Patent Application
20070031421
The present invention relates to compounds (including but not limited to nucleic acids and antibodies) useful for the treatment and/or prevention of metabolic bone disorders, a pharmaceutical composition for treating metabolic bone disorders characterized in that it contains the compound(s), including antibodies as an active ingredient, methods of detecting metabolic bone disorders; methods of screening for modulators of dendritic cell-specific transmembrane proteins, and a metabolic bone disorder detection kit.
Bone is known to be a dynamic organ which constantly cycles between formation and resorption for reconstruction in order to change its own morphology and to maintain blood calcium levels. Healthy bone maintains an equilibrium between bone formation by osteoblasts, bone resorption by osteoclasts, and its bone mass constant. In contrast, when the equilibrium between bone formation and bone resorption is lost, a metabolic bone disorder such as osteoporosis can develop (Endocrinological Review 13, 66-80, 1992; and Principles of Bone Biology pp. 87-102, 1996, Academic Press, New York).
Many factors involved in regulation of bone metabolism have been reported, including systemic hormones and local cytokines, and they serve together to form and maintain bone (Endocrinological Review 13, 66-80, 1992; and Endocrinological Review 17, 308-332, 1996). A change in bone tissue with aging is widely recognized as a cause of osteoporosis, but the mechanism of its development encompasses various factors, for example, a lower secretion of sex hormones and an abnormality of receptors for the hormones, expression of aging genes, failure to differentiate into osteoclasts and/or osteoblasts and dysfunction of those cells, and thus, as a physiological event due to aging, it is poorly understood. Osteoporosis is largely divided between osteoporosis after menopause due to a lower 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 development and to develop therapeutic agents.
Osteoclasts are multinucleate cells derived from hematopoietic stem cells, they release chloride and hydrogen ions on the bone surface to which they adhere to acidify the space between the bone surface and the cells themselves (American Journal of Physiology 260, C1315-C1324, 1991). This causes decomposition of calcium phosphate and activation of acid proteases, leading to 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 95, 3597-3602, 1998; and Cell 93, 165-176, 1998). It has been shown that RANKL is a membrane-bound factor produced by osteoblasts/stromal cells, its expression is regulated by a bone resorption factor; RANKL induces differentiation of preosteoclastic cells into multinucleate osteoclasts (Proceedings of the National Academy of Science of the United States of America 95, 3597-3602, 1998; and Journal of Bone and Mineral Research 23; S222, 1998). Furthermore, knockout mice devoid of RANKL have been found to develop a typical osteopetrosis, which has verified that RANKL is a physiological inducer for differentiation into osteoclasts (Nature 397, 315-323, 1999).
To treat a bone disorder or shorten the duration of treatment, bisphosphonates, activated vitamin D3, calcitonin and its derivatives, hormone preparations such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K2 (menatetrenone), calcium preparations and the like are currently used. However, these drugs have not always exhibited a satisfactory therapeutic effect, and thus there has been a desire to develop more potent drugs.
Dendritic cells (referred to as “DC” hereinafter) are specialized antigen-presenting cells of the immune system and distributed throughout the entire body. The dendritic cell-specific transmembrane protein (referred to as “DC-STAMP” hereinafter) is a protein extending across the cell membrane of dendritic cells, that has been cloned from the cDNA library of monocyte-derived DCs (European Journal of Immunology 30, 3585-3590, 2000). One human DC-STAMP cDNA has been reported (GenBank Accession No: NM—030788), its amino acid sequence is shown in SEQ ID NO: 2 in the Sequence Listing. The long nucleotide sequence containing the third exon of the cDNA for the murine DC-STAMP is registered as Accession No: AB109560_ with GenBank, and shown in SEQ ID NO: 3 in the sequence Listing; its amino acid sequence is shown in SEQ ID NO: 4 in the Sequence Listing. The nucleotide sequence having the short third exon of the splice variant cDNA for the murine DC-STAMP is registered as Accession No: AB109561 with GenBank, and shown in SEQ ID NO: 5 in the Sequence Listing; its amino acid sequence is shown in SEQ ID NO: 6 in the Sequence Listing.
An amino acid sequence homology of about 74% has been identified between the human DC-STAMP and the murine DC-STAMP. As a result of hydrophobicity analysis for the amino acid sequences, the DC-STAMPs are predicted to have seven transmembrane domains. The murine splice variant having a short third exon is considered to have the seventh transmembrane domain deleted, and is thus denoted by DC-STAMP ΔT7 hereinafter.
DC-STAMP is reported to be more highly expressed following inactivation of mononuclear phagocytes with IL-4, but less highly expressed following their inactivation with dexamethasone (Immunogenetics 53, 105-113, 2001). However, the association of DC-STAMP with differentiation into osteoclasts still remains to be elucidated.
Many metabolic bone disorders exist, including without limitation osteoporosis, rheumatoid arthritis, metastasis of cancer cells to bone, hypercalcaemia, osteolytic bone destruction associated with giant bone tumors (GCTs), and the like. As such, there is a need for methods of testing whether a substance can have an affect on such disorders. In particular, methods are needed to determine whether substances may inhibit specific genes that, upon expression, play a role in such disorders, such as for example, inhibiting certain osteoclastic activities. Through such methods, agents that may be used to prevent and/or treat such disorders can be identified. The present invention concerns the identification of such agents based on their capacity to prevent the differentiation, maturation, and activation of osteoclasts.
Accordingly, in one aspect, the invention provides compounds that inhibit DC-STAMP activity and thus suppresses osteoclastogenesis in a cell expressing the DC-STAMP protein. In one aspect, the compounds act at the protein level. In this aspect, the compounds include antibodies which a) specifically bind to a DC-STAMP protein selected from at least one member of the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; and b) suppress osteoclastogenesis in a cell expressing the DC-STAMP protein. In some aspects, the antibodies include chimeric antibodies (including humanized antibodies), human antibodies, monoclonal antibodies, and derivatives thereof), which can be IgG antibodies and preferably IgG1 antibodies. Other modulators (including inhibitors) that bind to DC-STAMP and suppress osteoclastogenesis are outlined herein.
