# Patent

## Title
Antibody targeting osteoclast-related protein Siglec-15

## Abstract

An isolated antibody capable of binding Siglec-15 which inhibits osteoclast formation and/or osteoclastic bone resorption, or a functional fragment thereof. The heavy chain of the antibody comprises a CDRH1 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 106, a CDRH2 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 107, and a CDRH3 comprising a sequence having at least 80% sequence identity to one of SEQ ID NOS: 35, 45, 55, 65 or 80. The light chain of the antibody comprises a CDRL1 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 73 or 83, a CDRL2 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 108, and a CDRL3 comprising a sequence having at least 80% sequence identity to one of SEQ ID NOS: 40, 50, 60, 70, 90, 100 or 109.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage application of PCT/EP2011/005219, filed Oct. 4, 2011, which claims priority from European application EP 10 251 744.8, filed Oct. 5, 2010.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 1, 2013, is named sequence.txt and is 179 KB.
TECHNICAL FIELD
The present invention relates to a substance useful as a therapeutic and/or preventive agent for abnormal bone metabolism. The present invention also relates to a pharmaceutical composition such as an antibody and a polynucleotide encoding such an antibody. In addition, the present invention relates to a method of treating and/or preventing abnormal bone metabolism.
BACKGROUND ART
Bone is known to be a dynamic organ which is continuously remodelled by repeated formation and resorption so as to change its own morphology and maintain blood calcium levels. Healthy bone maintains an equilibrium between bone formation by osteoblasts and bone resorption by osteoclasts, and the bone mass is maintained constant. In contrast, when the equilibrium between bone formation and bone resorption is lost, abnormal bone metabolism such as osteoporosis occurs (Endocrinological Review, (1992) 13, pp. 66-80, Principles of Bone Biology, Academic Press, New York, (1996) pp. 87-102).
As factors which regulate bone metabolism, many systemic hormones and local cytokines have been reported, and these factors collaborate with one another to form and maintain bone (Endocrinological Review, (1992) 13, pp. 66-80, Endocrinological Review, (1996) 17, pp. 308-332). As a change in bone tissue due to aging, the occurrence of osteoporosis is widely known, but the mechanism of its occurrence encompasses various factors such as a decrease in secretion of sex hormones and an abnormality in the receptors for the hormones, variation in cytokine expression locally in bone, expression of aging genes, and osteoclast or osteoblast differentiation failure or dysfunction, and thus, it is difficult to consider it as a simple age-related physiological phenomenon. Primary osteoporosis is largely divided into postmenopausal osteoporosis due to a decrease in secretion of estrogen and senile osteoporosis due to aging, but advancement of basic research on the mechanisms of regulation of bone formation and bone resorption is essential to elucidate the mechanism of its occurrence and to develop a therapeutic agent therefor.
Osteoclasts are multinucleated cells derived from hematopoietic stem cells, and by releasing chloride ions and hydrogen ions on a bone surface to which osteoclasts adhere, osteoclasts acidify a gap between the bone surface and the osteoclasts and also secrete cathepsin K which is an acid protease or the like (American Journal of Physiology, (1991) 260, C1315-C1324). This causes degradation of calcium phosphate, activation of acid proteases and degradation of bone matrix proteins, resulting in bone resorption.
Osteoclast precursor cells have been found to be differentiated into osteoclasts by stimulation with RANKL (receptor activator of NF-κB ligand) expressed on the cell membrane of osteoblasts/stromal cells present on the surface of bone (Proceedings of the National Academy of Science of the United States of America, (1998) 95, pp. 3597-3602, Cell, (1998) 93, pp. 165-176). It has been revealed that RANKL is a membrane protein produced by osteoblasts/stromal cells. Its expression being regulated by a bone resorption factor, RANKL induces differentiation of osteoclast precursor cells into multinucleated osteoclasts, and the like (Proceedings of the National Academy of Science of the United States of America, (1998) 95, pp. 3597-3602, Journal of Bone and Mineral Research, (1998) 23, S222). Further, knockout mice devoid of RANKL have been found to develop an osteopetrosis-like disease, and therefore, RANKL has been proved to be a physiological osteoclast differentiation-inducing factor (Nature, (1999) 397, pp. 315-323).
As drugs for treating bone metabolism diseases or shortening the duration of treatment, bisphosphonates, active vitamin D3, calcitonin and derivatives thereof, hormone preparations such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K2 (menatetrenone), PTH (parathyroid hormone) preparations, calcium preparations and the like are used. However, these drugs do not always exhibit a satisfactory therapeutic effect and the development of an agent with a more potent therapeutic effect has been demanded.
The cell membranes of immune cells are covered with a dense coating of various glycans, such as sialic acid, which are recognized by various glycan-binding proteins. Sialic-acid-binding immunoglobulin-like lectins (hereinafter referred to as “siglecs”) are a family of type I membrane proteins which recognize sialylated glycans and bind thereto. Many siglecs are expressed on the cell membranes of immune cells and recognize sialic acid similarly present on the cell membranes of immune cells and regulate cell interaction or cell function and are considered to be involved in the immune response (Nature Reviews Immunology, (2007) 7, pp. 255-266). However, there are also a lot of siglec molecules whose physiological functions have not been elucidated yet. Siglec-15 (Sialic-acid binding immunoglobulin-like lectin 15) is a molecule which has been newly reported to belong to the Siglecs (Glycobiology, (2007) 17, pp. 838-846) and is identical to a molecule called “CD33 antigen-like 3”, “CD33 molecule-like 3”, “CD33-like 3” or “CD33L3”. This molecule is highly evolutionarily conserved from fish to humans and has been found to be strongly expressed in dendritic cells and/or macrophages of human spleen and lymph nodes. Further, as a result of a binding test using a sialic acid probe, it has also been found that human Siglec-15 binds to Neu5Acα2-6GalNAc, and that mouse Siglec-15 binds to Neu5Acα2-3Galβ1-4Glc in addition to Neu5Acα2-6GalNAc (Glycobiology, (2007) 17, pp. 838-846). Until recently, the physiological role of Siglec-15 was not revealed, however, it has been reported that the expression of Siglec-15 increases with the differentiation and maturation of osteoclasts, and the differentiation of osteoclasts is inhibited by decreasing the expression of Siglec-15 by RNA interference (WO 2007/093042). It has also been reported that the Siglec-15 gene is specifically expressed in giant cell tumors and it has also been found that the expression level of the Siglec-15 gene increases when a monocyte-derived cell line differentiates into osteoclasts (WO2009/048072). Rabbit polyclonal and rat monoclonal anti-mouse Siglec-15 antibodies which can inhibit the differentiation of osteoclasts have been disclosed (WO2009/048072). Anti-Siglec-15 chimeric monoclonal antibodies which can inhibit the differentiation of osteoclasts have also been disclosed (US2010/0104575). However, human antibodies which can be administered to humans have not yet been disclosed.
DISCLOSURE OF THE INVENTION
Problems that the Invention is to Solve
An object of the invention is to provide a substance which inhibits the differentiation and maturation of osteoclasts and the activity thereof and a therapeutic and/or preventive agent for abnormal bone metabolism.
Means for Solving the Problems
The present inventors studied to elucidate the mechanism of osteoclast differentiation, maturation and activation in order to find a substance having a therapeutic and/or preventive effect on abnormal bone metabolism. As a result, they found that the expression of the Siglec-15 gene increases with the differentiation and maturation of osteoclasts and also found that the differentiation of osteoclasts is inhibited by an antibody which specifically binds to Siglec-15, and thus, the invention has been completed. In some instances, antibodies of the invention have a higher potency against osteoclasts than antibodies known in the art.
That is, the invention includes the following aspects.
According to a first aspect of the present invention, there is provided an isolated antibody capable of binding Siglec-15 which inhibits osteoclast formation and/or osteoclastic bone resorption, or a functional fragment thereof comprising (a) a heavy chain comprising a CDRH1 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71, a CDRH2 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 72, and a CDRH3 comprising a sequence having at least 80% sequence identity to one of SEQ ID NOS: 35, 45, 55 or 65; and (b) a light chain comprising a CDRL1 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 73, a CDRL2 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 74, and a CDRL3 comprising a sequence having at least 80% sequence identity to one of SEQ ID NOS: 40, 50, 60 or 70.
According to a second aspect of the present invention there is provided an isolated antibody capable of binding Siglec-15 which inhibits osteoclast formation and/or osteoclastic bone resorption, or a functional fragment thereof comprising (a) a heavy chain comprising a CDRH1 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 106, a CDRH2 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 107, and a CDRH3 comprising a sequence having at least 80% sequence identity to one of SEQ ID NOS: 55, 35, 45, 65, or 80; and (b) a light chain comprising a CDRL1 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 73 or 83, a CDRL2 comprising a sequence having at least 80% sequence identity to SEQ ID NO: 108, and a CDRL3 comprising a sequence having at least 80% sequence identity to one of SEQ ID NOS: 60, 40, 50, 70, 90, 100 or 109.
It is preferred that the CDRH3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 35 and the CDRL3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 40. Alternatively, the CDRH3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 45 and the CDRL3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 50. Alternatively, the CDRH3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 55 and the CDRL3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 60. Alternatively, the CDRH3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 65 and the CDRL3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 70. Alternatively, the CDRH3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 80 and the CDRL3 comprises a sequence having at least 80% sequence identity to SEQ ID NO: 109, preferably at least 80% sequence identity to any one of SEQ ID NO: 90 or 100.
Conveniently, the CDRH1 comprises a sequence having at least 80% sequence identity to one of SEQ ID NOS: 33, 43, 53, 63 or 78.
Preferably, the CDRH2 comprises a sequence having at least 80% sequence identity to one of SEQ ID NOS: 34, 44, 54, 64, 79, 94 or 104.
Advantageously, the CDRL1 comprises a sequence having at least 80% sequence identity to one of SEQ ID NOS: 38, 48, 58, 68 or 88.
Conveniently, the CDRL2 comprises a sequence having at least 80% sequence identity to one of SEQ ID NOS: 39, 49, 59, 69 or 89.
Preferably, the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise sequences having at least 80% sequence identity to SEQ. ID NOS: 33, 34, 35, 38, 39 and 40 respectively.
Alternatively, the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise sequences having at least 80% sequence identity to SEQ. ID NOS: 43, 44, 45, 48, 49 and 50 respectively.
Alternatively, the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise sequences having at least 80% sequence identity to SEQ. ID NOS: 53, 54, 55, 58, 59 and 60 respectively.
Alternatively, the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise sequences having at least 80% sequence identity to SEQ. ID NOS: 63, 64, 65, 68, 69, and 70, respectively.
Alternatively, the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise sequences having at least 80% sequence identity to SEQ. ID NOS: 78, 79, 80, 88, 89, and 90, respectively.
Alternatively, the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise sequences having at least 80% sequence identity to SEQ. ID NOS: 93, 94, 95, 98, 99, and 100, respectively.
Alternatively, the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise sequences having at least 80% sequence identity to SEQ. ID NOS: 103, 104, 105, 88, 89 and 90, respectively.
It is particularly preferred that the antibody or functional fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3 sequences having at least 90% or 95% sequence identity to the sequences set out above.
Preferably, the antibody comprises:
a heavy chain variable region selected from the group consisting of
a1) an amino acid sequence comprising: amino acid residues 20 to 136 of SEQ ID NO: 32, amino acid residues 20 to 135 of SEQ ID NO: 42, amino acid residues 20 to 137 of SEQ ID NO: 52, amino acid residues 20 to 136 of SEQ ID NO: 62, amino acid residues 20 to 138 of SEQ ID NO: 77, amino acid residues 20 to 138 of SEQ ID NO: 92 or amino acid residues 20 to 140 of SEQ ID NO: 102;

a2) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of a1);

a3) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of a1); and

a4) an amino acid sequence including addition, deletion or substitution of one or several amino acid residues in the amino acid sequence of a1); and

a light chain variable region selected form the group consisting of

b1) an amino acid sequence comprising: amino acid residues 20 to 129 of SEQ ID NO: 37, amino acid residues 20 to 129 of SEQ ID NO: 47, amino acid residues 20 to 129 of SEQ ID NO: 57, amino acid residues 20 to 129 of SEQ ID NO: 67, amino acid residues 20 to 134 of SEQ ID NO: 87 or amino acid residues 20 to 133 of SEQ ID NO: 97;

b2) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of b1);

b3) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of b1); and

b4) an amino acid sequence including addition, deletion or substitution of one or several amino acid residues in the amino acid sequence of b1).

