAACAGAC TTCTTCCCTG AAGACATTAC TGTGGAGTGG


 


1201
CAGTGGAATG GGCAGCCAGC GGAGAACTAC AAGAACACTC AGCCCATCAT


 


1251
GGACACAGAT GGCTCTTACT TCATCTACAG CAAGCTCAAT GTGCAGAAGA


 


1301
GCAACTGGGA GGCAGGAAAT ACTTTCACCT GCTCTGTGTT ACATGAGGGC


 


1351
CTGCACAACC ACCATACTGA GAAGAGCCTC TCCCACTCTC CTGGTAAATG


 


1401
A






Ab-13

The sequences of the Antibody 13 (also referred to herein as Ab-13) LC and HC are as follows:

Ab-13 Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-13 LC:















(SEQ ID NO: 205)


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-13 LC:









(SEQ ID NO: 206)










1
CAGATTGTTC TCACCCAGTC TCCAGCAATC ATGTCTGCAT CTCCAGGGGA



 


51
GAAGGTCACC ATGACCTGCA GGGCCAGCTC AAGTGTAACT TCCAGTTACT


 


101
TGAACTGGTA CCAGCAGAAG CCAGGATCTT CCCCCAAACT CTGGATTTAT


 


151
AGCACATCCA ACCTGGCTTC AGGAGTCCCA GCTCGCTTCA GTGGCAGTGG


 


201
GTCTGGGACC TCTTACTCTC TCACAATCAG CAGTGTGGAG GCTGAGGATG


 


251
CTGCCACTTA TTACTGCCAG CAGTATGATT TTTTCCCATC GACGTTCGGT


 


301
GGAGGCACCA AGCTGGAAAT CAAGCGGGCT GATGCTGCAC CAACTGTATC


 


351
CATCTTCCCA CCATCCAGTG AGCAGTTAAC ATCTGGAGGT GCCTCAGTCG


 


401
TGTGCTTCTT GAACAACTTC TACCCCAAAG ACATCAATGT CAAGTGGAAG


 


451
ATTGATGGCA GTGAACGACA AAATGGCGTC CTGAACAGTT GGACTGATCA


 


501
GGACAGCAAA GACAGCACCT ACAGCATGAG CAGCACCCTC ACGTTGACCA


 


551
AGGACGAGTA TGAACGACAT AACAGCTATA CCTGTGAGGC CACTCACAAG


 


601
ACATCAACTT CACCCATCGT CAAGAGCTTC AACAGGAATG AGTGT






Amino acid sequence of the Ab-13 LC including signal peptide:









(SEQ ID NO: 207)










1
MDSQVQIFSF LLISALVKMS RGQIVLTQSP AIMSASPGEK VTMTCRASSS



 


51
VTSSYLNWYQ QKPGSSPKLW IYSTSNLASG VPARFSGSGS GTSYSLTISS


 


101
VEAEDAATYY CQQYDFFPST FGGGTKLEIK RADAAPTVSI FPPSSEQLTS


 


151
GGASVVCFLN NFYPKDINVK WKIDGSERQN GVLNSWTDQD SKDSTYSMSS


 


201
TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNEC






Nucleic acid sequence of the Ab-13 LC including signal peptide encoding sequence:









(SEQ ID NO: 208)










1
ATGGATTCTC AAGTGCAGAT TTTCAGCTTC CTTCTAATCA GTGCCTTAGT



 


51
CAAAATGTCC AGAGGACAGA TTGTTCTCAC CCAGTCTCCA GCAATCATGT


 


101
CTGCATCTCC AGGGGAGAAG GTCACCATGA CCTGCAGGGC CAGCTCAAGT


 


151
GTAACTTCCA GTTACTTGAA CTGGTACCAG CAGAAGCCAG GATCTTCCCC


 


201
CAAACTCTGG ATTTATAGCA CATCCAACCT GGCTTCAGGA GTCCCAGCTC


 


251
GCTTCAGTGG CAGTGGGTCT GGGACCTCTT ACTCTCTCAC AATCAGCAGT


 


301
GTGGAGGCTG AGGATGCTGC CACTTATTAC TGCCAGCAGT ATGATTTTTT


 


351
CCCATCGACG TTCGGTGGAG GCACCAAGCT GGAAATCAAG CGGGCTGATG


 


401
CTGCACCAAC TGTATCCATC TTCCCACCAT CCAGTGAGCA GTTAACATCT


 


451
GGAGGTGCCT CAGTCGTGTG CTTCTTGAAC AACTTCTACC CCAAAGACAT


 


501
CAATGTCAAG TGGAAGATTG ATGGCAGTGA ACGACAAAAT GGCGTCCTGA


 


551
ACAGTTGGAC TGATCAGGAC AGCAAAGACA GCACCTACAG CATGAGCAGC


 


601
ACCCTCACGT TGACCAAGGA CGAGTATGAA CGACATAACA GCTATACCTG


 


651
TGAGGCCACT CACAAGACAT CAACTTCACC CATCGTCAAG AGCTTCAACA


 


701
GGAATGAGTG T






Ab-13 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-13 HC:















(SEQ ID NO: 209)


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-13 HC:









(SEQ ID NO: 210)










1
GAGGTCCAGC TGCAACAATC TGGACCTGAG CTGGTGAAGC CTGGGGCTTC



 


51
AGTGAAGATG TCCTGTAAGG CTTCTGGATA CACATTCACT GACTACTACA


 


101
TGAACTGGGT GAAGCAGAGC CATGGAGAGA GCCTTGAGTG GATTGGAGAT


 


151
ATTAATCCTT ACAACGATGA TACTACCTAC AACCACAAGT TCAAGGGCAA


 


201
GGCCACATTG ACTGTAGACA AATCCTCCAA CACAGCCTAC ATGCAGCTCA


 


251
ACAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC AAGAGAGACG


 


301
GCCGTTATTA CTACGAATGC TATGGACTAC TGGGGTCAAG GAACCTCAGT


 


351
CACCGTCTCC TCAGCCAAAA CGACACCCCC ATCTGTCTAT CCACTGGCCC


 


401
CTGGATCTGC TGCCCAAACT AACTCCATGG TGACCCTGGG ATGCCTGGTC


 


451
AAGGGCTATT TCCCTGAGCC AGTGACAGTG ACCTGGAACT CTGGATCCCT


 


501
GTCCAGCGGT GTGCACACCT TCCCAGCTGT CCTGCAGTCT GACCTCTACA


 


551
CTCTGAGCAG CTCAGTGACT GTCCCCTCCA GCACCTGGCC CAGCGAGACC


 


601
GTCACCTGCA ACGTTGCCCA CCCGGCCAGC AGCACCAAGG TGGACAAGAA


 


651
AATTGTGCCC AGGGATTGTG GTTGTAAGCC TTGCATATGT ACAGTCCCAG


 


701
AAGTATCATC TGTCTTCATC TTCCCCCCAA AGCCCAAGGA TGTGCTCACC


 


751
ATTACTCTGA CTCCTAAGGT CACGTGTGTT GTGGTAGACA TCAGCAAGGA


 


801
TGATCCCGAG GTCCAGTTCA GCTGGTTTGT AGATGATGTG GAGGTGCACA


 


851
CAGCTCAGAC GCAACCCCGG GAGGAGCAGT TCAACAGCAC TTTCCGCTCA


 


901
GTCAGTGAAC TTCCCATCAT GCACCAGGAC TGGCTCAATG GCAAGGAGTT


 


951
CAAATGCAGG GTCAACAGTG CAGCTTTCCC TGCCCCCATC GAGAAAACCA


 


1001
TCTCCAAAAC CAAAGGCAGA CCGAAGGCTC CACAGGTGTA CACCATTCCA


 


1051
CCTCCCAAGG AGCAGATGGC CAAGGATAAA GTCAGTCTGA CCTGCATGAT


 


1101
AACAGACTTC TTCCCTGAAG ACATTACTGT GGAGTGGCAG TGGAATGGGC


 


1151
AGCCAGCGGA GAACTACAAG AACACTCAGC CCATCATGGA CACAGATGGC


 


1201
TCTTACTTCA TCTACAGCAA GCTCAATGTG CAGAAGAGCA ACTGGGAGGC


 


1251
AGGAAATACT TTCACCTGCT CTGTGTTACA TGAGGGCCTG CACAACCACC


 


1301
ATACTGAGAA GAGCCTCTCC CACTCTCCTG GTAAA






Amino acid sequence of the Ab-13 HC including signal peptide:









(SEQ ID NO: 211)










1
MGWNWIFLFL LSGTAGVYSE VQLQQSGPEL VKPGASVKMS CKASGYTFTD



 


51
YYMNWVKQSH GESLEWIGDI NPYNDDTTYN HKFKGKATLT VDKSSNTAYM


 


101
QLNSLTSEDS AVYYCARETA VITTNAMDYW GQGTSVTVSS AKTTPPSVYP


 


151
LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD


 


201
LYTLSSSVTV PSSTWPSETV TCNVAHPASS TKVDKKIVPR DCGCKPCICT


 


251
VPEVSSVFIF PPKPKDVLTI TLTPKVTCVV VDISKDDPEV QFSWFVDDVE


 


301
VHTAQTQPRE EQFNSTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE


 


351
KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITDFF PEDITVEWQW


 


401
NGQPAENYKN TQPIMDTDGS YFIYSKLNVQ KSNWEAGNTF TCSVLHEGLH


 


451
NHHTEKSLSH SPGK






Nucleic acid sequence of the Ab-13 HC including signal peptide encoding sequence:









(SEQ ID NO: 212)










1
ATGGGATGGA ACTGGATCTT TCTCTTCCTC TTGTCAGGAA CTGCAGGTGT



 


51
CTACTCTGAG GTCCAGCTGC AACAATCTGG ACCTGAGCTG GTGAAGCCTG


 


101
GGGCTTCAGT GAAGATGTCC TGTAAGGCTT CTGGATACAC ATTCACTGAC


 


151
TACTACATGA ACTGGGTGAA GCAGAGCCAT GGAGAGAGCC TTGAGTGGAT


 


201
TGGAGATATT AATCCTTACA ACGATGATAC TACCTACAAC CACAAGTTCA


 


251
AGGGCAAGGC CACATTGACT GTAGACAAAT CCTCCAACAC AGCCTACATG


 


301
CAGCTCAACA GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG


 


351
AGAGACGGCC GTTATTACTA CGAATGCTAT GGACTACTGG GGTCAAGGAA


 


401
CCTCAGTCAC CGTCTCCTCA GCCAAAACGA CACCCCCATC TGTCTATCCA


 


451
CTGGCCCCTG GATCTGCTGC CCAAACTAAC TCCATGGTGA CCCTGGGATG


 


501
CCTGGTCAAG GGCTATTTCC CTGAGCCAGT GACAGTGACC TGGAACTCTG


 


551
GATCCCTGTC CAGCGGTGTG CACACCTTCC CAGCTGTCCT GCAGTCTGAC


 


601
CTCTACACTC TGAGCAGCTC AGTGACTGTC CCCTCCAGCA CCTGGCCCAG


 


651
CGAGACCGTC ACCTGCAACG TTGCCCACCC GGCCAGCAGC ACCAAGGTGG


 


701
ACAAGAAAAT TGTGCCCAGG GATTGTGGTT GTAAGCCTTG CATATGTACA


 


751
GTCCCAGAAG TATCATCTGT CTTCATCTTC CCCCCAAAGC CCAAGGATGT


 


801
GCTCACCATT ACTCTGACTC CTAAGGTCAC GTGTGTTGTG GTAGACATCA


 


851
GCAAGGATGA TCCCGAGGTC CAGTTCAGCT GGTTTGTAGA TGATGTGGAG


 


901
GTGCACACAG CTCAGACGCA ACCCCGGGAG GAGCAGTTCA ACAGCACTTT


 


951
CCGCTCAGTC AGTGAACTTC CCATCATGCA CCAGGACTGG CTCAATGGCA


 


1001
AGGAGTTCAA ATGCAGGGTC AACAGTGCAG CTTTCCCTGC CCCCATCGAG


 


1051
AAAACCATCT CCAAAACCAA AGGCAGACCG AAGGCTCCAC AGGTGTACAC


 


1101
CATTCCACCT CCCAAGGAGC AGATGGCCAA GGATAAAGTC AGTCTGACCT


 


1151
GCATGATAAC AGACTTCTTC CCTGAAGACA TTACTGTGGA GTGGCAGTGG


 


1201
AATGGGCAGC CAGCGGAGAA CTACAAGAAC ACTCAGCCCA TCATGGACAC


 


1251
AGATGGCTCT TACTTCATCT ACAGCAAGCT CAATGTGCAG AAGAGCAACT


 


1301
GGGAGGCAGG AAATACTTTC ACCTGCTCTG TGTTACATGA GGGCCTGCAC


 


1351
AACCACCATA CTGAGAAGAG CCTCTCCCAC TCTCCTGGTA AA






Ab-13 was humanized to generate Ab-14.
The sequences of the Antibody 14 (also referred to herein as Ab-14) LC and HC are as follows:
Ab-14 Light Chain:
Amino acid sequence of the mature form (signal peptide removed) of the Ab-14 LC:














(SEQ ID NO: 213)


 













Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-14 LC:









(SEQ ID NO: 214)










1
GACATCCAGC TGACCCAGAG CCCCAGCTTC CTTTCCGCAT CCGTTGGTGA



 


51
CCGAGTAACA ATCACATGCC GCGCCTCATC TTCAGTTACA TCTTCTTATC


 


101
TTAATTGGTA TCAACAAAAA CCAGGAAAAG CACCTAAACT TCTTATATAC


 


151
TCTACATCTA ATCTCGCATC AGGAGTTCCC TCTCGATTTT CAGGATCTGG


 


201
ATCAGGCACA GAATTTACAC TTACTATATC ATCACTCCAA CCAGAAGACT


 


251
TCGCCACTTA TTACTGCCAA CAATACGATT TTTTTCCAAG CACATTCGGA


 


301
GGAGGTACAA AAGTAGAAAT CAAGCGTACG GTGGCTGCAC CATCTGTCTT


 


351
CATCTTCCCG CCATCTGATG AGCAGTTGAA ATCTGGAACT GCCTCTGTTG


 


401
TGTGCCTGCT GAATAACTTC TATCCCAGAG AGGCCAAAGT ACAGTGGAAG


 


451
GTGGATAACG CCCTCCAATC GGGTAACTCC CAGGAGAGTG TCACAGAGCA


 


501
GGACAGCAAG GACAGCACCT ACAGCCTCAG CAGCACCCTG ACGCTGAGCA


 


551
AAGCAGACTA CGAGAAACAC AAAGTCTACG CCTGCGAAGT CACCCATCAG


 


601
GGCCTGAGCT CGCCCGTCAC AAAGAGCTTC AACAGGGGAG AGTGT






Amino acid sequence of the Ab-14 LC including signal peptide:









(SEQ ID NO: 215)










1
MDMRVPAQLL GLLLLWLPGA RCDIQLTQSP SFLSASVGDR VTITCRASSS



 


51
VTSSYLNWYQ QKPGKAPKLL IYSTSNLASG VPSRFSGSGS GTEFTLTISS


 


101
LQPEDFATYY CQQYDFFPST FGGGTKVEIK RTVAAPSVFI FPPSDEQLKS


 


151
GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS


 


201
TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC






Nucleic acid sequence of the Ab-14 LC including signal peptide encoding sequence:









(SEQ ID NO: 216)










1
ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTCCTGC TACTCTGGCT



 


51
CCCAGGTGCC AGATGTGACA TCCAGCTGAC CCAGAGCCCC AGCTTCCTTT


 


101
CCGCATCCGT TGGTGACCGA GTAACAATCA CATGCCGCGC CTCATCTTCA


 


151
GTTACATCTT CTTATCTTAA TTGGTATCAA CAAAAACCAG GAAAAGCACC


 


201
TAAACTTCTT ATATACTCTA CATCTAATCT CGCATCAGGA GTTCCCTCTC


 


251
GATTTTCAGG ATCTGGATCA GGCACAGAAT TTACACTTAC TATATCATCA


 


301
CTCCAACCAG AAGACTTCGC CACTTATTAC TGCCAACAAT ACGATTTTTT


 


351
TCCAAGCACA TTCGGAGGAG GTACAAAAGT AGAAATCAAG CGTACGGTGG


 


401
CTGCACCATC TGTCTTCATC TTCCCGCCAT CTGATGAGCA GTTGAAATCT


 


451
GGAACTGCCT CTGTTGTGTG CCTGCTGAAT AACTTCTATC CCAGAGAGGC


 


501
CAAAGTACAG TGGAAGGTGG ATAACGCCCT CCAATCGGGT AACTCCCAGG


 


551
AGAGTGTCAC AGAGCAGGAC AGCAAGGACA GCACCTACAG CCTCAGCAGC


 


601
ACCCTGACGC TGAGCAAAGC AGACTACGAG AAACACAAAG TCTACGCCTG


 


651
CGAAGTCACC CATCAGGGCC TGAGCTCGCC CGTCACAAAG AGCTTCAACA


 


701
GGGGAGAGTG T






Ab-14 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-14 HC:















(SEQ ID NO: 217)


 










Amino acid sequence of the mature form (signal peptide removed) of the Ab-14 HC without carboxy-terminal lysine:















(SEQ ID NO: 393)


 










Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-14 HC:









(SEQ ID NO: 218)










1
GAGGTGCAGC TGGTGCAGAG CGGCGCCGAG GTCAAGAAAC CTGGAGCAAG



 


51
CGTAAAGGTT AGTTGCAAAG CATCTGGATA CACATTTACC GACTACTACA


 


101
TGAATTGGGT ACGACAAGCC CCTGGACAAA GACTTGAATG GATGGGAGAC


 


151
ATTAACCCTT ATAACGACGA CACTACATAC AATCATAAAT TTAAAGGAAG


 


201
AGTTACAATT ACAAGAGATA CATCCGCATC AACCGCCTAT ATGGAACTTT


 


251
CCTCATTGAG ATCTGAAGAC ACTGCTGTTT ATTACTGTGC AAGAGAAACT


 


301
GCCGTTATTA CTACTAACGC TATGGATTAC TGGGGTCAAG GAACCACTGT


 


351
TACCGTCTCT AGTGCCTCCA CCAAGGGCCC ATCGGTCTTC CCCCTGGCGC


 


401
CCTGCTCCAG GAGCACCTCC GAGAGCACAG CGGCCCTGGG CTGCCTGGTC


 


451
AAGGACTACT TCCCCGAACC GGTGACGGTG TCGTGGAACT CAGGCGCTCT


 


501
GACCAGCGGC GTGCACACCT TCCCAGCTGT CCTACAGTCC TCAGGACTCT


 


551
ACTCCCTCAG CAGCGTGGTG ACCGTGCCCT CCAGCAACTT CGGCACCCAG


 


601
ACCTACACCT GCAACGTAGA TCACAAGCCC AGCAACACCA AGGTGGACAA


 


651
GACAGTTGAG CGCAAATGTT GTGTCGAGTG CCCACCGTGC CCAGCACCAC


 


701
CTGTGGCAGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC


 


751
CTCATGATCT CCCGGACCCC TGAGGTCACG TGCGTGGTGG TGGACGTGAG


 


801
CCACGAAGAC CCCGAGGTCC AGTTCAACTG GTACGTGGAC GGCGTGGAGG


 


851
TGCATAATGC CAAGACAAAG CCACGGGAGG AGCAGTTCAA CAGCACGTTC


 


901
CGTGTGGTCA GCGTCCTCAC CGTTGTGCAC CAGGACTGGC TGAACGGCAA


 


951
GGAGTACAAG TGCAAGGTCT CCAACAAAGG CCTCCCAGCC CCCATCGAGA


 


1001
AAACCATCTC CAAAACCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC


 


1051
CTGCCCCCAT CCCGGGAGGA GATGACCAAG AACCAGGTCA GCCTGACCTG


 


1101
CCTGGTCAAA GGCTTCTACC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA


 


1151
ATGGGCAGCC GGAGAACAAC TACAAGACCA CACCTCCCAT GCTGGACTCC


 


1201
GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG


 


1251
GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA


 


1301
ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A






Amino acid sequence of the Ab-14 HC including signal peptide:









(SEQ ID NO: 219)










1
MDWTWRILFL VAAATGAHSE VQLVQSGAEV KKPGASVKVS CKASGYTFTD



 


51
YYMNWVRQAP GQRLEWMGDI NPYNDDTTYN HKFKGRVTIT RDTSASTAYM


 


101
ELSSLRSEDT AVYYCARETA VITTNAMDYW GQGTTVTVSS ASTKGPSVFP


 


151
LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS


 


201
GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP


 


251
APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG


 


301
VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP


 


351
IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW


 


401
ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA


 


451
LHNHYTQKSL SLSPGK






Nucleic acid sequence of the Ab-14 HC including signal peptide encoding sequence:









(SEQ ID NO: 220)










1
ATGGACTGGA CCTGGAGGAT CCTCTTCTTG GTGGCAGCAG CCACAGGAGC



 


51
CCACTCCGAG GTGCAGCTGG TGCAGAGCGG CGCCGAGGTC AAGAAACCTG


 


101
GAGCAAGCGT AAAGGTTAGT TGCAAAGCAT CTGGATACAC ATTTACCGAC


 


151
TACTACATGA ATTGGGTACG ACAAGCCCCT GGACAAAGAC TTGAATGGAT


 


201
GGGAGACATT AACCCTTATA ACGACGACAC TACATACAAT CATAAATTTA


 


251
AAGGAAGAGT TACAATTACA AGAGATACAT CCGCATCAAC CGCCTATATG


 


301
GAACTTTCCT CATTGAGATC TGAAGACACT GCTGTTTATT ACTGTGCAAG


 


351
AGAAACTGCC GTTATTACTA CTAACGCTAT GGATTACTGG GGTCAAGGAA


 


401
CCACTGTTAC CGTCTCTAGT GCCTCCACCA AGGGCCCATC GGTCTTCCCC


 


451
CTGGCGCCCT GCTCCAGGAG CACCTCCGAG AGCACAGCGG CCCTGGGCTG


 


501
CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG


 


551
GCGCTCTGAC CAGCGGCGTG CACACCTTCC CAGCTGTCCT ACAGTCCTCA


 


601
GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAACTTCGG


 


651
CACCCAGACC TACACCTGCA ACGTAGATCA CAAGCCCAGC AACACCAAGG


 


701
TGGACAAGAC AGTTGAGCGC AAATGTTGTG TCGAGTGCCC ACCGTGCCCA


 


751
GCACCACCTG TGGCAGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA


 


801
GGACACCCTC ATGATCTCCC GGACCCCTGA GGTCACGTGC GTGGTGGTGG


 


851
ACGTGAGCCA CGAAGACCCC GAGGTCCAGT TCAACTGGTA CGTGGACGGC


 


901
GTGGAGGTGC ATAATGCCAA GACAAAGCCA CGGGAGGAGC AGTTCAACAG


 


951
CACGTTCCGT GTGGTCAGCG TCCTCACCGT TGTGCACCAG GACTGGCTGA


 


1001
ACGGCAAGGA GTACAAGTGC AAGGTCTCCA ACAAAGGCCT CCCAGCCCCC


 


1051
ATCGAGAAAA CCATCTCCAA AACCAAAGGG CAGCCCCGAG AACCACAGGT


 


1101
GTACACCCTG CCCCCATCCC GGGAGGAGAT GACCAAGAAC CAGGTCAGCC


 


1151
TGACCTGCCT GGTCAAAGGC TTCTACCCCA GCGACATCGC CGTGGAGTGG


 


1201
GAGAGCAATG GGCAGCCGGA GAACAACTAC AAGACCACAC CTCCCATGCT


 


1251
GGACTCCGAC GGCTCCTTCT TCCTCTACAG CAAGCTCACC GTGGACAAGA


 


1301
GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT GCATGAGGCT


 


1351
CTGCACAACC ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGTAAA






The CDR sequences in the variable region of the heavy chain of Ab-14 are:









(SEQ ID NO: 296)










CDR-H1:
DYYMN 


 









(SEQ ID NO: 297)










CDR-H2:
DINPYNDDTTYNHKFKG


 









(SEQ ID NO: 298)










CDR-H3:
ETAVITTNAMD






The light chain variable region CDR sequences of Ab-14 are:









(SEQ ID NO: 284)










CDR-L1:
RASSSVTSSYLN


 









(SEQ ID NO: 285)










CDR-L2:
STSNLAS


 









(SEQ ID NO: 286)










CDR-L3:
QQYDFFPST






Ab-14 Variable domains:

Ab-14 light chain variable domain amino acid sequence (without signal sequence):













(SEQ ID NO: 380)


 















Ab-14 light chain variable domain DNA sequence (without signal sequence):









(SEQ ID NO: 381)










1
GACATCCAGC TGACCCAGAG CCCCAGCTTC CTTTCCGCAT CCGTTGGTGA



 


51
CCGAGTAACA ATCACATGCC GCGCCTCATC TTCAGTTACA TCTTCTTATC


 


101
TTAATTGGTA TCAACAAAAA CCAGGAAAAG CACCTAAACT TCTTATATAC


 