In an additional aspect, the invention provides compounds that inhibit DC-STAMP by acting at the nucleic acid level, e.g. by preventing expression of the nucleic acid, for example through the use of siRNA.
In a further aspect, the invention provides pharmaceutical compositions comprising the antibodies of the invention and a pharmaceutically acceptable carrier.
In an additional aspect, the invention provides methods of screening for binding to human DC-STAMP comprising contacting a human DC-STAMP protein with a library of candidate agents and determining the presence or absence of binding of at least one candidate agent and the DC-STAMP protein. In some aspects, either the protein or the candidate agent can be immobilized on a solid support, including microspheres. Either component can be labeled, for example with fluorophores.
In a further aspect, the invention provides methods of screening for osteoclastogenesis suppressive activity in a population of cells expressing a human DC-STAMP protein comprising contacting the cells with a candidate agent to form a mixture and assaying for suppression of osteoclast formation. In some aspects, a library of candidate agents are tested, and in certain aspects, the candidate agents are antibodies. The method optionally includes adding osteoclastogenesis inducers to the mixture. Such inducers may be receptor activator of NF-κB ligand (RANKL) and/or Tumor Necrosis Factor-α (TNF-α).
In an additional aspect, the invention provides methods of inducing suppression of osteoclatogenesis in a cell expressing human DC-STAMP comprising adding an agent that inhibits DC-STAMP activity such that suppression of osteoclastogenesis is induced.
In a further aspect, the invention provides methods of detecting a metabolic bone disorder comprising measuring the amount of nucleic acid encoding DC-STAMP from a human test sample, measuring the amount of nucleic acid encoding DC-STAMP from a human healthy sample, and comparing the difference in the amounts to determine the presence of a bone disorder in the test sample.
In yet another aspect, the invention provides methods of detecting a metabolic bone disorder comprising measuring the amount of DC-STAMP protein from a human test sample, measuring the amount of DC-STAMP protein from a human healthy sample and comparing the difference in the amounts to determine the presence of a bone disorder in the test sample.
1. General Overview of the Invention
The present invention is directed to the finding that the dendritic cell-specific transmembrane protein (referred to hereinafter as “DC-STAMP”) is involved in the differentiation of hematopoietic stem cells into osteoclasts and their maturation and activation, and that a lower expression of DC-STAMP leads to a lower rate of differentiation of osteoclasts. For example, DC-STAMP is expressed at a higher level when a monocyte-derived cell strain is differentiated into osteoclasts and is specifically expressed in giant cell tumors. Thus, the present invention provides compositions, including antibodies capable of binding to DC-STAMP and suppressing osteoclastogenesis, pharmaceutical formulations comprising such compositions, as well as methods and kits for the diagnosis of a metabolic bone disorder. In addition, the present invention provides methods of screening for candidate agents that bind to and/or modulate the activity of DC-STAMP, including screening assays for candidate agents such as antibodies and/or other compounds for osteoclastogenesis suppressive activity of a cell that expresses DC-STAMP. As is more fully described below, these assays can take on a number of formats, including the use of substantially pure DC-STAMP (including fragments and derivatives thereof in homogeneous and heterogeneous assays, biochemical assays, and cellular assays that utilize cells expressing human DC-STAMP proteins.
The identification of the correlation between DC-STAMP and osteoclast differentiation, maturation and activation further allows a number of methods utilizing the DC-STAMP protein, including methods of screening for candidate agents that bind to and/or modulate the activity of DC-STAMP, including screening assays for candidate agents such as antibodies and/or other compounds for activity of cells that express DC-STAMP. As is more fully described below, these assays can take on a number of formats, including the use of substantially pure DC-STAMP proteins (including fragments and derivatives thereof in homogeneous and heterogeneous assays, biochemical assays, and cellular assays that utilize cells expressing human DC-STAMP proteins.
In addition, the invention provides for methods of suppressing differentiation, maturation, and/or activation of osteoclasts through the use of agents that inhibit the biological activity of DC-STAMP, including, but not limited to, antigen binding proteins such as antibodies that bind to DC-STAMP, and nucleic acids such as siRNAs that prevent translation of mRNA into the DC-STAMP protein.
Accordingly, in one embodiment, the present invention provides antigen binding proteins that bind to human DC-STAMP.
2. DC-STAMP Proteins
By “human dendritic cell-specific transmembrane protein” or “human DC-STAMP” herein is meant the protein sequence depicted in SEQ ID NO:2, 4, and 6, and allelic variations thereof, each having a biological activity comparable to DC-STAMP. In some embodiments, for example when the DC-STAMP is used in screening, fragments or derivatives of DC-STAMP (and nucleic acids, as outlined below) can be used. Thus, variants of human DC-STAMP can be used in some embodiments, both for screening and for the generation of antibodies, as well as other methods contemplated herein. As is more fully outlined below, it may be desirable to use DC-STAMP fusion proteins that contain labels such as epitope tags for attachment to surfaces for screening, or labels such as autofluorescent proteins (e.g. green fluorescent proteins). In some cases, for example in screening assays, it may be useful to use non-human DC-STAMP proteins, such as rodent or other non-human mammalian proteins.
The DC-STAMP protein of the present invention may be a product directly purified from monocytes, dendritic cells or bone marrow cells of human or non-human mammalian origin (e.g., guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.), a cell membrane fraction prepared from the above cells, a synthetic DC-STAMP made in vitro, or a genetically-engineered product formed in host cells, as further discussed below.
Specifically included in the definition of “DC-STAMP” proteins and nucleic acids are fragments, including fragments that delete the transmembrane domain, including the use of the extracellular domain (of particular use in screening assays) as outlined below.