It is particularly preferred that the heavy and light chain variable regions have at least 99% sequence identity to the sequences set out above.
Advantageously, the antibody comprises:
a heavy chain selected from the group consisting of
a1) an amino acid sequence comprising the sequence of one of SEQ ID NOS: 32, 42, 52, 62, 77, 92 or 102;
a2) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of a1);
a3) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of a1); and
a4) an amino acid sequence including addition, deletion or substitution of one or several amino acid residues in the amino acid sequence of a1); and a light chain selected from the group consisting of
b1) an amino acid sequence comprising the sequence of one of SEQ ID NOS: 37, 47, 57, 67, 87 or 97;
b2) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of b1);
b3) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of b1); and
b4) an amino acid sequence including addition, deletion or substitution of one or several amino acid residues in the amino acid sequence of b1).
It is particularly preferred that the heavy and light chains have at least 99% sequence identity to the sequences set out above.
Conveniently, the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 32 and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 37.
Alternatively, the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 42 and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 47.
Alternatively, the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 52 and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 57.
Alternatively, the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 62 and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 67.
Alternatively, the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 77 and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 87.
Alternatively, the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 92 and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 97.
Alternatively, the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 102 and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 87.
Conveniently, the functional fragment of the antibody of the invention is selected from the group consisting of Fab, F(ab′)2, Fab′ and Fv.
Preferably, the antibody is an scFv.
According to a third aspect of the invention there is provided an isolated antibody or a functional fragment thereof according to the first or second aspect of the invention for use in treating and/or preventing abnormal bone metabolism.
According to a fourth aspect of the present invention, there is provided the use of an isolated antibody or a functional fragment thereof according to the first or second aspect of the invention for the manufacture of a medicament for the treatment or prevention of abnormal bone metabolism.
According to a fifth aspect of the present invention, there is provided a method of treatment of a patient suffering from abnormal bone metabolism comprising administering a therapeutically effective amount of an isolated antibody or a functional fragment thereof according to the first or second aspect of the invention.
According to a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising at least one of the antibodies or functional fragments thereof according to the first or second aspect of the invention.
Conveniently, the pharmaceutical composition is a therapeutic and/or preventive agent for abnormal bone metabolism.
According to a seventh aspect of the present invention, there is provided a pharmaceutical composition for treating and/or preventing abnormal bone metabolism characterized by comprising at least one of the antibodies or functional fragments of the antibodies according to the first or second aspect of the invention and at least one member selected from the group consisting of bisphosphonates, active vitamin D3, calcitonin and derivatives thereof, hormone preparations such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K2 (menatetrenone), calcium preparations, PTH (parathyroid hormone) preparations, nonsteroidal anti-inflammatory agents, soluble TNF receptor preparations, anti-TNF-α antibodies or functional fragments of the antibodies, anti-PTHrP (parathyroid hormone-related protein) antibodies or functional fragments of the antibodies, IL-1 receptor antagonists, anti-IL-6 receptor antibodies or functional fragments of the antibodies, anti-RANKL antibodies or functional fragments of the antibodies and OCIF (osteoclastogenesis inhibitory factor).
Preferably, the abnormal bone metabolism is selected from the group consisting of: osteoporosis, bone destruction accompanying rheumatoid arthritis, cancerous hypercalcemia, bone destruction accompanying multiple myeloma or cancer metastasis to bone, giant cell tumor, tooth loss due to periodontitis, osteolysis around a prosthetic joint, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy and osteogenesis imperfecta.
Advantageously, the abnormal bone metabolism is osteoporosis, bone destruction accompanying rheumatoid arthritis or bone destruction accompanying cancer metastasis to bone.
Conveniently, the osteoporosis is postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis due to the use of a therapeutic agent such as a steroid or an immunosuppressant, or osteoporosis accompanying rheumatoid arthritis.
According to an eighth aspect of the present invention, there is provided a polynucleotide which comprises:
a) a nucleotide sequence that encodes one of the antibodies or functional fragments thereof according to the first or second aspect of the invention; or
b) a nucleotide sequence of a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of a) under stringent conditions.
Conveniently, the nucleotide sequences which encode the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise:
a) nucleotides 148 to 162 of SEQ ID NO: 31, nucleotides 205 to 255 of SEQ ID NO: 31, nucleotides 352 to 375 of SEQ ID NO: 31, nucleotides 124 to 156 of SEQ ID NO: 36, nucleotides 202 to 222 of SEQ ID NO: 36 and nucleotides 319 to 348 of SEQ ID NO: 36, respectively;

b) nucleotides 148 to 162 of SEQ ID NO: 41, nucleotides 205 to 255 of SEQ ID NO: 41, nucleotides 352 to 372 of SEQ ID NO: 41, nucleotides 124 to 156 of SEQ ID NO: 46, nucleotides 202 to 222 of SEQ ID NO: 46 and nucleotides 319 to 348 of SEQ ID NO: 46, respectively;

c) nucleotides 148 to 162 of SEQ ID NO: 51, nucleotides 205 to 255 of SEQ ID NO: 51, nucleotides 352 to 378 of SEQ ID NO: 51, nucleotides 124 to 156 of SEQ ID NO: 56, nucleotides 202 to 222 of SEQ ID NO: 56 and nucleotides 319 to 348 of SEQ ID NO: 56, respectively;

d) nucleotides 148 to 162 of SEQ ID NO: 61, nucleotides 205 to 255 of SEQ ID NO: 61, nucleotides 352 to 375 of SEQ ID NO: 61, nucleotides 124 to 156 of SEQ ID NO: 66, nucleotides 202 to 222 of SEQ ID NO: 66 and nucleotides 319 to 348 of SEQ ID NO: 66, respectively;

e) nucleotides 148 to 162 of SEQ ID NO: 76, nucleotides 205 to 255 of SEQ ID NO: 76, nucleotides 352 to 381 of SEQ ID NO: 76, nucleotides 124 to 165 of SEQ ID NO: 86, nucleotides 211 to 231 of SEQ ID NO: 86 and nucleotides 328 to 363 of SEQ ID NO: 86, respectively;

f) nucleotides 148 to 162 of SEQ ID NO: 91, nucleotides 205 to 255 of SEQ ID NO: 91, nucleotides 352 to 381 of SEQ ID NO: 91, nucleotides 124 to 165 of SEQ ID NO: 96, nucleotides 211 to 231 of SEQ ID NO: 96 and nucleotides 328 to 360 of SEQ ID NO: 96, respectively; or

g) nucleotides 148 to 162 of SEQ ID NO: 101, nucleotides 205 to 261 of SEQ ID NO: 101, nucleotides 358 to 387 of SEQ ID NO: 101, nucleotides 124 to 165 of SEQ ID NO: 86, nucleotides 211 to 231 of SEQ ID NO: 86 and nucleotides 328 to 363 of SEQ ID NO: 86, respectively.

Preferably, the polynucleotide comprises:
a nucleotide sequence encoding a heavy chain variable region selected from the group consisting of
a1) a nucleotide sequence comprising: nucleotides 58 to 408 of SEQ ID NO: 31, nucleotides 58 to 405 of SEQ ID NO: 41, nucleotides 58 to 411 of SEQ ID NO: 51, nucleotides 58 to 408 of SEQ ID NO: 61, nucleotides 58 to 414 of SEQ ID NO: 76, nucleotides 58 to 414 of SEQ ID NO: 91 or nucleotides 58 to 420 of SEQ ID NO: 101;

a2) a nucleotide sequence of a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of a1) under stringent conditions; and

a3) a nucleotide sequence including addition, deletion or substitution of one or several nucleotides in the nucleotide sequence of a1); and

a nucleotide sequence encoding a light chain variable region selected from the group consisting of

b1) a nucleotide sequence comprising: nucleotides 58 to 387 of SEQ ID NO: 36, nucleotides 58 to 387 of SEQ ID NO: 46, nucleotides 58 to 387 of SEQ ID NO: 56, nucleotides 58 to 387 of SEQ ID NO: 66, nucleotides 58 to 402 of SEQ ID NO: 86 or nucleotides 58 to 399 of SEQ ID NO: 96;

b2) a nucleotide sequence of a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of b1) under stringent conditions; and

b3) a nucleotide sequence including addition, deletion or substitution of one or several nucleotides in the nucleotide sequence of b1).

Advantageously, the polynucleotide comprises:
a nucleotide sequence encoding a heavy chain selected from the group consisting of
a1) a nucleotide sequence the sequence of one of SEQ ID NOS: 31, 41, 51, 61, 76, 91 or 101;
a2) a nucleotide sequence of a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of a1) under stringent conditions; and
a3) a nucleotide sequence including addition, deletion or substitution of one or several nucleotides in the nucleotide sequence of a1); and
a nucleotide sequence encoding a light chain selected from the group consisting of
b1) a nucleotide sequence comprising the sequence of one of SEQ ID NOS: 36, 46, 56, 66, 86 or 96;
b2) a nucleotide sequence of a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of b1) under stringent condition; and
b3) a nucleotide sequence including addition, deletion or addition of one or several nucleotides in the nucleotide sequence of b1).
According to a ninth aspect of the present invention, there is provided a vector comprising at least one polynucleotide according to the eighth aspect of the invention.
According to a tenth aspect of the present invention, there is provided a transformed cell comprising at least one polynucleotide according to the eighth aspect of the invention.
According to an eleventh aspect of the present invention, there is provided a transformed cell comprising at least one vector according to the ninth aspect of the invention.
According to a twelfth aspect of the present invention, there is provided a method of producing antibodies according to the first or second aspect of the invention, comprising the steps of culturing a transformed cell according to the tenth or eleventh aspects of the invention, collecting culturing materials and purifying said antibodies from said culturing materials.
In this specification, the percentage “identity” between two sequences is determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402) using default parameters. In particular, the BLAST algorithm can be accessed on the internet using the URL www.ncbi.nlm.nih.gov/blast.
Advantage of the Invention
According to the invention, a therapeutic and/or preventive agent for abnormal bone metabolism whose mechanism of action is to inhibit the differentiation and maturation of osteoclasts and the activity thereof can be obtained.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the results of an ELISA assay to measure binding of antibodies MOR09281, MOR09892, MOR09898, MOR10110, MOR09012, MOR08656, MOR08662 and human IgG (negative control) to human Siglec-15-Fc, mouse Siglec-15-Fc and human Fc.
FIG. 2 is a graph showing the TRAP activity of mouse osteoclasts formed in the presence of antibody MOR09281.
FIG. 3 is a graph showing the TRAP activity of mouse osteoclasts formed in the presence of antibody MOR09012 (parent antibody of MOR09892).
FIG. 4 is a graph showing the TRAP activity of mouse osteoclasts formed in the presence of antibody MOR09892.
FIG. 5 is a graph showing the TRAP activity of mouse osteoclasts formed in the presence of antibody MOR08656 (parent antibody of MOR09898).
FIG. 6 is a graph showing the TRAP activity of mouse osteoclasts formed in the presence of antibody MOR08662 (parent antibody of MOR10110).
FIG. 7 is a graph showing the TRAP activity of mouse osteoclasts formed in the presence of antibody MOR09898.
FIG. 8 is a graph showing the TRAP activity of mouse osteoclasts formed in the presence of antibody MOR10110.
FIG. 9 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibody MOR09281.
FIG. 10 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibody MOR08656 (parent antibody of MOR09898).
FIG. 11 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibody MOR09898.
FIG. 12 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibodies MOR09012 (parent antibody of MOR09892) and MOR09892.
FIG. 13 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibodies MOR08662 (parent antibody of MOR10110) and MOR10110.
FIG. 14 is the DNA sequence (SEQ ID NO: 1) encoding the heavy chain of antibody MOR08656.
FIG. 15 is the amino acid sequence (SEQ ID NO: 2) for the heavy chain of antibody MOR08656.
FIG. 16 is the DNA sequence (SEQ ID NO: 6) encoding the light chain of antibody MOR08656.
FIG. 17 is the amino acid sequence (SEQ ID NO: 7) for the light chain of antibody MOR08656.
FIG. 18 is the DNA sequence (SEQ ID NO: 11) encoding the heavy chain of antibody MOR08662.
FIG. 19 is the amino acid sequence (SEQ ID NO: 12) for the heavy chain of antibody MOR08662.
FIG. 20 is the DNA sequence (SEQ ID NO: 16) encoding the light chain of antibody MOR08662.
FIG. 21 is the amino acid sequence (SEQ ID NO: 17) for the light chain of antibody MOR08662.
FIG. 22 is the DNA sequence (SEQ ID NO: 21) encoding the heavy chain of antibody MOR09012.
FIG. 23 is the amino acid sequence (SEQ ID NO: 22) for the heavy chain of antibody MOR09012.
FIG. 24 is the DNA sequence (SEQ ID NO: 26) encoding the light chain of antibody MOR09012.
FIG. 25 is the amino acid sequence (SEQ ID NO: 27) for the light chain of antibody MOR09012.
FIG. 26 is the DNA sequence (SEQ ID NO: 31) encoding the heavy chain of antibody MOR09281.
FIG. 27 is the amino acid sequence (SEQ ID NO: 32) for the heavy chain of antibody MOR09281.
FIG. 28 is the DNA sequence (SEQ ID NO: 36) encoding the light chain of antibody MOR09281.
FIG. 29 is the amino acid sequence (SEQ ID NO: 37) for the light chain of antibody MOR09281.
FIG. 30 is the DNA sequence (SEQ ID NO: 41) encoding the heavy chain of antibody MOR09892.
FIG. 31 is the amino acid sequence (SEQ ID NO: 42) for the heavy chain of antibody MOR09892.
FIG. 32 is the DNA sequence (SEQ ID NO: 46) encoding the light chain of antibody MOR09892.
FIG. 33 is the amino acid sequence (SEQ ID NO: 47) for the light chain of antibody MOR09892.
FIG. 34 is the DNA sequence (SEQ ID NO: 51) encoding the heavy chain of antibody MOR09898.
FIG. 35 is the amino acid sequence (SEQ ID NO: 52) for the heavy chain of antibody MOR09898.
FIG. 36 is the DNA sequence (SEQ ID NO: 56) encoding the light chain of antibody MOR09898.
FIG. 37 is the amino acid sequence (SEQ ID NO: 57) for the light chain of antibody MOR09898.
FIG. 38 is the DNA sequence (SEQ ID NO: 61) encoding the heavy chain of antibody MOR10110.
FIG. 39 is the amino acid sequence (SEQ ID NO: 62) for the heavy chain of antibody MOR10110.
FIG. 40 is the DNA sequence (SEQ ID NO: 66) encoding the light chain of antibody MOR10110.
FIG. 41 is the amino acid sequence (SEQ ID NO: 67) for the light chain of antibody MOR10110.
FIG. 42 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibody MOR09268.
FIG. 43 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibody MOR12574.
FIG. 44 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibody MOR13154.
FIG. 45 is a graph showing the release of collagen fragments from the plate degraded by human osteoclast precursor cells when cultured in the presence of antibody MOR13156.
FIG. 46 is the DNA sequence (SEQ ID NO: 76) encoding the heavy chain of antibodies MOR09268 and MOR12574.
FIG. 47 is the amino acid sequence (SEQ ID NO: 77) for the heavy chain of antibodies MOR09268 and MOR12574.
FIG. 48 is the DNA sequence (SEQ ID NO: 81) encoding the light chain of antibody MOR09268.
FIG. 49 is the amino acid sequence (SEQ ID NO: 82) for the light chain of antibody MOR09268.
FIG. 50 is the DNA sequence (SEQ ID NO: 86) encoding the light chain of antibodies MOR12574 and MOR13156.
FIG. 51 is the amino acid sequence (SEQ ID NO: 87) for the light chain of antibodies MOR12574 and MOR13156.
FIG. 52 is the DNA sequence (SEQ ID NO: 91) encoding the heavy chain of antibody MOR13154.
FIG. 53 is the amino acid sequence (SEQ ID NO: 92) for the heavy chain of antibody MOR13154.
FIG. 54 is the DNA sequence (SEQ ID NO: 96) encoding the light chain of antibody MOR13154.
FIG. 55 is the amino acid sequence (SEQ ID NO: 97) for the light chain of antibody MOR13154.
FIG. 56 is the DNA sequence (SEQ ID NO: 101) encoding the heavy chain of antibody MOR13156.
FIG. 57 is the amino acid sequence (SEQ ID NO: 102) for the heavy chain of antibody MOR13156.