151
TCTACATCTA ATCTCGCATC AGGAGTTCCC TCTCGATTTT CAGGATCTGG


 


201
ATCAGGCACA GAATTTACAC TTACTATATC ATCACTCCAA CCAGAAGACT


 


251
TCGCCACTTA TTACTGCCAA CAATACGATT TTTTTCCAAG CACATTCGGA


 


301
GGAGGTACAA AAGTAGAAAT CAAG






Ab-14 heavy chain variable domain amino acid sequence (without signal sequence):









(SEQ ID NO: 382)














Ab-14 heavy chain variable domain DNA sequence (without signal sequence):









(SEQ ID NO: 383)










1
GAGGTGCAGC TGGTGCAGAG CGGCGCCGAG GTCAAGAAAC CTGGAGCAAG



 


51
CGTAAAGGTT AGTTGCAAAG CATCTGGATA CACATTTACC GACTACTACA


 


101
TGAATTGGGT ACGACAAGCC CCTGGACAAA GACTTGAATG GATGGGAGAC


 


151
ATTAACCCTT ATAACGACGA CACTACATAC AATCATAAAT TTAAAGGAAG


 


201
AGTTACAATT ACAAGAGATA CATCCGCATC AACCGCCTAT ATGGAACTTT


 


251
CCTCATTGAG ATCTGAAGAC ACTGCTGTTT ATTACTGTGC AAGAGAAACT


 


301
GCCGTTATTA CTACTAACGC TATGGATTAC TGGGGTCAAG GAACCACTGT


 


351
TACCGTCTCT AGT






Ab-3 was humanized to generate Ab-15.
Ab-15
The sequences of the Antibody 15 (also referred to herein as Ab-15) LC and HC are as follows:
Ab-15 Light Chain:
Amino acid sequence of the mature form (signal peptide removed) of the Ab-15 LC:








(SEQ ID NO: 221)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-15 LC:









(SEQ ID NO: 222)










1
GACATCCAGA TGACCCAGTC TCCATCCTCC CTCTCAGCAT CCGTAGGCGA



 


51
TAGAGTTACA ATAACATGCA GCGTATCATC AACTATATCA TCAAATCATC


 


101
TTCATTGGTT CCAACAGAAA CCCGGCAAAG CACCTAAATC ACTTATATAC


 


151
GGCACATCAA ATCTCGCATC AGGCGTTCCT TCAAGATTTT CAGGCTCTGG


 


201
CTCAGGCACC GACTTTACTC TTACAATATC CTCCCTCCAA CCCGAAGACT


 


251
TCGCAACCTA TTACTGTCAA CAATGGTCCT CATATCCACT CACATTTGGC


 


301
GGCGGCACAA AAGTAGAAAT TAAACGTACG GTGGCTGCAC CATCTGTCTT


 


351
CATCTTCCCG CCATCTGATG AGCAGTTGAA ATCTGGAACT GCCTCTGTTG


 


401
TGTGCCTGCT GAATAACTTC TATCCCAGAG AGGCCAAAGT ACAGTGGAAG


 


451
GTGGATAACG CCCTCCAATC GGGTAACTCC CAGGAGAGTG TCACAGAGCA


 


501
GGACAGCAAG GACAGCACCT ACAGCCTCAG CAGCACCCTG ACGCTGAGCA


 


551
AAGCAGACTA CGAGAAACAC AAAGTCTACG CCTGCGAAGT CACCCATCAG


 


601
GGCCTGAGCT CGCCCGTCAC AAAGAGCTTC AACAGGGGAG AGTGT






Amino acid sequence of the Ab-15 LC including signal peptide:









(SEQ ID NO: 223)










1
MDMRVPAQLL GLLLLWLRGA RCDIQMTQSP SSLSASVGDR VTITCSVSST



 


51
ISSNHLHWFQ QKPGKAPKSL IYGTSNLASG VPSRFSGSGS GTDFTLTISS


 


101
LQPEDFATYY CQQWSSYPLT FGGGTKVEIK RTVAAPSVFI FPPSDEQLKS


 


151
GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS


 


201
TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC






Nucleic acid sequence of the Ab-15 LC including signal peptide encoding sequence:









(SEQ ID NO: 224)










1
ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTCCTGC TACTCTGGCT



 


51
CCGAGGTGCC AGATGTGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTCT


 


101
CAGCATCCGT AGGCGATAGA GTTACAATAA CATGCAGCGT ATCATCAACT


 


151
ATATCATCAA ATCATCTTCA TTGGTTCCAA CAGAAACCCG GCAAAGCACC


 


201
TAAATCACTT ATATACGGCA CATCAAATCT CGCATCAGGC GTTCCTTCAA


 


251
GATTTTCAGG CTCTGGCTCA GGCACCGACT TTACTCTTAC AATATCCTCC


 


301
CTCCAACCCG AAGACTTCGC AACCTATTAC TGTCAACAAT GGTCCTCATA


 


351
TCCACTCACA TTTGGCGGCG GCACAAAAGT AGAAATTAAA CGTACGGTGG


 


401
CTGCACCATC TGTCTTCATC TTCCCGCCAT CTGATGAGCA GTTGAAATCT


 


451
GGAACTGCCT CTGTTGTGTG CCTGCTGAAT AACTTCTATC CCAGAGAGGC


 


501
CAAAGTACAG TGGAAGGTGG ATAACGCCCT CCAATCGGGT AACTCCCAGG


 


551
AGAGTGTCAC AGAGCAGGAC AGCAAGGACA GCACCTACAG CCTCAGCAGC


 


601
ACCCTGACGC TGAGCAAAGC AGACTACGAG AAACACAAAG TCTACGCCTG


 


651
CGAAGTCACC CATCAGGGCC TGAGCTCGCC CGTCACAAAG AGCTTCAACA


 


701
GGGGAGAGTG T






Ab-15 Heavy Chain

Amino acid sequence of the mature form (signal peptide removed) of Ab-15 HC.









(SEQ ID NO: 225)














Amino acid sequence of the mature form (signal peptide removed) of Ab-15 HC without carboxy-terminal lysine:









(SEQ ID NO: 394)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-15 HC:









(SEQ ID NO: 226)










1
GAGGTGCAGC TGGTGCAGTC TGGGGCTGAG GTGAAGAAGC CTGGGGCCTC



 


51
AGTGAAGGTC TCCTGCAAGG CTTCTGACTT CAACATTAAA GACTTCTATC


 


101
TACACTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATTGGAAGG


 


151
ATTGATCCTG AGAATGGTGA TACTTTATAT GACCCGAAGT TCCAGGACAA


 


201
GGTCACCATG ACCACAGACA CGTCCACCAG CACAGCCTAC ATGGAGCTGA


 


251
GGAGCCTGAG ATCTGACGAC ACGGCCGTGT ATTACTGTGC GAGAGAGGCG


 


301
GATTATTTCC ACGATGGTAC CTCCTACTGG TACTTCGATG TCTGGGGCCG


 


351
TGGCACCCTG GTCACCGTCT CTAGTGCCTC CACCAAGGGC CCATCGGTCT


 


401
TCCCCCTGGC GCCCTGCTCC AGGAGCACCT CCGAGAGCAC AGCGGCCCTG


 


451
GGCTGCCTGG TCAAGGACTA CTTCCCCGAA CCGGTGACGG TGTCGTGGAA


 


501
CTCAGGCGCT CTGACCAGCG GCGTGCACAC CTTCCCAGCT GTCCTACAGT


 


551
CCTCAGGACT CTACTCCCTC AGCAGCGTGG TGACCGTGCC CTCCAGCAAC


 


601
TTCGGCACCC AGACCTACAC CTGCAACGTA GATCACAAGC CCAGCAACAC


 


651
CAAGGTGGAC AAGACAGTTG AGCGCAAATG TTGTGTCGAG TGCCCACCGT


 


701
GCCCAGCACC ACCTGTGGCA GGACCGTCAG TCTTCCTCTT CCCCCCAAAA


 


751
CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA CGTGCGTGGT


 


801
GGTGGACGTG AGCCACGAAG ACCCCGAGGT CCAGTTCAAC TGGTACGTGG


 


851
ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCACGGGA GGAGCAGTTC


 


901
AACAGCACGT TCCGTGTGGT CAGCGTCCTC ACCGTTGTGC ACCAGGACTG


 


951
GCTGAACGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA GGCCTCCCAG


 


1001
CCCCCATCGA GAAAACCATC TCCAAAACCA AAGGGCAGCC CCGAGAACCA


 


1051
CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA AGAACCAGGT


 


1101
CAGCCTGACC TGCCTGGTCA AAGGCTTCTA CCCCAGCGAC ATCGCCGTGG


 


1151
AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACACCTCCC


 


1201
ATGCTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAAGC TCACCGTGGA


 


1251
CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC GTGATGCATG


 


1301
AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT


 


1351
AAA






Amino acid sequence of the Ab-15 HC including signal peptide:









(SEQ ID NO: 227)



  1 MDWTWRILFL VAAATGAHSE VQLVQSGAEV KKPGASVKVS CKASDFNIKD



 


 51 FYLHWVRQAP GQGLEWIGRI DPENGDTLYD PKFQDKVTMT TDTSTSTAYM


 


101 ELRSLRSDDT AVYYCAREAD YFHDGTSYWY FDVWGRGTLV TVSSASTKGP


 


151 SVFPLAPCSR STSESTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV


 


201 LQSSGLYSLS SVVTVPSSNF GTQTYTCNVD HKPSNTKVDK TVERKCCVEC


 


251 PPCPAPPVAG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVQFNW


 


301 YVDGVEVHNA KTKPREEQFN STFRVVSVLT VVHQDWLNGK EYKCKVSNKG


 


351 LPAPIEKTIS KTKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI


 


401 AVEWESNGQP ENNYKTTPPM LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV


 


451 MHEALHNHYT QKSLSLSPGK






Nucleic acid sequence of the Ab-15 HC including signal peptide encoding sequence:









(SEQ ID NO: 228)



   1 ATGGACTGGA CCTGGAGGAT CCTCTTCTTG GTGGCAGCAG CCACAGGAGC



 


  51 CCACTCCGAG GTGCAGCTGG TGCAGTCTGG GGCTGAGGTG AAGAAGCCTG


 


 101 GGGCCTCAGT GAAGGTCTCC TGCAAGGCTT CTGACTTCAA CATTAAAGAC


 


 151 TTCTATCTAC ACTGGGTGCG ACAGGCCCCT GGACAAGGGC TTGAGTGGAT


 


 201 TGGAAGGATT GATCCTGAGA ATGGTGATAC TTTATATGAC CCGAAGTTCC


 


 251 AGGACAAGGT CACCATGACC ACAGACACGT CCACCAGCAC AGCCTACATG


 


 301 GAGCTGAGGA GCCTGAGATC TGACGACACG GCCGTGTATT ACTGTGCGAG


 


 351 AGAGGCGGAT TATTTCCACG ATGGTACCTC CTACTGGTAC TTCGATGTCT


 


 401 GGGGCCGTGG CACCCTGGTC ACCGTCTCTA GTGCCTCCAC CAAGGGCCCA


 


 451 TCGGTCTTCC CCCTGGCGCC CTGCTCCAGG AGCACCTCCG AGAGCACAGC


 


 501 GGCCCTGGGC TGCCTGGTCA AGGACTACTT CCCCGAACCG GTGACGGTGT


 


 551 CGTGGAACTC AGGCGCTCTG ACCAGCGGCG TGCACACCTT CCCAGCTGTC


 


 601 CTACAGTCCT CAGGACTCTA CTCCCTCAGC AGCGTGGTGA CCGTGCCCTC


 


 651 CAGCAACTTC GGCACCCAGA CCTACACCTG CAACGTAGAT CACAAGCCCA


 


 701 GCAACACCAA GGTGGACAAG ACAGTTGAGC GCAAATGTTG TGTCGAGTGC


 


 751 CCACCGTGCC CAGCACCACC TGTGGCAGGA CCGTCAGTCT TCCTCTTCCC


 


 801 CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACGT


 


 851 GCGTGGTGGT GGACGTGAGC CACGAAGACC CCGAGGTCCA GTTCAACTGG


 


 901 TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CACGGGAGGA


 


 951 GCAGTTCAAC AGCACGTTCC GTGTGGTCAG CGTCCTCACC GTTGTGCACC


 


1001 AGGACTGGCT GAACGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGGC


 


1051 CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAACCAAAG GGCAGCCCCG


 


1101 AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGAGGAG ATGACCAAGA


 


1151 ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTACCC CAGCGACATC


 


1201 GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC


 


1251 ACCTCCCATG CTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA


 


1301 CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG


 


1351 ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC


 


1401 TCCGGGTAAA






The CDR sequences in the variable region of the heavy chain of Ab-15 are:









(SEQ ID NO: 290)



CDR-H1: DFYLH



 



(SEQ ID NO: 291)



CDR-H2: RIDPENGDTLYDPKFQD



 



(SEQ ID NO: 292)



CDR-H3: EADYFHDGTSYWYFDV






The light chain variable region CDR sequences of Ab-15 are:









(SEQ ID NO: 278)



CDR-L1: SVSSTISSNHLH



 



(SEQ ID NO: 279)



CDR-L2: GTSNLAS



 



(SEQ ID NO: 280)



CDR-L3: QQWSSYPLT






Ab-15 Variable domains:

Ab-15 light chain variable domain amino acid sequence (without signal sequence):









(SEQ ID NO: 384)














Ab-15 light chain variable domain DNA sequence (without signal sequence):









(SEQ ID NO: 385)



  1 GACATCCAGA TGACCCAGTC TCCATCCTCC CTCTCAGCAT CCGTAGGCGA



 


 51 TAGAGTTACA ATAACATGCA GCGTATCATC AACTATATCA TCAAATCATC


 


101 TTCATTGGTT CCAACAGAAA CCCGGCAAAG CACCTAAATC ACTTATATAC


 


151 GGCACATCAA ATCTCGCATC AGGCGTTCCT TCAAGATTTT CAGGCTCTGG


 


201 CTCAGGCACC GACTTTACTC TTACAATATC CTCCCTCCAA CCCGAAGACT


 


251 TCGCAACCTA TTACTGTCAA CAATGGTCCT CATATCCACT CACATTTGGC


 


301 GGCGGCACAA AAGTAGAAAT TAAA






Ab-15 heavy chain variable domain amino acid sequence (without signal sequence):









(SEQ ID NO: 386)














Ab-15 heavy chain variable domain DNA sequence (without signal sequence):









(SEQ ID NO: 387)



  1 GAGGTGCAGC TGGTGCAGTC TGGGGCTGAG GTGAAGAAGC CTGGGGCCTC



 


 51 AGTGAAGGTC TCCTGCAAGG CTTCTGACTT CAACATTAAA GACTTCTATC


 


101 TACACTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATTGGAAGG


 


151 ATTGATCCTG AGAATGGTGA TACTTTATAT GACCCGAAGT TCCAGGACAA


 


201 GGTCACCATG ACCACAGACA CGTCCACCAG CACAGCCTAC ATGGAGCTGA


 


251 GGAGCCTGAG ATCTGACGAC ACGGCCGTGT ATTACTGTGC GAGAGAGGCG


 


301 GATTATTTCC ACGATGGTAC CTCCTACTGG TACTTCGATG TCTGGGGCCG


 


351 TGGCACCCTG GTCACCGTCT CTAGT






Ab-11 was humanized to generate Ab-16.
Ab-16
The sequences of the Antibody 16 (also referred to herein as Ab-16) LC and HC are as follows:
Ab-16 Light Chain:
Amino acid sequence of the mature form (signal peptide removed) of the Ab-16 LC:








(SEQ ID NO: 229)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-16 LC:









(SEQ ID NO: 230)



  1 GACATCCAGT TGACCCAGTC TCCATCCTTC CTGTCTGCAT CTGTAGGAGA



 


 51 CAGAGTCACC ATCACTTGCA GGGCCAGCTC AAGTATAAGT TACATACACT


 


101 GGTATCAGCA AAAACCAGGG AAAGCCCCTA AGCTCCTGAT CTATGCCACA


 


151 TCCAACCTGG CTTCTGGGGT CCCATCAAGG TTCAGCGGCA GTGGATCTGG


 


201 GACAGAATTC ACTCTCACAA TCAGCAGCCT GCAGCCTGAA GATTTTGCAA


 


251 CTTATTACTG TCAGCAGTGG AGTAGTGACC CACTCACGTT CGGCGGAGGG


 


301 ACCAAGGTGG AGATCAAACG TACGGTGGCT GCACCATCTG TCTTCATCTT


 


351 CCCGCCATCT GATGAGCAGT TGAAATCTGG AACTGCCTCT GTTGTGTGCC


 


401 TGCTGAATAA CTTCTATCCC AGAGAGGCCA AAGTACAGTG GAAGGTGGAT


 


451 AACGCCCTCC AATCGGGTAA CTCCCAGGAG AGTGTCACAG AGCAGGACAG


 


501 CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG AGCAAAGCAG


 


551 ACTACGAGAA ACACAAAGTC TACGCCTGCG AAGTCACCCA TCAGGGCCTG


 


601 AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGT






Amino acid sequence of the Ab-16 LC including signal peptide:









(SEQ ID NO: 231)



  1 MDMRVPAQLL GLLLLWLPGA RCDIQLTQSP SFLSASVGDR VTITCRASSS



 


 51 ISYIHWYQQK PGKAPKLLIY ATSNLASGVP SRFSGSGSGT EFTLTISSLQ


 


101 PEDFATYYCQ QWSSDPLTFG GGTKVEIKRT VAAPSVFIFP PSDEQLKSGT


 


151 ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL


 


201 TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC






Nucleic acid sequence of the Ab-16 LC including signal peptide encoding sequence:









(SEQ ID NO: 232)



  1 ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTCCTGC TGCTCTGGCT



 


 51 CCCAGGTGCC AGATGTGACA TCCAGTTGAC CCAGTCTCCA TCCTTCCTGT


 


101 CTGCATCTGT AGGAGACAGA GTCACCATCA CTTGCAGGGC CAGCTCAAGT


 


151 ATAAGTTACA TACACTGGTA TCAGCAAAAA CCAGGGAAAG CCCCTAAGCT


 


201 CCTGATCTAT GCCACATCCA ACCTGGCTTC TGGGGTCCCA TCAAGGTTCA


 


251 GCGGCAGTGG ATCTGGGACA GAATTCACTC TCACAATCAG CAGCCTGCAG


 


301 CCTGAAGATT TTGCAACTTA TTACTGTCAG CAGTGGAGTA GTGACCCACT


 


351 CACGTTCGGC GGAGGGACCA AGGTGGAGAT CAAACGTACG GTGGCTGCAC


 


401 CATCTGTCTT CATCTTCCCG CCATCTGATG AGCAGTTGAA ATCTGGAACT


 


451 GCCTCTGTTG TGTGCCTGCT GAATAACTTC TATCCCAGAG AGGCCAAAGT


 


501 ACAGTGGAAG GTGGATAACG CCCTCCAATC GGGTAACTCC CAGGAGAGTG


 


551 TCACAGAGCA GGACAGCAAG GACAGCACCT ACAGCCTCAG CAGCACCCTG


 


601 ACGCTGAGCA AAGCAGACTA CGAGAAACAC AAAGTCTACG CCTGCGAAGT


 


651 CACCCATCAG GGCCTGAGCT CGCCCGTCAC AAAGAGCTTC AACAGGGGAG


 


701 AGTGT






Ab-16 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-16 HC:









(SEQ ID NO: 233)














Amino acid sequence of the mature form (signal peptide removed) of the Ab-16 HC without carboxy-terminal lysine:









(SEQ ID NO: 395)














Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-16 HC:









(SEQ ID NO: 234)



   1 GAGGTGCAGC TGGTGCAGTC TGGGGCTGAG GTGAAGAAGC CTGGGGCCTC



 


  51 AGTGAAGGTC TCCTGCAAGG CTTCTGGATT CGACATTAAG GACTACTATA


 


 101 TACACTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATCGGAAGG


 


 151 GTTGATCCTG ACAATGGTGA GACTGAATTT GCCCCGAAGT TCCCGGGCAA


 


 201 GGTCACCATG ACCACAGACA CGTCCATCAG CACAGCCTAC ATGGAGCTGA


 


 251 GCAGGCTGAG ATCTGACGAC ACGGCCGTGT ATTACTGTGC GAGAGAAGAC


 


 301 TACGATGGTA CCTACACCTG GTTTCCTTAT TGGGGCCAAG GGACTCTGGT


 


 351 CACCGTCTCT AGTGCCTCCA CCAAGGGCCC ATCGGTCTTC CCCCTGGCGC


 


 401 CCTGCTCCAG GAGCACCTCC GAGAGCACAG CGGCCCTGGG CTGCCTGGTC


 


 451 AAGGACTACT TCCCCGAACC GGTGACGGTG TCGTGGAACT CAGGCGCTCT


 


 501 GACCAGCGGC GTGCACACCT TCCCAGCTGT CCTACAGTCC TCAGGACTCT


 


 551 ACTCCCTCAG CAGCGTGGTG ACCGTGCCCT CCAGCAACTT CGGCACCCAG


 


 601 ACCTACACCT GCAACGTAGA TCACAAGCCC AGCAACACCA AGGTGGACAA


 


 651 GACAGTTGAG CGCAAATGTT GTGTCGAGTG CCCACCGTGC CCAGCACCAC


 


 701 CTGTGGCAGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC


 


 751 CTCATGATCT CCCGGACCCC TGAGGTCACG TGCGTGGTGG TGGACGTGAG


 


 801 CCACGAAGAC CCCGAGGTCC AGTTCAACTG GTACGTGGAC GGCGTGGAGG


 


 851 TGCATAATGC CAAGACAAAG CCACGGGAGG AGCAGTTCAA CAGCACGTTC


 


 901 CGTGTGGTCA GCGTCCTCAC CGTTGTGCAC CAGGACTGGC TGAACGGCAA


 


 951 GGAGTACAAG TGCAAGGTCT CCAACAAAGG CCTCCCAGCC CCCATCGAGA


 


1001 AAACCATCTC CAAAACCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC


 


1051 CTGCCCCCAT CCCGGGAGGA GATGACCAAG AACCAGGTCA GCCTGACCTG


 


1101 CCTGGTCAAA GGCTTCTACC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA


 


1151 ATGGGCAGCC GGAGAACAAC TACAAGACCA CACCTCCCAT GCTGGACTCC


 


1201 GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG


 


1251 GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA


 


1301 ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A






Amino acid sequence of the Ab-16 HC including signal peptide:









(SEQ ID NO: 235)



  1 MDWTWRILFL VAAATGAHSE VQLVQSGAEV KKPGASVKVS CKASGFDIKD



 


 51 YYIHWVRQAP GQGLEWIGRV DPDNGETEFA PKFPGKVTMT TDTSISTAYM


 


101 ELSRLRSDDT AVYYCAREDY DGTYTWFPYW GQGTLVTVSS ASTKGPSVFP


 


151 LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS


 


201 GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP


 


251 APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG


 


301 VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP


 


351 IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW


 


401 ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA


 


451 LHNHYTQKSL SLSPGK






Nucleic acid sequence of the Ab-16 HC including signal peptide encoding sequence:









(SEQ ID NO: 236)



   1 ATGGACTGGA CCTGGAGGAT CCTCTTCTTG GTGGCAGCAG CCACAGGAGC



 


  51 CCACTCCGAG GTGCAGCTGG TGCAGTCTGG GGCTGAGGTG AAGAAGCCTG


 


 101 GGGCCTCAGT GAAGGTCTCC TGCAAGGCTT CTGGATTCGA CATTAAGGAC


 


 151 TACTATATAC ACTGGGTGCG ACAGGCCCCT GGACAAGGGC TTGAGTGGAT


 


 201 CGGAAGGGTT GATCCTGACA ATGGTGAGAC TGAATTTGCC CCGAAGTTCC


 


 251 CGGGCAAGGT CACCATGACC ACAGACACGT CCATCAGCAC AGCCTACATG


 


 301 GAGCTGAGCA GGCTGAGATC TGACGACACG GCCGTGTATT ACTGTGCGAG


 


 351 AGAAGACTAC GATGGTACCT ACACCTGGTT TCCTTATTGG GGCCAAGGGA


 


 401 CTCTGGTCAC CGTCTCTAGT GCCTCCACCA AGGGCCCATC GGTCTTCCCC


 


 451 CTGGCGCCCT GCTCCAGGAG CACCTCCGAG AGCACAGCGG CCCTGGGCTG


 


 501 CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG


 


 551 GCGCTCTGAC CAGCGGCGTG CACACCTTCC CAGCTGTCCT ACAGTCCTCA


 


 601 GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAACTTCGG


 


 651 CACCCAGACC TACACCTGCA ACGTAGATCA CAAGCCCAGC AACACCAAGG


 


 701 TGGACAAGAC AGTTGAGCGC AAATGTTGTG TCGAGTGCCC ACCGTGCCCA


 


 751 GCACCACCTG TGGCAGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA


 