In addition, DC-STAMP proteins include proteins with a biological activity comparable to DC-STAMP.
2A. DC-STAMP Variants
In some embodiments, depending on the use of the DC-STAMP protein, variants can be used. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the DC-STAMP protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant DC-STAMP protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the DC-STAMP amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
Specifically included within the definition of “DC-STAMP” variants are deletion variants that delete the transmembrane domain of DC-STAMP. Additional useful deletion variants include the extracellular domain and the cytoplasmic domain.
While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed DC-STAMP variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of DC-STAMP protein activities.
Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger, for example when the transmembrane domain and/or cytoplasmic domain are to be deleted, e.g. for screening for compounds that bind and/or modulate the activity of DC-STAMP.
Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the DC-STAMP protein are desired, substitutions are generally made in accordance with the following chart:
Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the DC-STAMP proteins as needed.
3. Modulators of DC-STAMP Activity
The invention provides modulators of DC-STAMP activity. “Modulation” in this context includes both inhibition and activation (e.g. agonists and antagonists), with inhibition being preferred in some embodiments. Modulation can occur via binding to the DC-STAMP protein, as outlined below for antigen binding proteins, or through inhibiting expression at the nucleic acid level, e.g. through the use of siRNA.
The invention provides antigen binding proteins that bind to DC-STAMP. By “antigen binding protein” as used herein is meant a protein that specifically binds a specified antigen; the antigen in the present invention is human DC-STAMP.
The antigen binding proteins of the invention specifically bind to human DC-STAMP. “Specifically binds” as used herein means the equilibrium dissociation constant is <10−7 to 10−10 M, more preferably <10−8 to <10−10 M, even more preferably <10−9 to <10−10 M. In a specific embodiment, the antigen binding protein binds to the human DC-STAMP having the amino acid sequence of SEQ ID NO:1 and/or SEQ ID NO:2.
Antigen binding proteins include antibodies, as outlined below, as well as other proteins (including peptides) that bind and/or modulate DC-STAMP activity.
3A. Antibodies as Antigen Binding Proteins
In one embodiment, the present invention provides antigen binding proteins that are antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and fragments of each, respectively.
3 A i) Antibody Structures
Traditional antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. Each of the heavy chains and light chains has a repeat structure, in which an amino acid sequence formed of about 110 residues are conserved, this constitutes a basic unit (hereinafter referred to as a “domain”) of the three dimensional structure of IgG. The heavy chain and light chain constitute 4 and 2 independent continuous domains, respectively. In both the heavy chain and the light chain, the variation in the amino terminal domain between different antibodies is greater than the variation in the other domains. This domain is called a variable domain (hereinafter referred to as a “V domain”). At the amino terminus of IgG, the V domains of the heavy chain and light chain are complementarily associated to form a variable region.
According to the results of X-ray crystallography, a domain has a longitudinal cylindrical structure in which two layers of antiparallel β-sheets each formed of 3 to 5 β chains are superposed. In the variable region, three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a “CDR”), in which the variation in the amino acid sequence is most significant. The portions other than the CDRs of the variable region generally play a role in supporting the structure of the CDR, and are thus called the “framework”. Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E. A. Kabat et al.). Furthermore, the frameworks are classified into a plurality of subgroups based on common features of the amino acid sequences. Furthermore, it was found that there is a corresponding framework between a human and a mouse.
The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. The constant domain has a sequence intrinsic to each animal species. For example, the constant region of mouse IgG differs from that of human IgG. Therefore, mouse IgG is recognized as a foreign substance by the human immune system. As a result, a human anti-mouse antibody response (hereinafter referred to as “HAMA”) is raised (see Schroff et al., Cancer Res., 45, 879-85 (1985). Because of this, mouse antibodies are generally not administered repeatedly to a human subject.
Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. The antibodies of the invention may be of any type including IgM, IgG (including IgG1, IgG2, IgG3, IgG4), IgD, IgA, or IgE antibody. In specific embodiments, the antigen binding protein is an IgG type antibody, with specific embodiments including antibodies with IgG1 sequences All subclasses are contemplated within the present invention.
Within light and heavy chains, the variable and constant regions are joined by a “J” region of about twelve (12) or more amino acids, with the heavy chain also including a “D” region of about ten (10) more amino acids. See, generally, Paul, W., ed., 1989, Fundamental Immunology Ch. 7, 2nd ed. Raven Press, N.Y. The variable regions of each light/heavy chain pair form the antibody binding site.
The variable regions of the heavy and light chains typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs are the hypervariable regions of an antibody (or antigen binding protein, as outlined herein), that are responsible for antigen recognition and binding. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest. Chothia et al., 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883.
CDRs constitute the major surface contact points for antigen binding. See, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. Further, CDR3 of the light chain and, especially, CDR3 of the heavy chain may constitute the most important determinants in antigen binding within the light and heavy chain variable regions. See, e.g., Chothia and Lesk, 1987, supra; Desiderio et al., 2001, J. Mol. Biol. 310:603-615; Xu and Davis, 2000, Immunity 13:37-45; Desmyter et al., 2001, J. Biol. Chem. 276:26285-26290; and Muyldermans, 2001, J. Biotechnol. 74:277-302. In some antibodies, the heavy chain CDR3 appears to constitute the major area of contact between the antigen and the antibody. Desmyter et al., 2001, supra. In vitro selection schemes in which CDR3 alone is varied can be used to vary the binding properties of an antibody. Muyldermans, 2001, supra; Desiderio et al., 2001, supra.
Naturally occurring antibodies typically include a signal sequence, which directs the antibody into the cellular pathway for protein secretion and which is not present in the mature antibody. A polynucleotide encoding an antibody of the invention may encode a naturally occurring signal sequence or a heterologous signal sequence as described below.