BEST MODE FOR CARRYING OUT THE INVENTION
The term “gene” as used herein includes not only DNA, but also mRNA, cDNA and cRNA.
The term “polynucleotide” as used herein is used in the same meaning as a nucleic acid and also includes DNA, RNA, probes, oligonucleotides and primers.
The terms “polypeptide” and “protein” as used herein are used without distinction.
The term “RNA fraction” as used herein refers to a fraction containing RNA.
The term “cell” as used herein also includes cells in an animal individual and cultured cells.
The term “Siglec-15” as used herein is used in the same meaning as Siglec-15 protein.
The term “osteoclast formation” as used herein is used in the same meaning as “osteoclast differentiation” or “osteoclast maturation”.
The term “functional fragment of an antibody” as used herein refers to a partial fragment of an antibody having an antigen-binding activity and includes Fab, F(ab′)2, scFv and the like. The term also encompasses Fab′ which is a monovalent fragment of a variable region of an antibody obtained by treating F(ab′)2 under reducing conditions. However, the term is not limited to these molecules as long as the fragment has a binding affinity for an antigen. Further, these functional fragments include not only a fragment obtained by treating a full-length molecule of an antibody protein with an appropriate enzyme, but also a protein produced in an appropriate host cell using a genetically modified antibody gene.
It is known that a heavy or light chain of an antibody has three complementarity determining regions (CDRs). Complementarity Determining Regions are also known as hypervariable domains and these regions are hypervariable in the primary structure of a heavy or a light chain variable region. Such hypervariable domains exist as three divergent portions in the polypeptide of a heavy or light chain of an antibody. In this specification, the CDRs of a heavy chain are termed CDRH1, CDRH2 and CDRH3 in order from the amino terminus (N-terminus) of the polypeptide of the heavy chain. CDRs of a light chain are termed CDRL1, CDRL2 and CDRL3 in order from the amino terminus of the polypeptide of the light chain. These 6 CDRs are in close proximity within the tertiary structure of an antibody and determine the specificity of the binding to an antigen.
The phrase “hybridization is performed under stringent conditions” as used herein refers to hybridization being performed under the conditions under which identification can be effected by performing hybridization at 68° C. in a commercially available hybridization solution, such as ExpressHyb Hybridization Solution (manufactured by Clontech, Inc.) or performing hybridization at 68° C. in the presence of 0.7 to 1.0 M NaCl using a filter having DNA immobilized thereon, followed by performing washing at 68° C. using 0.1 to 2×SSC solution (1×SSC solution is composed of 150 mM NaCl and 15 mM sodium citrate) or under conditions equivalent thereto.
The term “capable of binding” when used in relation to an antibody with respect to Siglec-15 means that the antibody is able to bind the Siglec-15 preferentially under physiological conditions. In some embodiments, this is synonymous with the term “specific for”. Thus in some embodiments, the antibody has greater affinity for Siglec-15 than for other related polypeptides, for example 1.5 fold, 2-fold, 5-fold, 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, 106-fold greater affinity.
The term “several” as used herein means a number from 2 to 4 in some embodiments.
1. Siglec-15
Where Siglec-15 is to be used as an immunogen, Siglec-15 may be directly purified from monocytes or bone marrow cells of humans, non-human mammals (such as guinea pigs, rats, mice, rabbits, pigs, sheep, cattle or monkeys) or chickens and used, or a cell membrane fraction of the above-mentioned cells may be prepared and can be used. Furthermore, Siglec-15 can be obtained by in vitro synthesis thereof or production thereof in a host cell through genetic engineering. In such genetic engineering, more specifically, Siglec-15 cDNA is integrated into a vector capable of expressing Siglec-15 cDNA, and Siglec-15 is synthesized in a solution containing enzymes, substrates, and energy substances required for transcription and translation, or another prokaryotic or eucaryotic host cell is transformed to express Siglec-15, whereby the protein can be obtained.
The nucleotide sequence of human Siglec-15 cDNA has been registered in GenBank with the accession number of NM—213602. The nucleotide sequence of mouse Siglec-15 cDNA has been registered in GenBank with the accession number of XM—884636.
2. Anti-Siglec-15 Antibody
The general structure of antibodies is known in the art and will only be briefly summarised here. An immunoglobulin monomer comprises two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is paired with one of the light chains to which it is directly bound via a disulfide bond. Each heavy chain comprises a constant region (which varies depending on the isotype of the antibody) and a variable region. The variable region comprises three hypervariable regions (or complementarity determining regions) which are designated CDRH1, CDRH2 and CDRH3 and which are supported within framework regions. Each light chain comprises a constant region and a variable region, with the variable region comprising three hypervariable regions (designated CDRL1, CDRL2 and CDRL3) supported by framework regions in an analogous manner to the variable region of the heavy chain.
The hypervariable regions of each pair of heavy and light chains mutually cooperate to provide an antigen binding site that is capable of binding a target antigen. The binding specificity of a pair of heavy and light chains is defined by the sequence of CDR1, CDR2 and CDR3 of the heavy and light chains. Thus once a set of CDR sequences (i.e. the sequence of CDR1, CDR2 and CDR3 for the heavy and light chains) is determined which gives rise to a particular binding specificity, the set of CDR sequences can, in principle, be inserted into the appropriate positions within any other antibody framework regions linked with any antibody constant regions in order to provide a different antibody with the same antigen binding specificity.
In preferred embodiments, there are provided antibodies MOR09281, MOR09892, MOR09898, MOR10110, MOR12574, MOR13154 and MOR13156, details of whose amino acid sequences are set forth in Tables 4 to 7 and 10 to 12. However, the present invention is not limited to these preferred antibodies.
In general terms, in the anti-Siglec 15 antibodies of the present invention, the sequences of CDR1 and CDR2 of the heavy and light chains conform with the consensus sequences set out in SEQ ID NOS: 71 to 74. Alternatively, the sequences of the CDR1 and CDR2 sequences of the heavy chain may conform with the consensus sequences set out in SEQ ID NOS: 106 and 107; the sequence of the CDR2 sequence of the light chain may conform with SEQ ID NO: 108 and the sequence of the CDR1 sequence of the light chain may conform with either SEQ ID NO: 73 or 83.
The sequence of the CDR3 sequence of the light chain may conform with the consensus sequence set out in SEQ ID NO: 109. It is particularly preferred that the antibody has the CDR3 sequence of the light chain conforming with the consensus sequence of SEQ ID NO: 109 and the CDR3 sequence of the heavy chain has the sequence of SEQ ID NO: 80.
Specific CDR1, CDR2 and CDR3 sequences from some of the preferred antibodies (MOR09281, MOR09892, MOR09898 and MOR10110) of the invention are set out in Table 8. Specific CDR1, CDR2 and CDR3 sequences from the other preferred antibodies (MOR12574, MOR13154 and MOR13156) of the invention are set out in Table 13. Thus there are provided antibodies comprising CDRs 1 to 3 having the sequences from these preferred antibodies. However, since there is a high level of sequence identity between sequences of the preferred antibodies of the invention, it is also within the scope of the invention to provide antibodies with CDR sequences from different preferred antibodies. For example, an antibody may be provided comprising a heavy chain having the sequence of CDR1 from MOR09281, CDR2 from MOR09892 and CDR3 from MOR09898 and light chain having the sequence of CDR1 from MOR10110, CDR2 from MOR09281 and CDR3 from MOR09892.
It is also to be understood that in some embodiments of the invention, the amino acid sequence of CDRs 1 to 3 is varied from those sequences provided in Tables 8 and 13 while still retaining at least 80% (or, in some embodiments, at least 90% or 95%) sequence identity therewith. In some embodiments, an amino acid residue at a position in a CDR of one antibody is swapped with the amino acid residue at the corresponding position in the same CDR of another antibody. For example, an antibody may be provided comprising a heavy chain in which the sequence of CDR1 comprises the amino acid sequence NYAMS (SEQ ID NO. 75) i.e. the sequence of CDR1 of the heavy chain of MOR09281 with the amino acid at position 3 substituted with the amino acid at position 3 from CDR1 of the heavy chain of MOR09892. In other embodiments, one or more amino acid residues in a CDR are substituted with another amino acid. The substitution may be “conservative” in the sense of being a substitution within the same family of amino acids. The naturally occurring amino acids may be divided into the following four families and conservative substitutions will take place within those families.
1) Amino acids with basic side chains: lysine, arginine, histidine.
2) Amino acids with acidic side chains: aspartic acid, glutamic acid
3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine.
4) Amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.
In still further embodiments, one or more amino acid residues are added to or deleted from one or more CDRs of an antibody. Such additions or deletions occur at the N or C termini of the CDR or at a position within the CDR.
By varying the amino acid sequence of the CDRs of an antibody by addition, deletion or substitution of amino acids, various effects such as increased binding affinity for the target antigen or reduced aggregation may be obtained.
It is to be appreciated that antibodies of the invention comprising such varied CDR sequences still bind Siglec-15 and inhibit osteoclast formation and/osteoclastic bone resorption. This may be tested by way of the binding assays and biological activity assays disclosed in Examples 5 and 6 herein.
The constant regions of antibodies may also be varied from those specifically disclosed for antibodies MOR09281, MOR09892, MOR09898, MOR10110, MOR12574, MOR13154 and MOR13156. For example, antibodies may be provided with Fc regions of any isotype: IgA, IgD, IgE, IgG or IgM.
It is preferred that the antibody is a human antibody.
Antibodies, their manufacture and uses are well known and disclosed in, for example, Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999.
The antibodies may be generated using standard methods known in the art. Examples of antibodies include (but are not limited to) monoclonal, single chain, and functional fragments of antibodies.
A “functional fragment” of an antibody includes any fragment of an antibody which is capable of binding a target antigen and thus includes Fab fragments, F(ab′)2 fragments, Fab′ fragments and Fv fragments.
Antibodies may be produced in a range of hosts, for example goats, rabbits, rats, mice, humans, and others. They may be immunized by injection with a target antigen (in this invention Siglec-15) or a fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase an immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are particularly useful.
Monoclonal antibodies, to Siglec-15 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Koehler et al., 1975, Nature, 256: 495-497; Kosbor et al., 1983, Immunol. Today 4: 72; Cote et al., 1983, PNAS USA, 80: 2026-2030; Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., New York, pp. 77-96).
Alternatively, techniques for the production of single chain antibodies may be used. Single chain antibodies (scFvs) comprise a heavy chain variable region and a light chain variable region connected with a linker peptide (typically around 5 to 25 amino acids in length). scFvs may be synthesised using recombinant techniques, for example by expression of a vector encoding the scFv in a host organism such as E. coli. 
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents (Orlandi et al., 1989, PNAS USA, 86: 3833-3837; Winter, G. et al., 1991, Nature, 349: 293-299).
Antigen binding fragments may also be generated, for example the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., 1989, Science, 256: 1275-1281).
Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between Siglec-15, or any fragment or oligopeptide thereof and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies specific to two non-interfering Siglec-15 epitopes may be used, but a competitive binding assay may also be employed (Maddox et al., 1983, J. Exp. Med., 158: 1211-1216).
3. Medicine Containing Anti-Siglec-15 Antibody
The anti-Siglec-15 antibodies described in the above item “2. Anti-Siglec-15 antibody”, neutralize the biological activity of Siglec-15. Such an antibody, which neutralizes the biological activity of Siglec-15, inhibits the biological activity of Siglec-15 in vivo (i.e., the differentiation and/or maturation of osteoclasts) and therefore can be used as a therapeutic and/or preventive agent for abnormal bone metabolism caused by abnormal differentiation and/or maturation of osteoclasts. The abnormal bone metabolism may be any disorder characterized by net bone loss (osteopenia or osteolysis). In general, the therapeutic and/or preventive effect of the anti-Siglec-15 antibody is applied in cases where inhibition of bone resorption is required. Examples of abnormal bone metabolism which can be treated and/or prevented by the anti-Siglec-15 antibody include osteoporosis (postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis due to the use of a therapeutic agent such as a steroid or an immunosuppressant, or osteoporosis accompanying rheumatoid arthritis), bone destruction accompanying rheumatoid arthritis, cancerous hypercalcemia, bone destruction accompanying multiple myeloma or cancer metastasis to bone, giant cell tumor, tooth loss due to periodontitis, osteolysis around a prosthetic joint, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy and osteogenesis imperfecta. However, the abnormal bone metabolism is not limited to these examples as long as it is a disease accompanied by net bone loss caused by osteoclasts.
The in vitro activity of the anti-Siglec-15 antibody in neutralizing the biological activity of Siglec-15 can be determined by, for example, the activity of inhibiting the differentiation of the cells which overexpress Siglec-15 into osteoclasts. For example, the anti-Siglec-15 antibody is added to RAW 264.7 cells or Raw 264 cells, which are a mouse monocyte-derived cell line, at various concentrations, and the activity of inhibiting the differentiation into osteoclasts by stimulation with RANKL or TNF-α can be determined. In a further example, the anti-Siglec-15 antibody is added to bone marrow-derived primary cultured cells at various concentrations, and the activity of inhibiting the differentiation into osteoclasts by stimulation with RANKL, TNF-α or active vitamin D3 can be determined. In a further example, the anti-Siglec-15 antibody is added to normal human osteoclast precursor cells (Normal Human Natural Osteoclast Precursor Cells, available from Sanko Junyaku Co., Ltd., Cat. No. 2T-110) at various concentrations, and the activity of inhibiting the differentiation into osteoclasts by stimulation with RANKL or M-CSF can be determined. Such an inhibitory effect on osteoclast differentiation can be determined by using the inhibition of tartrate-resistant acid phosphatase (TRAP) activity of osteoclasts as an index. In a further example, the inhibitory effect on osteoclast differentiation can also be determined by using the inhibition of formation of TRAP-positive multinucleated osteoclasts, i.e., the inhibition of cell fusion of osteoclasts as an index. In a further example, in an experiment of a pit assay (Takada et al., Bone and Mineral, (1992) 17, 347-359) using femur- and/or tibia-derived cells, the in vitro activity of inhibiting bone resorption by osteoclasts can be determined by adding the anti-Siglec-15 antibody to femur- and/or tibia-derived cells at various concentrations, and observing pit formation on a dentine slice. As a system for determining the in vitro activity of inhibiting bone resorption by osteoclasts, it is also possible to use a plate coated with human collagen conjugated to europium. The in vivo therapeutic or preventive effect of the anti-Siglec-15 antibody on abnormal bone metabolism in an experimental animal can be confirmed by administering the anti-Siglec-15 antibody to a model animal of osteoporosis or a transgenic animal which overexpresses siglec-15 and measuring a change in osteoclasts.