 801 GGACACCCTC ATGATCTCCC GGACCCCTGA GGTCACGTGC GTGGTQGTGG


 


 851 ACGTGAGCCA CGAAGACCCC GAGGTCCAGT TCAACTGGTA CGTGGACGGC


 


 901 GTGGAGGTGC ATAATGCCAA GACAAAGCCA CGGGAGGAGC AGTTCAACAG


 


 951 CACGTTCCGT GTGGTCAGCG TCCTCACCGT TGTGCACCAG GACTGGCTGA


 


1001 ACGGCAAGGA GTACAAGTGC AAGGTCTCCA ACAAAGGCCT CCCAGCCCCC


 


1051 ATCGAGAAAA CCATCTCCAA AACCAAAGGG CAGCCCCGAG AACCACAGGT


 


1101 GTACACCCTG CCCCCATCCC GGGAGGAGAT GACCAAGAAC CAGGTCAGCC


 


1151 TGACCTGCCT GGTCAAAGGC TTCTACCCCA GCGACATCGC CGTGGAGTGG


 


1201 GAGAGCAATG GGCAGCCGGA GAACAACTAC AAGACCACAC CTCCCATGCT


 


1251 GGACTCCGAC GGCTCCTTCT TCCTCTACAG CAAGCTCACC GTGGACAAGA


 


1301 GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT GCATGAGGCT


 


1351 CTGCACAACC ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGTAAA






The CDR sequences in the variable region of the heavy chain of Ab-16 are:









(SEQ ID NO: 293)



CDR-H1: DYYIH



 



(SEQ ID NO: 294)



CDR-H2: RVDPDNGETEFAPKFPG



 



(SEQ ID NO: 295)



CDR-H3: EDYDGTYTWFPY






The light chain variable region CDR sequences of Ab-16 are:









(SEQ ID NO: 281)



CDR-L1: RASSSISYIH



 



(SEQ ID NO: 282)



CDR-L2: ATSNLAS



 



(SEQ ID NO: 283)



CDR-L3: QQWSSDPLT






Ab-16 Variable domains:

Ab-16 light chain variable domain amino acid sequence (without signal sequence):









(SEQ ID NO: 388)














Ab-16 light chain variable domain DNA sequence (without signal sequence):









(SEQ ID NO: 389)



  1 GACATCCAGT TGACCCAGTC TCCATCCTTC CTGTCTGCAT CTGTAGGAGA



 


 51 CAGAGTCACC ATCACTTGCA GGGCCAGCTC AAGTATAAGT TACATACACT


 


101 GGTATCAGCA AAAACCAGGG AAAGCCCCTA AGCTCCTGAT CTATGCCACA


 


151 TCCAACCTGG CTTCTGGGGT CCCATCAAGG TTCAGCGGCA GTGGATCTGG


 


201 GACAGAATTC ACTCTCACAA TCAGCAGCCT GCAGCCTGAA GATTTTGCAA


 


251 CTTATTACTG TCAGCAGTGG AGTAGTGACC CACTCACGTT CGGCGGAGGG


 


301 ACCAAGGTGG AGATCAAA






Ab-16 heavy chain variable domain amino acid sequence (without signal sequence):









(SEQ ID NO: 390)














Ab-16 heavy chain variable domain DNA sequence (without signal sequence):









(SEQ ID NO: 391)










  1
GAGGTGCAGC TGGTGCAGTC TGGGGCTGAG GTGAAGAAGC CTGGGGCCTC



 


 51
AGTGAAGGTC TCCTGCAAGG CTTCTGGATT CGACATTAAG GACTACTATA


 


101
TACACTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATCGGAAGG


 


151
GTTGATCCTG ACAATGGTGA GACTGAATTT GCCCCGAAGT TCCCGGGCAA


 


201
GGTCACCATG ACCACAGACA CGTCCATCAG CACAGCCTAC ATGGAGCTGA


 


251
GCAGGCTGAG ATCTGACGAC ACGGCCGTGT ATTACTGTGC GAGAGAAGAC


 


301
TACGATGGTA CCTACACCTG GTTTCCTTAT TGGGGCCAAG GGACTCTGGT


 


351
CACCGTCTCT AGT






Additional antibodies are referred to herein as Antibodies 17-22 (also referred to herein as Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, and Ab-22). The Kappa Constant region for all VK regions of Ab-17, Ab-19, and Ab-21 is as follows:







(SEQ ID NO: 323)


TDAAPTVSIFPFSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNG


 


VLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS


 


FNRNEC






The Heavy Constant Region for all VH regions of antibodies 17, 19 and 21 is as follows:







(SEQ ID NO: 324)


AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGV


 


HTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR


 


DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEV


 


QFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRV


 


NSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFF


 


PEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTF


 


TCSVLHEGLHNHHTEKSLSHSPGK






In the following antibody amino acid sequences, the boxed-shaded amino acids represent complement-determining regions (CDRs) and the underlined amino acids represent signal peptide.
Ab-17
Amino acid sequence of the Ab-17 LC including signal peptide:







(SEQ ID NO: 299)












Nucleic acid sequence of the Ab-17 LC including signal peptide:








(SEQ ID NO: 300)


ATGGATTTTCAGGTGCAGATTTTCAGCTTCATGCTAATCAGTGTCACAGT


 


CATATTGTCCAGTGGAGAAATTGTGCTCACCCAGTCTCCAGCACTCATGG


 


CTGCATCTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCGAGT


 


ATAAGTTCCAGCAACTTACACTGGTCCCAGCAGAAGTCAGGAACCTCCCC


 


CAAACTCTGGATTTATGGCACATCCAACCTTGCTTCTGGAGTCCCTGTTC


 


GCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGC


 


ATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGACTACTAC


 


GTATACGTTCGGATCGGGGACCAAGCTGGAGCTGAAACGT






Amino acid sequence of the Ab-17 HC including signal peptide:








(SEQ ID NO: 301)












Nucleic acid sequence of the Ab-17 HC including signal peptide:








(SEQ ID NO: 302)


ATGGGATGGAACTGGATCATCTTCTTCCTGATGGCAGTGGTTACAGGGGT


 


CAATTCAGAGGTGCAGTTGCGGCAGTCTGGGGCAGACCTTGTGAAGCCAG


 


GGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGAC


 


TACTATATACACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGAT


 


TGGAAGGATTGATCCTGATAATGGTGAAAGTACATATGTCCCGAAGTTCC


 


AGGGCAAGGCCACTATAACAGCAGACACATCATCCAACACAGCCTACCTA


 


CAACTCAGAAGCCTGACATCTGAGGACACTGCCATCTATTATTGTGGGAG


 


AGAGGGGCTCGACTATGGTGACTACTATGCTGTGGACTACTGGGGTCAAG


 


GAACCTCGGTCACAGTCTCGAGC






Ab-17 was humanized to generate Ab-18.

Ab-18

Amino acid sequence of the Ab-18 LC including signal peptide:








(SEQ ID NO: 303)












Nucleic acid sequence of the Ab-18 LC including signal peptide:








(SEQ ID NO: 104)


ATGGATATGCGCGTGCCGGCGCAGCTGCTGGGCCTGCTGCTGCTGTGGCT


 


GCCGGGCGCGCGCTGCGATATTCAGCTGACCCAGAGCCCGAGCTTTCTGA


 


GCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCAGCGTGAGCAGCAGC


 


ATTAGCAGCAGCAACCTGCATTGGTATCAGCAGAAACCGGGCAAAGCGCC


 


GAAACTGCTGATTTATGGCACCAGCAACCTGGCGAGCGGCGTGCCGAGCC


 


GCTTTAGCGGCAGCGGCAGCGGCACCGAATTTACCCTGACCATTAGCAGC


 


CTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGTGGACCACCAC


 


CTATACCTTTGGCCAGGGCACCAAACTGGAAATTAAACGT






Amino acid sequence of the Ab-18 HC including signal peptide:








(SEQ ID NO: 305)












Nucleic acid sequence of the Ab-18 HC including signal peptide:








(SEQ ID NO: 306)


ATGGATTGGACCTGGAGCATTCTGTTTCTGGTGGCGGCGCCGACCGGCGC


 


GCATAGCGAAGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGG


 


GCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGTTTAACATTAAAGATT


 


ATTATATTCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATG


 


GGCCGCATTGATCCGGATAACGGCGAAAGCACCTATGTGCCGAAATTTCA


 


GGGCCGCGTGACCATGACCACCGATACCAGCACCAGCACCGCGTATATGG


 


AACTGCGCAGCCTGCGCAGCGATGATACCGCGGTGTATTATTGCGCGCGC


 


GAAGGCCTGGATTATGGCGATTATTATGCGGTGGATTATTGGGGCCAGGG


 


CACCCTGGTGACCGTCTCGAGC






Ab-18 light chain variable domain amino acid sequence (without signal sequence):













(SEQ ID NO: 368)


 












Ab-18 light chain variable domain DNA sequence (without signal sequence):








(SEQ ID NO: 369)


GATATTCAGCTGACCCAGAGCCCGAGCTTTCTGAGCGCGAGCGTGGGCGA


 


TCGCGTGACCATTACCTGCAGCGTGAGCAGCAGCATTAGCAGCAGCAACC


 


TGCATTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTAT


 


GGCACCAGCAACCTGGCGAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGG


 


CAGCGGCACCGAATTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATT


 


TTGCGACCTATTATTGCCAGCAGTGGACCACCACCTATACCTTTGGCCAG


 


GGCACCAAACTGGAAATTAAACGT






Ab-18 heavy chain variable domain amino acid sequence (without signal sequence):













(SEQ ID NO: 370)


 












Ab-18 heavy chain variable domain DNA sequence (without signal sequence):








(SEQ ID NO: 371)


GAAGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCGCGAG


 


CGTGAAAGTGAGCTGCAAAGCGAGCGGCTTTAACATTAAAGATTATTATA


 


TTCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCCGC


 


ATTGATCCGGATAACGGCGAAAGCACCTATGTGCCGAAATTTCAGGGCCG


 


CGTGACCATGACCACCGATACCAGCACCAGCACCGCGTATATGGAACTGC


 


GCAGCCTGCGCAGCGATGATACCGCGGTGTATTATTGCGCGCGCGAAGGC


 


CTGGATTATGGCGATTATTATGCGGTGGATTATTGGGGCCAGGGCACCCT


 


GGTGACCGTCTCGAGC






Ab-19

Amino acid sequence of the Ab-19 LC including signal peptide:













(SEQ ID NO: 307)


 












Nucleic acid sequence of the Ab-19 LC including signal peptide:








(SEQ ID NO: 308)


ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGG


 


TACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCT


 


CTCTGGGAGACAGAGTCAACATCAGCTGCAGGGCAAGTCAGGACATTAGC


 


AGTTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCT


 


GATCTACTCCACATCAAGATTAAACTCAGGAGTCCCATCAAGGTTCAGTG


 


GCAGTGGGTCTGGGACAGATTATTCTCTCACTATTAGCAACCTGGCACAA


 


GAAGATATTGCCACTTACTTTTGCCAACAGGATATTAAGCATCCGACGTT


 


CGGTGGAGGCACCAAGTTGGAGCTGAAACGT






Amino acid sequence of the Ab-19 HC including signal peptide:













(SEQ ID NO: 309)


 












Nucleic acid sequence of the Ab-19 HC including signal peptide:








(SEQ ID NO: 310)


ATGGAATGGATCTGGATATTTCTCTTCCTCCTGTCAGGAACTGCAGGTGT


 


CCACTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTG


 


GGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGGTTCACATTCACTGAC


 


TACATTATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGAT


 


TGGATATATTAATCCTTACAATGATGATACTGAATACAATGAGAAGTTCA


 


AAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATG


 


GATCTCAGCAGTCTGACCTCTGAGGGCTCTGCGGTCTATTACTGTGCAAG


 


ATCGATTTATTACTACGATGCCCCGTTTGCTTACTGGGGCCAAGGGACTC


 


TGGTCACAGTCTCGAGC






Ab-19 was humanized to generate Antibody 20 (also referred to herein as Ab-20) and Antibody 23 (also referred to herein as Ab-23).

Ab-20

IgG4 version

Amino acid sequence of the Ab-20 LC including signal peptide:













(SEQ ID NO: 311)


 












Nucleic acid sequence of the Ab-20 LC including signal peptide:








(SEQ ID NO: 312)


ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGG


 


TACCAGATGTGATATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAT


 


CTGTAGGTGACCGTGTCACCATCACTTGCCGCGCAAGTCAGGATATTAGC


 


AGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT


 


GATCTATTCTACTTCCCGTTTGAATAGTGGGGTCCCATCACGCTTCAGTG


 


GCAGTGGCTCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT


 


GAAGATTTTGCAACTTACTACTGTCAACAGGATATTAAACACCCTACGTT


 


CGGTCAAGGCACCAAGGTGGAGATCAAACGT






Amino acid sequence of the Ab-20 HC including signal peptide:













(SEQ ID NO: 313)


 












Nucleic acid sequence of the Ab-20 HC including signal peptide:








(SEQ ID NO: 349)


ATGGAATGGATCTGGATATTTCTCTTCCTCCTGTCAGGAACTGCAGGTGT


 


CCACTCTGAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG


 


GGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGTTTTACCTTCACCGAC


 


TATATTATGCACTGGGTGCGTCAGGCCCCTGGTCAAGGGCTTGAGTGGAT


 


GGGCTATATCAACCCTTATAATGATGACACCGAATACAACGAGAAGTTCA


 


AGGGCCGTGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATG


 


GAGCTGAGCAGCCTGCGCTCTGAGGACACGGCCGTGTATTACTGTGCGCG


 


TTCGATTTATTACTACGATGCCCCGTTTGCTTACTGGGGCCAAGGGACTC


 


TGGTCACAGTCTCGAGC






Ab-23

IgG2 version

Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-23 LC:















(SEQ ID NO: 341)


 










Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-23 LC:









(SEQ ID NO: 342)










 1
GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGTGA



 


 51
CCGTGTCACC ATCACTTGCC GCGCAAGTCA GGATATTAGC AGCTATTTAA


 


101
ATTGGTATCA GCAGAAACCA GGGAAAGCCC CTAAGCTCCT GATCTATTCT


 


151
ACTTCCCGTT TGAATAGTGG GGTCCCATCA CGCTTCAGTG GCAGTGGCTC


 


201
TGGGACAGAT TTCACTCTCA CCATCAGCAG TCTGCAACCT GAAGATTTTG


 


251
CAACTTACTA CTGTCAACAG GATATTAAAC ACCCTACGTT CGGTCAAGGC


 


301
ACCAAGGTGG AGATCAAACG TACGGTGGCT GCACCATCTG TCTTCATCTT


 


351
CCCGCCATCT GATGAGCAGT TGAAATCTGG AACTGCCTCT GTTGTGTGCC


 


401
TGCTGAATAA CTTCTATCCC AGAGAGGCCA AAGTACAGTG GAAGGTGGAT


 


451
AACGCCCTCC AATCGGGTAA CTCCCAGGAG AGTGTCACAG AGCAGGACAG


 


501
CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG AGCAAAGCAG


 


551
ACTACGAGAA ACACAAAGTC TACGCCTGCG AAGTCACCCA TCAGGGCCTG


 


601
AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGT






Amino acid sequence of the Ab-23 LC including signal peptide:









(SEQ ID NO: 343)










  1
MDMRVPAQLL GLLLLWLRGA RCDIQMTQSP SSLSASVGDR VTITCRASQD



 


 51
ISSYLNWYQQ KPGKAPKLLI YSTSRLNSGV PSRFSGSGSG TDFTLTISSL


 


101
QPEDFATYYC QQDIKHPTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT


 


151
ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL


 


201
TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC






Nucleic acid sequence of the Ab-23 LC including signal peptide encoding sequence:









(SEQ ID NO: 344)










  1
ATGGACATGA GGGTGCCCGC TCAGCTCCTG GGGCTCCTGC TGCTGTGGCT



 


 51
GAGAGGTGCC AGATGTGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT


 


101
CTGCATCTGT AGGTGACCGT GTCACCATCA CTTGCCGCGC AAGTCAGGAT


 


151
ATTAGCAGCT ATTTAAATTG GTATCAGCAG AAACCAGGGA AAGCCCCTAA


 


201
GCTCCTGATC TATTCTACTT CCCGTTTGAA TAGTGGGGTC CCATCACGCT


 


251
TCAGTGGCAG TGGCTCTGGG ACAGATTTCA CTCTCACCAT CAGCAGTCTG


 


301
CAACCTGAAG ATTTTGCAAC TTACTACTGT CAACAGGATA TTAAACACCC


 


351
TACGTTCGGT CAAGGCACCA AGGTGGAGAT CAAACGTACG GTGGCTGCAC


 


401
CATCTGTCTT CATCTTCCCG CCATCTGATG AGCAGTTGAA ATCTGGAACT


 


451
GCCTCTGTTG TGTGCCTGCT GAATAACTTC TATCCCAGAG AGGCCAAAGT


 


501
ACAGTGGAAG GTGGATAACG CCCTCCAATC GGGTAACTCC CAGGAGAGTG


 


551
TCACAGAGCA GGACAGCAAG GACAGCACCT ACAGCCTCAG CAGCACCCTG


 


601
ACGCTGAGCA AAGCAGACTA CGAGAAACAC AAAGTCTACG CCTGCGAAGT


 


651
CACCCATCAG GGCCTGAGCT CGCCCGTCAC AAAGAGCTTC AACAGGGGAG


 


701
AGTGT






Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-23 HC:















(SEQ ID NO: 345)


 










Amino acid sequence of the mature form (signal peptide removed) of the Ab-23 HC without carboxy-terminal lysine:















(SEQ ID NO: 396)


 










Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-23 HC:









(SEQ ID NO: 346)










   1
GAGGTGCAGC TGGTGCAGTC TGGGGCTGAG GTGAAGAAGC CTGGGTCCTC



 


  51
GGTGAAGGTC TCCTGCAAGG CTTCTGGTTT TACCTTCACC GACTATATTA


 


 101
TGCACTGGGT GCGTCAGGCC CCTGGTCAAG GGCTTGAGTG GATGGGCTAT


 


 151
ATCAACCCTT ATAATGATGA CACCGAATAC AACGAGAAGT TCAAGGGCCG


 


 201
TGTCACGATT ACCGCGGACA AATCCACGAG CACAGCCTAC ATGGAGCTGA


 


 251
GCAGCCTGCG CTCTGAGGAC ACGGCCGTGT ATTACTGTGC GCGTTCGATT


 


 301
TATTACTACG ATGCCCCGTT TGCTTACTGG GGCCAAGGGA CTCTGGTCAC


 


 351
CGTCTCTAGT GCCTCCACCA AGGGCCCATC GGTCTTCCCC CTGGCGCCCT


 


 401
GCTCCAGGAG CACCTCCGAG AGCACAGCGG CCCTGGGCTG CCTGGTCAAG


 


 451
GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCTCTGAC


 


 501
CAGCGGCGTG CACACCTTCC CAGCTGTCCT ACAGTCCTCA GGACTCTACT


 


 551
CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAACTTCGG CACCCAGACC


 


 601
TACACCTGCA ACGTAGATCA CAAGCCCAGC AACACCAAGG TGGACAAGAC


 


 651
AGTTGAGCGC AAATGTTGTG TCGAGTGCCC ACCGTGCCCA GCACCACCTG


 


 701
TGGCAGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA GGACACCCTC


 


 751
ATGATCTCCC GGACCCCTGA GGTCACGTGC GTGGTGGTGG ACGTGAGCCA


 


 801
CGAAGACCCC GAGGTCCAGT TCAACTGGTA CGTGGACGGC GTGGAGGTGC


 


 851
ATAATGCCAA GACAAAGCCA CGGGAGGAGC AGTTCAACAG CACGTTCCGT


 


 901
GTGGTCAGCG TCCTCACCGT TGTGCACCAG GACTGGCTGA ACGGCAAGGA


 


 951
GTACAAGTGC AAGGTCTCCA ACAAAGGCCT CCCAGCCCCC ATCGAGAAAA


 


1001
CCATCTCCAA AACCAAAGGG CAGCCCCGAG AACCACAGGT GTACACCCTG


 


1051
CCCCCATCCC GGGAGGAGAT GACCAAGAAC CAGGTCAGCC TGACCTGCCT


 


1101
GGTCAAAGGC TTCTACCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG


 


1151
GGCAGCCGGA GAACAACTAC AAGACCACAC CTCCCATGCT GGACTCCGAC


 


1201
GGCTCCTTCT TCCTCTACAG CAAGCTCACC GTGGACAAGA GCAGGTGGCA


 


1251
GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC


 


1301
ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGTAAA






Amino acid sequence of the Ab-23 HC including signal peptide:









(SEQ ID NO: 347)










  1
MDWTWRILFL VAAATGAHSE VQLVQSGAEV KKPGSSVKVS CKASGFTFTD



 


 51
YIMHWVRQAP GQGLEWMGYI NPYNDDTEYN EKFKGRVTIT ADKSTSTAYM


 


101
ELSSLRSEDT AVYYCARSIY YYDAPFAYWG QGTLVTVSSA STKGPSVFPL


 


151
APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG


 


201
LYSLSSVVTV PSSNFGTQTY TCNVDHKPSN TKVDKTVERK CCVECPPCPA


 


251
PPVAGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VQFNWYVDGV


 


301
EVHNAKTKPR EEQFNSTFRV VSVLTVVHQD WLNGKEYKCK VSNKGLPAPI


 


351
EKTISKTKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE


 


401
SNGQPENNYK TTPPMLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL


 


451
HNHYTQKSLS LSPGK






Nucleic acid sequence of the Ab-23 HC including signal peptide encoding sequence:









(SEQ ID NO: 348)










   1
ATGGACTGGA CCTGGAGGAT CCTCTTCTTG GTGGCAGCAG CCACAGGAGC



 


  51
CCACTCCGAG GTGCAGCTGG TGCAGTCTGG GGCTGAGGTG AAGAAGCCTG


 


 101
GGTCCTCGGT GAAGGTCTCC TGCAAGGCTT CTGGTTTTAC CTTCACCGAC


 


 151
TATATTATGC ACTGGGTGCG TCAGGCCCCT GGTCAAGGGC TTGAGTGGAT


 


 201
GGGCTATATC AACCCTTATA ATGATGACAC CGAATACAAC GAGAAGTTCA


 


 251
AGGGCCGTGT CACGATTACC GCGGACAAAT CCACGAGCAC AGCCTACATG


 


 301
GAGCTGAGCA GCCTGCGCTC TGAGGACACG GCCGTGTATT ACTGTGCGCG


 


 351
TTCGATTTAT TACTACGATG CCCCGTTTGC TTACTGGGGC CAAGGGACTC


 


 401
TGGTCACCGT CTCTAGTGCC TCCACCAAGG GCCCATCGGT CTTCCCCCTG


 


 451
GCGCCCTGCT CCAGGAGCAC CTCCGAGAGC ACAGCGGCCC TGGGCTGCCT


 


 501
GGTCAAGGAC TACTTCCCCG AACCGGTGAC GGTGTCGTGG AACTCAGGCG


 


 551
CTCTGACCAG CGGCGTGCAC ACCTTCCCAG CTGTCCTACA GTCCTCAGGA


 


 601
CTCTACTCCC TCAGCAGCGT GGTGACCGTG CCCTCCAGCA ACTTCGGCAC


 


 651
CCAGACCTAC ACCTGCAACG TAGATCACAA GCCCAGCAAC ACCAAGGTGG


 


 701
ACAAGACAGT TGAGCGCAAA TGTTGTGTCG AGTGCCCACC GTGCCCAGCA


 


 751
CCACCTGTGG CAGGACCGTC AGTCTTCCTC TTCCCCCCAA AACCCAAGGA


 


 801
CACCCTCATG ATCTCCCGGA CCCCTGAGGT CACGTGCGTG GTGGTGGACG


 


 851
TGAGCCACGA AGACCCCGAG GTCCAGTTCA ACTGGTACGT GGACGGCGTG


 


 901
GAGGTGCATA ATGCCAAGAC AAAGCCACGG GAGGAGCAGT TCAACAGCAC


 


 951
GTTCCGTGTG GTCAGCGTCC TCACCGTTGT GCACCAGGAC TGGCTGAACG


 


1001
GCAAGGAGTA CAAGTGCAAG GTCTCCAACA AAGGCCTCCC AGCCCCCATC


 


1051
GAGAAAACCA TCTCCAAAAC CAAAGGGCAG CCCCGAGAAC CACAGGTGTA


 


1101
CACCCTGCCC CCATCCCGGG AGGAGATGAC CAAGAACCAG GTCAGCCTGA


 


1151
CCTGCCTGGT CAAAGGCTTC TACCCCAGCG ACATCGCCGT GGAGTGGGAG


 


1201
AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCACACCTC CCATGCTGGA


 


1251
CTCCGACGGC TCCTTCTTCC TCTACAGCAA GCTCACCGTG GACAAGAGCA


 


1301
GGTGGCAGCA GGGGAACGTC TTCTCATGCT CCGTGATGCA TGAGGCTCTG


 