3 A i) a) Chimeric and Humanized Antibodies
In some embodiments, however, the scaffold components can be a mixture from different species. As such, if the antigen binding protein is an antibody, such antibody may be a chimeric antibody and/or a humanized antibody. In general, both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from more than one species. For example, “chimeric antibodies” traditionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human. “Humanized antibodies” generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies is described in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536. Humanized antibodies can also be generated using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20:639-654.
From studies of the structural features of Immunoglobulin G (“IgG”), a method of preparing a humanized antibody has been conceived as described below.
Initially, a chimeric antibody was proposed in which the variable region of an antibody derived from a mouse is connected to a constant region derived from a human (see Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855, (1984)). However, such a chimeric antibody still contains many non-human amino acid residues. Therefore, when the chimeric antibody is administered over a long period of time, a HAMA response may possibly be induced (see Begent et al., Br. J. Cancer, 62, 487, (1990)).
As a method for further reducing the amino acid residues derived from a non-human mammalian source, which may possibly cause a HAMA response in humans, a method of integrating only the CDR portion into a human-derived antibody was proposed (see Nature, 321, 522-525, (1986)). However, to maintain immunoglobulin activity against an antigen, transplantation of the CDR alone was generally insufficient.
Chothia et al., found the following based on data obtained by X-ray crystallography in 1987:
(i) in the amino acid sequence of the CDR, there is a site which binds directly to an antigen and a site responsible for maintaining the structure of the CDR itself, and the three dimensional structures of the CDR that can be adopted are classified into a plurality of typical patterns (canonical structures); and
(ii) the classes of canonical structures are determined not only by the CDR, but also by the type of amino acid present in a specific site of the framework portion (see J. Mol. Biol., 196, 901-917, (1987)).
Based on these findings, it was suggested that when the CDR transplantation method is employed, amino acid residues of a part of the framework must be transplanted into a human antibody in addition to the sequence of the CDR (see Japanese National Publication of International Patent Application No. 4-502408).
Generally, a non-human mammalian-derived antibody from which the CDR is to be transplanted is defined as a “donor”, whereas the human antibody into which the CDR is transplanted is defined as an “acceptor”. The present invention follows these definitions.
A point which should be considered in carrying out the CDR transplantation is that the activity of the immunoglobulin molecule is maintained by preserving the CDR structure as much as possible. To achieve this, attention must be paid to the following two points:
(i) which subgroup of antibodies the acceptor is selected from; and
(ii) which amino acid residue is selected from the framework of the donor.
Queen et al. proposed a design method for transplanting an amino acid residue into an acceptor together with the CDR sequence when the amino acid residue of the framework of the donor corresponds to at least one of the following references (see Japanese National Publication of International Patent Application No. 4-502408).
(a) the amino acid is rarely present at the position within the framework of an acceptor, whereas the corresponding amino acid of a donor is usually present at the equivalent position;
(b) the amino acid is present near one of the CDRs; and it is predicted that the amino acid has a side chain atom within about 3 angstroms from the CDR in its three dimensional immunoglobulin model and that the side main atom can interact with an antigen or the CDR of a humanized antibody.
3. A i) b) Bispecific Antibodies
In one embodiment, the DC-STAMP antigen binding protein is a multispecific antibody, and notably a bispecific antibody, also sometimes referred to as “diabodies”. These are antibodies that bind to two (or more) different antigens. Diabodies can be manufactured in a variety of ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449), e.g., prepared chemically or from hybrid hybridomas.
3. A i) c) Minibodies
In one embodiment, the DC-STAMP antigen binding protein is a minibody. Minibodies are minimized antibody-like proteins comprising a scFv joined to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061.
3. A i) d) Domain Antibodies
In one embodiment, the DC-STAMP antigen binding protein is a domain antibody; see for example U.S. Pat. No. 6,248,516. Domain antibodies (dAbs) are functional binding domains of antibodies, corresponding to the variable regions of either the heavy (VH) or light (VL) chains of human antibodies dABs have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a full antibody. dABs are well expressed in a variety of hosts including bacterial, yeast, and mammalian cell systems. In addition, dAbs are highly stable and retain activity even after being subjected to harsh conditions, such as freeze-drying or heat denaturation. See, for example, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; US Serial No. 2004/0110941; European Patent 0368684; U.S. Pat. No. 6,696,245, WO04/058821, WO04/003019 and WO03/002609.
3. A i) e) Antibody Fragments
In one embodiment, the DC-STAMP antigen binding protein is an antibody fragment, that is a fragment of any of the antibodies outlined herein that retain binding specificity to DC-STAMP.
Specific antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546) which consists of a single variable, (v) isolated CDR regions, (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883), (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies” or “triabodies”, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448). The antibody fragments may be modified. For example, the molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14:1239-1245).
3. A i) f) Human Antibodies
In one embodiment, the DC-STAMP antigen binding protein is a fully human antibody. “Fully human antibody” or “complete human antibody” refers to a human antibody having only the gene sequence of an antibody derived from a human chromosome. The anti-human DC-STAMP complete human antibody can be obtained by a method using a human antibody-producing mouse having a human chromosome fragment containing the genes for a heavy chain and light chain of a human antibody [see Tomizuka, K. et al., Nature Genetics, 16, p. 133-143, 1997; Kuroiwa, Y. et al., Nuc. Acids Res., 26, p. 3447-3448, 1998; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p. 69-73 (Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA, 97, 722-727, 2000, etc.] or obtained by a method for obtaining a human antibody derived from a phage display selected from a human antibody library [see Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science. 43(7), p. 2301-8, 2002; Carmen, S. et al., Briefings in Functional Genomics and Proteomics, 1 (2), p. 189-203, 2002; Siriwardena, D. et al., Ophthalmology, 109(3), p. 427-431, 2002, etc.]