The above-described antibody which neutralizes the biological activity of Siglec-15 is useful as a medicine, particularly as a pharmaceutical composition for treating or preventing abnormal bone metabolism such as osteoporosis, bone destruction accompanying rheumatoid arthritis or bone destruction accompanying cancer metastasis to bone, or as an antibody for immunological diagnosis of such a disease.
In the treatment of rheumatoid arthritis (RA), a major problem is bone loss accompanying the occurrence of the disease. It has been reported that in this bone loss accompanying RA, osteoclasts play a primary role. The cytokines considered to be most important for osteoclast induction (differentiation and maturation) and activation and the cause of bone destruction in RA are RANKL and TNF-α (Romas E. et al., Bone 30, pp. 340-346, 2002). OCIF/OPG which is a decoy receptor for RANKL can inhibit osteoclast formation induced by RANKL but does not inhibit osteoclast formation induced by TNF-α. On the other hand, the anti-Siglec-15 antibody according to the invention effectively inhibits osteoclast formation induced by both RANKL and TNF-α. Therefore, it is expected that the anti-Siglec-15 antibody of the invention can inhibit bone loss and bone destruction induced by TNF-α in RA or the like more strongly than an RANKL blocker (OCIF/OPG, an anti-RANKL antibody or the like).
As one example, for the treatment or prevention of abnormal bone metabolism, the anti-Siglec-15 antibody is administered alone or along with at least one other therapeutic agent for a bone-related disease. As another example, the anti-Siglec-15 antibody can be administered along with a therapeutically effective amount of a therapeutic agent for abnormal bone metabolism. The anti-Siglec-15 antibody and the therapeutic agent may be administered simultaneously, separately or sequentially. Examples of the therapeutic agent which can be administered along with the anti-Siglec-15 antibody include, but are not limited to, bisphosphonates, active vitamin D3, calcitonin and derivatives thereof, hormone preparations such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K2 (menatetrenone), calcium preparations, PTH (parathyroid hormone) preparations, nonsteroidal anti-inflammatory agents, soluble TNF receptor preparations, anti-TNF-α antibodies or functional fragments of the antibodies, anti-PTHrP (parathyroid hormone-related protein) antibodies or functional fragments of the antibodies, IL-1 receptor antagonists, anti-IL-6 receptor antibodies or functional fragments of the antibodies, anti-RANKL antibodies or functional fragments of the antibodies and OCIF (osteoclastogenesis inhibitory factor). In another example, the therapeutic agent is a second different anti-Siglec-15 antibody of the present invention. That is to say, two different anti-Siglec-15 antibodies of the invention are administered. Depending on the state of abnormal bone metabolism or the intended degree of the treatment and/or prevention, two or three, or more types of therapeutic agents can be administered, and these therapeutic agents can be supplied all together by encapsulating them in the same preparation. These therapeutic agents and the anti-Siglec-15 antibody can be supplied all together by encapsulating them in the same preparation. Further, these therapeutic agents can be supplied all together by encapsulating them as a kit to be used for treatment and/or prevention. Further, these therapeutic agents and the anti-Siglec-15 antibody can be supplied separately. In the case of administration in gene therapy, a gene of a proteinaceous therapeutic agent for a bone disease and a gene of the anti-Siglec-15 antibody can be inserted downstream of the same promoter region or different promoter regions, and can be introduced into the same vector or different vectors.
By conjugating a therapeutic agent for a bone disease to the anti-Siglec-15 antibody or a fragment thereof, a targeted drug conjugate as described in M. C. Garnet “Targeted drug conjugates: principles and progress”, Advanced Drug Delivery Reviews, (2001) 53, 171-216 can be produced. For achieving this purpose, other than the antibody molecule, any antibody fragment can be applied as long as it does not completely lose the ability to recognize osteoclasts, and examples thereof include fragments such as Fab, F(ab′)2, and Fv. In the invention, the antibody and the fragment can be used in the same manner. The conjugate formed by the anti-Siglec-15 antibody or a fragment thereof and a therapeutic agent for a bone disease can be any of various forms described in M. C. Garnet “Targeted drug conjugates: principles and progress”, Advanced Drug Delivery Reviews, (2001) 53, 171-216, G. T. Hermanson “Bioconjugate Techniques” Academic Press, California (1996), Putnam and J. Kopecek “Polymer Conjugates with Anticancer Activity” Advances in Polymer Science (1995) 122, 55-123 and the like. That is, a conjugate form in which the anti-Siglec-15 antibody and a therapeutic agent for a bone disease are conjugated to each other chemically and directly or via a spacer such as an oligopeptide and a conjugate formed via an appropriate drug carrier can be exemplified. Examples of the drug carrier include a liposome and a water-soluble polymer. More specific examples of the conjugate formed via such a drug carrier include a conjugate form in which the antibody and a therapeutic agent for a bone disease are incorporated in a liposome and the liposome and the antibody are conjugated to each other, and a conjugate form in which a therapeutic agent for a bone disease is conjugated to a water-soluble polymer (a compound having a molecular weight of from about 1000 to 100000) chemically and directly or via a spacer such as an oligopeptide and the antibody is conjugated to the water-soluble polymer. The conjugation of the antibody (or a fragment thereof) to a therapeutic agent for a bone disease or a drug carrier such as a liposome or a water-soluble polymer can be effected by a method known to those skilled in the art such as the method described in G. T. Hermanson “Bioconjugate Techniques” Academic Press, California (1996), Putnam and J. Kopecek “Polymer Conjugates with Anticancer Activity” Advances in Polymer Science (1995) 122, 55-123. The incorporation of a therapeutic agent for a bone disease in a liposome can be effected by a method known to those skilled in the art such as the method described in D. D. Lasic “Liposomes: From Physics to Applications” Elsevier Science Publishers B. V., Amsterdam (1993) or the like. The conjugation of a therapeutic agent for a bone disease to a water-soluble polymer can be effected by a method known to those skilled in the art such as the method described in D. Putnam and J. Kopecek “Polymer Conjugates with Anticancer Activity” Advances in Polymer Science (1995) 122, 55-123. A conjugate between the antibody (or a fragment thereof) and a proteinaceous therapeutic agent for a bone disease (or a fragment thereof) can be produced by a method known to those skilled in the art through genetic engineering other than the above-mentioned method.
The invention also provides a pharmaceutical composition containing a therapeutically and/or preventively effective amount of the anti-Siglec-15 antibody and a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifying agent, preservative and/or adjuvant.
The invention also provides a pharmaceutical composition containing a therapeutically and/or preventively effective amount of the anti-Siglec-15 antibody, a therapeutically and/or preventively effective amount of at least one therapeutic agent for a bone disease, and a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifying agent, preservative and/or adjuvant. Examples of the therapeutic agent for a bone disease include, but are not limited to, bisphosphonates, active vitamin D3, calcitonin and derivatives thereof, hormone preparations such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K2 (menatetrenone), calcium preparations, PTH (parathyroid hormone) preparations, nonsteroidal anti-inflammatory agents, soluble TNF receptor preparations, anti-TNF-α antibodies or functional fragments of the antibodies, anti-PTHrP (parathyroid hormone-related protein) antibodies or functional fragments of the antibodies, IL-1 receptor antagonists, anti-IL-6 receptor antibodies or functional fragments of the antibodies, anti-RANKL antibodies or functional fragments of the antibodies and OCIF (osteoclastogenesis inhibitory factor). In another example, the therapeutic agent is a second, different anti-Siglec-15 antibody of the invention. That is to say that the pharmaceutical composition comprises two different anti-Siglec-15 antibodies of the invention.
A substance to be used in a preparation acceptable in a pharmaceutical composition according to the present invention is preferably non-toxic to a person to whom the pharmaceutical composition is to be administered, in terms of the dose and concentration.
The pharmaceutical composition of the invention can contain a substance for pharmaceutical use which is capable of changing or maintaining the pH, osmotic pressure, viscosity, transparency, color, isotonicity, color, aseptic condition, stability, solubility, release rate, absorption rate, or permeability of the pharmaceutical composition or its components. Examples of the substance for pharmaceutical use include, but are not limited to, amino acids such as glycine, alanine, glutamine, asparagine, arginine and lysine; antimicrobial agents; antioxidants such as ascorbic acid, sodium sulfate and sodium hydrogen sulfite; buffers such as phosphate, citrate, borate buffers, bicarbonate and Tris-HCl solutions; fillers such as mannitol and glycine; chelating agents such as ethylenediamine tetraacetate (EDTA); complexing agents such as caffeine, polyvinylpyrrolidine, p-cyclodextrin and hydroxypropyl-β-cyclodextrin; expanders such as glucose, mannose and dextrin; other carbohydrates such as monosaccharides and disaccharides; coloring agents; flavors; diluents; emulsifying agents; hydrophilic polymers such as polyvinylpyrrolidine; preservatives such as low molecular weight polypeptides, base forming counter ions, benzalkonium chloride, benzoate, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide; solvents such as glycerin, propylene glycol and polyethylene glycol; sugar alcohols such as mannitol and sorbitol; suspending agents; surfactants such as sorbitan ester, polysorbates including polysorbate 20 and polysorbate 80, Triton, tromethamine, lecithin and cholesterol; stability enhancing agents such as sucrose and sorbitol; elasticity enhancing agents such as sodium chloride, potassium chloride and mannitol and sorbitol; transport agents; diluents; excipients; and/or pharmaceutical adjuvants. The addition amount of these substances for pharmaceutical use is preferably from 0.01 to 100 times, particularly preferably from 0.1 to 10 times the weight of the anti-Siglec-15 antibody. Those skilled in the art can appropriately determine a preferred formulation of the pharmaceutical composition in a preparation depending on the disease to be applied, the route of administration to be applied or the like.
The excipient or carrier in the pharmaceutical composition may be in the form of a liquid or a solid. An appropriate excipient or carrier may be injectable water, physiological saline, an artificial cerebral spinal fluid or other substance commonly used for parenteral administration. Further, neutral physiological saline or physiological saline containing serum albumin can also be used as a carrier. The pharmaceutical composition may contain a Tris buffer of pH 7.0 to 8.5 or an acetate buffer of pH 4.0 to 5.5 which may be supplemented with sorbitol or another compound. Examples of the pharmaceutical composition of the invention include a pharmaceutical composition containing the anti-Siglec-15 antibody and a pharmaceutical composition containing the anti-Siglec-15 antibody and at least one therapeutic agent for a bone disease. The pharmaceutical composition of the invention is prepared in the form of a lyophilized product or a liquid as a medicinal agent having a selected composition and a required purity. The pharmaceutical composition containing the anti-Siglec-15 antibody and the pharmaceutical composition containing the anti-Siglec-15 antibody and at least one therapeutic agent for abnormal bone metabolism can also be formed into a lyophilized product using an appropriate excipient such as sucrose.
The pharmaceutical composition of the invention can be prepared for parenteral administration or for gastrointestinal absorption through oral administration. The composition and concentration of a preparation can be determined depending on the administration method. To the extent that the affinity of the anti-Siglec-15 antibody contained in the pharmaceutical composition of the invention for Siglec-15 is higher, that is, as the dissociation constant (Kd value) for Siglec-15 is lower, the anti-Siglec-15 antibody can exhibit its drug efficacy at a lower dose for humans, and therefore, the dose of the pharmaceutical composition of the invention for humans can also be determined based on this result. As for the dose, in the case where a human anti-Siglec-15 antibody is administered to humans, the antibody may be administered at a dose of from about 0.1 to 100 mg/kg once per one to 180 days.
Examples of the dosage form of the pharmaceutical composition of the invention include injections, infusions, suppositories, transnasal agents, sublingual agents and percutaneous absorbents.
4. Other Products of the Invention
There is also provided a polynucleotide which comprises a sequence encoding an anti-Siglec-15 antibody described in the above item “2. Anti-Siglec-15 Antibody”. Details of the nucleotide sequence encoding the preferred antibodies (MOR09281, MOR09892, MOR09898, MOR10110, MOR12574, MOR13154 and MOR13156) are provided in Tables 4 to 7 and 10 to 12. However, it is to be understood that the polynucleotides of the present invention are not limited to those set forth in Tables 4 to 7 and 10 to 12. In particular, as explained in above item 2, anti-Siglec-15 antibodies of the invention include those which comprise CDRs from different preferred antibodies and also includes antibodies which comprise CDRs comprising one or more amino acid additions, deletions or substitutions. The polynucleotides of the present invention include those which encode such antibodies. Furthermore, owing to the degeneracy of the nucleic acid code, nucleotide sequences aside from those set forth in Tables 4 to 7 and 10 to 12 also encode the same amino acid sequences.
Also provided is a polynucleotide which comprises a nucleotide sequence of a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence that encodes one of the antibodies set forth in above item 2, under stringent conditions. In this regard, the phrase “hybridization is performed under stringent conditions” is defined above.
The polynucleotide may also comprise other nucleotide sequences such as a promoter and/or other expression-regulating sequences, sequences which encode a polyadenylation tail, minimal untranslated regions and a Kozak consensus sequence.
In some embodiments, a vector comprising such a polynucleotide is provided. Exemplary vectors include plasmids, cosmids and viral vectors, including bacteriophage. In addition to the polynucleotide, itself, such vectors may include sequences such as an origin of replication, genetic markers, antibiotic resistance genes, cos sites, cloning sites and targeting sequences. In many embodiments, the vector comprises sufficient elements to replicate in a host cell and to express the polynucleotide sequence encoding the anti-Siglec-15 antibody in the host cell.
Thus in further embodiments, there is provided a transformed cell comprising a polynucleotide as described above (for example, incorporated into the genome of the cell) or comprising a vector as described above. Suitable host cells include bacterial cells such as E. coli and eukaryotic cells such as yeast cells or human cell lines. Such transformed cells may be used to synthesize a Siglec-15 antibody of the invention by culturing the cells, expressing nucleotide sequences encoding the antibody and then purifying the antibody from the culture medium.
EXAMPLES