1351
CACAACCACT ACACGCAGAA GAGCCTCTCC CTGTCTCCGG GTAAA






The CDR (complementarity determining region) sequences in the variable region of the heavy chain of Ab-23 are as follows:









(SEQ ID NO: 269)



CDR-H1: DYIMH



 



(SEQ ID NO: 270)



CDR-H2: YINPYNDDTEYNEKFKG



 



(SEQ ID NO: 271)



CDR-H3: SIYYYDAPFAY






The light chain variable region CDR sequences of Ab-23 are:









(SEQ ID NO: 239)



CDR-L1: RASQDISSYLN



 



(SEQ ID NO: 240)



CDR-L2: STSRLNS



 



(SEQ ID NO: 241)



CDR-L3: QQDIKHPT






Ab-23 Variable domains:

Ab-23 light chain variable domain amino acid sequence (without signal sequence):










(SEQ ID NO: 364)



DIQMTQSPSS LSASVGDRVT ITCRASQDIS SYLNWYQQKP



 



GKAPKLLIYS TSRLNSGVPS RFSGSGSGTD FTLTISSLQP



 



EDFATYYCQQ DIKHPTFGQG TKVEIK






Ab-23 light chain variable domain DNA sequence (without signal sequence):








(SEQ ID NO: 365)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGTGA


 


CCGTGTCACC ATCACTTGCC GCGCAAGTCA GGATATTAGC


 


AGCTATTTAAATTGGTATCA GCAGAAACCA GGGAAAGCCC


 


CTAAGCTCCT GATCTATTCTACTTCCCGTT TGAATAGTGG


 


GGTCCCATCA CGCTTCAGTG GCAGTGGCTCTGGGACAGAT


 


TTCACTCTCA CCATCAGCAG TCTGCAACCT


 


GAAGATTTTGCAACTTACTA CTGTCAACAG GATATTAAAC


 


ACCCTACGTT CGGTCAAGGCACCAAGGTGG AGATCAAA






Ab-23 heavy chain variable domain amino acid sequence (without signal sequence):










(SEQ ID NO: 366)



EVQLVQSGAE VKKPGSSVKV SCKASGFTFT DYIMHWVRQA



 



PGQGLEWMGYINPYNDDTEY NEKFKGRVTI TADKSTSTAY



 



MELSSLRSED TAVYYCARSIYYYDAPFAYW GQGTLVTVSS






Ab-23 heavy chain variable domain DNA sequence (without signal sequence):








(SEQ ID NO: 367)


GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC


 


GGTGAAGGTC TCCTGCAAGG CTTCTGGTTT TACCTTCACC


 


GACTATATTATGCACTGGGT GCGTCAGGCC CCTGGTCAAG


 


GGCTTGAGTG GATGGGCTATATCAACCCTT ATAATGATGA


 


CACCGAATAC AACGAGAAGT TCAAGGGCCGTGTCACGATT


 


ACCGCGGACA AATCCACGAG CACAGCCTAC


 


ATGGAGCTGAGCAGCCTGCG CTCTGAGGAC ACGGCCGTGT


 


ATTACTGTGC GCGTTCGATTTATTACTACG ATGCCCCGTT


 


TGCTTACTGG GGCCAAGGGACTCTGGTCACCGTCTCTAGT






Ab-21

Amino acid sequence of the Ab-21 LC including signal peptide:













(SEQ ID NO: 315)












Nucleic acid sequence of the Ab-21 LC including signal peptide:








(SEQ ID NO: 316)


ATGAAGTCACAGACCCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGG


 


TGTTGAAGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACGT


 


CAGTAGGAGACAGGGTCACCATCACCTGCAAGGCCAGTCAGGATGTCTTT


 


ACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACT


 


GATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAG


 


GCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCT


 


GAAGACTTGGCAGATTATTTCTGTCAACAATATAGCAGCTATCCTCTCAC


 


GTTCGGTGCTGGGACCAAGTTGGAGCTGAAACGT






Amino acid sequence of the Ab-21 HC including signal peptide:













(SEQ ID NO: 317)












Nucleic acid sequence of the Ab-21 HC including signal peptide:








(SEQ ID NO: 318)


ATGGGATGGAACTGGATCATCTTCTTCCTGATGGCAGTGGTTACAGGGGT


 


CAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAG


 


GGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAATATTAAAGAC


 


TACTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGAT


 


TGGAAGGATTGATCCTGAGAATGGTGATATTATATATGACCCGAAGTTCC


 


AGGGCAAGGCCAGTATAACAACAGACACATCCTCCAACACAGCCTACCTG


 


CAGCTCAGCAGCCTGACGTCTGAGGACACTGCCGTCTATTACTGTGCTTA


 


CGATGCTGGTGACCCCGCCTGGTTTACTTACTGGGGCCAAGGGACTCTGG


 


TCACCGTCTCGAGC






Ab-21 was humanized to yield Ab-22.

Ab-22

Amino acid sequence of the Ab-22 LC including signal peptide:













(SEQ ID NO: 319)












Nucleic acid sequence of the Ab-22 LC including signal peptide:








(SEQ ID NO: 320)


ATGGATATGCGCGTGCCGGCGCAGCTGCTGGGCCTGCTGCTGCTGTGGCT


 


GCGCGGCGCGCGCTGCGATATCCAGATGACCCAGAGCCCGAGCAGCCTGA


 


GCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCAAAGCGAGCCAGGAT


 


GTGTTTACCGCGGTGGCGTGGTATCAGCAGAAACCGGCTCAAAGCGCCGA


 


AACTGCTGATTTATTGGGCGAGCACCCGCCATACCGGCGTGCCGAGTCGC


 


TTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCT


 


GCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGTATAGCAGCTATC


 


CGCTGACCTTTGGCGGCGGCACCAAAGTGGAAATTAAACGT






Amino acid sequence of the Ab-22 HC including signal peptide:













(SEQ ID NO: 321)












Nucleic acid sequence of the Ab-22 HC including signal peptide:








(SEQ ID NO: 322)


ATGGATTGGACCTGGAGCATTCTGTTTCTGGTGGCGGCGCCGACCGGCGC


 


GCATAGCGAAGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGG


 


GCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCTTTAACATTAAAGAT


 


TATTATATGCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGAT


 


CGGCCGCATTGATCCGGAAAACGGCGATATTATTTATGATCCGAAATTTC


 


AGGGCCGCGTGACCATGACCACCGATACCAGCACCAGCACCGCGTATATG


 


GAACTGCGCAGCCTGCGCAGCGATGATACCGCGGTGTATTATTGCGCGTA


 


TGATGCGGGCGATCCGGCGTGGTTTACCTATTGGGGCCAGGGCACCCTGG


 


TGACCGTCTCGAGC






Ab-22 light chain variable domain amino acid sequence (without signal sequence):










(SEQ ID NO: 336)



DIQMTQSPSS LSASVGDRVT ITCKASQDVF TAVAWYQQKP



 



GKAPKLLIYW ASTRHTGVPS RFSGSGSGTD FTLTISSLQP



 



EDFATYYCQQ YSSYPLTFGG GTKVEIKR






Ab-22 light chain variable domain DNA sequence (without signal sequence):








(SEQ ID NO: 337)


GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGA


 


TCGCGTGACCATTACCTGCAAAGCGAGCCAGGATGTGTTTACCGCGGTGG


 


CGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATTGG


 


GCGAGCACCCGCCATACCGGCGTGCCGAGTCGCTTTAGCGGCAGCGGCAG


 


CGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTG


 


CGACCTATTATTGCCAGCAGTATAGCAGCTATCCGCTGACCTTTGGCGGC


 


GGCACCAAAGTGGAAATTAAACGT






Ab-22 heavy chain variable domain amino acid sequence (without signal sequence):










(SEQ ID NO: 338)



EVQLVQSGAE VKKPGASVKV SCKASGFNIK DYYMHWVRQA



 



PGQGLEWIGRIDPENGDIIY DPKFQGRVTM TTDTSTSTAY



 



MELRSLRSDD TAVYYCAYDAGDPAWFTYWG QGTLVTVSS






Ab-22 heavy chain variable domain DNA sequence (without signal sequence):








(SEQ ID NO: 339)


GAAGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCGCGAG


 


CGTGAAAGTGAGCTGCAAAGCGAGCGGCTTTAACATTAAAGATTATTATA


 


TGCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATCGGCCGC


 


ATTGATCCGGAAAACGGCGATATTATTTATGATCCGAAATTTCAGGGCCG


 


CGTGACCATGACCACCGATACCAGCACCAGCACCGCGTATATGGAACTGC


 


GCAGCCTGCGCAGCGATGATACCGCGGTGTATTATTGCGCGTATGATGCG


 


GGCGATCCGGCGTGGTTTACCTATTGGGGCCAGGGCACCCTGGTGACCGT


 


CTCGAGC.






For Ab-18, Ab-20, and Ab-22, the light chain human kappa constant region is as follows:








(SEQ ID NO: 325)


TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN


 


SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS


 


FNRGEC*






and the heavy chain human gamma-4 constant region is as follows:








(SEQ ID NO: 326)


ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV


 


HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES


 


KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED


 


PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK


 


CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG


 


NVFSCSVMHEALHNHYTQKSLSLSLGK*






The hinge region contains the Ser-241-Pro mutation to improve hinge stability (Angal S et al, (1993), Mol Immunol, 30(1), 105-108).

Ab-24

The sequences of Antibody 24 (also referred to herein as Ab-24) LC and HC are as follows:

Light Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-24 LC:















(SEQ ID NO: 350)


 










Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-24 LC:








(SEQ ID NO: 354)










  1
GACATTGTGT TGACCCAGTC TCCAGCTTCT TTGGCTGTGT CTCTAGGGCA



 


 51
GAGGGCCACC ATCGCCTGCA AGGCCAGCCA AAGTGTTGAT TATGATGGTA


 


101
CTAGTTATAT GAATTGGTAC CAACAGAAAC CAGGACAGCC ACCCAAACTC


 


151
CTCATCTATG CTGCATCCAA TCTAGAATCT GAGATCCCAG CCAGGTTTAG


 


201
TGGCACTGGG TCTGGGACAG ACTTCACCCT CAACATCCAT CCTGTGGAGG


 


251
AGGAGGATAT CACAACCTAT TACTGTCAGC AAAGTAATGA GGATCCGTTC


 


301
ACGTTCGGAG GGGGGACCAA GTTGGAAATA AAACGGGCTG ATGCTGCACC


 


351
AACTGTATCC ATCTTCCCAC CATCCAGTGA GCAGTTAACA TCTGGAGGTG


 


401
CCTCAGTCGT GTGCTTCTTG AACAACTTCT ACCCCAAAGA CATCAATGTC


 


451
AAGTGGAAGA TTGATGGCAG TGAACGACAA AATGGCGTCC TGAACAGTTG


 


501
GACTGATCAG GACAGCAAAG ACAGCACCTA CAGCATGAGC AGCACCCTCA


 


551
CGTTGACCAA GGACGAGTAT GAACGACATA ACAGCTATAC CTGTGAGGCC


 


601
ACTCACAAGA CATCAACTTC ACCCATTGTC AAGAGCTTCA ACAGGAATGA


 


651
GTGTTAG






Amino acid sequence of the Ab-24 LC including signal peptide:









(SEQ ID NO: 355)










  1
METDTILLWV LLLWVPGSTG DIVLTQSPAS LAVSLGQRAT IACKASQSVD



 


 51
YDGTSYMNWY QQKPGQPPKL LIYAASNLES EIPARFSGTG SGTDFTLNIH


 


101
PVEEEDITTY YCQQSNEDPF TFGGGTKLEI KRADAAPTVS IFPPSSEQLT


 


151
SGGASVVCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS


 


201
STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC






Nucleic acid sequence of the Ab-24 LC including signal peptide encoding sequence:








(SEQ ID NO: 356)










  1
ATGGAGACAG ACACAATCCT GCTATGGGTG CTGCTGCTCT GGGTTCCAGG



 


 51
CTCCACTGGT GACATTGTGT TGACCCAGTC TCCAGCTTCT TTGGCTGTGT


 


101
CTCTAGGGCA GAGGGCCACC ATCGCCTGCA AGGCCAGCCA AAGTGTTGAT


 


151
TATGATGGTA CTAGTTATAT GAATTGGTAC CAACAGAAAC CAGGACAGCC


 


201
ACCCAAACTC CTCATCTATG CTGCATCCAA TCTAGAATCT GAGATCCCAG


 


251
CCAGGTTTAG TGGCACTGGG TCTGGGACAG ACTTCACCCT CAACATCCAT


 


301
CCTGTGGAGG AGGAGGATAT CACAACCTAT TACTGTCAGC AAAGTAATGA


 


351
GGATCCGTTC ACGTTCGGAG GGGGGACCAA GTIGGAAATA AAACGGGCTG


 


401
ATGCTGCACC AACTGTATCC ATCTTCCCAC CATCCAGTGA GCAGTTAACA


 


451
TCTGGAGGTG CCTCAGTCGT GTGCTTCTTG AACAACTTCT ACCCCAAAGA


 


501
CATCAATGTC AAGTGGAAGA TTGATGGCAG TGAACGACAA AATGGCGTCC


 


551
TGAACAGTTG GACTGATCAG GACAGCAAAG ACAGCACCTA CAGCATGAGC


 


601
AGCACCCTCA CGTTGACCAA GGACGAGTAT GAACGACATA ACAGCTATAC


 


651
CTGTGAGGCC ACTCACAAGA CATCAACTTC ACCCATTGTC AAGAGCTTCA


 


701
ACAGGAATGA GTGTTAG






Ab-24 Heavy Chain:

Amino acid sequence of the mature form (signal peptide removed) of the Ab-24 HC:















(SEQ ID NO: 357)


 










Nucleic acid sequence encoding the mature form (signal peptide removed) of the Ab-24 HC:









(SEQ ID NO: 361)










   1
CAGGTCCAAC TACAGCAGCC TGGGACTGAG CTGGTGAGGC CTGGAACTTC



 


  51
AGTGAAGTTG TCCTGTAAGG CTTCTGGCTA CATCTTCACC ACCTACTGGA


 


 101
TGAACTGGGT GAAACAGAGG CCTGGACAAG GCCTTGAGTG GATTGGCATG


 


 151
ATTCATCCTT CCGCAAGTGA AATTAGGTTG GATCAGAAAT TCAAGGACAA


 


 201
GGCCACATTG ACTCTTGACA AATCCTCCAG CACAGCCTAT ATGCACCTCA


 


 251
GCGGCCCGAC ATCTGTGGAT TCTGCGGTCT ATTACTGTGC AAGATCAGGG


 


 301
GAATGGGGGT CTATGGACTA CTGGGGTCAA GGAACCTCAG TCACCGTCTC


 


 351
CTCAGCCAAA ACGACACCCC CATCTGTCTA TCCACTGGCC CCTGGATCTG


 


 401
CTGCCCAAAC TAACTCCATG GTGACCCTGG GATGCCTGGT CAAGGGCTAT


 


 451
TTCCCTGAGC CAGTGACAGT GACCTGGAAC TCTGGATCCC TGTCCAGCGG


 


 501
TGTGCACACC TTCCCAGCTG TCCTGCAGTC TGACCTCTAC ACTCTGAGCA


 


 551
GCTCAGTGAC TGTCCCCTCC AGCACCTGGC CCAGCGAGAC CGTCACCTGC


 


 601
AACGTTGCCC ACCCGGCCAG CAGCACCAAG GTGGACAAGA AAATTGTGCC


 


 651
CAGGGATTGT GGTTGTAAGC CTTGCATATG TACAGTCCCA GAAGTATCAT


 


 701
CTGTCTTCAT CTTCCCCCCA AAGCCCAAGG ATGTGCTCAC CATTACTCTG


 


 751
ACTCCTAAGG TCACGTGTGT TGTGGTAGAC ATCAGCAAGG ATGATCCCGA


 


 801
GGTCCAGTTC AGCTGGTTTG TAGATGATGT GGAGGTGCAC ACAGCTCAGA


 


 851
CGCAACCCCG GGAGGAGCAG TTCAACAGCA CTTTCCGCTC AGTCAGTGAA


 


 901
CTTCCCATCA TGCACCAGGA CTGGCTCAAT GGCAAGGAGT TCAAATGCAG


 


 951
GGTCAACAGT GCAGCTTTCC CTGCCCCCAT CGAGAAAACC ATCTCCAAAA


 


1001
CCAAAGGCAG ACCGAAGGCT CCACAGGTGT ACACCATTCC ACCTCCCAAG


 


1051
GAGCAGATGG CCAAGGATAA AGTCAGTCTG ACCTGCATGA TAACAGACTT


 


1101
CTTCCCTGAA GACATTACTG TGGAGTGGCA GTGGAATGGG CAGCCAGCGG


 


1151
AGAACTACAA GAACACTCAG CCCATCATGG ACACAGATGG CTCTTACTTC


 


1201
ATCTACAGCA AGCTCAATGT GCAGAAGAGC AACTGGGAGG CAGGAAATAC


 


1251
TTTCACCTGC TCTGTGTTAC ATGAGGGCCT GCACAACCAC CATACTGAGA


 


1301
AGAGCCTCTC CCACTCTCCT GGTAAATGA






Amino acid sequence of the Ab-24 HC including signal peptide:









(SEQ ID NO: 362)










  1
MGWSSIILFL VATATGVHSQ VQLQQPGTEL VRPGTSVKLS CKASGYIFTT



 


 51
YWMNWVKQRP GQGLEWIGMI HPSASEIRLD QKFKDKATLT LDKSSSTAYM


 


101
HLSGPTSVDS AVYYCARSGE WGSMDYWGQG TSVTVSSAKT TPPSVYPLAP


 


151
GSAAQTNSMV TLGCLVKGYF PEPVTVTWNS GSLSSGVHTF PAVLQSDLYT


 


201
LSSSVTVPSS TWPSETVTCN VAHPASSTKV DKKIVPRDCG CKPCICTVPE


 


251
VSSVFIFPPK PKDVLTITLT PKVTCVVVDI SKDDPEVQFS WFVDDVEVHT


 


301
AQTQPREEQF NSTFRSVSEL PIMHQDWLNG KEFKCRVNSA AFPAPIEKTI


 


351
SKTKGRPKAP QVYTIPPPKE QMAKDKVSLT CMITDFFPED ITVEWQWNGQ


 


401
PAENYKNTQP IMDTDGSYFI YSKLNVQKSN WEAGNTFTCS VLHEGLHNHH


 


451
TEKSLSHSPG K






Nucleic acid sequence of the Ab-24 HC including signal peptide encoding sequence:









(SEQ ID NO: 363)










   1
ATGGGATGGA GCTCTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTGT



 


  51
CCACTCCCAG GTCCAACTAC AGCAGCCTGG GACTGAGCTG GTGAGGCCTG


 


 101
GAACTTCAGT GAAGTTGTCC TGTAAGGCTT CTGGCTACAT CTTCACCACC


 


 151
TACTGGATGA ACTGGGTGAA ACAGAGGCCT GGACAAGGCC TTGAGTGGAT


 


 201
TGGCATGATT CATCCTTCCG CAAGTGAAAT TAGGTTGGAT CAGAAATTCA


 


 251
AGGACAAGGC CACATTGACT CTTGACAAAT CCTCCAGCAC AGCCTATATG


 


 301
CACCTCAGCG GCCCGACATC TGTGGATTCT GCGGTCTATT ACTGTGCAAG


 


 351
ATCAGGGGAA TGGGGGTCTA TGGACTACTG GGGTCAAGGA ACCTCAGTCA


 


 401
CCGTCTCCTC AGCCAAAACG ACACCCCCAT CTGTCTATCC ACTGGCCCCT


 


 451
GGATCTGCTG CCCAAACTAA CTCCATGGTG ACCCTGGGAT GCCTGGTCAA


 


 501
GGGCTATTTC CCTGAGCCAG TGACAGTGAC CTGGAACTCT GGATCCCTGT


 


 551
CCAGCGGTGT GCACACCTTC CCAGCTGTCC TGCAGTCTGA CCTCTACACT


 


 601
CTGAGCAGCT CAGTGACTGT CCCCTCCAGC ACCTGGCCCA GCGAGACCGT


 


 651
CACCTGCAAC GTTGCCCACC CGGCCAGCAG CACCAAGGTG GACAAGAAAA


 


 701
TTGTGCCCAG GGATTGTGGT TGTAAGCCTT GCATATGTAC AGTCCCAGAA


 


 751
GTATCATCTG TCTTCATCTT CCCCCCAAAG CCCAAGGATG TGCTCACCAT


 


 801
TACTCTGACT CCTAAGGTCA CGTGTGTTGT GGTAGACATC AGCAAGGATG


 


 851
ATCCCGAGGT CCAGTTCAGC TGGTTTGTAG ATGATGTGGA GGTGCACACA


 


 901
GCTCAGACGC AACCCCGGGA GGAGCAGTTC AACAGCACTT TCCGCTCAGT


 


 951
CAGTGAACTT CCCATCATGC ACCAGGACTG GCTCAATGGC AAGGAGTTCA


 


1001
AATGCAGGGT CAACAGTGCA GCTTTCCCTG CCCCCATCGA GAAAACCATC


 


1051
TCCAAAACCA AAGGCAGACC GAAGGCTCCA CAGGTGTACA CCATTCCACC


 


1101
TCCCAAGGAG CAGATGGCCA AGGATAAAGT CAGTCTGACC TGCATGATAA


 


1151
CAGACTTCTT CCCTGAAGAC ATTACTGTGG AGTGGCAGTG GAATGGGCAG


 


1201
CCAGCGGAGA ACTACAAGAA CACTCAGCCC ATCATGGACA CAGATGGCTC


 


1251
TTACTTCATC TACAGCAAGC TCAATGTGCA GAAGAGCAAC TGGGAGGCAG


 


1301
GAAATACTTT CACCTGCTCT GTGTTACATG AGGGCCTGCA CAACCACCAT


 


1351
ACTGAGAAGA GCCTCTCCCA CTCTCCTGGT AAATGA






The CDR sequences in the variable region of the light chain of Ab-24 are as follows:










(SEQ ID NO: 351)



CDR-L1: KASQSVDYDGTSYMN



 



(SEQ ID NO: 352)



CDR-L2: AASNLES



 



(SEQ ID NO: 353)



CDR-L3: QQSNEDPFT






The CDR sequences in the variable region of the heavy chain of Ab-24 are as follows:










(SEQ ID NO: 358)



CDR-H1: TYWMN



 



(SEQ ID NO: 359)



CDR-H2: MIHPSASEIRLDQKFKD



 



(SEQ ID NO: 360)



CDR-H3: SGEWGSMDY






Table 1 below provides the SEQ ID NOs and amino acid sequences of the CDR's of Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24. L1, L2, and L3 refer to light chain CDR's 1, 2, and 3, and H1, H2, and H3 refer to heavy chain CDR's 1, 2, and 3 according to the Kabat numbering system (Kabat et at, 1987 in Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA).