In one embodiment, the DC-STAMP antigen binding protein is an antibody analog, sometimes referred to as “synthetic antibodies.” For example, a variety of recent work utilizes either alternative protein scaffolds or artificial scaffolds with grafted CDRs. Such scaffolds include, but are not limited to, mutations introduced to stabilize the three-dimensional structure of the binding protein as well as wholly synthetic scaffolds consisting for example of biocompatible polymers. See, for example, Korndorfer et a., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics (“PAMs) can be used, as well as work based on antibody mimetics utilizing fibronection components as a scaffold.
3. A i) g) Antibody Conjugates
In one embodiment, the DC-STAMP antigen binding protein is an antibody fusion protein (sometimes referred to herein as an “antibody conjugate”). The conjugate partner can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups on the antigen binding protein (see the discussion on covalent modifications of the antigen binding proteins) and on the conjugate partner. For example linkers are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see, 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
Suitable conjugates include, but are not limited to, labels as described below, drugs and cytotoxic agents including, but not limited to, cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antigen binding proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antigen binding protein. Additional embodiments utilize calicheamicin, auristatins, geldanamycin and maytansine.
Additional fusion proteins are discussed below with particular reference to screening assays.
Conjugation of an anti-DC-STAMP antibody or a fragment thereof with a therapeutic agent for bone disease can be used to make a targeted drug conjugate as described in M. C. Garnet, “Targeted drug conjugates: principles and progress”, Advanced Drug Delivery Reviews, (2001) 53, 171-216. The antibody molecule or fragments thereof may be used for this purpose, unless the fragment completely loses its ability to recognize osteoclasts, Fab, F(ab′)2 and Fv are exemplary fragments. The above antibody or fragments thereof can be also used for this purpose in the present invention. The mode of conjugation of the anti-DC-STAMP antibody or a fragment thereof with a therapeutic agent for bone disease may be any of the 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 so forth. Specifically, the anti-DC-STAMP antibody and the therapeutic agent for bone disease may be chemically conjugated directly, or via a spacer, such as an oligopeptide, or conjugated via a suitable drug carrier. Examples of drug carriers include liposomes and aqueous polymers. More specifically, the drug carrier may be used, for example, for encapsulation of both the antibody and the therapeutic agent for bone disease into a liposome, for conjugation of the antibody with the liposome, by chemical bonding of the therapeutic agent of bone disease directly, or via a spacer, such as an oligopeptide, with an aqueous polymer (a compound with a molecular weight of about 1,000 to 100,000), or conjugation of the antibody with an aqueous polymer. The antibody (or fragment thereof, the therapeutic agent for bone disease, and/or the drug carrier, such as a liposome, or aqueous polymer, may bound to each other according to the methods 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, or the like, which are all well known to those skilled in the art. Encapsulation of the therapeutic agent for bone disease into the liposome may be carried out according to the methods described in D. D. Lasic, “Liposomes: From Physics to Applications”, Elsevier Science Publishers B. V., Amsterdam (1993), or the like, which are well known to those skilled in the art. Attachment of the therapeutic agent for bone disease to an aqueous polymer may be carried out according to the methods described in D. Putnam and J. Kopecek, “Polymer Conjugates with Anticancer Activity”, Advances in Polymer Science (1995) 122, 55-123, or the like, which are well known to those skilled in the art. The antibody (or fragment thereof) and a proteinaceous therapeutic agent for bone disease (or fragment thereof may form a fusion protein according to genetic engineering methods well known to those skilled in the art, in addition to the above-described methods.
3. A ii) Covalent Modifications of Antigen Binding Proteins such as Antibodies
Covalent modifications of antigen binding proteins, as well as the DC-STAMP proteins themselves, are included within the scope of this invention, and are generally, but not always, done post-translationally. For example, several types of covalent modifications of the antigen binding protein are introduced into the molecule by reacting specific amino acid residues of the antigen binding protein with an organic derivatizing agent that is capable of reacting with selected side chains or the N— or C-terminal residues.
Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.
Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125I or 131I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
Derivatization with bifunctional agents is useful for crosslinking antigen binding proteins to a water-insoluble support matrix or surface for use in a variety of methods, in addition to methods described below. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the antigen binding protein included within the scope of this invention comprises altering the glycosylation pattern of the protein. As is known in the art, glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Particular expression systems are discussed below.
In another embodiment, the antigen binding proteins disclosed herein can be modified to include one or more engineered glycoforms. By “engineered glycoform” as used herein is meant a carbohydrate composition that is covalently attached to an antigen binding protein, wherein said carbohydrate composition differs chemically from that of a parent antigen binding protein. Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by a variety of methods known in the art (Uma{umlaut over (n)}a et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; WO 00/61739A1; WO 01/29246A1; WO 02/31140A1; WO 02/30954A1, all incorporated by reference in their entirety; (Potelligent™ technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylation engineering technology [Glycart Biotechnology AG, Zurich, Switzerland]). Many of these techniques are based on controlling the level of fucosylated and/or bisecting oligosaccharides that are covalently attached to the Fc region, for example by expressing an IgG in various organisms or cell lines, engineered or otherwise (for example Lec-13 CHO cells or rat hybridoma YB2/0 cells), by regulating enzymes involved in the glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase] and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifying carbohydrate(s) after the IgG has been expressed. Engineered glycoform typically refers to the different carbohydrate or oligosaccharide; thus an IgG variant, for example an antibody (including Fc fusions), can include an engineered glycoform. Alternatively, engineered glycoform may refer to the antibody, e.g. IgG, variant that comprises the different carbohydrate or oligosaccharide. As is known in the art, glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Particular expression systems are discussed below.
Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tri-peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tri-peptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the antigen binding protein is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tri-peptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). For ease, the antigen binding protein amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the antigen binding protein is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell that has glycosylation capabilities for N— and O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both incorporated by reference in their entirety.