Hereinafter, the invention will be more specifically described with reference to Examples, however, the invention is not limited thereto. Note that the respective operations regarding gene manipulation in the following Examples were performed according to the methods described in “Molecular Cloning” (written by Sambrook, J., Fritsch, E. F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989), or in the case of using commercially available reagents or kits, performed according to the protocols attached thereto.
Example 1
Cell Culture and Transient Transfection
Human embryonic kidney (HEK) 293FreeStyleTM cells were grown in Freestyle 293 Medium (Invitrogen). For cell pannings, HEK 293FreeStyleTM cells were transfected with plasmid DNAs such as plasmid pcDNA3.1 (+)/human Siglec-15 (encoding human Siglec-15, accession number, Q6ZMC9), plasmid pcDNA3.1 (+)/mouse Siglec-15 (encoding mouse Siglec-15, accession number, NP—001094508) or pcDNA3.1(+) using 293fectin (Invitrogen) according to the supplier's instructions. Transfected HEK 293FreeStyleTM cells were harvested after 2 days-cultivation for further cell panning.
Recombinant Human Siglec-15-Fc and Mouse Siglec-15-Fc Preparation
An expression vector for soluble human Siglec-15/phIgFc was constructed by the Gateway technology (Invitrogen). A DNA fragment encoding the extracellular domain of human Siglec-15 (accession number, Q6ZMC9, 1-260) was amplified by PCR to construct an entry clone into pDNOR221 (Invitrogen). Next, an LR reaction was performed between this entry clone and a destination vector harboring a DNA encoding human Fc sequence. The DNA sequence of the human Siglec-15-Fc in the constructed plasmid was confirmed by DNA sequence analysis. Human Siglec-15-Fc was transiently expressed in HEK 293FreeStyleTM harboring the expression vector of soluble human Siglec-15/phIgFc. 1.5 L of the culture solution of HEK 293FreeStyleTM cells expressing soluble human Siglec-15-Fc was filtered through a Sterivex-GV filter (Millipore), then the filtrate was applied to a HiTrap Protein A column (Amersham Biosciences) which was previously equilibrated with Dulbecco's PBS (D-PBS, manufactured by Invitrogen, Inc.). Human Siglec-15-Fc was eluated with 0.1 M sodium citrate buffer (pH 3.0) and neutralized with 1 M Tris. A 2.5 ml aliquot of human Siglec-15-Fc fraction was applied to a PD-10 desalting column (Amersham Biosciences) which had previously been equilibrated with phosphate-buffered saline containing 50 mM arginine hydrochloride (pH 7.0, A-PBS), followed by elution with A-PBS, whereby 3.5 ml of a sample whose solvent was replaced with A-PBS was obtained. The samples prepared by the above-mentioned procedure were cryopreserved at −80° C. until use.
An expression vector for soluble mouse Siglec-15/phIgFc was constructed by the Gateway technology (Invitrogen). A DNA fragment encoding the extracellular domain of mouse Siglec-15 (Accession number, NP—001094508, 1-258) was amplified by PCR to construct an entry clone into pDNOR221 (Invitrogen). Next an LR reaction was performed between this entry clone and a destination vector harboring DNA encoding a human Fc sequence. The DNA sequence of the mouse Siglec-15-Fc in the constructed plasmid was confirmed by DNA sequence analysis. Mouse Siglec-15-Fc was transiently expressed in HEK 293FreeStyleTM harboring the expression vector of soluble mouse Siglec-15/phIgFc. 1.8 L of the culture solution of mouse Siglec-15-Fc-expressing HEK 293FreeStyleTM cells was filtered through a Sterivex-GV filter (Millipore), and the filtrate was applied to a HiTrap Protein A column (Amersham Biosciences) which had previously been equilibrated with Dulbecco's PBS (D-PBS, Invitrogen). Mouse Siglec-15-Fc was eluated with 0.1 M sodium citrate buffer (pH 3.0) and neutralized with 1 M Tris. The collected mouse Siglec-15-Fc fraction was concentrated to 2.5 ml with a centrifugal membrane concentrator Amicon Ultra-15 (Millipore). The buffer was exchanged with Otsuka Physiological Saline for Infection (Otsuka Pharmaceutical) containing 0.01% Tween-20. The sample was cryopreserved at −80° C. until use.
Antibody Generation from HuCAL Gold Libraries
A. Phage Display Library
For the generation of therapeutic antibodies against human Siglec-15, selections with the MorphoSys HuCAL GOLD phage display library were carried out. HuCAL GOLD® is a Fab library based on the HuCAL® concept (Knappik et al. (J. Mol. Biol., 296, 57-86, 2000); Krebs et al., J. Immunol. Methods, 254, 67-84, 2001; Rothe et al., J. Mol. Biol., 376(4):1182-200, 2008), in which all six CDRs are diversified, and which employs the CysDisplayTM technology for linking Fab fragments to the phage surface (WO 01/05950).
B. Pannings with HuCAL GOLD®
MOR09012, MOR08656 and MOR08662 were isolated as follows. For the selections, the HuCAL GOLD® antibody library was subjected to whole cell panning using HEK 293FreeStyleTM cells transfected with either plasmid pcDNA3.1 (+)/human Siglec-15 or plasmid pcDNA3.1 (+)/mouse Siglec-15 using 293 fectin (Invitrogen) followed by pH-elution and a post-adsorption step on Siglec-15-negative HEK 293FreeStyleTM cells for depletion of irrelevant antibody-phages. Finally, the remaining antibody phages were used to infect E. coli cells which were then plated on agar plates and incubated overnight. The next day, the bacterial colonies were scraped off from the plates, the phages were rescued and amplified. The HuCAL GOLD® antibody library was subjected to capture panning using recombinant human Siglec-15-Fc captured by goat anti-human Fc antibody (Jackson Immuno Research, cat. No. 109-005-098) on 96-well Maxi-sorp plate (Nunc, clear). In order to eliminate human Fc and goat IgG binders, both human IgG (Jackson Immuno Research, cat No. 009-000-003) and Goat IgG (Jackson Immuno Research, cat No. 005-000-003) were added to the selection solution. After washing with Tween-20/PBS and PBS, bound phages were eluted. Mid-log phase E. coli cells were infected with the eluted phages and plated onto LB-agar supplemented with glucose and chloramphenicol (LB-CG). After overnight incubation, colonies were scraped off and kept in LB-CG containing 15% glycerol.
MOR09281 was isolated as follows. To obtain Fab with improved affinities, phage resulting from the solid phase Fc capture and differential whole cell pannings were selected for LCDR3 diversification followed by additional selection rounds. Output from all panning strategies was screened in a primary ELISA on recombinant human Siglecl5-hFc. Positive clones were subjected to secondary ELISA and FACS screenings. Selected clones were consolidated and purified.
C. Micro Expression of HuCAL GOLD® Fab Antibodies in E. coli 
Micro expression of Fab fragments encoded by pMORPH® x23_Fab in E. coli was carried out in a round bottom 96-well culture plate (Corning). E. coli colonies were inoculated in medium. The culture plates were incubated until the culture became slightly turbid. Then, additional medium was added. The culture plate was further incubated over night. Next day, BEL buffer (protease inhibitor cocktail (Roche, cat No. 11873580001)) containing 2.5 mg/ml lysozyme (Roche) was added to each well of the culture plate and the culture plate was shaken. After lysis of the E. coli, skim milk/TBS (Takara Bio) solution was added and the culture plate was further shaken. The prepared lysate of E. coli was used for ELISA screening for human Siglec-15 binders.
D. ELISA Screening of Siglec-15 Binders
Wells of a 96-well MaxiSorpTM microtiter plate (Nunc) were coated with 1.25 μg/ml of goat anti-human Fc antibody (Jackson Immuno Research, cat No. 109-005-098) overnight. Next day, after washing with PBS the microtiter plate was blocked with skim milk/PBS. After washing with PBS, 1 μg/ml of recombinant human Siglec-15-Fc was added to the microtiter plate. The wells were washed three times with Tween-20/Tris buffered saline (Takara Bio, (TBST)) before adding the E. coli lysate as described above. The plate was shaken gently followed by washing with TBST. Then, alkaline phosphatase conjugated anti-human Fab (Jackson Immuno Research, Cat No. 109-035-097) was diluted with TBST and was added to the plate. The plate was shaken gently followed by washing with TBST. Binding was detected by Attophos Substrate Set (Roche) and fluorescence (Excitation: 440 nm; Emission: 550 nm) was measured with a plate reader (Molecular Devices, SpectraMax M5).
Example 2
Construction, Expression and Purification OF HuCAL® IgG2
A. Conversion to IgG2
In order to express full length IgG, variable domain fragments of heavy (VH) and light chains (VL) were subcloned from pMORPHx® 9 Fab expression vectors into appropriate pMOPRH® 2_h_Ig vectors for human IgG2. Restriction enzymes MfeI and BlpI were used for subcloning of the VH domain fragment into pMORP® 2_h_IgG2, EcoRV and BsiWI for subcloning of the VL domain fragment into pMORPH® 2_h_Igκ, and EcoRV and HpaI for subcloning of the VL domain fragment into pMORPH® 2_h_Igλ2. Subsequent to ligation, transformation and DNA preparation, the resulting IgG expression plasmids were characterized by restriction analysis and sequencing.
B. Transient Expression and Purification of Human IgG2
Eukaryotic HKB11 cells were transfected with equal amounts of IgG heavy and light chain expression vector (pMORPH® 2). The cell culture supernatant was generally harvested from 3 to 7 days post transfection. After sterile filtration, the solution was subjected to standard protein A affinity chromatography (MabSelect SURE, GE Healthcare). If not otherwise stated, buffer exchange was performed either to 1× Dulbecco's PBS (pH 7.2, Invitrogen) or to citrate buffer (100 mM citrate, 150 mM NaCl, pH 5.0) and samples were sterile filtered (0.2 μm pore size). Protein concentrations were determined by UV-spectrophotometry. Purity of IgG was analyzed under denaturing, reducing and non-reducing conditions in SDS-PAGE or by using Agilent BioAnalyzer and in native state by HP-SEC.
Example 3
Screening and Antibody Characterization
A. Capture ELISA on Siglec-15-FC
Wells of a 384-well MaxiSorpTM microtiter plate were coated with goat anti-human IgG (Dianova, Cat#109-005-098, diluted 1:1,000 in PBS). The next day, wells were washed and then blocked with MPBST on a microtiter plate shaker. The wells were washed with PBST before addition of recombinant human or mouse Siglec15-human Fc fusion protein. After incubation, pre-blocked BEL extracts (for preparations refer to Example 1C) or purified Fab/IgG were added. The plate was incubated on a microtiter plate shaker and then washed with PBST.
For detection of the antigen bound to the captured Fab, the plate was washed with PBST and anti-Fab-AP (Dianova, Cat#109-055-097, 1:5000 diluted in PBS) was added.
After incubation the plate was washed with TBST, Attophos (AttoPhos Substrate Set, Roche #11681982001, diluted 1:10 in TBS) was added and fluorescence was measured in a TECAN microtiter plate reader (emission: 535 nm; excitation: 430 nm).
B. FACS Analysis
HEK293T cells were detached with Versene (Invitrogen #15040066), washed and resuspended in FACS buffer (PBS/3% FCS). The non-adherent HEK293 FreeStyleTM cells were washed and resuspended in FACS buffer. 1×105-1×106 cells/well were transferred into a 96 round bottom well plate, pelleted for 5 min at 300×g and resuspended in diluted BEL extract (see Example 1C) or purified antibody. Cell-antibody mixtures were incubated for 1 h at 4° C. The cells were washed twice by centrifugation for 5 min at 300×g and subsequent suspension in FACS buffer. (During the course of the project, the number of washing steps was increased to five to prevent antibody binding to untransfected cells.) Cells were resuspended in detection antibody solution (diluted 1:100 in FACS buffer). After 1 h incubation at 4° C., cells were washed twice with FACS buffer, resuspended in FACS buffer and analyzed via FACS Array device. Data were analyzed via FlowJo software. MOR03207 (anti-lysozyme) antibody was used as negative control. Human antibodies were detected with goat anti-human PE (Jackson ImmunoResearch #109-116-097).
Example 4
Affinity Maturation of Candidate Antibodies
A. Generation of Affinity Maturation Libraries
To increase affinity and biological activity of selected antibody fragments, CDR3L and CDRH2 regions were optimized in parallel by cassette mutagenesis using trinucleotide directed mutagenesis (Virnekas et al. 1994), while the framework regions were kept constant. Prior to cloning for affinity maturation, all parental Fab fragments were transferred from the corresponding expression vector (pMORPH® x9_FH) into the CysDisplay® vector pMORPH® 25_LHC via XbaI/EcoRI. After CDRL3 or CDRH2 diversification, ligation mixtures of the resulting seven different libraries were electroporated into 4 ml E. coli cells (Invitrogen, Carlsbad, Calif., USA) yielding  ̃107 to 108 independent colonies per library. This library size ensured coverage of the theoretical diversity. Amplification of the library was performed as described before (Rauchenberger et al. 2003). For quality control, single clones were randomly picked and sequenced.
B. Solid-Phase Fc Capture Pannings
1×1012 phage rescued from the affinity maturation libraries as described above, were introduced to the selection via solid phase Fc capture panning as described below.
The appropriate number of wells of a MaxiSorpTM plate (F96 MaxiSorpTM, Nunc #442402) was coated overnight with 330 μl Fcγ fragment specific anti-human IgG, (10 μg/ml in PBS; Jackson Immuno Research, Cat#109-005-098). The following day, coated wells were washed with PBS and blocked with MPBST on a microtiter plate shaker. Subsequently, wells were washed with PBS and incubated with 330 μl antigen solution. The phage were pre-blocked in PBS solution containing milk powder, Tween 20 and 0.1 mg/ml goat gammaglobulin (Jackson Immuno Research, Cat#009-000-002) for 2 h at room temperature on a rotator. For the selection process the antigen solution was removed from the MaxiSorpTM plate and wells were washed with PBS. Pre-blocked phage were added to the corresponding wells and the plate was incubated on a microtiter plate shaker. Following incubation, phage solution was removed and wells were washed several times with PBST, and PBS. PBS was removed after the last washing step before continuing with elution. Specifically bound phage were eluted via DTT in Tris/HCl, pH 8.0 and a 10 min incubation period at room temperature. Eluates were used to infect log phase E. coli TG1 F+ cultures. Infected E. coli were plated onto LB agar plates supplemented with chloramphenicol and glucose and were incubated. The following day, bacterial colonies were scraped off and resuspended in YT media.
Selection rounds were applied in CDRL3 maturation pannings, and in CDRH2 maturation pannings. Panning stringency was increased in each selection round by decreasing antigen concentration and increasing the number and intensity of washing steps.
C. Differential Whole Cell Panning
Differential whole cell maturation pannings were essentially carried out as described below.
Differential whole cell pannings (dWCPs) were designed by alternation of solid phase Fc capture pannings on recombinant antigen (see Example 4B) and whole cell pannings on transfected cells as described below.
Whole cell pannings (WCPs) were performed using HEK293 FreeStyleTM cells transiently transfected with human Siglec-15. The Siglec-15-expressing cells were washed, resuspended and pre-blocked.  ̃1×1012 phage rescued from each maturation library were pre-blocked. After blocking, 1×106 Siglec-15-expressing cells were pelleted, resuspended in pre-blocked phage solution and incubated. The cell number was reduced to 5×105 in the subsequent selection round. Non-specific phage were removed by washing steps, whereby washing stringency was increased during the panning process. For washing, cells were pelleted by centrifugation, resuspended and incubated on a rotator. After washing, the specifically bound phage were eluted from the cells. After centrifugation, the supernatant was removed from the cell pellet and its pH neutralized with 2 M Tris base (unbuffered). Infection of E. coli TG1 F+ with the selected phage was performed as described for solid phase pannings (see Example 4B). Rounds of selection were performed as Fc capture pannings, or as whole cell panning.
D. Parent Antibodies
MOR09012, MOR08656 and MOR08662 are the parent antibodies of MOR09892, MOR09898 or MOR10110, respectively. MOR09281 was selected as a candidate for the further analysis without the step of Affinity Maturation.
Information concerning the amino acid sequences of the antibodies, the nucleic acid sequences encoding them and the structures thereof is summarized in Tables 1 to 7.