TABLE 1





SEQ




ID

AMINO ACID


NO
DESCRIPTION
SEQUENCE







 54
Ab-A and Ab-1 CDR-L1
QSSQSVYDNNWLA


 


 55
Ab-A and Ab-1 CDR-L2
DASDLAS


 


 56
Ab-A and Ab-1 CDR-L3
QGAYNDVIYA


 


 51
Ab-A and Ab-1 CDR-H1
SYWMN


 


 52
Ab-A and Ab-1 CDR-H2
TIDSGGRTDYASWAKG


 


 53
Ab-A and Ab-1 CDR-H3
NWNL


 


 60
Ab-B CDR-L1
SASSSVSFVD


 


 61
Ab-B CDR-L2
RTSNLGF


 


 62
Ab-B CDR-L3
QQRSTYPPT


 


 57
Ab-B CDR-H1
TSGMGVG


 


 58
Ab-B CDR-H2
HIWWDDVKRYNPVLKS


 


 59
Ab-B CDR-H3
EDFDYDEEYYAMDY


 


 48
Ab-C CDR-L1
KASQSVDYDGDSYMN


 


 49
Ab-C CDR-L2
AASNLES


 


 50
Ab-C CDR-L3
QQSNEDPWT


 


 45
Ab-C CDR-H1
DCYMN


 


 46
Ab-C CDR-H2
DINPFNGGTTYNQKFKG


 


 47
Ab-C CDR-H3
SHYYFDGRVPWDAMDY


 


 42
Ab-D CDR-L1
QASQGTSINLN


 


 43
Ab-D CDR-L2
GSSNLED


 


 44
Ab-D CDR-L3
LQHSYLPYT


 


 39
Ab-D CDR-H1
DHYMS


 


 40
Ab-D CDR-H2
DINPYSGETTYNQKFKG


 


 41
Ab-D CDR-H3
DDYDASPFAY


 


275
Ab-2 CDR-L1
RASSSVYYYMH


 


276
Ab-2 CDR-L2
ATSNLAS


 


277
Ab-2 CDR-L3
QQWSSDPLT


 


287
Ab-2 CDR-H1
DYFIH


 


288
Ab-2 CDR-H2
RLDPEDGESDYAPKFQD


 


289
Ab-2 CDR-H3
EDYDGTYTFFPY


 


278
Ab-3 and Ab-15 CDR-L1
SVSSTISSNHLH


 


279
Ab-3 and Ab-15 CDR-L2
GTSNLAS


 


280
Ab-3 and Ab-15 CDR-L3
QQWSSYPLT


 


290
Ab-3 and Ab-15 CDR-H1
DFYLH


 


291
Ab-3 and Ab-15 CDR-H2
RIDPENGDTLYDPKFQD


 


292
Ab-3 and Ab-15 CDR-H3
EADYFHDGTSYWYFDV


 


 78
Ab-4 and Ab-5 CDR-L1
RASQDISNYLN


 


 79
Ab-4 and Ab-5 CDR-L2
YTSRLLS


 


 80
Ab-4 and Ab-5 CDR-L3
QQGDTLPYT


 


245
Ab-4 and Ab-5 CDR-H1
DYNMH


 


246
Ab-4 and Ab-5 CDR-H2
EINPNSGGAGYNQKFKG


 


247
Ab-4 and Ab-5 CDR-H3
LGYDDIYDDWYFDV


 


 81
Ab-6 CDR-L1
RASQDISNYLN


 


 99
Ab-6 CDR-L2
YTSRLHS


 


100
Ab-6 CDR-L3
QQGDTLPYT


 


248
Ab-6 CDR-H1
DYNMH


 


249
Ab-6 CDR-H2
EINPNSGGSGYNQKFKG


 


250
Ab-6 CDR-H3
LVYDGSYEDWYFDV


 


101
Ab-7 CDR-L1
RASQVITNYLY


 


102
Ab-7 CDR-L2
YTSRLHS


 


103
Ab-7 CDR-L3
QQGDTLPYT


 


251
Ab-7 CDR-H1
DYNMH


 


252
Ab-7 CDR-H2
EINPNSGGAGYNQQFKG


 


253
Ab-7 CDR-H3
LGYVGNYEDWYFDV


 


104
Ab-8 CDR-L1
RASQDISNYLN


 


105
Ab-8 CDR-L2
YTSRLLS


 


106
Ab-8 CDR-L3
QQGDTLPYT


 


254
Ab-8 CDR-H1
DYNMH


 


255
Ab-8 CDR-H2
EINPNSGGAGYNQKFKG


 


256
Ab-8 CDR-H3
LGYDDIYDDWYFDV


 


107
Ab-9 CDR-L1
RASQDISNYLN


 


108
Ab-9 CDR-L2
YTSRLFS


 


109
Ab-9 CDR-L3
QQGDTLPYT


 


257
Ab-9 CDR-H1
DYNMH


 


258
Ab-9 CDR-H2
EINPNSGGAGYNQKFKG


 


259
Ab-9 CDR-H3
LGYDDIYDDWYFDV


 


110
Ab-10 CDR-L1
RASQDISNYLN


 


111
Ab-10 CDR-L2
YTSRLLS


 


112
Ab-10 CDR-L3
QQGDTLPYT


 


260
Ab-10 CDR-H1
DYNMH


 


261
Ab-10 CDR-H2
EINPNSGGAGYNQKFKG


 


262
Ab-10 CDR-H3
LGYDDIYDDWYFDV


 


281
Ab-11 and Ab-16 CDR-L1
RASSSISYIH


 


282
Ab-11 and Ab-16 CDR-L2
ATSNLAS


 


283
Ab-11 and Ab-16 CDR-L3
QQWSSDPLT


 


293
Ab-11 and Ab-16 CDR-H1
DYYIH


 


294
Ab-11 and Ab-16 CDR-H2
RVDPDNGETEFAPKFPG


 


295
Ab-11 and Ab-16 CDR-H3
EDYDGTYTWFPY


 


113
Ab-12 CDR-L1
RASQDISNYLN


 


114
Ab-12 CDR-L2
YTSTLQS


 


115
Ab-12 CDR-L3
QQGDTLPYT


 


263
Ab-12 CDR-H1
DYNMH


 


264
Ab-12 CDR-H2
EINPNSGGSGYNQKFKG


 


265
Ab-12 CDR-H3
LGYYGNYEDWYFDV


 


284
Ab-13 and Ab-14 CDR-L1
RASSSVTSSYLN


 


285
Ab-13 and Ab-14 CDR-L2
STSNLAS


 


286
Ab-13 and Ab-14 CDR-L3
QQYDFFPST


 


296
Ab-13 and Ab-14 CDR-H1
DYYMN


 


297
Ab-13 and Ab-14 CDR-H2
DINPYNDDTTYNHKFKG


 


298
Ab-13 and Ab-14 CDR-H3
ETAVITTNAMD


 


116
Ab-17 and Ab-18 CDR-L1
SVSSSISSSNLH


 


237
Ab-17 and Ab-18 CDR-L2
GTSNLAS


 


238
Ab-17 and Ab-18 CDR-L3
QQWTTTYT


 


266
Ab-17 and Ab-18 CDR-H1
DYYIH


 


267
Ab-17 and Ab-18 CDR-H2
RIDPDNGESTYVPKFQG


 


268
Ab-17 and Ab-18 CDR-H3
EGLDYGDYYAVDY


 


239
Ab-19, Ab-20 and Ab-23
RASQDISSYLN



CDR-L1



 


240
Ab-19, Ab-20 and Ab-23
STSRLNS



CDR-L2



 


241
Ab-19, Ab-20 and Ab-23
QQDIKHPT



CDR-L3



 


269
Ab-19, Ab-20 and Ab-23
DYIMH



CDR-H1



 


270
Ab-19, Ab-20 and Ab-23
YINPYNDDTEYNEKFKG



CDR-H2



 


271
Ab-19, Ab-20 and Ab-23
SIYYYDAPFAY



CDR-H3



 


242
Ab-21 and Ab-22 CDR-L1
KASQDVFTAVA


 


243
Ab-21 and Ab-22 CDR-L2
WASTRHT


 


244
Ab-21 and Ab-22 CDR-L3
QQYSSYPLT


 


272
Ab-21 and Ab-22 CDR-H1
DYYMH


 


273
Ab-21 and Ab-22 CDR-H2
RIDPENGDIIYDPKFQG


 


274
Ab-21 and Ab-22 CDR-H3
DAGDPAWFTY


 


351
Ab-24 CDR-L1
KASQSVDYDGTSYMN


 


352
Ab-24 CDR-L2
AASNLES


 


353
Ab-24 CDR-L3
QQSNEDPFT


 


358
Ab-24 CDR-H1
TYWMN


 


359
Ab-24 CDR-H2
MIHPSASEIRLDQKFKD


 


360
Ab-24 CDR-H3
SGEWGSMDY









An oligopeptide or polypeptide is within the scope of the invention if it has an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to least one of the CDR's of Table 1 above; and/or to a CDR of a sclerostin binding agent that cross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin, and/or is cross-blocked from binding to sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24; and/or to a CDR of a sclerostin binding agent wherein the binding agent can block the inhibitory effect of sclerostin in a cell based mineralization assay (i.e. a sclerostin neutralizing binding agent); and/or to a CDR of a sclerostin binding agent that binds to a Loop 2 epitope; and/or to a CDR of a sclerostin binding agent that binds to a T20.6 epitope; and/or to a CDR of a sclerostin binding agent that binds to a “T20.6 derivative (cystine-knot+4 arms)” epitope.
Sclerostin binding agent polypeptides and antibodies are within the scope of the invention if they have amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a variable region of at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24, and cross-block the binding of at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin, and/or are cross-blocked from binding to sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24; and/or can block the inhibitory effect of sclerostin in a cell based mineralization assay (i.e. a sclerostin neutralizing binding agent); and/or bind to a Loop 2 epitope; and/or bind to a T20.6 epitope; and/or bind to a “T20.6 derivative (cystine-knot+4 arms)” epitope.
Polynucleotides encoding sclerostin binding agents are within the scope of the invention if they have polynucleotide sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide encoding a variable region of at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24, and wherein the encoded sclerostin binding agents cross-block the binding of at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin, and/or are cross-blocked from binding to sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24; and/or can block the inhibitory effect of sclerostin in a cell based mineralization assay (i.e. a sclerostin neutralizing binding agent); and/or bind to a Loop 2 epitope; and/or bind to a T20.6 epitope; and/or bind to a “T20.6 derivative (cystine-knot+4 arms)” epitope.
Antibodies according to the invention may have a binding affinity for human sclerostin of less than or equal to 1×10−7 M, less than or equal to 1×10−8 M, less than or equal to 1×10−9M, less than or equal to 1×10−10 M, less than or equal to 1×10−11M, or less than or equal to 1×10−12M.
The affinity of a binding agent such as an antibody or binding partner, as well as the extent to which a binding agent (such as an antibody) inhibits binding, can be determined by one of ordinary skill in the art using conventional techniques, for example those described by Scatchard et al. (Ann. N.Y. Acad. Sci. 51:660-672 (1949)) or by surface plasmon resonance (SPR; BIAcore, Biosensor, Piscataway, N.J.). For surface plasmon resonance, target molecules are immobilized on a solid phase and exposed to ligands in a mobile phase running along a flow cell. If ligand binding to the immobilized target occurs, the local refractive index changes, leading to a change in SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rates of change of the SPR signal can be analyzed to yield apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values gives the apparent equilibrium constant (affinity) (see, e.g., Wolff et al., Cancer Res. 53:2560-65 (1993)).
An antibody according to the present invention may belong to any immunoglobin class, for example IgG, IgE, IgM, IgD, or IgA. It may be obtained from or derived from an animal, for example, fowl (e.g., chicken) and mammals, which includes but is not limited to a mouse, rat, hamster, rabbit, or other rodent, cow, horse, sheep, goat, camel, human, or other primate. The antibody may be an internalizing antibody. Production of antibodies is disclosed generally in U.S. Patent Publication No. 2004/0146888 A1.
Characterization Assays
In the methods described above to generate antibodies according to the invention, including the manipulation of the specific Ab-A, Ab-B, Ab-C, Ab-D, and Antibody 1-24 (Ab-1 to Ab-24) CDRs into new frameworks and/or constant regions, appropriate assays are available to select the desired antibodies or binding agents (i.e. assays for determining binding affinity to sclerostin; cross-blocking assays; Biacore-based “human sclerostin peptide epitope competition binding assay;” MC3T3-E1 cell based assay; in vivo assays).
Epitope Binding Assays
Mature form human sclerostin is a 190 amino acid glycoprotein with a cystine-knot structure (FIGS. 8 and 9). In addition to the cystine-knot structure, the protein is characterized as having three loops designated as Loop 1, Loop 2 and Loop 3. Human sclerostin was subjected to proteolytic digestion to produce fragments. Briefly, using different proteases, including trypsin, aspN, and lysC, fragments with various cleavage sites and sizes were generated. The sequences and mass for various human sclerostin peptides were determined. Antibody protection was evaluated to determine the effect on accessibility for proteolysis, including clipped site masking and peptide shifting. Finally, a BIAcore-based “human sclerostin peptide epitope competition assay” was performed.
Exposure of sclerostin to trypsin cleavage resulted in a pattern of peptide fragments as summarized in FIG. 13. The fragments are referred to as T19.2, T20, T20.6, and T21-22. As shown schematically in FIG. 19B, the T20.6 epitope is a complex of four separate peptide sequences which are joined by the three disulfide bonds of the cystine-knot region. Two of the peptides are joined by two disulfide bonds. The other two peptides are linked by one disulfide bond that, schematically, bisects the first two polypeptides.
The T20.6 epitope that was generated by trypsin digestion retains the cystine-knot structure of the native polypeptide and is recognized by antibodies Ab-C and Ab-D. A derivative of epitope T20.6 consists of the cystine-knot region and amino acids 58-64, 73-81, 112-117 and 138-141 in sequence position with reference to SEQ ID NO:1. This derivative epitope is shown in FIG. 21. An epitope comprising the cystine-knot region may have one or more amino acids that is present in the T20.6 epitope (FIG. 19B) but not present in the T20.6 derivative epitope (FIG. 21).
Another epitope-containing region was identified in the Loop 2 region of human sclerostin (FIG. 19A) and is recognized by antibodies Ab-A and Ab-B. A Loop 2 epitope comprises amino acids 86-111 of SEQ ID NO:1 (C4 GPARLLPNAIGRGKWWRPSGPDFRC5, SEQ ID NO:6). Sterically, with reference to full-length sclerostin of SEQ ID NO:1, the Loop 2-containing structure is defined at one end by a disulfide bond between cysteine at position 86 (C4) and cysteine at position 144 (C8), and at the other end by a disulfide bond between cysteine at position 111 (C5) and cysteine at position 57 (C1).
The peptides generated by aspN cleavage of human sclerostin are shown in FIG. 12. In the Figure, these peptides are designated as AspN14.6, AspN18.6, and AspN22.7-23.5, and are also referred to herein as N14.6, N18.6, and N22.7-23.5, respectively.
One group of antibodies exhibits a specific pattern of binding to certain epitopes as evidenced by a Biacore-based “human sclerostin peptide epitope competition binding assay.” Briefly, the antibody is preincubated with the epitope to be tested, at concentrations that will saturate the epitope-binding sites on the antibody. The antibody is then exposed to sclerostin bound to a chip surface. After the appropriate incubation and washing procedures, a pattern of competitive binding is established. As shown in FIG. 18, exemplary antibody Ab-D bound to sclerostin molecules attached to the surface of the chip. Preincubation of antibody Ab-D with sclerostin decreased the binding of the antibody to the sclerostin on the chip to close to zero. Preincubation with a peptide consisting of epitope T19.2 showed that T19.2 did not compete with sclerostin for antibody binding. However, preincubation with any one of the epitopes designated T20, T20.6, T21-22, or N22.7-23.5 abolished a large proportion of the binding of antibody to sclerostin on the chip. In contrast, preincubation of the antibody with any one of the epitopes designated T19.2, N14.6 or N18.6 did not abolish the ability of the antibody to bind to sclerostin. A second exemplary antibody with this binding profile (FIG. 17) is Ab-C.
Antibody Ab-D therefore is exemplary and representative of a group of antibodies that bind to the epitopes T20, T20.6, T21-22, and N22.7-23.5, and have minimal detectable binding to epitopes T19.2, N14.6 and N18.6, as measured by the ability to block antibody binding to sclerostin. Antibodies having this characteristic binding pattern may or may not share amino acid sequence in one or more regions of the antibody molecule. Antibody similarity is determined functionally such as by the ability to bind to sclerostin following preincubation with each of the epitopes described above. Antibodies that exhibit a binding pattern similar or identical to that of antibody Ab-D are included in the invention. By “similar to” is meant, for example, the antibody will exhibit binding to each of the polypeptides T20, T20.6, T21-22 and N22.7-23.5 whereby this binding will specifically compete out at least 50% of the antibody's binding to sclerostin that would otherwise occur in the absence of preincubation with sclerostin or a sclerostin peptide. The antibody will also exhibit little or no detectable binding to polypeptides T19.2, N14.6 and N18.6, resulting in a reduction of 30% or less of the binding that would occur in the absence of preincubation with sclerostin or a sclerostin peptide.
For example, without being bound by a particular mechanism, the antibody binding pattern of FIG. 18 suggests that the epitope space to which antibody Ab-D and other antibodies having the epitope binding pattern of Ab-D bind consists of a polypeptide comprising the cystine-knot region of sclerostin.
Thus, as disclosed herein and with reference to FIG. 19B, an exemplary T20.6 epitope comprises four peptide chains attached via three separate disulfide bonds. Peptide chain SAKPVTELVC3SGQC4 GPAR (SEQ ID NO:3) is attached to peptide chain LVASC7KC8KRLTR (SEQ ID NO:5) by disulfide bonds from C3 to C7, and from C4 to C8. Peptide chain DVSEYSC1RELHFTR (SEQ ID NO:2) is attached to peptide chain WWRPSGPDFRC5IPDRYR (SEQ ID NO:4) by a disulfide bond from C1 to C5. The polypeptides of SEQ ID NOs:3 and 5 remain associated with the polypeptides of SEQ ID NOs:2 and 4 through a steric construct whereby the C1-C5 bond crosses the plane of the C4-C8 and C3-C7 bonds and is located between them, as illustrated in FIG. 19B.
As disclosed herein and with reference to FIG. 21, an exemplary derivative epitope of T20.6 comprises four peptide chains attached via three separate disulfide bonds. Peptide chain SAKPVTELVC3SGQC4 (SEQ ID NO:70) is attached to peptide chain LVASC7KC8 (SEQ ID NO:71) by disulfide bonds from C3 to C7, and from C4 to C8. Peptide chain C1RELHFTR (SEQ ID NO:72) is attached to peptide chain C51PDRYR (SEQ ID NO:73) by a disulfide bond from C1 to C5. The polypeptides of SEQ ID NOs:70 and 71 remain associated with the polypeptides of SEQ ID NOs:72 and 73 through a steric construct whereby the C1-C5 bond crosses the plane of the C4-C8 and C3-C7 bonds and is located between them, as illustrated in FIG. 21.
Antibody Ab-A is exemplary and representative of a second group of antibodies that have a characteristic binding pattern to human sclerostin peptides that is distinct from that obtained for antibodies Ab-C and Ab-D. Ab-A and the group of antibodies it represents bind to the N22.7-23.5 epitope and have minimal detectable binding to epitopes T19.2, T20, T20.6, T21-22, N14.6 or N18.6, as measured by the ability to block antibody binding to sclerostin (FIG. 15). A second exemplary antibody with this binding profile (FIG. 16) is Ab-B. Antibodies having this characteristic binding pattern may or may not share amino acid sequence in one or more regions of the antibody molecule. Antibody similarity is determined functionally such as by the ability to bind to sclerostin following preincubation with each of the epitopes described above. Antibodies that exhibit a binding pattern similar or identical to that of antibody Ab-A are included in the invention. By “similar to” is meant, for example, the antibody will exhibit binding to the N22.7-23.5 polypeptide whereby this binding will specifically compete out at least 50% of the antibody's binding to sclerostin that would otherwise occur in the absence of preincubation with sclerostin or a sclerostin peptide. The antibody will also exhibit little or no detectable binding to polypeptides T19.2, T20, T20.6, T21-22, N14.6 and N18.6, resulting in a reduction of 30% or less of the binding that would occur in the absence of preincubation with sclerostin or a sclerostin peptide.
For example, without being bound by a particular mechanism, the antibody binding pattern of FIG. 15 suggests that the epitope space to which antibody Ab-A and other antibodies having the epitope binding pattern of Ab-A bind consists of a polypeptide comprising the Loop 2 region of sclerostin. Thus, as disclosed herein and with reference to FIG. 19A, the Loop 2 region can be described as a linear peptide, but it acquires a tertiary structure when it is present in native sclerostin or a cystine-knot-containing portion of sclerostin in which the native disulfide bond structure is maintained. The linear or tertiary structure of the Loop 2 epitope can affect antibody binding thereto, as discussed in the Examples. A Loop 2 region can comprise the following amino acid sequence: C4 GPARLLPNAIGRGKWWRPSGPDFRC5 (SEQ ID NO:6). “C4” refers to a cysteine residue located at position 86 with reference to SEQ ID NO:1. “C5” refers to a cysteine residue located at position 111 with reference to SEQ ID NO:1. In native sclerostin protein, C4 is linked to a cysteine at position 144 (C8) by a disulfide bond, and C5 is linked to a cysteine at position 57 (C1) by a disulfide bond. Epitopes derived from the Loop 2 region include CGPARLLPNAIGRGKWWRPS (SEQ ID NO:63); GPARLLPNAIGRGKWWRPSG (SEQ ID NO:64); PARLLPNAIGRGKWWRPSGP (SEQ ID NO:65); ARLLPNAIGRGKWWRPSGPD (SEQ ID NO:66); RLLPNAIGRGKWWRPSGPDF (SEQ ID NO:67); LLPNAIGRGKWWRPSGPDFR (SEQ ID NO:68); and LPNAIGRGKWWRPSGPDFRC (SEQ ID NO:69)
CROSS-BLOCKING ASSAYS
The terms “cross-block”, “cross-blocked” and “cross-blocking” are used interchangeably herein to mean the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to sclerostin.
The extent to which an antibody or other binding agent is able to interfere with the binding of another to sclerostin, and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays. One particularly suitable quantitative assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between antibodies or other binding agents in terms of their binding to sclerostin.
Biacore Cross-Blocking Assay
The following generally describes a suitable Biacore assay for determining whether an antibody or other binding agent cross-blocks or is capable of cross-blocking according to the invention. For convenience reference is made to two antibodies, but it will be appreciated that the assay can be used with any of the sclerostin binding agents described herein. The Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations.
Thus in one cross-blocking assay, sclerostin is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a sclerostin-coated surface. Typically 200-800 resonance units of sclerostin would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used).
The two antibodies (termed A* and B*) to be assessed for their ability to cross-block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture. When calculating the concentrations on a binding site basis the molecular weight of an antibody is assumed to be the total molecular weight of the antibody divided by the number of sclerostin binding sites on that antibody.
The concentration of each antibody in the test mix should be high enough to readily saturate the binding sites for that antibody on the sclerostin molecules captured on the Biacore chip. The antibodies in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis).
Separate solutions containing antibody A* alone and antibody B* alone are also prepared. Antibody A* and antibody B* in these solutions should be in the same buffer and at the same concentration as in the test mix.
The test mixture is passed over the sclerostin-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound antibodies without damaging the chip-bound sclerostin. Typically this is done by treating the chip with 30 mM HCl for 60 seconds.
The solution of antibody A* alone is then passed over the sclerostin-coated surface and the amount of binding recorded. The chip is again treated to remove all of the bound antibody without damaging the chip-bound sclerostin.
The solution of antibody B* alone is then passed over the sclerostin-coated surface and the amount of binding recorded.
The maximum theoretical binding of the mixture of antibody A* and antibody B* is next calculated, and is the sum of the binding of each antibody when passed over the sclerostin surface alone. If the actual recorded binding of the mixture is less than this theoretical maximum then the two antibodies are cross-blocking each other.
Thus, in general, a cross-blocking antibody or other binding agent according to the invention is one which will bind to sclerostin in the above Biacore cross-blocking assay such that during the assay and in the presence of a second antibody or other binding agent of the invention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two antibodies or binding agents in combination.
The Biacore assay described above is a primary assay used to determine if antibodies or other binding agents cross-block each other according to the invention. On rare occasions particular antibodies or other binding agents may not bind to sclerostin coupled via amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on sclerostin is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of Sclerostin, for example N-terminal His-tagged Sclerostin (R & D Systems, Minneapolis, Minn., USA; 2005 cat#1406-ST-025). In this particular format, an anti-His antibody would be coupled to the Biacore chip and then the His-tagged Sclerostin would be passed over the surface of the chip and captured by the anti-His antibody. The cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged sclerostin would be loaded back onto the anti-His antibody coated surface. In addition to the example given using N-terminal His-tagged Sclerostin, C-terminal His-tagged sclerostin could alternatively be used. Furthermore, various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin).
ELISA-Based Cross-Blocking Assay
The following generally describes an ELISA assay for determining whether an anti-sclerostin antibody or other sclerostin binding agent cross-blocks or is capable of cross-blocking according to the invention. For convenience, reference is made to two antibodies (Ab-X and Ab-Y), but it will be appreciated that the assay can be used with any of the sclerostin binding agents described herein.
The general principal of the assay is to have an anti-sclerostin antibody coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-sclerostin antibody is added in solution (i.e. not bound to the ELISA plate). A limited amount of sclerostin is then added to the wells. The coated antibody and the antibody in solution compete for binding of the limited number of sclerostin molecules. The plate is washed to remove sclerostin that has not been bound by the coated antibody and to also remove the second, solution phase antibody as well as any complexes formed between the second, solution phase antibody and sclerostin. The amount of bound sclerostin is then measured using an appropriate sclerostin detection reagent. An antibody in solution that is able to cross-block the coated antibody will be able to cause a decrease in the number of sclerostin molecules that the coated antibody can bind relative to the number of sclerostin molecules that the coated antibody can bind in the absence of the second, solution phase, antibody.
This assay is described in more detail further below for Ab-X and Ab-Y. In the instance where Ab-X is chosen to be the immobilized antibody, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of Ab-Y is then added to the ELISA plate such that the moles of Ab-Y sclerostin binding sites per well are at least 10 fold higher than the moles of Ab-X sclerostin binding sites that were used, per well, during the coating of the ELISA plate. Sclerostin is then added such that the moles of sclerostin added per well are at least 25-fold lower than the moles of Ab-X sclerostin binding sites that were used for coating each well. Following a suitable incubation period the ELISA plate is washed and a sclerostin detection reagent is added to measure the amount of sclerostin specifically bound by the coated anti-sclerostin antibody (in this case Ab-X). The background signal for the assay is defined as the signal obtained in wells with the coated antibody (in this case Ab-X), second solution phase antibody (in this case Ab-Y), sclerostin buffer only (i.e. no sclerostin) and sclerostin detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated antibody (in this case Ab-X), second solution phase antibody buffer only (i.e. no second solution phase antibody), sclerostin and sclerostin detection reagents. The ELISA assay needs to be run in such a manner so as to have the positive control signal be at least 6 times the background signal.
To avoid any artifacts (e.g. significantly different affinities between Ab-X and Ab-Y for sclerostin) resulting from the choice of which antibody to use as the coating antibody and which to use as the second (competitor) antibody, the cross-blocking assay needs to be run in two formats:

    
    
        1) format 1 is where Ab-X is the antibody that is coated onto the ELISA plate and Ab-Y is the competitor antibody that is in solution and
        2) format 2 is where Ab-Y is the antibody that is coated onto the ELISA plate and Ab-X is the competitor antibody that is in solution.
    