Specifically included within the definition of “antigen binding protein” are aglycosylated antigen binding proteins. By “aglycosylated antigen binding protein” as used herein is meant an antigen binding protein that lacks carbohydrate attached at position 297 of the Fc region, wherein numbering is according to the EU system as in Kabat. The aglycosylated antigen binding protein may be a deglycosylated antibody, that is an antigen binding protein for which the Fc carbohydrate has been removed, for example chemically or enzymatically. Alternatively, the aglycosylated antigen binding protein may be a nonglycosylated or unglycosylated antibody, that is an antigen binding protein that was expressed without Fc carbohydrate, for example by mutation of one or residues that encode the glycosylation pattern or by expression in an organism that does not attach carbohydrates to proteins, for example bacteria.
Removal of carbohydrate moieties present on the starting antigen binding protein may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 138:350, incorporated by reference herein in its entirety. Glycosylation at potential glycosylation sites may be prevented by the use of the compound tunicamycin as described by Duskin et al., 1982, J. Biol. Chem. 257:3105, incorporated by reference herein in its entirety. Tunicamycin blocks the formation of protein-N-glycoside linkages.
Another type of covalent modification of the antigen binding protein comprises linking the antigen binding protein to various nonproteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, as is known in the art, amino acid substitutions may be made in various positions within the antigen binding protein to facilitate the addition of polymers such as PEG.
3. A iii) Labeled Antibodies
In some embodiments, the covalent modification of the antigen binding proteins of the invention comprises the addition of one or more labels. In some cases, these are considered antibody fusions.
The term “labelling group” means any detectable label. In some embodiments, the labelling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labelling proteins are known in the art and may be used in performing the present invention.
In general, labels fall into a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labelling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and may be used in performing the present invention.
Specific labels include optical dyes, including, but not limited to, chromophores, phosphors and fluorophores, with the latter being specific in many instances. Fluorophores can be either “small molecule” fluores, or proteinaceous fluores.
By “fluorescent label” is meant any molecule that may be detected via its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.
Suitable proteinaceous fluorescent labels also include, but are not limited to, green fluorescent protein, including a Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463, WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, 5,925,558). All of the above-cited references are expressly incorporated herein by reference.
3. B. Nucleic Acids as Modulators of DC-STAMP Activity
In addition to compounds including antigen binding proteins, certain nucleic acids can also be used to modulate DC-STAMP activity.
In one embodiment, the nucleic acids that are used to modulate (e.g. inhibit) activity of DC-STAMP and thus osteoclastogenesis are antisense nucleic acids. By “nucleic acid” or “oligonucleotide” or grammatical equivalents herein is meant at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds (e.g. for synthesis of DC-STAMP proteins), although in some cases, as outlined herein, for example in the use of nucleic acids as candidate agents in screeing assays, siRNA and antisense utilities, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with bicyclic structures including locked nucleic acids, Koshkin et al., J. Am. Chem. Soc. 120:13252-3 (1998); positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of labels, or to increase the stability and half-life of such molecules in physiological environments.
As will be appreciated by those in the art, all of these nucleic acid analogs may find use in the present invention. In addition, mixtures of naturally occurring nucleic acids and analogs can be made, as well as mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. As used herein, the term “nucleoside” includes nucleotides as well as nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, “nucleoside” includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
In one embodiment, the nucleic acids are short interfering nucleic acid (siNA) molecules that act by invoking RNA interference. RNA interference mechanisms recognize RNA as “foreign” due to its existence in a double-stranded form. This results in the degradation of the double-stranded RNA, along with single-stranded RNA having the same sequence. Short interfering RNAs, or “siRNAs”, are an intermediate in the RNAi process in which the long double-stranded RNA has been cut up into short (˜21 nucleotides) double-stranded RNA. The siRNA stimulates the cellular machinery to cut up other single-stranded RNA having the same sequence as the siRNA.
In some embodiments, the siNAs are siRNAs; in others, nucleotide analogs can be used. See the extensive discussion in US publication 2006/0160757, hereby incorporated by reference in its entirety, with particular reference to suitable chemically modified nucleosides, and the use of “blunt” and/or “overhang” sequences. In some embodiments, the siNAs are directed to a portion of the transmembrane domain (e.g. the 1st, 2nd, 3rd, 4th, 5th, 6th or 7th transmembrane spanning region), a portion of the extracellular domain, a portion of the cytoplasmic domain, or any junction thereof. A siNA of the invention can be unmodified or chemically-modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized.
In one embodiment of the invention a siNA molecule comprises an antisense strand having about 19 to about 29 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides, wherein the antisense strand is complementary to a RNA sequence encoding a DC-STAMP protein, and wherein said siNA further comprises a sense strand having about 19 to about 29 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences with at least about 19 complementary nucleotides (these are referred to as double stranded siNAs, or dssiNAs).
In one embodiment, the compounds of the invention that serve to inhibit the activity of DC-STAMP are antisense nucleic acids, as are generally described in US publication 2006/0172957, hereby incorporated by reference in its entirety, and with particular reference to suitable chemically modified nucleosides and nucleic acids for use in antisense technologies and mechanisms. Antisense mechanisms are processes in which the antisense compound specifically hybridizes to it's target RNA to form a duplex. The formation of this duplex prevents the RNA from functioning normally and from producing a protein product. In general, antisense molecules can be from 5 to 100 basepairs in length, with from about 8 to about 50 bases being preferred.
4. Nucleic Acids Encoding DC-STAMP and DC-STAMP Antigen Binding Proteins
The invention provides nucleic acids that encode DC-STAMP and DC-STAMP antigen binding proteins, as outlined herein (in addition to nucleic acids as candidate agents, as described below). It should be noted that the following discussion can also apply to nucleic acids encoding DC-STAMP antigen binding proteins, nucleic acids used as candidate agents (e.g. antisense or siRNA molecules), or to nucleic acids encoding proteinaceous candidate agents, as described below.