TABLE 1







MOR08656










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
 1
 2
 6
 7



FIG.
14
15
16
17



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-411
 20-137
 58-384
 20-128



Region*



CDR1*
148-162
50-54
124-156
42-52



CDR2*
205-255
69-85
202-222
68-74



CDR3*
352-378
118-126
319-345
107-115

















TABLE 2







MOR08662










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
11
12
16
17



FIG.
18
19
20
21



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-408
 20-136
 58-387
 20-129



Region*



CDR1*
148-162
50-54
124-156
42-52



CDR2*
205-255
69-85
202-222
68-74



CDR3*
352-375
118-125
319-348
107-116

















TABLE 3







MOR09012










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
21
22
26
27



FIG.
22
23
24
25



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-405
 20-135
 58-390
 20-130



Region*



CDR1*
148-162
50-54
124-156
42-52



CDR2*
205-255
69-85
202-222
68-74



CDR3*
352-372
118-124
319-351
107-117

















TABLE 4







MOR09281










Chain













Heavy

Light











Sequence Type
















Amino

Amino




Nucleotide
Acid
Nucleotide
Acid







SEQ ID NO
31
32
36
37



FIG.
26
27
28
29



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-408
 20-136
 58-387
 20-129



Region*



CDR1*
148-162
50-54
124-156
42-52



CDR2*
205-255
69-85
202-222
68-74



CDR3*
352-375
118-125
319-348
107-116

















TABLE 5







MOR09892










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
41
42
46
47



FIG.
30
31
32
33



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-405
 20-135
 58-387
 20-129



Region*



CDR1*
148-162
50-54
124-156
42-52



CDR2*
205-255
69-85
202-222
68-74



CDR3*
352-372
118-124
319-348
107-116

















TABLE 6







MOR09898










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
51
52
56
57



FIG.
34
35
36
37



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-411
 20-137
 58-387
 20-129



Region*



CDR1*
148-162
50-54
124-156
42-52



CDR2*
205-255
69-85
202-222
68-74



CDR3*
352-378
118-126
319-348
107-116

















TABLE 7







MOR10110










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
61
62
66
67



FIG.
38
39
40
41



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-408
 20-136
 58-387
 20-129



Region*



CDR1*
148-162
50-54
124-156
42-52



CDR2*
205-255
69-85
202-222
68-74



CDR3*
352-375
118-125
319-348
107-116







*range of nucleotides or amino acid residues






The amino acid sequences of the complementarity determining regions (CDRs) of each of the antibodies are set out in Table 8.











TABLE 8









SEQ






ID


Antibody
Chain
CDR
Sequence
NO.







MOR08656
Heavy
CDR1
NYWMT
 3




CDR2
FISYSGSTTYYADSVKG
 4




CDR3
EGTSSMFDV
 5


 



Light
CDR1
SGDALRSYYAS
 8




CDR2
DDNKRPS
 9




CDR3
GSYDGTVHV
10


 


MOR08662
Heavy
CDR1
NYAMN
13




CDR2
TISYIGSNTYYADSVKG
14




CDR3
GAGLGYDV
15


 



Light
CDR1
SGDNLRSKYVY
18




CDR2
DTNDRPS
19




CDR3
QTYDMTSQDV
20


 


MOR09012
Heavy
CDR1
SYAMH
23




CDR2
YISYSGSNTYYADSVKG
24




CDR3
GWGGFDY
25


 



Light
CDR1
SGDNLPNRYVH
28




CDR2
DDNNRPS
29




CDR3
QTYDMFSMSDV
30


 


MOR09281
Heavy
CDR1
NYWMS
33




CDR2
LISYSGSTTYYADSVKG
34




CDR3
DTPIGMDF
35


 



Light
CDR1
SGDNLGSYYAY
38




CDR2
GDNDRPS
39




CDR3
SSYDIVQPYV
40


 


MOR09892
Heavy
CDR1
SYAMH
43




CDR2
YISYSGSNTYYADSVKG
44




CDR3
GWGGFDY
45


 



Light
CDR1
SGDNLPNRYVH
48




CDR2
DDNNRPS
49




CDR3
QSRDLHYSPV
50


 


MOR09898
Heavy
CDR1
NYWMT
53




CDR2
FISYSGSTTYYADSVKG
54




CDR3
EGTSSMFDV
55


 



Light
CDR1
SGDALRSYYAS
58




CDR2
DDNKRPS
59




CDR3
ASFTYMSDFV
60


 


MOR10110
Heavy
CDR1
NYAMN
63




CDR2
YISYSSSNTYYADSVKG
64




CDR3
GAGLGYDV
65


 



Light
CDR1
SGDNLRSKYVY
68




CDR2
DTNDRPS
69




CDR3
QTYDMTSQDV
70









The amino acid sequences of CDR1 and CDR2 of antibodies MOR09281, MOR09892, MOR09898 and MOR10110 conform to the consensus sequences set out below.
Heavy chain CDR1 consensus sequence is










X1YX2MX3
(SEQ ID NO: 71)






wherein X1 is N or S, X2 is W or A and X3 is S, H, T or N.

Heavy chain CDR2 consensus sequence is










X4ISYSX5SX6TYYADSVKG
(SEQ ID NO: 72)






wherein X4 is L, Y or F, X5 is S or G and X6 is T or N.

Light chain CDR1 consensus sequence is










SGDX7LX8X9X10YX11X12
(SEQ ID NO: 73)






wherein X7 is N or A, X9 is G, P or R, X9 is S or N, X10 is Y, R or K, X11 is A or V and X12 is Y, H or S.

Light chain CDR2 consensus sequence is










X13X14NX15RPS
(SEQ ID NO: 74)






wherein X13 is G or D, X14 is T or D and X15 is N, D or K.

Example 5
Binding Activity Analysis
A. Binding Activity Analysis by ELISA
0.1 mL of 1 μg/ml recombinant human Siglec-15-Fc, mouse Siglec-15-Fc or human Fc (Jackson Immuno Research, #009-000-008) was added to the wells of a 96-well Immobilizer amino plate (Nunc) and the plate was kept overnight at 4° C. Next day, after washing with 0.05% Tween-20/PBS (PBST), the plate was blocked with 5% BSA/PBS for 1.5 hours at room temperature. After washing with PBST, 0.1 mL of 1 μg/ml anti-Siglec-15 antibodies (MOR09281, MOR09892, MOR09898, MOR10110, MOR09012, MOR08656, MOR08662) and human IgG (Jackson Immuno Research, Cat. No. 009-000-003 as a negative control) were added to the plate and the plate was kept for one hour at room temperature. After washing with PBST, 0.1 mL of peroxidise conjugated anti-human Fab antibody (Jackson Immuno Research, Cat. No. 112-035-097) diluted by 5,000 fold with PBST was added to the plate and the plate was kept for 1.5 hours at room temperature. After washing with PBST, binding was detected using ELISA POD ABTS Kit (Nacalai Tesque, Cat. No. 02893-60). Reaction time was 4 min. Absorbance of 450 nm and 650 nm was measured with a plate reader (Molecular Devices, SpectraMax M5). FIG. 1 shows the ELISA results. It was found that MOR09281, MOR09892, MOR09898, MOR10110, MOR09012, MOR08656 and MOR08662 bound to both recombinant human Siglec-15-Fc and mouse Siglec-15-Fc but not human Fc.
Example 6
Biological Assays
A. Mouse Osteoclast Formation Assay
A-1. Preparation of Mouse Bone Marrow Nonadherent Cells
The femur and tibia were resected from a male ddY mouse at the age of 5 to 8 weeks and soft tissues were removed. Both ends of the femur or tibia were cut off, and D-PBS was injected using a syringe with a 23-gauge injection needle to push out bone marrow cells. Centrifugation was performed at room temperature for 5 minutes at 1,000 rpm, and the supernatant was removed. To the cell pellet, 1 ml of a hemolytic buffer (Red Blood Cell Lysing Buffer, manufactured by Sigma Co., Ltd.) was added to suspend it, and the resulting suspension was left at room temperature for 5 minutes. Twenty ml of MEM-α medium (manufactured by Invitrogen, Inc.) containing 10% fetal bovine serum (FBS) was added thereto, and the suspension was centrifuged at room temperature for 5 minutes at 1,000 rpm, and the supernatant was removed. To the cell pellet, 10 ml of MEM-α medium containing 5 ng/ml of M-CSF (manufactured by R&D systems, Inc.) and 10% FBS was added to suspend it. Then, the resulting suspension was passed through a cell strainer (40 μm Nylon, manufactured by BD Falcon) to remove aggregates. The resulting cells were transferred to a 75 cm2-T flask (for the use of adherent cells) and cultured overnight in a CO2 incubator. After the overnight culture, the cells which did not adhere to the T-flask were recovered and used as mouse bone marrow nonadherent cells.
A-2. Effect of Anti-Siglec-15 Antibody on Osteoclast Differentiation from Mouse Bone Marrow Nonadherent Cells (Stimulation with RANKL)
The effect of anti-Siglec-15 antibodies produced in Examples 1 and 4, was evaluated by an osteoclast differentiation assay by using mouse bone marrow nonadherent cells. Mouse bone marrow nonadherent cells prepared by the above-mentioned method in Example 6A-1 were prepared at 1.5×105 cells/ml in α-MEM medium containing 10% FBS and 10 ng/ml of M-CSF, and the resulting cell preparation was seeded in each well of a 96-well plate in an amount of 200 μl and the cells were cultured for 2 days in a CO2 incubator. The old culture medium in the 96-well plate was removed, and 100 μl of MEM-α medium containing 10% FBS, 20 ng/ml human RANKL (RANKL, manufactured by Peprotech, Inc.), and 10 ng/ml M-CSF was added to each well. To the cell culture medium, each anti-Siglec-15 antibody (MOR09281, MOR09012, MOR09892, MOR08656, MOR09898, MOR08662, and MOR10110) was added at concentrations from 3 to 100 ng/ml, and the cells were cultured for an additional 3 days in a CO2 incubator. After completion of the culturing, the activity of tartrate-resistant acid phosphatase (TRAP) of the formed osteoclasts was measured by the following procedure. The culture medium in each well of the 96-well plate was removed by suction, and 50 μl of 50 mM sodium citrate buffer (pH 6.1) containing 1% Triton X-100 was added to each well. Then, the plate was shaken for 5 minutes on a plate shaker to lyse the cells. To each well, 50 μl of a substrate solution (50 mM sodium citrate buffer (pH 6.1) containing 5 mg/ml p-nitrophenyl phosphate and 0.46% sodium tartrate) was added, and the plate was incubated at room temperature for 10 minutes. After the incubation, 50 μl of a 1 N sodium hydroxide solution was added to each well of the 96-well plate to stop the enzymatic reaction. After stopping the enzymatic reaction, an absorbance of each well at 405 nm was measured as an index of TRAP activity. The results are shown in FIGS. 2 to 8. A significant inhibition of TRAP activity was not observed for MOR09012 (parent antibody of MOR09892), MOR08656 (parent antibody of MOR09898), and MOR08662 (parent antibody for MOR10110). On the other hand, a significant inhibition of TRAP activity was observed for MOR09281, MOR09892, MOR09898, and MOR10110 at 13 ng/ml or higher concentrations in a dose-dependent manner. From the above results, it was shown that the anti-Siglec-15 antibodies (MOR09281, MOR09892, MOR09898, and MOR10110) have a potent inhibitory effect on osteoclast formation (osteoclast differentiation and maturation). Furthermore, it was also shown that affinity maturation of the parent antibodies (MOR09012, MOR08656, and MOR08662) produced antibodies (MOR09892, MOR09898, and MOR10110, respectively) that have higher inhibitory activity on osteoclast formation.
B. OsteoLyse Assay with Human Osteoclasts
B) Effect of Addition of Human Anti-Human Siglec-15 Monoclonal Antibodies on Bone Resorption Activity of Normal Human Osteoclast Precursor Cells (Evaluation Using Collagen-Coated Plate)
It is known that osteoclasts release a protease such as cathepsin K and degrade type I collagen which is a constitutional component of bone tissue. OsteoLyse Assay Kit (manufactured by Lonza, Inc., Cat. No. PA-1500) provides a 96-well plate coated with europium-conjugated human collagen (96-well OsteoLyse cell culture plate), and it is possible to evaluate the bone resorption activity of osteoclasts in vitro by measuring the amount of fluorescent collagen fragments released in the supernatant when osteoclasts are cultured in the plate.
Normal human osteoclast precursor cells (Normal Human Natural Osteoclast Precursor Cells, purchased from Sanko Junyaku Co., Ltd., Cat. No. 2T-110) were seeded in a 96-well OsteoLyse cell culture plate at 1×104 cells/well according to the protocol attached to the cells. As the medium, a minimal essential medium for osteoclast precursor cells (OPBM, purchased from Sanko Junyaku Co., Ltd., Cat. No. PT-8201) supplemented with an OPGM supplement set (Osteoclast SingleQuotTM Kit, purchased from Sanko Junyaku Co., Ltd., Cat. No. PT-9501) containing fetal bovine serum (final concentration: 10%), human RANKL (final concentration: 10 or 20 ng/ml), human M-CSF (final concentration: 33 ng/ml) and the like was used. To the resulting culture supernatant, the human anti-human Siglec-15 monoclonal antibodies (MOR09281, 09012, 09892, 08656, 09898, 08662 and 10110) prepared in Examples 1 and 4 was added to give a final concentration of 4, 20, 100, or 500 ng/ml, and the cells were cultured for 4 or 5 days in a CO2 incubator. A 10 μl aliquot of the culture supernatant was collected, and 200 μl of Fluorophore Releasing Reagent included in the OsteoLyse Assay Kit was added thereto, and a fluorescence intensity (RFU) was measured using a time-resolved fluorescence fluorimeter (ALVO MX, manufactured by Perkin Elmer Inc., with excitation at 340 nm and emission at 615 nm), whereby the amount of free fluorescent collagen fragments released in the culture supernatant was determined. The results are shown in FIGS. 9 to 13. As a result, the amount of fluorescent collagen fragments increased by the addition of RANKL was reduced by the anti-Siglec-15 antibodies in a concentration-dependent manner within the range of from 4 ng/ml to 500 ng/ml. The key finding is that the affinity-improved antibodies (MOR09892, 09898 and 10110) inhibited the bone resorption activity at lower concentrations than respective parental antibodies (MOR09012, 08656 and 08662). These results suggest that improvement of the binding affinity of the antibody leads to the improvement of bioactivity as well.
Example 7
Preparation and Assays of Additional Antibodies
A. Preparation of Antibodies
MOR09268 was prepared in the same manner as in Example 1 in which MOR09281 was obtained and it was converted to IgG2 in accordance with Example 2. MOR09268 is the parental antibody of MOR12574, MOR13154 and MOR13156. Affinity maturations to generate these three antibodies were performed in accordance with the protocol described in Example 4.
Information concerning the amino acid sequences of the antibodies, the nucleic acid sequences encoding them and the structures thereof is summarized in Tables 9 to 12.