    


Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-sclerostin antibody is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the sclerostin detection signal (i.e. the amount of sclerostin bound by the coated antibody) as compared to the sclerostin detection signal obtained in the absence of the solution phase anti-sclerostin antibody (i.e. the positive control wells).
An example of such an ELISA-based cross blocking assay can be found in Example 7 (“ELISA-based cross-blocking assay”).
Cell Based Neutralization Assay
Mineralization by osteoblast-lineage cells in culture, either primary cells or cell lines, is used as an in vitro model of bone formation. Mineralization takes from about one to six weeks to occur beginning with the induction of osteoblast-lineage cell differentiation by one or more differentiation agents. The overall sequence of events involves cell proliferation, differentiation, extracellular matrix production, matrix maturation and finally deposition of mineral, which refers to crystallization and/or deposition of calcium phosphate. This sequence of events starting with cell proliferation and differentiation, and ending with deposition of mineral is referred to herein as mineralization. Measurement of calcium (mineral) is the output of the assay.
MC3T3-E1 cells (Sudo H, Kodama H-A, Amagai Y, Yamamoto S, Kasai S. 1983. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 96:191-198) and subclones of the original cell line can form mineral in culture upon growth in the presence of differentiating agents. Such subclones include MC3T3-E1-BF (Smith E, Redman R, Logg C, Coetzee G, Kasahara N, Frenkel B. 2000. Glucocorticoids inhibit developmental stage-specific osteoblast cell cycle. J. Biol. Chem. 275:19992-20001). For both the MC3T3-E1-BF subclone as well as the original MC3T3-E1 cells, sclerostin can inhibit one or more of the sequence of events leading up to and including mineral deposition (i.e. sclerostin inhibits mineralization). Anti-sclerostin antibodies that are able to neutralize sclerostin's inhibitory activity allow for mineralization of the culture in the presence of sclerostin such that there is a statistically significant increase in deposition of calcium phosphate (measured as calcium) as compared to the amount of calcium measured in the sclerostin-only (i.e. no antibody) treatment group. The antibodies used in the cell based mineralization assay experiments shown in FIGS. 22, 23 and 24 have molecular weights of about 145 Kd and have 2 sclerostin binding sites per antibody molecule.
When running the assay with the goal of determining whether a particular anti-sclerostin antibody or anti-sclerostin binding agent can neutralize sclerostin (i.e., is a sclerostin neutralizing antibody or derivative thereof, or is a sclerostin neutralizing binding agent), the amount of sclerostin used in the assay needs to be the minimum amount of sclerostin that causes at least a 70%, statistically significant, reduction in deposition of calcium phosphate (measured as calcium) in the sclerostin-only group, as compared to the amount of calcium measured in the no sclerostin group. An anti-sclerostin neutralizing antibody or an anti-sclerostin neutralizing binding agent is defined as one that causes a statistically significant increase in deposition of calcium phosphate (measured as calcium) as compared to the amount of calcium measured in the sclerostin-only (i.e. no antibody, no binding agent) treatment group. To determine whether an anti-sclerostin antibody or an anti-sclerostin binding agent is neutralizing or not, the amount of anti-sclerostin antibody or anti-sclerostin binding agent used in the assay needs to be such that there is an excess of moles of sclerostin binding sites per well as compared to the number of moles of sclerostin per well. Depending on the potency of the antibody, the fold excess that may be required can be 24, 18, 12, 6, 3, or 1.5, and one of skill is familiar with the routine practice of testing more than one concentration of binding agent. For example, a very potent anti-sclerostin neutralizing antibody or anti-sclerostin neutralizing binding agent will be able to neutralize sclerostin even when there is less than a 6-fold excess of moles of sclerostin binding sites per well as compared to the number of moles of sclerostin per well. A less potent anti-sclerostin neutralizing antibody or anti-sclerostin neutralizing binding agent will be able to neutralize sclerostin only at a 12, 18 or 24 fold excess. Sclerostin binding agents within this full range of potencies are suitable as neutralizing sclerostin binding agents. Exemplary cell based mineralization assays are described in detail in Example 8.
Anti-sclerostin antibodies and derivatives thereof that can neutralize human sclerostin, and sclerostin binding agents that can neutralize human sclerostin may be of use in the treatment of human conditions/disorders that are caused by, associated with, or result in at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength.
In Vivo Neutralization Assay
Increases in various parameters associated with, or that result from, the stimulation of new bone formation can be measured as an output from in vivo testing of sclerostin binding agents in order to identify those binding agents that are able to neutralize sclerostin and thus able to cause stimulation of new bone formation. Such parameters include various serum anabolic markers [e.g. osteocalcin, P1NP (n-terminal propeptide of type 1 procollagen)], histomorphometric markers of bone formation (e.g. osteoblast surface/bone surface; bone formation rate/bone surface; trabecular thickness), bone mineral density, bone mineral content, bone mass, bone quality and bone strength. A sclerostin neutralizing binding agent is defined as one capable of causing a statistically significant increase, as compared to vehicle treated animals, in any parameter associated with, or that results from, the stimulation of new bone formation. Such in vivo testing can be performed in any suitable mammal (e.g. mouse, rat, monkey). An example of such in vivo testing can be found in Example 5 (“In vivo testing of anti-sclerostin monoclonal antibodies”).
Although the amino acid sequence of sclerostin is not 100% identical across mammalian species (e.g. mouse sclerostin is not 100% identical to human sclerostin), it will be appreciated by one skilled in the art that a sclerostin binding agent that can neutralize, in vivo, the sclerostin of a certain species (e.g. mouse) and that also can bind human sclerostin in vitro is very likely to be able to neutralize human sclerostin in vivo. Thus, such a human sclerostin binding agent (e.g. anti-human sclerostin antibody) may be of use in the treatment of human conditions/disorders that are caused by, associated with, or result in at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength. Mice in which homologous recombination had been used to delete the mouse sclerostin gene and insert the human sclerostin gene in its place (i.e. human sclerostin gene knock-in mice or human SOST knock-in mice) would be an example of an additional in vivo system.
Pharmaceutical compositions are provided, comprising one of the above-described binding agents such as at least one of antibody Ab-A, Ab-B, Ab-C, Ab-D and Ab-1 to Ab-24 to human sclerostin, along with a pharmaceutically or physiologically acceptable carrier, excipient, or diluent. Pharmaceutical compositions and methods of treatment are disclosed in copending application Ser. No. 10/868,497, filed Jun. 16, 2004, which claims priority to Ser. No. 60/478,977, both of which are incorporated by reference herein.
The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., subcutaneous, oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, is well known in the art, some of which are briefly discussed below for general purposes of illustration.
In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein subcutaneously, parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.
Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Remington's Pharmaceutical Sciences, 15th ed., pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards.
In another embodiment of the invention, the compositions disclosed herein may be formulated in a neutral or salt form. Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like, are used for the introduction of the compositions of the present invention into suitable host cells/organisms. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Alternatively, compositions of the present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles.
The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see for example, Lasic, Trends Biotechnol. 16(7):307-21, 1998; Takakura, Nippon Rinsho 56(3):691-95, 1998; Chandran et al., Indian J. Exp. Biol. 35(8):801-09, 1997; Margalit, Crit. Rev. Ther. Drug Carrier Syst. 12(2-3):233-61, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporated herein by reference in its entirety). The use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery. In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)).
Alternatively, in other embodiments, the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (see, for example, Quintanar-Guerrero et al., Drug Dev. Ind. Pharm. 24(12):1113-28, 1998). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) may be designed using polymers able to be degraded in vivo. Such particles can be made as described, for example, by Couvreur et al., Crit. Rev. Ther. Drug Carrier Syst. 5(1):1-20, 1988; zur Muhlen et al., Eur. J. Pharm. Biopharm. 45(2):149-55, 1998; Zambaux et al., J. Controlled Release 50(1-3):31-40, 1998; and U.S. Pat. No. 5,145,684.
In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.
The dose administered may range from 0.01 mg/kg to 100 mg/kg of body weight. As will be evident to one of skill in the art, the amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth. Typically, the compositions may be administered by a variety of techniques, as noted above.
Increases in bone mineral content and/or bone mineral density may be determined directly through the use of X-rays (e.g., Dual Energy X-ray Absorptometry or “DEXA”), or by inference through the measurement of 1) markers of bone formation and/or osteoblast activity, such as, but not limited to, osteoblast specific alkaline phosphatase, osteocalcin, type 1 procollagen C′ propeptide (PICP), total alkaline phosphatase (see Cornier, Curr. Opin. in Rheu. 7:243 (1995)) and serum procollagen 1 N-terminal propeptide (P1NP) and/or 2) markers of bone resorption and/or osteoclast activity including, but not limited to, pyridinoline, deoxypryridinoline, N-telopeptide, urinary hydroxyproline, plasma tartrate-resistant acid phosphatases, and galactosyl hydroxylysine; (see Comier, id), serum TRAP 5b (tartrate-resistant acid phosphatase isoform 5b) and serum cross-linked C-telopeptide (sCTXI). The amount of bone mass may also be calculated from body weights or by using other methods (see Guinness-Hey, Metab. Bone Dis. Relat. Res. 5:177-181, 1984). Animals and particular animal models are used in the art for testing the effect of the compositions and methods of the invention on, for example, parameters of bone loss, bone resorption, bone formation, bone strength or bone mineralization that mimic conditions of human disease such as osteoporosis and osteopenias. Examples of such models include the ovariectomized rat model (Kalu, D. N., The ovariectomized rat model of postmenopausal bone loss. Bone and Mineral 15:175-192 (1991); Frost, H. M. and Jee, W. S. S. On the rat model of human osteopenias and osteoporosis. Bone and Mineral 18:227-236 (1992); and Jee, W. S. S, and Yao, W., Overview: animal models of osteopenia and osteoporosis. J. Musculoskel. Neuron. Interact. 1:193-207 (2001)).
Particular conditions which may be treated by the compositions of the present invention include dysplasias, wherein growth or development of bone is abnormal and a wide variety of causes of osteopenia, osteoporosis and bone loss. Representative examples of such conditions include achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher's Disease, hypophosphatemic rickets, Marfan's syndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, pseudoarthrosis, and pyogenic osteomyelitis, periodontal disease, anti-epileptic drug induced bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism syndromes, weightlessness induced bone loss, osteoporosis in men, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative disorders of bone, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis, metabolic bone diseases, mastocytosis, sickle cell anemia/disease, organ transplant related bone loss, kidney transplant related bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritides, thalassemia, mucopolysaccharidoses, fabry disease, turner syndrome, Down Syndrome, Klinefelter Syndrome, leprosy, Perthes' Disease, adolescent idiopathic scoliosis, infantile onset multi-system inflammatory disease, Winchester Syndrome, Menkes Disease, Wilson's Disease, ischemic bone disease (such as Legg-Calve-Perthes disease, regional migratory osteoporosis), anemic states, conditions caused by steroids, glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, idiopathic osteopenia or osteoporosis, congenital osteopenia or osteoporosis, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis, osteomalacia, bone loss associated with joint replacement, HIV associated bone loss, bone loss associated with loss of growth hormone, bone loss associated with cystic fibrosis, fibrous dysplasia, chemotherapy associated bone loss, tumor induced bone loss, cancer-related bone loss, hormone ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, disease associated facial bone loss, disease associated cranial bone loss, disease associated bone loss of the jaw, disease associated bone loss of the skull, and bone loss associated with space travel. Further conditions relate to bone loss associated with aging, including facial bone loss associated with aging, cranial bone loss associated with aging, jaw bone loss associated with aging, and skull bone loss associated with aging.
Compositions of the present invention may also be useful for improving outcomes in orthopedic procedures, dental procedures, implant surgery, joint replacement, bone grafting, bone cosmetic surgery and bone repair such as fracture healing, nonunion healing, delayed union healing and facial reconstruction. One or more compositions may be administered before, during and/or after the procedure, replacement, graft, surgery or repair.
The invention also provides a diagnostic kit comprising at least one anti-sclerostin binding agent according to the present invention. The binding agent may be an antibody. In addition, such a kit may optionally comprise one or more of the following:

    
    
        (1) instructions for using the one or more binding agent(s) for screening, diagnosis, prognosis, therapeutic monitoring or any combination of these applications;
        (2) a labeled binding partner to the anti-sclerostin binding agent(s);
        (3) a solid phase (such as a reagent strip) upon which the anti-sclerostin binding agent(s) is immobilized; and
        (4) a label or insert indicating regulatory approval for screening, diagnostic, prognostic or therapeutic use or any combination thereof.

If no labeled binding partner to the binding agent(s) is provided, the binding agent(s) itself can be labeled with one or more of a detectable marker(s), e.g. a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.

    
    


The following examples are offered by way of illustration, and not by way of limitation.
EXAMPLES
Example 1
Recombinant Expression of Sclerostin
Recombinant human sclerostin/SOST is commercially available from R&D
Systems (Minneapolis, Minn., USA; 2006 cat#1406-ST-025). Additionally, recombinant mouse sclerostin/SOST is commercially available from R&D Systems (Minneapolis, Minn., USA; 2006 cat#1589-ST-025).
Alternatively, the different species of sclerostin can be expressed transiently in serum-free suspension adapted 293T or 293EBNA cells. Transfections can be performed as 500 mL or 1L cultures. The following reagents and materials are available from Gibco BRL (now Invitrogen, Carlsbad, Calif.). Catalog numbers are listed in parentheses: serum-free DMEM (21068-028); DMEM/F12 (3:1) (21068/11765); 1X Insulin-Transferrin-Selenium Supplement (51500-056); 1X Pen Strep Glut (10378-016); 2 mM 1-Glutamine (25030-081); 20 mM HEPES (15630-080); 0.01% Pluronic F68 (24040-032). Briefly, the cell inoculum (5.0-10.0×105 cells/mL×culture volume) is centrifuged at 2,500 RPM for 10 minutes at 4° C. to remove the conditioned medium.
The cells are resuspended in serum-free DMEM and centrifuged again at 2,500 RPM for 10 minutes at 4° C. After aspirating the wash solution, the cells are resuspended in growth medium [DMEM/F12 (3:1)+1X Insulin-Transferrin-Selenium Supplement+1X Pen Strep Glut+2 mM L-Glutamine+20 mM HEPES+0.01% Pluronic F68] in a 1L or 3L spinner flask culture. The spinner flask culture is maintained on magnetic stir plate at 125 RPM which is placed in a humidified incubator maintained at 37° C. and 5% CO2. The mammalian expression plasmid DNA (e.g. pcDNA3.1, pCEP4, Invitrogen Life Technologies, Carlsbad, Calif.), containing the complete coding region (and stop codon) of sclerostin with a Kozak consensus sequence (e.g., CCACC) directly 5′ of the start site ATG, is complexed to the transfection reagent in a 50 mL conical tube.
The DNA-transfection reagent complex can be prepared in 5-10% of the final culture volume in serum-free DMEM or OPTI-MEM. The transfection reagents that can be used for this purpose include X-tremeGene RO-1539 (Roche Applied Science, Indianapolis, Ind.), FuGene6 (Roche Applied Science, Indianapolis, Ind.), Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) and 293fectin (Invitrogen, Carlsbad, Calif.). 1-5 μg plasmid DNA/mL culture is first added to serum-free DMEM, followed by 1-5 μl transfection reagent/mL culture. The complexes can be incubated at room temperature for approximately 10-30 minutes and then added to the cells in the spinner flask. The transfection/expression can be performed for 4-7 days, after which the conditioned medium (CM) is harvested by centrifugation at 4,000 RPM for 60 minutes at 4° C.
Example 2
Purification of Recombinant Sclerostin
Recombinant sclerostin was purified from mammalian host cells as follows. All purification processes were carried out at room temperature. One purification scheme was used to purify various species of sclerostin, including murine and human sclerostin. The purification scheme used affinity chromatography followed by cation exchange chromatography.
Heparin Chromatography
The mammalian host cell conditioned medium (CM) was centrifuged in a Beckman J6-M1 centrifuge at 4000 rpm for 1 hour at 4° C. to remove cell debris. The CM supernatant was then filtered through a sterile 0.2 μm filter. (At this point the sterile filtered CM may be optionally stored frozen until purification.) If the CM was frozen, it was thawed at the following temperatures, or combination thereof: 4° C., room temperature or warm water. Following thawing the CM was filtered through a sterile 0.2 μm filter and optionally concentrated by tangential flow ultrafiltration (TFF) using a 10 kD molecular weight cut-off membrane. The CM concentrate was filtered through a sterile 0.2 μm filter and then loaded onto a Heparin High Performance (Heparin HP) column (GE Healthcare, formerly Amersham Biosciences) equilibrated in PBS. Alternatively, the filtered CM supernatant may be loaded directly onto the Heparin HP column equilibrated in PBS.
After loading, the Heparin HP column was washed with PBS until the absorbance at 280 nm of the flow-through returned to baseline (i.e., absorbance measured before loading CM supernatant). The sclerostin was then eluted from the column using a linear gradient from 150 mM to 2M sodium chloride in PBS. The absorbance at 280 nm of the eluate was monitored and fractions containing protein were collected. The fractions were then assayed by Coomassie-stained SDS-PAGE to identify fractions containing a polypeptide that migrates at the size of glycosylated sclerostin. The appropriate fractions from the column were combined to make the Heparin HP pool.
Cation Exchange Chromatography
The sclerostin eluted from the Heparin HP column was further purified by cation exchange chromatography using SP High Performance (SPHP) chromatography media (GE Healthcare, formerly Amersham Biosciences). The Heparin HP pool was buffer exchanged into PBS by dialysis using 10,000 MWCO membranes (Pierce Slide-A-Lyzer). The dialyzed Heparin HP pool was then loaded onto an SPHP column equilibrated in PBS. After loading, the column was washed with PBS until the absorbance at 280 nm of the flow-through returned to baseline. The sclerostin was then eluted from the SPHP column using a linear gradient from 150 mM to 1 M sodium chloride in PBS. The absorbance at 280 nm of the eluate was monitored and the eluted sclerostin was collected in fractions. The fractions were then assayed by Coomassie-stained SDS-PAGE to identify fractions containing a polypeptide that migrates at the size of glycosylated sclerostin. The appropriate fractions from the column were combined to make the SPHP pool.
Formulation
Following purification, the SPHP pool was formulated in PBS by dialysis using 10,000 MWCO membranes (Pierce Slide-A-Lyzer). If concentration of sclerostin was necessary, a centrifugal device (Amicon Centricon or Centriprep) with a 10,000 MWCO membrane was used. Following formulation the sclerostin was filtered through a sterile 0.2 μm filter and stored at 4° C. or frozen.
Example 3
Peptide Binding ELISA
A series of overlapping peptides (each peptide being approximately 20-25 amino acids long) were synthesized based on the known amino acid sequence of rat sclerostin (SEQ ID NO:98). The peptides were designed such that they all contained a reduced cysteine residue; an additional cysteine was included at the C-terminus of each peptide which did not already contain one in its sequence. This enabled the peptides to be bound to the assay plates by covalent coupling, using commercially available sulfhydryl binding plates (Costar), at a concentration of 1 μg/ml, in phosphate buffered saline (PBS: pH 6.5) containing 1 mM EDTA. Following incubation for 1 hour at room temperature, the plates were washed three times with PBS containing 0.5% Tween 20. The plates were blocked by incubation with a PBS solution containing 0.5% fish skin gelatin (Sigma) for 30 minutes at room temperature and then washed three times in PBS containing 0.5% Tween 20.
Antibodies to be tested were diluted to 1 μg/ml in PBS containing 0.5% fish skin gelatin and incubated with the peptide-coated plates for 1 hour at room temperature. Excess antibody was removed by three washes with PBS, 0.5% Tween 20. The plates were then incubated with an appropriate secondary antibody conjugated to horseradish peroxidase (diluted appropriately in PBS containing 0.5% Tween 20) and capable of binding to the antibody of interest. The plates were then washed three times: once with PBS containing 0.5% Tween 20, and twice with PBS. Finally the plates were incubated with a horseradish peroxidase chromogenic substrate (TMB-Stable Stop, RDI) for 5 minutes at room temperature, the color development was stopped with acid, and the plates' optical density measured at 450 nm.
Materials
Costar's Sulfhydryl Binding Plates (VWR #29442-278)
Coating Buffer: 1×PBS PH 6.5+1 mM EDTA
Blocking Buffer: 1×PBS+0.5% Fish Skin Gelatin (PBS from CS; FSG from Sigma# G 7765)
Wash Buffer: 1X PBS+0.5% Tween 20
Rat Sclerostin peptides
Antibody Samples Transient Ab, Purified recombinant Ab, rabbit Serum, etc.
Appropriate secondary Ab: Goat-anti-Rabbit/Mouse-HRP (Jackson Immuno Research, 115-036-072)
TMB-Stable Stop (RDI# RDI-TMBSX-1L)
0.5M HCl
Methods were as follows:


    1. Coat plates with 100 μl/well of rat sclerostin peptide diluted in 1×PBS PH 6.5+1 mM EDTA at 1 μg/ml. Incubate plates 1 hour at room temperature. (Plates should be used within 30 minutes of opening).
    2. Wash plates 3× with wash buffer.
    3. Block plates with 200 ul/well blocking buffer. Incubate plates 30 minutes at room temp.
    4. Repeat washing as described in (2).
    5. Incubate plates with 50 ul/well of samples diluted in blocking buffer—Serum titers starting at 1:100; Transient Recombinant Ab use neat; Purified recombinant Ab use at 1 μg/ml (all samples run in duplicates). Incubate plates 1 h at room temp.
    6. Wash plates as described in (2).
    7. Incubate plates with 50 μl/well of appropriate Secondary Antibody (HRP labeled) diluted 1:1600 in Blocking Buffer. Incubate plates 1 hour at room temperature.
    8. Wash plates 1× wash buffer, 2×PBS
    9. Incubate plates with 50 μl/well of TMB, 5 minutes at room temp.
    10. Stop reaction with 50 μl/well 0.5M HCl.
    11. Read plates at 450 nm wavelength.


The following peptides sequences were screened as described above:









(SEQ ID NO: 82)



QGWQAFKNDATEIIPGLREYPEPP



 



(SEQ ID NO: 83)



TEIIPGLREYPEPPQELENN



 



(SEQ ID NO: 84)



PEPPQELENNQTMNRAENGG



 



(SEQ ID NO: 85)



ENGGRPPHHPYDTKDVSEYS



 



(SEQ ID NO: 86)



CRELHYTRFVTDGP



 



(SEQ ID NO: 87)



CRELHYTRFVTDGPSRSAKPVTELV



 



(SEQ ID NO: 88)



CRSAKPVTELVSSGQSGPRARLL



 



(SEQ ID NO: 89)



CGPARLLPNAIGRVKWWRPNGPDFR



 



(SEQ ID NO: 90)



RAQRVQLLCPGGAAPRSRKV



 



(SEQ ID NO: 91)



PGGAAPRSRKVRLVAS



 



(SEQ ID NO: 92)



KRLTRFHNQSELKDFGPETARPQ



 



(SEQ ID NO: 93)



IPDRYAQRVQLLSPGG



 



(SEQ ID NO: 94)



SELKDFGPETARPQKGRKPRPRAR



 



(SEQ ID NO: 95)



KGRKPRPRARGAKANQAELENAY



 



(SEQ ID NO: 96)



PNAIGRVKWWRPNGPDFR



 



(SEQ ID NO: 97)



KWWRPNGPDFRCIPDRYRAQRV.