As will be appreciated by those in the art, the depiction of a single strand (“Watson”) also defines the sequence of the other strand (“Crick”); thus the nucleic acid sequences depicted herein (e.g. SEQ ID NOS:1, 3, and 5) also include the complement of these sequences. By the term “recombinant nucleic acid” herein is meant nucleic acid, described below, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature. Thus an isolated DC-STAMP nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the DC-STAMP antigen binding proteins and DC-STAMP proteins of the present invention. Thus, having identified a particular amino acid sequence those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the encoded protein.
In general, DC-STAMP nucleic acids hybridize to the sequences depicted in SEQ ID NOS:1, 3, and 5 under high stringency conditions. The term “hybridizes under stringent conditions” refers to hybridization which is performed at 68° C. in a commercially available hybridization solution, namely ExpressHyb (manufactured by Clontech), or hybridization which is performed at 68° C. in the presence of NaCl at 0.7 to 1.0 M using a filter having DNA immobilized thereon, followed by washing at 68° C. with 0.1 to 2×SSC solution (1×SSC solution contains 150 mM NaCl and 15 mM sodium citrate), resulting in hybridization. The above term also includes hybridization under conditions equivalent to those above.
In some embodiments, the DC-STAMP proteins (or antigen binding proteins, in some instances) are isolated proteins or substantially pure proteins. An “isolated” protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 5%, more preferably at least about 50% by weight of the total protein in a given sample. The definition of “isolated” includes the production of a protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. A “substantially pure” protein comprises at least about 75% by weight of the total protein, with at least about 80% being specific, and at least about 90% being particularly specific. Thus, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as described herein. Methods and techniques for the production of recombinant proteins are well known in the art and described below in the “nucleic acid” section.
4 A. Preparation of DC-STAMP Proteins
There are a variety of techniques useful in obtaining DC-STAMP nucleic acids, as is well known in the art. Generally, probe polymerase chain reaction (PCR) primer sequences, based on the sequences of SEQ ID NO:1, SEQ ID NO:3, and/or SEQ ID NO:5, may be used to clone expression vectors, as well as find other related DC-STAMP proteins from other organisms as needed. As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the DC-STAMP nucleic acid sequence. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are well known in the art. Other nucleic acid amplification techniques may also be used, including NASBA, TMA, QBReplicase, SDA, etc.
Once the DC-STAMP protein is amplified, it can be cloned and, if necessary, its constituent parts recombined to form the entire DC-STAMP nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant DC-STAMP nucleic acid can be expressed as outlined below.
4 B. Nucleic Acids Encoding DC-STAMP Antigen Binding Proteins such as Antibodies
In the case of antigen binding proteins such as antibodies, nucleic acids, particularly DNA, encoding heavy chains and/or light chains of the anti human DC-STAMP monoclonal antibodies (or other types of antibodies, as outlined herein) of the present invention can be obtained by preparing mRNA from a hybridoma cell producing the anti-human DC-STAMP monoclonal antibody, converting the mRNA into cDNA using reverse transcriptase, and isolating each DNA encoding the heavy chain or light chain of the antibody.
In extracting mRNA, the guanidine thiocyanate—hot phenol method, and guanidine thiocyanate guanidine—hydrochloride method may be employed; however, the guanidine thiocyanate cesium chloride method is also suitable. Preparation of mRNA from a cell is performed by first preparing total RNA and purifying the mRNA using a poly(a)+ RNA purification carrier such as oligo (dT) cellulose or oligo (dT) latex beads or directly purifying mRNA from a cell lysate by use of the carrier. For preparing total RNA, use may be made of the alkaline sucrose density-gradient centrifugation method [see Dougherty, W. G. and Hiebert, E. (1980) Virology 101, 466-474], the guanidine thiocyanate-phenol method, the guanidine thiocyanate-trifluoro-cesium method, and the phenol SDS method and the like; however, the method using guanidine thiocyanate and cesium chloride is also suitable [see Chirgwin, J. M., et al. (1979) Biochemistry 18, 5294-5299].
After a single-stranded cDNA is synthesized by a reverse transcriptase reaction using the poly(a)+ RNA obtained as mentioned above as a template, double-stranded cDNA can be synthesized from the single-stranded cDNA. This method may be the S1 nuclease method [see Efstratiadis, A., et al. (1976) Cell, 7, 279-288], the Gubler/Hoffmann method [see Gubler, U. and Hoffman, B. J. (1983) Gene 25, 263-269], the Okayama/Berg method [see Okayama, H. and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170] or others; however, suitably used in the present invention is the so-called RT-PCR method in which a polymerase chain reaction (hereinafter referred to as a “PCR”) [see Saiki, R. K., et al. (1988) Science 239, 487-49] is performed using a single-stranded cDNA as a template.
The double-stranded cDNA thus obtained is integrated into a cloning vector to obtain a recombinant vector, which is then introduced into a microorganism, such as Escherichia coli, to form a transformant. The transformant can be selected by using tetracycline resistance or ampicillin resistance as a marker. Escherichia coli can be transformed by the Hanahan method [see Hanahan, D. (1983) J. Mol. Biol. 166, 557-580], more specifically, by preparing a competent cell in the presence of calcium chloride, magnesium chloride or rubidium chloride, and adding the recombinant DNA vector to the competent cell. Note that when a plasmid is used as a vector, the plasmid must have any one of the drug resistance genes as mentioned above. Needless to say, a cloning vector other than a plasmid, such as a lambda group phage, may be used.
As a method of selecting a strain having a cDNA, which encodes each of the subunits of a desired anti-human DC-STAMP monoclonal antibody from the transformant strain obtained above, any of the methods described below can be employed. When a desired cDNA is specifically amplified by the RT-PCR method, such operation of the method can be skipped.
4 C. Methods of Producing Proteins including DC-STAMP and Antigen Binding Proteins
The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes which comprise at least one nucleic acid, as outlined herein. In addition, the invention provides host cells comprising such expression systems or constructs. As outlined herein, these nucleic acids can encode DC-STAMP proteins, DC-STAMP antigen binding proteins such as anti-DC-STAMP antibodies, or candidate agents, as defined below.