TABLE 9







MOR09268










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
76
77
81
82



FIG.
46
47
48
49



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-414
 20-138
 58-399
 20-133



Region*



CDR1*
148-162
50-54
124-165
42-55



CDR2*
205-255
69-85
211-231
71-77



CDR3*
352-381
118-127
328-360
110-120

















TABLE 10







MOR12574










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
76
77
86
87



FIG.
46
47
50
51



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-414
 20-138
 58-402
 20-134



Region*



CDR1*
148-162
50-54
124-165
42-55



CDR2*
205-255
69-85
211-231
71-77



CDR3*
352-381
118-127
328-363
110-121

















TABLE 11







MOR13154










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
91
92
96
97



FIG.
52
53
54
55



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-414
 20-138
 58-399
 20-133



Region*



CDR1*
148-162
50-54
124-165
42-55



CDR2*
205-255
69-85
211-231
71-77



CDR3*
352-381
118-127
328-360
110-120

















TABLE 12







MOR13156










Chain













Heavy

Light











Sequence Type














Amino

Amino



Nucleotide
Acid
Nucleotide
Acid

















SEQ ID NO
101
102
86
87



FIG.
 56
 57
50
51



Signal
 1-57
 1-19
 1-57
 1-19



Sequence*



Variable
 58-420
 20-140
 58-402
 20-134



Region*



CDR1*
148-162
50-54
124-165
42-55



CDR2*
205-261
69-87
211-231
71-77



CDR3*
358-387
120-129
328-363
110-121







*range of nucleotides or amino acid residues






n.b. The heavy chains of antibodies MOR09268 and MOR12574 are identical. The light chains of antibodies MOR12574 and MOR13156 are identical.

The amino acid sequences of the complementarity determining regions (CDRs) of each of the antibodies are set out in Table 13.











TABLE 13









SEQ






ID


Antibody
Chain
CDR
Sequence
NO.







MOR09268
Heavy
CDR1
TYAMH
 78




CDR2 
NIAYSGSVTYYADSVKG
 79




CDR3
RGPGMGNMDI
 80


 



Light
CDR1
TGTSSDVGDYNYVS
 83




CDR2
YVTNRPS
 84




CDR3
QSYDTGSFAMV
 85


 


MOR12574
Heavy
CDR1
TYAMH
 78




CDR2
NIAYSGSVTYYADSVKG
 79




CDR3
RGPGMGNMDI
 80


 



Light
CDR1
TGTSSDVGDYNYVS
 88




CDR2
YVTNRPS
 89




CDR3
QSYAPLPSSHIV
 90


 


MOR13154
Heavy
CDR1
TYAMH
 93




CDR2
TIFGSSSSTYYADSVKG
 94




CDR3
RGPGMGNMDI
 95


 



Light
CDR1
TGTSSDVGDYNYVS
 98




CDR2
YVTNRPS
 99




CDR3
QSYAGASFNLV
100


 


MOR13156
Heavy
CDR1
TYAMH
103




CDR2
TIEIKEAGYATNYAAGVKG
104




CDR3
RGPGMGNMDI
105


 



Light
CDR1
TGTSSDVGDYNYVS
 88




CDR2
YVTNRPS
 89




CDR3
QSYAPLPSSHIV
 90









The amino acid sequences of CDR1 and CDR2 of the heavy chain and CDR2 of the light chain of antibodies MOR09281, MOR09892, MOR09898, MOR10110, MOR09268, MOR12574, MOR13154 and MOR13156 conform to the consensus sequences set out below.
Heavy chain CDR1 consensus sequence is










X16YX17MX18
(SEQ ID NO: 106)






wherein X16 is N, S or T, X17 is W or A and X18 is S, H, T or N.

Heavy chain CDR2 consensus sequence is









(SEQ ID NO: 107)



X19IX20X21X22X23X24X25X26X27TX28YAX29X30VKG






wherein X19 is L, Y, F, N or T, X20 is S, A, F or E, X21 is Y, G or I, X22 is K or no amino acid, X23 is E or no amino acid, X24 is S or A, X25 is S or G, X26 is S or Y, X27 is T, N, V, S or A, X28 is Y or N, X29 is D or A and X30 is S or G.

Light chain CDR2 consensus sequence is










X31X32X33X34RPS
(SEQ ID NO: 108)






wherein X31 is G, D or Y, X32 is T, D or V, X33 is N or T and X34 is N, D or K.

Antibodies MOR12574, MOR13154 and MOR13156 all have the same light chain CDR1 sequence of:










TGTSSDVGDYNYVS
(SEQ ID NO: 83)






Antibodies MOR12574, MOR13154 and MOR13156 all have the same light chain CDR2 sequence of:










YVTNRPS
(SEQ ID NO: 89)






Antibodies MOR12574, MOR13154 and MOR13156 all have the same heavy chain CDR3 sequence of:










RGPGMGNMDI
(SEQ ID NO: 80)






The amino acid sequences of CDR3 of the light chain of antibodies MOR12574, MOR13154 and MOR13156 conform to the consensus sequences set out below.










QSYAX35X36X37X38X39X40X41V
(SEQ ID NO: 109)






wherein X35 is P or G, X36 is L or A, X37 is S or P, X38 is S or F, X39 is S or N, X40 is H or no amino acid and X41 is I or L.

B. OsteoLyse Assay with Human Osteoclasts

Effect of human anti-human Siglec-15 monoclonal antibodies on bone resorption activity of normal human osteoclast precursor cells (Evaluation using collagen-coated plate)
Normal human osteoclast precursor cells (Normal Human Natural Osteoclast Precursor Cells, purchased from Sanko Junyaku Co., Ltd., Cat. No. 2T-110) were seeded in a 96-well OsteoLyse cell culture plate (Lonza, Inc., Cat. No. PA-1500) at 1×104 cells/well according to the protocol attached to the cells. As the medium, a minimal essential medium for osteoclast precursor cells (OPBM, purchased from Sanko Junyaku Co., Ltd., Cat. No. PT-8201) supplemented with an OPGM supplement set (Osteoclast SingleQuotTM Kit, purchased from Sanko Junyaku Co., Ltd., Cat. No. PT-9501) containing fetal bovine serum (final concentration: 10%), human RANKL (final concentration: 10 or 20 ng/ml), human M-CSF (final concentration: 33 ng/ml) and the like was used. To the resulting culture supernatant, MOR09268 was added to give a final concentration of 3 or 30 μg/ml, and the cells were cultured for 6 days in a CO2 incubator. Likewise MOR12574, MOR13154 or MOR13156 were added to the culture supernatant, to give a final concentration of 4, 20, 100, or 500 ng/ml, and the cells were cultured for 5 days in a CO2 incubator. A 10 μl aliquot of the culture supernatant was collected, and 200 μl of Fluorophore Releasing Reagent included in the OsteoLyse Assay Kit was added thereto. Fluorescence intensity (RFU) was measured using a time-resolved fluorescence fluorimeter (ALVO MX, manufactured by Perkin Elmer Inc., with excitation at 340 nm and emission at 615 nm), whereby the amount of free fluorescent collagen fragments released in the culture supernatant was determined. The results for MOR09268, MOR12574, MOR13154 and MOR13156 are shown in FIGS. 42, 43, 44 and 45, respectively. As shown in FIGS. 42 to 45, the number of fluorescent collagen fragments increased by the addition of RANKL was reduced by the anti-Siglec-15 antibodies in a concentration-dependent manner.
Example 8
Evaluation of Biological Activity of Anti-Siglec-15 Monoclonal Antibody Using Ovariectomized Rats 
A. Protocol of Animal Experiment
The ovaries on both sides are removed from female F344 rats (obtained from Charles River Laboratories Japan, Inc.) at the age of 12 weeks, and the rats are divided into two groups: a vehicle administration group; and an anti-Siglec-15 monoclonal antibody administration group. Further, as a sham operation group, one group is prepared. In the antibody administration group, the anti-Siglec-15 monoclonal antibody in Example 2 or 7 is intraperitoneally administered at a dose of 1 mg/kg three times a week repeatedly for 4 weeks from the next day of the operation. In the vehicle administration group and the sham operation group, PBS containing 0.01% Tween 20 is intraperitoneally administered as the vehicle. At 4 weeks after the initiation of administration, urine is collected for 24 hours under fasting conditions, and the urine samples are stored at −80° C. until measurement. After completion of the urine collection, the rats are euthanized, and the lumbar spine is excised from each rat.
B. Measurement of Lumbar Spine Bone Density
Soft tissues adhered to the excised lumbar spine are removed, and the 4th to 6th lumbar vertebrae are extracted. The extracted lumbar vertebrae are degreased and dehydrated by being shaken in ethanol and then air-dried, and the bone density is measured using a bone densitometer (DCS-600EX, manufactured by Aloka Co., Ltd.). A significant decrease in lumbar spine bone density will be observed in the ovariectomized group as compared with the sham operation group, however, in the antibody administration groups, a decrease in bone density due to ovariectomy will be significantly inhibited.
C. Measurement of Urinary Deoxypyridinoline Excretion
A variety of type I collagen crosslinked metabolites sharply reflect bone metabolic turnover, particularly bone resorption. Above all, deoxypyridinoline is localized mainly in bone collagen, and therefore, is considered to have high reliability as an index of bone resorption.
The cryopreserved urine sample is thawed, and insoluble matter is precipitated by a centrifugal operation, whereby a supernatant is obtained. The amount of deoxypyridinoline contained in this supernatant is measured using Osteolinks “DPD” (manufactured by DS Pharma Biomedical Co., Ltd.). Further, by using Creatinine Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), the content of creatinine in the supernatant is also measured, and the amount of deoxypyridinoline corrected for creatinine is calculated. The urinary deoxypyridinoline excretion will be significantly increased in the ovariectomized group as compared with the sham group, and therefore, it will be indicated that in the ovariectomized rats, osteoclastic bone resorption is increased. On the other hand, in the antibody administration groups, an increase in deoxypyridinoline excretion due to ovariectomy will be inhibited. From this result, it will also be confirmed in the animal models that the monoclonal antibodies specifically binding to Siglec-15 inhibit osteoclastic bone resorption, and it will be strongly indicated that due to the inhibitory effect on bone resorption, a decrease in lumbar spine bone density in the ovariectomized rats is inhibited.
INDUSTRIAL APPLICABILITY
The anti-Siglec-15 antibody of the invention has the ability to inhibit osteoclast differentiation or bone resorption activity, and a pharmaceutical composition containing the anti-Siglec-15 antibody can be a therapeutic or preventive agent for a disease of abnormal bone metabolism.