A high-affinity neutralizing antibody (Ab-19) bound to two overlapping peptide sequences: PNAIGRVKWWRPNGPDFR (SEQ ID NO:96) and KWWRPNGPDFRCIPDRYRAQRV (SEQ ID NO:97).
This procedure allows the recognition of epitopes for antibodies that react with apparent linear epitopes. Peptides that contain all or part of the antibody binding site will bind antibody and thus be detected.
Example 4
Identification of Human Sclerostin Epitopes
Sclerostin Structure
Mature form (signal peptide removed) human sclerostin is a 190 amino acid protein (FIG. 8). FIG. 9 shows a schematic of the general structure of sclerostin with an N-terminal arm (from the N-terminal Q to Cysteine1) and a C-terminal arm (from Cysteine-8 to the terminal Y). Sandwiched in between these two arms there is the cystine-knot structure and three loops which are designated Loop1, Loop2 and Loop 3. The four disulfide bonds in sclerostin are Cys1 at sequence position 57 linked to Cys5 at sequence position 111 (referred to as C1-C5), Cys2 at sequence position 71 linked to Cys6 at sequence position 125 (referred to as C2-C6), Cys3 at sequence position 82 linked to Cys7 at sequence position 142 (referred to as C3-C7), Cys4 at sequence position 86 linked to Cys8 at sequence position 144 (referred to as C4-C8). The eight-membered ring structure is formed via C3-C7 and C4-C8 disulfide bonding. This ring structure, together with the C1-C5 disulfide bond penetrating through the ring, forms a typical cystine-knot. C2-C6, which is not part of the cystine-knot, brings two large loop structures, loop 1 (residues 57 to 82) and loop 3 (residues 111 to 142) close together. Loop 2 goes from C4 (residue 86) to C5 (residue 111).
EXPERIMENTAL
The general approach for characterizing the epitopes bound by anti-sclerostin monoclonal antibodies involved fragmenting human Sclerostin into peptides with different proteases, determining the sequence of the various human sclerostin peptides, isolating these peptides and testing each of them for their ability to bind to a particular monoclonal antibody using a Biacore-based “human sclerostin peptide epitope competition binding assay.”. The resulting data permitted the location of the binding epitope to be determined.
The peptide digests were subjected to HPLC peptide mapping; the individual peaks were collected, and the peptides identified and mapped by matrix assisted laser desorption mass spectrometry (MALDI-MS) and electrospray ionization LC-MS (ESI-LC-MS) analyses and/or by N-terminal sequencing. All HPLC analyses for these studies were performed using a reverse-phase C8 column (2.1 mm i.d.×15 cm length). HPLC peptide mapping was performed with a linear gradient from 0.05% trifloroacetic acid (mobile phase A) to 90% acetonitrile in 0.05% trifluoroacetic acid. Columns were developed over 50 minutes at a flow rate of 0.2 ml/min.
Trypsin and AspN Endoproteinase Digestions
Mature form human sclerostin was digested with trypsin, which cleaves after arginine and lysine, or with AspN. About 200 μg of sclerostin at 0.5-1.0 mg/ml was incubated in PBS (pH 7.2) for 20 hrs at 37° C. with 8 μg of either trypsin or AspN.
Trypsin Digestion
HPLC chromatography of the trypsin digests yielded several major peaks (FIG. 10A). Sequence analysis was conducted on the peptide peaks recovered from HPLC after trypsin digestion. On-line ESI LC-MS analysis of the peptide digest was also performed to determine the precise mass of the peptides that were separated by HPLC. The identity of the peptides present in the peptide peaks was thus determined (FIG. 11). FIG. 13 shows the alignment of various peptide sequences (T19.2, T20, T20.6, T21-22) along the sclerostin sequence. The number following each T (e.g., T19.2) reflects the retention time. T19.2 contains two peptides (one from loop 1 and one from loop 3) linked by the C2-C6 disulfide bond. T20 contains two peptides held together by the cystine-knot structure, with intact loops 1 and 3 held together by the C2-C6 disulfide and with most of loop 2 absent. T20.6 contains four sequences held together by the cystine-knot structure, but is missing part of loop 1 and 3 (the T19.2 part) and is missing most of loop 2. T21-22 is almost identical to T20 but has 3 additional amino acids in the loop 2 region.
AspN Digestion
HPLC chromatography of the AspN digests yielded several major peaks (FIG. 10B). Sequence analysis was conducted on the peptide peaks recovered from HPLC. On-line ESI LC-MS analysis of the peptide digest was also performed to determine the precise mass of the peptides that were separated by HPLC. The identity of the peptides present in the peptide peaks from the AspN digestion was thus determined (FIG. 12). FIG. 14 shows the alignment of various peptide sequences (AspN14.6, AspN18.6, AspN22.7-23.5) along the sclerostin sequence. The number following each AspN (e.g. AspN18.6) reflects the retention time. AspN14.6 contains three short peptides from both the N- and C-terminal arms of sclerostin, while AspN18.6 is a larger peptide from the N-terminal arm of sclerostin. AspN22.7-23.5 contains a single peptide fragment of 104 amino acids the encompasses all eight cysteines (the four disulfide bonds), the cystine-knot and all of loops 1, 2 and 3.
The strategy for characterizing the epitopes was to use these various trypsin and AspN generated human sclerostin peptides and determine which peptides could still be bound by the various Antibodies (Ab-A, Ab-B, Ab-C and Ab-D). Specifically this was tested in a Biacore-based “human sclerostin peptide epitope competition binding assay” where the binding of a particular monoclonal antibody to human sclerostin immobilized on the Biacore chip was determine in the presence or absence of each of the various isolated trypsin and AspN HPLC peptide fractions. In the absence of any competing peptides, the particular monoclonal antibody was able to bind the human sclerostin on the chip and produce a resonance unit, RU, response. Preincubation of the particular monoclonal antibody with intact human sclerostin in solution, followed by testing of binding to the chip, demonstrated that the binding of the Mab to human sclerostin in solution prevented the binding of the Mab to the human sclerostin on the chip, thus validating the general principal of this competition assay.
This general procedure was repeated individually for each peptide. A robust RU response was taken to indicate that the particular peptide being tested could not bind the Mab in solution (hence the Mab was free to bind the human sclerostin that had been immobilized on the chip). Conversely, the absence of a robust RU response indicated that the Mab was able to bind the sclerostin peptide in solution. These binding patterns, couple with the known identity of the various sclerostin peptides, were used to determine the epitopes of sclerostin that were bound by anti-sclerostin antibodies Ab-A, Ab-B, Ab-C and Ab-D.
Biacore-Based Human Sclerostin Peptide Epitope Competition Binding Assay
Preparation of Human Sclerostin Surface:
Immobilization of mature form human sclerostin to a BIAcore sensor chip (CM5) surface was performed according to manufacturer's instructions. Briefly, carboxyl groups on the sensor chip surfaces were activated by injecting 60 μL of a mixture containing 0.2 M N-ethyl-N′-(dimethylaminopropyl) carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS). Human sclerostin was diluted in 10 mM sodium acetate, pH 4.0 at a concentration of 20 μg/mL followed by injecting over the activated CM5 surface. Excess reactive groups on the surfaces were deactivated by injecting 60 μL of 1 M ethanolamine. Final immobilized levels were  ̃5000 resonance units (RU) for the human sclerostin surface. A blank, mock-coupled reference surface was also prepared on the sensor chips.
Binding Specificity Analysis:
1X Phosphate-buffered saline without calcium chloride or magnesium chloride was from Gibco/Invitrogen, Carlsbad, Calif. Bovine serum albumin, fraction V, IgG-free was from Sigma-Aldrich, St. Louis, Mo. Each Mab (2 nM) was separately incubated with 20 nM human sclerostin or a particular human sclerostin peptide (note: there are 3 unlinked peptides in AspN14.6) in sample buffer (1X PBS+0.005% P-20+0.1 mg/mL BSA) before injection over the immobilized human sclerostin surface. The flow rate for sample injection was 5 μL/min followed by surface regeneration using 1 M NaCl in 8 mM Glycine, pH 2.0 at 30 μL/min for 30 seconds. The data was analyzed using BIAevaluation 3.2, and is presented in FIG. 15 (Ab-A), FIG. 16 (Ab-B), FIG. 17 (Ab-C) and FIG. 18 (Ab-D).
Loop 2 and T20.6 Epitopes:
The sclerostin peptide binding pattern for two representative antibodies (Ab-A and Ab-B) were virtually identical (FIG. 15 and FIG. 16) and showed that both of these Antibodies could only bind the AspN22.7-23.5 peptide. The unique difference between AspN22.7-23.5 and all the other sclerostin peptides is that AspN22.7-23.5 contains an intact loop 2. This shows that Ab-A and Ab-B bind the loop 2 region of sclerostin thus defining the loop 2 epitope (FIG. 19A). The sclerostin peptide binding pattern for Ab-C and Ab-D were virtually identical to each other (FIG. 17 and FIG. 18) but completely distinct from that found for Ab-A and Ab-B. Of the peptides tested in this Example, the most diminutive peptide that Ab-C and Ab-D could bind to was the T20.6 peptide. This result defines the T20.6 epitope (FIG. 19B).
Protease Protection Assay:
The general principle of this assay is that binding of a Mab to sclerostin can result in protection of certain specific protease cleavage sites and this information can be used to determine the region of sclerostin to where the Mab binds.
“T20.6 Derivative 1 (Cystine-Knot+4 Arms)” Epitope:
FIG. 20 shows the HPLC peptide maps for a human sclerostin Ab-D complex (FIG. 20A: human sclerostin was preincubated at a 1:1 molar ratio with Ab-D prior to digestion with trypsin as described above) and human sclerostin alone (FIG. 20B: human sclerostin was digested with trypsin as described above). The peptide peaks of T19.2 and T20.6 in FIG. 20A showed a clear reduction in their respective peak height, as compared to FIG. 20B. This reduction in peak heights was accompanied by an increase in peak height for peptides T20 and T21-22. These data indicate that basic amino acid residues in loop 1 and loop 3, which in the absence of Ab-D were cleaved by trypsin to generate peptides T19.2 and T20.6, were resistant to cleavage by trypsin when Ab-D was prebound to sclerostin. The presence of T20, T20.6 and T21-22 indicates that loop 2 was still cleaved efficiently when Ab-D was prebound to sclerostin. These data indicate that Ab-D bound on the loop 1 and loop 3 side of the T20.6 epitope thus defining the smaller “T20.6 derivative 1 (cystine-knot+4 arms)” epitope shown in FIG. 21.
Example 5
In Vivo Testing of Anti-Sclerostin Monoclonal Antibodies in Mice
Four week-old BDF1 male mice were obtained from Charles River Laboratories (Raleigh, N.C.) and housed in clean caging, five animals per cage. Room temperature was maintained between 68 and 72° F., and relative humidity was maintained between 34 and 73%. The laboratory housing the cages had a 12-hour light/dark cycle and met all AAALAC specifications. Clinical observations of all mice on study occurred once daily.
Purified anti-sclerostin monoclonal antibodies (Ab-A FIG. 1; Ab-B FIG. 2; Ab-C FIG. 3; Ab-D FIG. 4) were diluted in sterile Dulbecco's phosphate buffered saline. Mice were injected with anti-sclerostin Antibodies or PBS vehicle subcutaneously at 21 μl per gram body weight, two times per week (Monday and Thursday) at 25 mg/kg. Human PTH (1-34) was diluted in PTH buffer (0.001 N HCl, 0.15 M NaCl, 2% BSA), and dosed subcutaneously at 21 μl per gram body weight five times per week (Monday, Tuesday, Wednesday, Thursday, Friday) at 100 μg/kg as a positive control (FIGS. 5 and 6). Number of mice per group was N=5 in FIGS. 5 and 6, and N=6 in FIG. 7.
PIXImus In Vivo Bone Densitometry
Bone mineral density (BMD) was determined weekly at the proximal tibial metaphysis and lumbar vertebrae by peripheral Dual Energy X-ray Absorptometry (pDEXA) with the PIXImus2 system from GE/Lunar Medical Systems, Madison, Wis. A 25 mm2 region of interest (ROI) was placed to include the proximal articular surface, the epiphysis, and the proximal end on the metaphysis of the tibia. A region of interest (ROI) was placed to include the lumbar vertebrae (L1-L5). The proximal tibia and lumbar regions were analyzed to determine total bone mineral density. Group means were reported±Standard Deviation and compared to the vehicle treatment group for statistical analysis.
Statistical Analysis
Statistical analysis was performed with a Dunnett's and Tukey-Kramer (using MS Excel and JMP v. 5.0. for the BMD data). Group means for each data set were considered significantly different when the P value was less than 0.05 (P<0.05).
Sclerostin Neutralizing Activity of Antibodies
The statistically significant increases in BMD as compared to vehicle seen for each of Ab-A (FIG. 5), Ab-B (FIG. 5), Ab-C (FIG. 6) and Ab-D (FIG. 7) demonstrates that these four antibodies are sclerostin neutralizing antibodies. Furthermore this data shows that, for anti-sclerostin antibodies that bind mouse sclerostin, treatment and analysis of mice as described above can be used to identify sclerostin neutralizing antibodies.
Example 6
Screening Assay for Antibodies that Block Binding of an Antibody to Human Sclerostin
Human sclerostin was coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a sclerostin coated surface. 300 resonance units of sclerostin were coupled to the surface.
The antibodies to be tested were diluted to a concentration of 200 ug/ml in HBS-EP buffer (being 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) Surfactant P20) and then mixed in a one to one molar ratio (on a binding site basis) to generate the test mixture. This test mixture thus contained each antibody at a concentration of 100 ug/ml (1.3 um on a binding site basis). Separate solutions containing each of the antibodies in the test mix alone were also prepared. These solutions contained the individual antibodies in HBS-EP buffer at a concentration of 100 ug/ml (1.3 um on a binding site basis).
20 μL of the test mixture was passed over the sclerostin-coated chip at a flow rate of 10 μL/min and the amount of binding recorded. The chip was then treated with two 60 second pulses of 30 mM HCl to remove all of the bound antibody. A solution containing only one of the antibodies of the test mixture (at 1.3 μM in the same buffer as the test mixture on a binding site basis) was then passed over the chip in the same manner as the test mixture and the amount of binding recorded. The chip was again treated to remove all of the bound antibody and finally a solution containing the other antibody from the test mixture alone (at 1.3 μM in the same buffer as the test mixture on a binding site basis) was passed over the chip and the amount of binding recorded.
The table below show the results from cross-blocking assays on a range of different antibodies. The values in each square of the table represent the amount of binding (in RU) seen when the antibodies (at 1.3 μM on a binding site basis) or buffer indicated in the top row of the table were mixed with the antibodies (at 1.3 uM on a binding site basis) or buffer indicated in the first column of the table.


















Buffer
Ab-4
Ab-13
Ab-A
Ab-3
Ab-19






















Buffer
−0.5
693
428.5
707.3
316.1
649.9


Ab-4
687.7
795.1
1018.2
860.5
869.3
822.5


Ab-13
425.6
1011.3
442.7
1108.4
431.9
1042.4


Ab-A
692.4
833.1
1080.4
738.5
946.2
868.1


Ab-3
305.5
845.1
428.2
952.2
344.4
895.7


Ab-19
618.1
788.6
1022.5
863.3
891.5
658.7









Using the mean binding value (in RU) for each combination of antibodies in the above table (since each combination appears twice) it is possible to calculate the percentage of the theoretical binding shown by each combination of antibodies. The theoretical binding being calculated as the sum of the average values for the components of each test mixture when assayed alone (i.e., antibody and buffer).


















Buffer
Ab-4
Ab-13
Ab-A
Ab-3
Ab-19





















Buffer







Ab-4

90.75
60.45
85.4
60.75


Ab-13


96.9
58.0
97.0


Ab-A



93.5
65.0


Ab-3




94.4


Ab-19









From the above data it is clear that Ab-4, Ab-A and Ab-19 cross-block each other. Similarly Ab-13 and Ab-3 cross block each other.
Example 7
ELISA-Based Cross-Blocking Assay
Liquid volumes used in this example would be those typically used in 96-well plate ELISAs (e.g. 50-200 μl/well). Ab-X and Ab-Y, in this example are assumed to have molecular weights of about 145 Kd and to have 2 sclerostin binding sites per antibody molecule. An anti-sclerostin antibody (Ab-X) is coated (e.g. 50μ of 1 μg/ml) onto a 96-well ELISA plate [e.g. Corning 96 Well EIA/RIA Flat Bottom Microplate (Product #3590), Corning Inc., Acton, Mass.] for at least one hour. After this coating step the antibody solution is removed, the plate is washed once or twice with wash solution (e.g., PBS and 0.05% Tween 20) and is then blocked using an appropriate blocking solution (e.g., PBS, 1% BSA, 1% goat serum and 0.5% Tween 20) and procedures known in the art. Blocking solution is then removed from the ELISA plate and a second anti-sclerostin antibody (Ab-Y), which is being tested for it's ability to cross-block the coated antibody, is added in excess (e.g. 50 μl of 10 μg/ml) in blocking solution to the appropriate wells of the ELISA plate. Following this, a limited amount (e.g. 50 μl of 10 ng/ml) of sclerostin in blocking solution is then added to the appropriate wells and the plate is incubated for at least one hour at room temperature while shaking. The plate is then washed 2-4 times with wash solution. An appropriate amount of a sclerostin detection reagent [e.g., biotinylated anti-sclerostin polyclonal antibody that has been pre-complexed with an appropriate amount of a streptavidin-horseradish peroxidase (HRP) conjugate] in blocking solution is added to the ELISA plate and incubated for at least one hour at room temperature. The plate is then washed at least 4 times with wash solution and is developed with an appropriate reagent [e.g. HRP substrates such as TMB (colorimetric) or various HRP luminescent substrates]. The background signal for the assay is defined as the signal obtained in wells with the coated antibody (in this case Ab-X), second solution phase antibody (in this case Ab-Y), sclerostin buffer only (i.e. no sclerostin) and sclerostin detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated antibody (in this case Ab-X), second solution phase antibody buffer only (i.e. no second solution phase antibody), sclerostin and sclerostin detection reagents. The ELISA assay needs to be run in such a manner so as to have the positive control signal be at least 6 times the background signal.
To avoid any artifacts (e.g. significantly different affinities between Ab-X and Ab-Y for sclerostin) resulting from the choice of which antibody to use as the coating antibody and which to use as the second (competitor) antibody, the cross-blocking assay needs to be run in two formats:
1) format 1 is where Ab-X is the antibody that is coated onto the ELISA plate and Ab-Y is the competitor antibody that is in solution
and
2) format 2 is where Ab-Y is the antibody that is coated onto the ELISA plate and Ab-X is the competitor antibody that is in solution.
Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-sclerostin antibody is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the sclerostin detection signal (i.e. the amount of sclerostin bound by the coated antibody) as compared to the sclerostin detection signal obtained in the absence of the solution phase anti-sclerostin antibody (i.e. the positive control wells).
In the event that a tagged version of sclerostin is used in the ELISA, such as a N-terminal His-tagged Sclerostin (R&D Systems, Minneapolis, Minn., USA; 2005 cat#1406-ST-025) then an appropriate type of sclerostin detection reagent would include an HRP labeled anti-H is antibody. In addition to using N-terminal His-tagged Sclerostin, one could also use C-terminal His-tagged Sclerostin. Furthermore, various other tags and tag binding protein combinations that are known in the art could be used in this ELISA-based cross-blocking assay (e.g., HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin).
Example 8
Cell Based Mineralization Assay for Identifying Agents able to Antagonize Sclerostin Activity
Introduction
Mineralization by osteoblast-lineage cells in culture, either primary cells or cell lines, is used as an in vitro model of bone formation. Mineralization takes from about one to six weeks to occur beginning with the induction of osteoblast-lineage cell differentiation by one or more differentiation agents. The overall sequence of events involves cell proliferation, differentiation, extracellular matrix production, matrix maturation and finally deposition of mineral, which refers to crystallization and/or deposition of calcium phosphate. This sequence of events starting with cell proliferation and differentiation, and ending with deposition of mineral is referred to herein as mineralization. Measurement of calcium (mineral) is the output of the assay.
Deposition of mineral has a strong biophysical characteristic, in that once mineral “seeds” begin to form, the total amount of mineral that will be deposited in the entire culture can sometimes be deposited quite rapidly, such as within a few days thereafter. The timing and extent of mineral deposition in culture is influenced, in part, by the particular osteoblast-lineage cells/cell-line being used, the growth conditions, the choice of differentiation agents and the particular lot number of serum used in the cell culture media. For osteoblast-lineage cell/cell-line mineralization cultures, at least eight to fifteen serum lots from more than one supplier should be tested in order to identify a particular serum lot that allows for mineralization to take place.
MC3T3-E1 cells (Sudo H et al., In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 96:191-198) and subclones of the original cell line can form mineral in culture upon growth in the presence of differentiating agents. Such subclones include MC3T3-E1-BF (Smith E, Redman R, Logg C, Coetzee G, Kasahara N, Frenkel B. 2000. Glucocorticoids inhibit developmental stage-specific osteoblast cell cycle. J Biol Chem 275:19992-20001).
Identification of Sclerostin Neutralizing Antibodies
MC3T3-E1-BF cells were used for the mineralization assay. Ascorbic acid and B-glycerophosphate were used to induce MC3T3-E1-BF cell differentiation leading to mineral deposition. The specific screening protocol, in 96-well format, involved plating cells on a Wednesday, followed by seven media changes (as described further below) over a 12-day period with most of the mineral deposition taking place in the final approximately eighteen hours (e.g. Sunday night through Monday). For any given treatment, 3 wells were used (N=3). The specific timing, and extent, of mineral deposition may vary depending, in part, on the particular serum lot number being used. Control experiments will allow such variables to be accounted for, as is well know in the art of cell culture experimentation generally.
In this assay system sclerostin inhibited one or more of the sequence of events leading up to and including mineral deposition (i.e., sclerostin inhibited mineralization). Anti-sclerostin antibodies that were able to neutralize sclerostin's inhibitory activity allowed for mineralization of the culture in the presence of sclerostin such that there was a statistically significant increase in deposition of calcium phosphate (measured as calcium) as compared to the amount of calcium measured in the sclerostin-only (i.e., no antibody) treatment group. For statistical analysis (using MS Excel and JMP) a 1-way-ANOVA followed by Dunnett's comparison was used to determine differences between groups. Group means for each data set were considered significantly different when the P value was less than 0.05 (P<0.05). A representative result from running this assay is shown in FIG. 22. In the absence of recombinant mouse sclerostin, the sequence of events leading up to and including mineral deposition proceeded normally. Calcium levels in each treatment group are shown as means±Standard Error of the Mean (SEM). In this exemplary experiment calcium levels from the calcium assay were  ̃31 μg/ml. However, addition of recombinant mouse sclerostin caused inhibition of mineralization, and calcium was reduced by  ̃85%. Addition of anti-sclerostin monoclonal antibody Ab-19 or Ab-4 along with the recombinant sclerostin resulted in a statistically significant increase in mineral deposition, as compared to the sclerostin-only group, because the inhibitory activity of sclerostin was neutralized by either antibody. The results from this experiment indicate that Ab-19 and Ab-4 are sclerostin neutralizing monoclonal antibodies (Mabs).
FIG. 23 shows a very similar result using recombinant human sclerostin and two humanized anti-sclerostin Mabs. FIG. 24 also shows a very similar result using recombinant human sclerostin and mouse and humanized anti-sclerostin Mabs as indicated.
The antibodies used for the experiments shown in FIGS. 22, 23 and 24 have molecular weights of about 145 Kd and have 2 sclerostin binding sites per antibody molecule.
A detailed MC3T3-E1-BF cell culture protocol is described below.
Reagents and Medias












Reagents
Company
Catalog #







Alpha-MEM
Gibco-Invitrogen
12571-048


Ascorbic acid
Sigma
A4544


Beta-glycerophosphate
Sigma
G6376


100X PenStrepGlutamine
Gibco-Invitrogen
10378-016


Dimethylsulphoxide (DMSO)
Sigma
D5879 or D2650


Fetal bovine serum (FBS)
Cansera
CS-C08-500




(lot # SF50310)


or Fetal bovine serum (FBS)
TerraCell Int.
CS-C08-1000A




(lot # SF-20308)









Alpha-MEM is usually manufactured with a 1 year expiration date. Alpha-MEM that was not older than 6-months post-manufacture date was used for the cell culture.

Expansion Medium (Alpha-MEM/10% FBS/PenStrepGlu) was prepared as follows: A 500 ml bottle of FBS was thawed and filter sterilized through a 0.22 micron filter. 100 mls of this FBS was added to 1 liter of Alpha-MEM followed by the addition of 10 mls of 100× PenStrepGlutamine. Unused FBS was aliquoted and refrozen for later use.

Differentiation Medium (Alpha-MEM/10% FBS/PenStrepGlu, +50 μg/ml ascorbic acid, +10 mM beta-glycerophosphate) was prepared as follows:

100 mls of Differentiation Medium was prepared by supplementing 100 mls of Expansion Medium with ascorbic acid and beta-glycerophosphate as follows:
















Stock conc





(see below)
Volume
Final Conc.



















Ascorbic acid
10 mg/ml
0.5 mls
100 μg/ml





(50 ug/ml + 50 μg/ml)


β-glycerophosphate
1M
1.0 mls
10 mM









Differentiation Medium was made by supplementing Expansion Medium only on the day that the Differentiation media was going to be used for cell culture. The final concentration of ascorbic acid in Differentiation medium is 100 μg/ml because Alpha-MEM already contains 50 μg/ml ascorbic acid. Ascorbic acid stock solution (10 mg/ml) was made and aliquoted for freezing at −80° C. Each aliquot was only used once (i.e. not refrozen). Beta-glycerophosphate stock solution (1 M) was made and aliquoted for freezing at −20° C. Each aliquot was frozen and thawed a maximum of 5 times before being discarded.
Cell Culture for expansion of MC3T3-E1-BF cells.
Cell culture was performed at 37° C. and 5% CO2. A cell bank was generated for the purposes of screening for sclerostin neutralizing antibodies. The cell bank was created as follows:
One vial of frozen MC3T3-E1-BF cells was thawed by agitation in a 37° C. water bath. The thawed cells were put into 10 mls of Expansion Medium (Alpha-MEM/10% FBS/PenStrepGlu) in a 50 ml tube and gently spun down for 5 minutes. The cells were then resuspended in 4 mls of Alpha-MEM/10% FBS/PenStrepGlu. After determining the number of cells using trypan blue and hemacytometer, 1×106 cells were plated in 50 mls Alpha-MEM/10% FBS/PenStrepGlu media in one T175 flask.
When this passage was confluent (at approximately 7 days), the cells were trypsinized with trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spun down for 5 minutes and then resuspended in 5 mls Alpha-MEM/10% FBS/PenStrepGlu. After determining the number of cells using trypan blue and hemacytometer, cells were plated at 1×106 cells in 50 mls Alpha-MEM/10% FBS/PenStrepGlu media per one T175 flask. The number of T175 flasks used for plating at this point depended upon the total cell number available and the desired number of flasks that were to be taken forward to the next passage. Extra cells were frozen down at 1−2×106 live cells/ml in 90% FBS/10% DMSO.
When this passage was confluent (about 3-4 days), the cells were trypsinized with trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spun down for 5 minutes and then resuspended in 5 mls Alpha-MEM/10% FBS/PenStrepGlu. After determining the number of cells using trypan blue and hemacytometer, cells were plated at 1×106 cells in 50 mls Alpha-MEM/10% FBS/PenStrepGlu media per one T175 flask. The number of T175 flasks used for plating at this point depended upon the total cell number available and the desired number of flasks that were to be taken forward to the next passage. Extra cells were frozen down at 1−2×106 live cells/ml in 90% FBS/10% DMSO.
When this passage was confluent (about 3-4 days), the cells were trypsinized with trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spun down for 5 minutes and then resuspended in 5 mls Alpha-MEM/10% FBS/PenStrepGlu. After determining the number of cells using trypan blue and hemacytometer, cells were plated at 1×106 cells in 50 mls Alpha-MEM/10% FBS/PenStrepGlu media per one T175 flask. The number of T175 flasks used for plating at this point depended upon the total cell number available and the desired number of flasks that were to be taken forward to the next passage. Extra cells were frozen down at 1−2×106 live cells/ml in 90% FBS/10% DMSO.
When this passage was confluent (about 3-4 days), the cells were trypsinized with trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spun down for 5 minutes and then resuspended in 5 mls Alpha-MEM/10% FBS/PenStrepGlu. After determining the number of cells using trypan blue and hemacytometer, the cells were frozen down at 1−2×106 live cells/ml in 90% FBS/10% DMSO. This “final passage” of frozen cells was the passage that was used for the screening assay.
Cell Culture for Mineralizing MC3T3-E1-BF Cells.
Cell culture was performed at 37° C. and 5% CO2. It is desirable to minimize temperature and % CO2 fluctuations during the mineralization cell culture procedure. This can be achieved by minimizing the time that plates spend out of the incubator during feeding and also by minimizing the number of times the incubator door is opened and closed during the mineralization cell culture procedure. In this regard having a tissue culture incubator that is dedicated exclusively for the mineralization cell culture (and thus not opened and closed more than is necessary) can be helpful.
An appropriate number of “final passage” vials prepared as described above were thawed by agitation in a 37° C. water bath. The thawed cells were put into 10 mls of Expansion Medium (Alpha-MEM/10% FBS/PenStrepGlu) in a 50 ml tube and gently spun down for 5 minutes. The cells were then resuspended in 4 mls of Alpha-MEM/10% FBS/PenStrepGlu. After determining the number of cells by trypan blue and hemacytometer, 2500 cells were plated in 200 microliters of Expansion media per well on collagen I coated 96-well plates (Becton Dickinson Labware, cat #354407).
To avoid a mineralization plate-edge effect, cells were not plated in the outermost row/column all the way around the plate. Instead 200 microliters of PBS was added to these wells.
Exemplary Cell Culture Procedure
In the following procedure, the starting day for plating the cells is indicated to be a Wednesday. If a different day of the week is used as the starting day for plating the cells, that day will trigger the daily schedule for removing and adding media during the entire process as indicated below. For example, if the cells are plated on a Tuesday, media should not be removed and added on the first Friday and Saturday, nor on the second Friday and Saturday. With a Tuesday start, the plates would be prepared for the calcium assay on the final Sunday.
Cells were plated on a Wednesday at 2500 cells in 200 μl of Expansion media.
On Thursday all of the Expansion media was removed and 200 μl of Differentiation Media was added.
On Friday 100 μl of media was removed and 100 μl of fresh Differentiation Media was added.
On Monday 100 μl of media was removed and 100 μl of fresh Differentiation Media was added.
On Tuesday 100 μl of media was removed and 100 μl of fresh Differentiation Media was added.
On Wednesday 100 μl of media was removed and 100 μl of fresh Differentiation Media was added.
On Thursday 100 μl of media was removed and 100 μl of fresh Differentiation Media was added.
On Friday 100 μl of media was removed and 100 μl of fresh Differentiation Media was added.
On the following Monday plates were prepared for the calcium assay as follows:
Plates were washed once with 10 mM Tris, HCl pH 7-8.
Working under a fume hood, 200 μl of 0.5 N HCl was added per well. Plates were then frozen at −80° C.
Just prior to measuring calcium, the plates were freeze-thawed twice, and then trituration with a multichannel pipette was used to disperse the contents of the plate. The contents of the plate was then allowed to settle at 4° C. for 30 minutes at which point an appropriate amount of supernatant was removed for measuring calcium using a commercially available calcium kit. An exemplary and not-limiting kit is Calcium (CPC) Liquicolor, Cat. No. 0150-250, Stanbio Laboratory, Boerne, Tex.
In this cell based assay, sclerostin inhibits one or more of the sequence of events leading up to and including mineral deposition (i.e. sclerostin inhibits mineralization). Thus, in experiments where sclerostin was included in the particular cell culture experiment, the recombinant sclerostin was added to the media starting on the first Thursday and every feeding day thereafter. In cases where an anti-sclerostin monoclonal antibody (Mab) was being tested for the ability to neutralize sclerostin, i.e. allow for mineralization by neutralizing sclerostin's ability to inhibit mineralization, the Mab was added to the media starting on the first Thursday and every feeding day thereafter. According to the protocol, this was accomplished as follows: the Mab was preincubated with the recombinant sclerostin in Differentiation media for 45-60 minutes at 37° C. and then this media was used for feeding the cells.
Described above is a 12-day mineralization protocol for MC3T3-E1-BF cells. Using the same reagents and feeding protocol, the original MC3T3-E1 cells (Sudo H, Kodama H-A, Amagai Y, Yamamoto S, Kasai S. 1983. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191-198) which we obtained from the RIKEN Cell Bank (RCB 1126, RIKEN BioResource Center 3-1-1 Koyadai, Tsukuba-shi, Ibaraki 305-0074 Japan) took longer to mineralize (20 days total for mineralization) than the MC3T3-E1-BF cells. Mineralization of the original MC3T3-E1 cells was inhibited by recombinant sclerostin and this inhibition was blocked using a sclerostin neutralizing antibody.
Example 9
Anti-Sclerostin Antibody Protects from Inflammation-Induced Bone Loss in the CD4 CD45RBHI Transfer Model of Colitis in SCID Mice
Summary of Model
Injection of the CD45RBhigh subset of CD4+ T cells into C.B-17 scid mice results in chronic intestinal inflammation with characteristics similar to those of human inflammatory bowel disease (IBD). Diarrhoea and wasting disease is noted 3-5 weeks after cell transfer with severe leukocyte infiltration into the colon accompanied by epithelial cell hyperplasia and granuloma formation. C.B-17 scid mice which receive the reciprocal subset of CD4+ cells, those which express CD45RBlow, do not exhibit colitis and have a weight gain indistinguishable from uninjected scid mice. In addition to colitis symptoms, the CD4+ CD45RBhigh T cell transfer model of colitis is accompanied by a reduction in bone mineral density (BMD), thought to be primarily through inflammatory mechanisms rather than dietary malabsorption (Byrne, F. R. et al., Gut 54:78-86, 2005).
Induction of Colitis and Inflammation-Induced Bone Loss
Spleens were taken from female balb/c mice and disrupted through a 70 μm cell strainer. The CD4+ population was then enriched by negative selection with Dynabeads using antibodies against B220, MAC-1, CD8 and I-Ad. The enriched population was then stained with FITC conjugated anti-CD4 and PE conjugated anti-CD45RB and fractionated into CD4+CD45RBhigh and CD4+CD45RBlow populations by two-color sorting on a Moflo (Dakocytomation). The CD45RBhigh and CD45RBlow populations were defined as the brightest staining 40% and the dullest staining 20% of CD4+ cells respectively. 5×105 cells were then injected i.p. into C.B-17 scid mice on day 0 and the development of colitis was monitored through the appearance of soft stools or diarrhoea and weight loss. Bone mineral density measurements were taken at the termination of the study (day 88).
Effect of Anti-Sclerostin Treatment on Colitis Symptoms and BMD
Ab-A IgG was dosed at 10 mg/kg s.c. from the day prior to CD4+CD45RBhigh cell transfer and compared with mice which received the negative control antibody 101.4 also dosed at 10 mg/kg s.c. The antibodies were dosed weekly thereafter. A group of mice which received non-pathogenic CD4+CD45RBlow cells and were dosed with 10 mg/kg 101.4 was studied as a control. At the termination of the study (day 88) the bone mineral density was measured and sections of the colon taken for analysis of cell infiltration and assessment of histological damage.
a) No Effect on Colitis Symptoms
Typical colitis symptoms such as weight loss and infiltration of inflammatory cells into the colon were unaffected by treatment with Ab-A. Similarly there was no improvement of histological damage to the colon after treatment with Ab-A.
b) Inhibition of Inflammation-Induced Loss of Bone Mineral Density.
On day 88 after transfer of cells into C.B-17 scid mice, the bone mineral density was measured (total BMD, vertebrae BMD and femur BMD). In comparison to control mice which received CD4+CD45RBlow non-pathogenic cells, mice which received CD4+CD45RBhigh T cells and the negative control antibody 101.4 had reduced bone mineral density, as shown in FIG. 25. In contrast, no reduction in BMD was noted after treatment with Ab-A. Total, vertebrae and femur measurements of BMD were significantly higher in mice receiving CD4+ CD45RBhigh T cells and treated with Ab-A than mice receiving CD4+ CD45RBhigh T cells and treated with 101.4 (P<0.001 by Bonferroni multiple comparison test).
Example 10
KinExa-Based Determination of Affinity (KD) of Anti-Sclerostin Antibodies for Human Sclerostin
The affinity of several anti-sclerostin antibodies to human sclerostin was assessed by a solution equilibrium binding analysis using KinExA® 3000 (Sapidyne Instruments Inc., Boise, Id.). For these measurements, Reacti-Gel 6× beads (Pierce, Rockford, Ill.) were pre-coated with 40 μg/ml human sclerostin in 50 mM Na2CO3, pH 9.6 at 4° C. overnight. The beads were then blocked with 1 mg/ml BSA in 1 M Tris-HCl, pH 7.5 at 4° C. for two hours. 10 pM, 30 pM, or 100 pM of the antibody was mixed with various concentrations of human sclerostin, ranging in concentration from 0.1 pM to 1 nM, and equilibrated at room temperature for over 8 hours in PBS with 0.1 mg/ml BSA and 0.005% P20. The mixtures were then passed over the human sclerostin coated beads. The amount of bead-bound anti-sclerostin antibody was quantified using fluorescent Cy5-labeled goat anti-mouse-IgG or fluorescent Cy5-labeled goat anti-human-IgG antibodies (Jackson Immuno Research, West Grove, Pa.) for the mouse or human antibody samples, respectively. The amount of fluorescent signal measured was proportional to the concentration of free anti-sclerostin antibody in each reaction mixture at equilibrium. The dissociation equilibrium constant (KD) was obtained from nonlinear regression of the competition curves using a n-curve one-site homogeneous binding model provided in the KinExA Pro software. Results of the KinExA assays for the selected antibodies are summarized in the table below.













Antibodies
Antigen
KD (pM)
95% confidence interval



















Ab-13
Human Sclerostin
0.6
0.4~0.8
pM


Ab-4
Human Sclerostin
3
1.8~4
pM


Ab-19
Human Sclerostin
3
1.7~4
pM


Ab-14
Human Sclerostin
1
0.5~2
pM


Ab-5
Human Sclerostin
6
4.3~8
pM


Ab-23
Human Sclerostin
4
2.1~8
pM









Example 11
Biacore Method for Determining the Affinity of Humanised Anti-Sclerostin Antibodies for Human Sclerostin
The BIAcore technology monitors the binding between biomolecules in real time and without the requirement for labelling. One of the interactants, termed the ligand, is either immobilised directly or captured on the immobilised surface while the other, termed the analyte, flows in solution over the captured surface. The sensor detects the change in mass on the sensor surface as the analyte binds to the ligand to form a complex on the surface. This corresponds to the association process. The dissociation process is monitored when the analyte is replaced by buffer. In the affinity BIAcore assay, the ligand is the anti-sclerostin antibody and the analyte is sclerostin.
Instrument
Biacore® 3000, Biacore AB, Uppsala, Sweden
Sensor Chip
CM5 (research grade) Catalogue Number: BR-1001-14, Biacore AB, Uppsala, Sweden. Chips were stored at 4° C.
BIAnormalising Solution
70% (w/w) Glycerol. Part of BIAmaintenance Kit Catalogue Number: BR-1002-51, Biacore AB, Uppsala, Sweden. The BIAmaintenance kit was stored at 4° C.
Amine Coupling Kit
Catalogue Number: BR-1000-50, Biacore AB, Uppsala, Sweden.
Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Made up to 75 mg/mL in distilled water and stored in 200 μL aliquots at −70° C.
N-Hydroxysuccinimide (NHS). Made up to 11.5 mg/mL in distilled water and stored in 200 μL aliquots at −70° C.
1 M Ethanolamine hydrochloride-NaOH pH 8.5. Stored in 200 μL aliquots at −70° C.
Buffers
Running buffer for immobilising capture antibody: HBS-EP (being 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20). Catalogue Number: BR-1001-88, Biacore AB, Uppsala, Sweden. Buffer stored at 4° C.
Immobilisation buffer: Acetate 5.0 (being 10 mM sodium acetate pH 5.0). Catalogue number: BR-1003-51, Biacore AB, Uppsala, Sweden. Buffer stored at 4° C.
Running buffer for binding assay: HBS-EP (being 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Catalogue Number: BR-1001-88, Biacore AB, Uppsala, Sweden) with CM-Dextran added at 1 mg/mL (Catalogue Number 27560, Fluka BioChemika, Buchs, Switzerland). Buffer stored at 4° C.

Ligand Capture

Affinipure F(ab′)2 fragment goat anti-human IgG, Fc fragment specific. Jackson ImmunoResearch Inc (Pennsylvania, USA) Catalogue number: 109-006-098. Reagent stored at 4° C.

Ligand

Humanised anti-human sclerostin antibodies Ab5, Ab14 and Ab20.

Analyte

Recombinant human sclerostin. Aliquots stored at −70° C. and thawed once for each assay.

Regeneration Solution

40 mM HCl prepared by dilution with distilled water from an 11.6 M stock solution (BDH, Poole, England. Catalogue number: 101254H).

5 mM NaOH prepared by dilution with distilled water from a 50 mM stock solution. Catalogue number: BR-1003-58, Biacore AB, Uppsala, Sweden.

Assay Method

The assay format was capture of the anti-sclerostin antibody by immobilised anti-human IgG-Fc then titration of the sclerostin over the captured surface.
An example of the procedure is given below:
BIA (Biamolecular Interaction Analysis) was performed using a BIAcore 3000 (BIAcore AB). Affinipure F(ab′)2 Fragment goat anti-human IgG, Fc fragment specific (Jackson ImmunoResearch) was immobilised on a CM5 Sensor Chip via amine coupling chemistry to a capture level of ≈4000 response units (RUs). HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, BIAcore AB) containing 1 mg/mL CM-Dextran was used as the running buffer with a flow rate of 10 μl/min. A 10 μl injection of the anti-sclerostin antibody at  ̃5 μg/mL was used for capture by the immobilised anti-human IgG-Fc. Antibody capture levels were typically 100-200 RU. Sclerostin was titrated over the captured anti-sclerostin antibody at various concentrations at a flow rate of 30 μL/min. The surface was regenerated by two 10 μL injections of 40 mM HCl, followed by a 5 μL injection of 5 mM NaOH at a flowrate of 10 μL/min.

Background subtraction binding curves were analyzed using the BIAevaluation software (version 3.2) following standard procedures. Kinetic parameters were determined from the fitting algorithm.

The kinetic data and calculated dissociation constants are given in Table 2.








TABLE 2







Affinity of anti-sclerostin antibodies for sclerostin












Antibody
ka (1/Ms)
kd (1/s)
Kd (pM)
















Ab-5
1.78E+06
1.74E−04
97.8



Ab-14
3.30E+06
4.87E−06
1.48



Ab-20
2.62E+06
4.16E−05
15.8










Example 12
In Vivo Testing of Anti-Sclerostin Monoclonal Antibodies in Cynomolgous Monkeys
Thirty-three, approximately 3-5 year old, female cynomolgus monkeys (Macaca fascicularis) were used in this 2-month study. The study contained 11 groups:
Group 1: vehicle (N=4)
Group 2: Ab-23 (N=2, dose 3 mg/kg)
Group 3: Ab-23 (N=3, dose 10 mg/kg)
Group 4: Ab-23 (N=3, dose 30 mg/kg)
Group 5: Ab-5 (N=3, dose 3 mg/kg)
Group 6: Ab-5 (N=3, dose 10 mg/kg)
Group 7: Ab-5 (N=3, dose 30 mg/kg)
Group 8: Ab-14 (N=3, dose 3 mg/kg)
Group 9: Ab-14 (N=3, dose 10 mg/kg)
Group 10: Ab-14 (N=3, dose 30 mg/kg)
Group 11: Parathyroid Hormone (1-34) [PTH (1-34)] (N=3, dose 10 ug/kg)
All dosing was subcutaneous. PTH (1-34) was dosed everyday, monoclonal antibodies (Mabs) were dosed twice (first dose at the beginning of the study and second dose at the one month time point). For assessment of bone parameters (e.g. bone mineral density) pQCT (peripheral quantitative computed tomography) and DXA (dual energy X-ray absorptiometry) scans were performed prior to the beginning of the study (to obtain baseline values) and after a month (prior to the second dose of Mab) and finally at the end of the study (2-month time point) at which point the monkeys were necropsied for further analysis (e.g. histomorphometric analysis).

Animals were fluorochrome labeled (days 14, 24, 47, and 57) for dynamic histomorphometry. Serum was collected at various time points during the study [day 1 pre-dose (the day of the first Mab dose), day 1 twelve hours post-dose, day 2, day 3, day 5, day 7, day 14, day 21, day 28, day 29 twelve hours post-dose (day 29 was the day of the second and final Mab dose), day 30, day 31, day 33, day 35, day 42, day 49 and day 56].

Three bone-related serum biomarkers were measured using commercially available kits:

Osteocalcin (OC) (DSL Osteocalcin Radioimmunoassay Kit; Diagnostic Systems Laboratories, Inc., Webster, Tex., USA)

N-terminal Propeptide of Type I Procollagen (P1NP) (P1NP Radioimmunoassay Kit; Orion Diagnostica, Espoo, Finland)

C-telopeptide fragments of collagen type I at chains (sCTXI) (Serum CrossLaps® ELISA; Nordic Bioscience Diagnostics A/S, Herlev, Denmark).

pQCT and MCA scans yielded data on various bone parameters (including bone mineral density (BMD) and bone mineral content) across numerous skeletal sites (including tibial metaphysis and diaphysis, radial metaphysis and diaphysis, femur neck, lumbar vertebrae). Analysis of this bone data (percent change from baseline for each animal) and the anabolic (OC, P1NP) serum biomarker data (percent change from baseline for each animal) revealed statistically significant increases, versus the vehicle group, in some parameters at some of the time points and doses for each Mab. This bone parameter data, serum biomarker data, as well as the histomorphometric data, indicated that each of the 3 Mabs (Ab-23, Ab-5 and Ab-14) was able to neutralize sclerostin in cynomolgous monkeys. This activity was most robust for Ab-23 and Ab-5, particularly at the highest dose (30 mg/kg), with a clear increase in bone formation (anabolic effect) as well as net gains in bone (e.g. BMD). Statistically significant increases in bone parameters and anabolic histomorphometric parameters were also found for the positive control group (PTH (1-34)).
Serum bone formation markers (P1NP, osteocalcin) were increased (p<0.05 vs vehicle (VEH)) at various time points and doses, but particularly in the 30 mg/kg groups for Ab-23 and Ab-5. Histomorphometric analysis revealed dramatic increases (p<0.05 vs VEH) in bone formation rates in cancellous bone at lumbar vertebra and proximal tibia (up to 5-fold increase), as well as at the endocortical surface of the femur midshaft (up to 10-fold increase) at the higher doses of Ab-23 and Ab-5. Trabecular thickness was increased with high dose Ab-23 and Ab-5 in lumbar vertebrae (>60%, p<0.05 vs VEH). By study end (2 months), areal BMD, as percent change from baseline, was increased (p<0.05 vs VEH) at the femur neck, ultra-distal radius (Ab-23, 30 mg/kg), and lumbar vertebrae (Ab-5, 30 mg/kg). The increases in areal BMD at the lumbar vertebrae were accompanied by increases in vertebral strength (97% increase in vertebral maximal load for Ab-23, 30 mg/kg; p<0.05 vs VEI-1); baseline values for lumbar areal BMD prior to Mab dosing were statistically similar across all groups. In summary, short-term administration of sclerostin-neutralizing Mabs in cynomolgous monkeys resulted, in part, in increases in bone formation, BMD and vertebral bone strength.
From the foregoing, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All publications, published patent applications, and patent documents disclosed herein are hereby incorporated by reference.

## Claims

1. An IgG antibody that binds sclerostin of SEQ ID NO: 1 and comprises CDRH-1, CDR-H2, CDR-H3, CDR-L1 CDR-L2 and CDR-L3 wherein CDR-H1 is SEQ ID NO:245, CDR-H2 is SEQ ID NO:246, CDR-H3 is SEQ ID NO:247, CDR-L1 is SEQ ID NO:78, CDR-L2 is SEQ ID NO:79 and CDR-L3 is SEQ ID NO:80.

2. An antibody that binds sclerostin of SEQ ID NO: 1 and comprises a heavy chain comprising a polypeptide having the sequence provided in SEQ ID NO:145 or 392, and a light chain comprising a polypeptide having the sequence provided in SEQ ID NO: 141.

3. A pharmaceutical composition comprising an antibody according to claim 2 in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier.

4. A pharmaceutical composition according to claim 3, additionally comprising other active ingredients.

5. A method of increasing at least one of bone formation, bone mineral content, bone mass, bone mineral density, or bone strength in a mammalian subject comprising administering to the mammalian subject an antibody comprising a heavy chain comprising CDRs of SEQ ID NOs: 245, 246 and 247 and a light chain comprising CDRs of SEQ ID NOs: 78, 79 and 80, wherein the antibody demonstrates a binding affinity for sclerostin of SEQ ID NO: 1 of less than or equal to 1×10−7 M.

6. The method of claim 5, wherein the heavy chain of the antibody comprises SEQ ID NO: 378 and the light chain of the antibody comprises SEQ ID NO: 376.

7. The method of claim 5, wherein the heavy chain of the antibody comprises SEQ ID NO: 141 and the light chain of the antibody comprises SEQ ID NO: 145 or SEQ ID NO: 392.

8. The antibody of claim 2, which is an IgG antibody.

9. The method of claim 5, wherein the antibody is a chimeric antibody or a humanized antibody.

10. The method of claim 5, wherein the antibody is an IgG antibody.

11. The method of claim 5, wherein the antibody is a monoclonal antibody.

12. The method of claim 5, wherein the antibody demonstrates a binding affinity for sclerostin of SEQ ID NO: 1 of less than or equal to 1×10−8 M.

13. The method of claim 5, wherein the antibody demonstrates a binding affinity for sclerostin of SEQ ID NO: 1 of less than or equal to 1×10−9 M.

14. The method of claim 5, wherein the subject is a postmenopausal woman.

15. A method of treating osteoporosis or osteopenia comprising administering to a mammalian subject in need thereof an IgG antibody comprising heavy chain CDRs of SEQ ID NOs: 245, 246 and 247 and light chain CDRs of SEQ ID NOs: 78, 79 and 80, wherein the antibody demonstrates a binding affinity for sclerostin of SEQ ID NO: 1 of less than or equal to 1×10−7 M.

16. The method of claim 15, wherein the subject is a postmenopausal woman.