## Claims

1. An isolated antibody capable of binding Siglec-15 which inhibits osteoclast formation and/or osteoclastic bone resorption, or an antigen-binding fragment thereof comprising (a) a heavy chain comprising a CDRH1 comprising a sequence of SEQ ID NO: 106, a CDRH2 comprising a sequence of SEQ ID NO: 107, and a CDRH3 comprising a sequence of any one of SEQ ID NOS: 80, 55, 65, 35 or 45; and (b) a light chain comprising a CDRL1 comprising a sequence of SEQ ID NO: 83 or 73, a CDRL2 comprising a sequence of SEQ ID NO: 108, and a CDRL3 comprising a sequence of any one of SEQ ID NOS: 90, 60, 100, 70, 40, 50 or 109, wherein the antibody or antigen-binding fragment binds to and inhibits Siglec-15, thereby inhibiting osteoclast formation and/or osteoclastic bone resorption.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the CDRH1 comprises a sequence of any one of SEQ ID NOS: 78, 53, 63, 33 or 43.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the CDRH2 comprises a sequence of any one of SEQ ID NOS: 104, 54, 94, 64, 34, 44 or 79.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the CDRL1 comprises a sequence of any one of SEQ ID NOS: 88, 58, 68, 38 or 48.

5. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the CDRL2 comprises a sequence of any one of SEQ ID NOS: 89, 59, 69, 39 or 49.

6. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 in the antibody comprise:
a) SEQ ID NOS: 103, 104, 105, 88, 89 and 90, respectively;
b) SEQ ID NOS: 53, 54, 55, 58, 59 and 60, respectively;
c) SEQ ID NOS: 93, 94, 95, 98, 99 and 100, respectively;
d) SEQ ID NOS: 63, 64, 65, 68, 69 and 70, respectively;
e) SEQ ID NOS: 33, 34, 35, 38, 39 and 40, respectively;
f) SEQ ID NOS: 43, 44, 45, 48, 49 and 50, respectively; or
g) SEQ ID NOS: 78, 79, 80, 88, 89 and 90, respectively.

7. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises:
a heavy chain variable region selected from the group consisting of
a1) an amino acid sequence comprising: amino acid residues 20 to 140 of SEQ ID NO: 102, amino acid residues 20 to 137 of SEQ ID NO: 52, amino acid residues 20 to 138 of SEQ ID NO: 92, amino acid residues 20 to 136 of SEQ ID NO: 62, amino acid residues 20 to 136 of SEQ ID NO: 32, amino acid residues 20 to 135 of SEQ ID NO: 42, or amino acid residues 20 to 138 of SEQ ID NO: 77;
and
a light chain variable region selected from the group consisting of
b1) an amino acid sequence comprising: amino acid residues 20 to 134 of SEQ ID NO: 87, amino acid residues 20 to 129 of SEQ ID NO: 57, amino acid residues 20 to 133 of SEQ ID NO: 97, amino acid residues 20 to 129 of SEQ ID NO: 67, amino acid residues 20 to 129 of SEQ ID NO: 37, or amino acid residues 20 to 129 of SEQ ID NO: 47.

8. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises:
a heavy chain selected from the group consisting of
a1) an amino acid sequence comprising the sequence of any one of SEQ ID NOS: 102, 52, 92, 62, 32, 42 or 77;
and
a light chain selected from the group consisting of
b1) an amino acid sequence comprising the sequence of any one of SEQ ID NOS: 87, 57, 97, 67, 37 or 47.

9. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 102, and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 87.

10. The isolated antibody or functional fragment thereof according to claim 1, wherein the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 52, and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 57.

11. The isolated antibody or functional fragment thereof according to claim 1, wherein the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 92, and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 97.

12. The isolated antibody or functional fragment thereof according to claim 1, wherein the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 62, and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 67.

13. The isolated antibody or functional fragment thereof according to claim 1, wherein the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 32, and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 37.

14. The isolated antibody or functional fragment thereof according to claim 1, wherein the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 42, and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 47.

15. The isolated antibody or functional fragment thereof according to claim 1, wherein the antibody comprises a heavy chain consisting of an amino acid sequence comprising SEQ ID NO: 77, and a light chain consisting of an amino acid sequence comprising SEQ ID NO: 87.

16. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab′)2, Fab′ and Fv.

17. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is a scFv.

18. A method for treating a patient suffering from abnormal bone metabolism comprising administering a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the abnormal bone metabolism is characterized by insufficient bone mass or density.

19. A pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof according to claim 1.

20. The pharmaceutical composition according to claim 19, wherein the composition is a therapeutic agent for abnormal bone metabolism, wherein the abnormal bone metabolism is characterized by insufficient bone mass or density.

21. A pharmaceutical composition for treating abnormal bone metabolism comprising at least one antibody or antigen-binding fragment thereof according to claim 1, and at least one member selected from the group consisting of bisphosphonates, active vitamin D3, calcitonin, hormones, ipriflavone, vitamin K2 (menatetrenone), calcium, PTH (parathyroid hormone), nonsteroidal anti-inflammatory agents, soluble TNF receptor, anti-TNF-α antibodies or antigen-binding fragments thereof, anti-PTHrP (parathyroid hormone-related protein) antibodies or antigen-binding fragments thereof, anti-IL-6 receptor antibodies or antigen-binding fragments thereof, anti-RANKL antibodies or antigen-binding fragments thereof, and OCIF (osteoclastogenesis inhibitory factor).

22. The method of claim 18, wherein the abnormal bone metabolism is selected from the group consisting of: osteoporosis, bone destruction accompanying rheumatoid arthritis, cancerous hypercalcemia, bone destruction accompanying multiple myeloma or cancer metastasis to bone, giant cell tumor, tooth loss due to periodontitis, osteolysis around a prosthetic joint, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy and osteogenesis imperfecta.

23. The method according to claim 22, characterized in that the abnormal bone metabolism is osteoporosis, bone destruction accompanying rheumatoid arthritis or bone destruction accompanying cancer metastasis to bone.

24. The method according to claim 23, characterized in that the osteoporosis is postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis due to the use of a therapeutic agent, or osteoporosis accompanying rheumatoid arthritis.

25. A polynucleotide which comprises a nucleotide sequence that encodes the antibody or functional antigen-binding fragment thereof according to claim 1.

26. The polynucleotide according to claim 25, wherein the nucleotide sequences encoding CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise:
a) nucleotides 148 to 162 of SEQ ID NO: 101, nucleotides 205 to 261 of SEQ ID NO: 101, nucleotides 358 to 387 of SEQ ID NO: 101, nucleotides 124 to 165 of SEQ ID NO: 86, nucleotides 211 to 231 of SEQ ID NO: 86 and nucleotides 328 to 363 of SEQ ID NO: 86, respectively;
b) nucleotides 148 to 162 of SEQ ID NO: 51, nucleotides 205 to 255 of SEQ ID NO: 51, nucleotides 352 to 378 of SEQ ID NO: 51, nucleotides 124 to 156 of SEQ ID NO: 56, nucleotides 202 to 222 of SEQ ID NO: 56 and nucleotides 319 to 348 of SEQ ID NO: 56, respectively;
c) nucleotides 148 to 162 of SEQ ID NO: 91, nucleotides 205 to 255 of SEQ ID NO: 91, nucleotides 352 to 381 of SEQ ID NO: 91, nucleotides 124 to 165 of SEQ ID NO: 96, nucleotides 211 to 231 of SEQ ID NO: 96 and nucleotides 328 to 360 of SEQ ID NO: 96, respectively;
d) nucleotides 148 to 162 of SEQ ID NO: 61, nucleotides 205 to 255 of SEQ ID NO: 61, nucleotides 352 to 375 of SEQ ID NO: 61, nucleotides 124 to 156 of SEQ ID NO: 66, nucleotides 202 to 222 of SEQ ID NO: 66 and nucleotides 319 to 348 of SEQ ID NO: 66, respectively;
e) nucleotides 148 to 162 of SEQ ID NO: 31, nucleotides 205 to 255 of SEQ ID NO: 31, nucleotides 352 to 375 of SEQ ID NO: 31, nucleotides 124 to 156 of SEQ ID NO: 36, nucleotides 202 to 222 of SEQ ID NO: 36 and nucleotides 319 to 348 of SEQ ID NO: 36, respectively;
f) nucleotides 148 to 162 of SEQ ID NO: 41, nucleotides 205 to 255 of SEQ ID NO: 41, nucleotides 352 to 372 of SEQ ID NO: 41, nucleotides 124 to 156 of SEQ ID NO: 46, nucleotides 202 to 222 of SEQ ID NO: 46 and nucleotides 319 to 348 of SEQ ID NO: 46, respectively; or
g) nucleotides 148 to 162 of SEQ ID NO: 76, nucleotides 205 to 255 of SEQ ID NO: 76, nucleotides 352 to 381 of SEQ ID NO: 76, nucleotides 124 to 165 of SEQ ID NO: 86, nucleotides 211 to 231 of SEQ ID NO: 86 and nucleotides 328 to 363 of SEQ ID NO: 86, respectively.

27. The polynucleotide according to claim 25, wherein the polynucleotide comprises:
a nucleotide sequence encoding a heavy chain variable region comprising: nucleotides 58 to 420 of SEQ ID NO: 101, nucleotides 58 to 411 of SEQ ID NO: 51, nucleotides 58 to 414 of SEQ ID NO: 91, nucleotides 58 to 408 of SEQ ID NO: 61, nucleotides 58 to 408 of SEQ ID NO: 31, nucleotides 58 to 405 of SEQ ID NO: 41 or nucleotides 58 to 414 of SEQ ID NO: 76; and
a nucleotide sequence encoding a light chain variable region comprising: nucleotides 58 to 402 of SEQ ID NO: 86, nucleotides 58 to 387 of SEQ ID NO: 56, nucleotides 58 to 399 of SEQ ID NO: 96, nucleotides 58 to 387 of SEQ ID NO: 66, nucleotides 58 to 387 of SEQ ID NO: 36 or nucleotides 58 to 387 of SEQ ID NO: 46.

28. The polynucleotide according to claim 27, wherein the polynucleotide comprises:
a nucleotide sequence encoding a heavy chain comprising the nucleotide sequence of any one of SEQ ID NOS: 101, 51, 91, 61, 31, 41 or 76; and
a nucleotide sequence encoding a light chain comprising the nucleotide sequence of any one of SEQ ID NOS: 86, 56, 96, 66, 36 or 46.

29. A vector comprising at least one polynucleotide according to claim 25.

30. A transformed cell comprising at least one polynucleotide according to claim 25.

31. A transformed cell comprising at least one vector according to claim 29.

32. A method of producing the isolated antibody or antigen-binding fragment of claim 1, comprising (i)-culturing a transformed cell that comprises at least one polynucleotide encoding (a) a heavy chain comprising a CDRH1 comprising a sequence of SEQ ID NO: 106, a CDRH2 comprising a sequence of SEQ ID NO: 107, and a CDRH3 comprising a sequence of any one of SEQ ID NOS: 80, 55, 65, 35 or 45; and (b) a light chain comprising a CDRL1 comprising a sequence of SEQ ID NO: 83 or 73, a CDRL2 comprising a sequence of SEQ ID NO: 108, and a CDRL3 comprising a sequence of any one of SEQ ID NOS: 90, 60, 100, 70, 40, 50 or 109, (ii) collecting culturing materials, and (iii) purifying said antibody from said culturing materials.

33. The pharmaceutical composition according to claim 20, wherein the abnormal bone metabolism is selected from the group consisting of: osteoporosis, bone destruction accompanying rheumatoid arthritis, cancerous hypercalcemia, bone destruction accompanying multiple myeloma or cancer metastasis to bone, giant cell tumor, tooth loss due to periodontitis, osteolysis around a prosthetic joint, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy and osteogenesis imperfecta.

34. The pharmaceutical composition according to claim 21, wherein the abnormal bone metabolism is selected from the group consisting of: osteoporosis, bone destruction accompanying rheumatoid arthritis, cancerous hypercalcemia, bone destruction accompanying multiple myeloma or cancer metastasis to bone, giant cell tumor, tooth loss due to periodontitis, osteolysis around a prosthetic joint, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy and osteogenesis imperfecta.

35. The pharmaceutical composition according to claim 33, wherein the abnormal bone metabolism is osteoporosis, bone destruction accompanying rheumatoid arthritis or bone destruction accompanying cancer metastasis to bone.

36. The pharmaceutical composition according to claim 34, wherein the abnormal bone metabolism is osteoporosis, bone destruction accompanying rheumatoid arthritis or bone destruction accompanying cancer metastasis to bone.

37. The pharmaceutical composition according to claim 35, wherein the osteoporosis is postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis due to the use of a therapeutic agent, or osteoporosis accompanying rheumatoid arthritis.

38. The pharmaceutical composition according to claim 36, wherein the osteoporosis is postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis due to the use of a therapeutic agent, or osteoporosis accompanying rheumatoid arthritis.

39. The pharmaceutical composition according to claim 21, wherein the hormone is estradiol.

40. The pharmaceutical composition according to claim 24, wherein the secondary osteoporosis is due to the use of a steroid or an immunosuppressant.

41. The pharmaceutical composition according to claim 37, wherein the secondary osteoporosis is due to the use of a steroid or an immunosuppressant.

42. The pharmaceutical composition according to claim 38, wherein the secondary osteoporosis is due to the use of a steroid or an immunosuppressant.