Typically, expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.
Optionally, the vector may contain a “tag”-encoding sequence, i.e., an oligonucleotide molecule located at the 5′ or 3′ end of the DC-STAMP antigen binding protein coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another “tag” such as FLAG, HA (hemaglutinin influenza virus), or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification or detection of the DC-STAMP antigen binding protein from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified DC-STAMP antigen binding protein by various means such as using certain peptidases for cleavage.
Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.
Flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.
Whether all or only a portion of the flanking sequence is known, it may be obtained using polymerase chain reaction (PCR) and/or by screening a genomic library with a suitable probe such as an oligonucleotide and/or flanking sequence fragment from the same or another species. Where the flanking sequence is not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, Calif.), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, Mass.) is suitable for most gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the virus early promoter).
A transcription termination sequence is typically located 3′ to the end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.
A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene may also be used for selection in both prokaryotic and eukaryotic host cells.
Other selectable genes may be used to amplify the gene that will be expressed. Amplification is the process wherein genes that are required for production of a protein critical for growth or cell survival are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thyrnidine kinase genes. Mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively increased, thereby leading to the amplification of both the selectable gene and the DNA that encodes another gene, such as an antigen binding protein antibody that binds to DC-STAMP polypeptide. As a result, increased quantities of a polypeptide such as an DC-STAMP antigen binding protein are synthesized from the amplified DNA.
A ribosome-binding site is usually necessary for translation initiation of rnRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3′ to the promoter and 5′ to the coding sequence of the polypeptide to be expressed.
In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, one may manipulate the various pre- or prosequences to improve glycosylation or yield. For example, one may alter the peptidase cleavage site of a particular signal peptide, or add prosequences, which also may affect glycosylation. The final protein product may have, in the −1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression, which may not have been totally removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the amino-terminus. Alternatively, use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.
Expression and cloning vectors of the invention will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the DC-STAMP antigen binding protein. Promoters are untranscribed sequences located upstream (i.e., 5′) to the start codon of a structural gene (generally within about 100 to 1000 bp) that control transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, uniformly transcribe gene to which they are operably linked, that is, with little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding heavy chain or light chain comprising an DC-STAMP antigen binding protein of the invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.
Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.
Additional promoters which may be of interest include, but are not limited to: SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. U.S.A. 81:659-663); the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from the metallothionine gene Prinster et a., 1982, Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region that is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495); the albumin gene control region that is active in liver (Pinkert et al., 1987, Genes and Devel. 1 :268-276); the alpha-feto-protein gene control region that is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); the alpha 1-antitrypsin gene control region that is active in liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); the beta-globin gene control region that is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
An enhancer sequence may be inserted into the vector to increase transcription of DNA encoding light chain or heavy chain comprising an DC-STAMP antigen binding protein of the invention by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent, having been found at positions both 5′ and 3′ to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be positioned in the vector either 5′ or 3′ to a coding sequence, it is typically located at a site 5′ from the promoter. A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into an expression vector, to promote extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cells in which the antibody is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846.
Expression vectors of the invention may be constructed from a starting vector such as a commercially available vector as outlined above. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art.
After the vector has been constructed and a nucleic acid molecule encoding all or part of the DC-STAMP antigen binding protein or DC-STAMP protein has been inserted into the proper site of the vector (or plurality of vectors, as the case may be), the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression. The transformation of an expression vector for an DC-STAMP antigen binding protein into a selected host cell may be accomplished by well known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al., 2001, supra.
A host cell, when cultured under appropriate conditions, synthesizes an DC-STAMP antigen binding protein that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. In certain embodiments, cell lines may be selected through determining which cell lines have high expression levels and constitutively produce antigen binding proteins with DC-STAMP binding properties. In another embodiment, a cell line from the B cell lineage that does not make its own antibody but has a capacity to make and secrete a heterologous antibody can be selected.
5. Use Of DC-STAMP Antigen Binding Proteins For Diagnostic, Therapeutic and Screening Purposes
In the specification of the present invention, a compound having a therapeutic effect is a compound suitable for the prevention and/or treatment of a metabolic bone disorder, which has a suppressive effect on osteoclastogenesis in a cell expressing DC-STAMP. By “metabolic bone disorder”, is meant a condition in an individual where bone resorption levels are increased to a degree greater than that found in a healthy individual not suffering from a bone disorder. In the specification of the present invention, the terms “metabolic bone disorder” and “bone disorder” have the same meaning. The term “osteoclastogenesis” of a cell used herein refers to the formation of an osteoclast by a cell expressing DC-STAMP. Osteoclastogenesis involves the differentiation, including maturation and/or activation, of a cell expressing DC-STAMP into an osteoclast following appropriate induction, such as for example, stimulation by RANKL. The cell undergoing osteoclastogenesis and expressing DC-STAMP may be an a mononuclear osteoclast precursor cell of monocyte or macrophage lineage, which may fuse or undergo multinucleation to form osteoclasts.
In the specification, a protein having the same function as that of human DC-STAMP, such as the induction of osteoclastogenesis of a cell, is also referred to as a “human DC-STAMP”. Note that the term “pre-osteoclast” as used in the present invention includes a mononuclear osteoclast precursor cell.
The present invention provides agents that confer and/or induce the suppression or inhibition of osteoclastogenesis in a cell expressing DC-STAMP. The term “inhibition” or “suppression”, including grammatical equivalents, as used herein refers to the delay and/or prevention of osteoclast formation in a cell expressing DC-STAMP.
The correlation of the connection between DC-STAMP and osteoclastogenesis allows a number of utilities, including diagnostic methods and kits, therapeutic uses, including metabolic bone disorders, and screening technologies for modulation of DC-STAMP activity.
5 A. Diagnosis Utilizing DC-STAMP and DC-STAMP Antigen Binding Proteins
As is described herein, DC-STAMP has been shown to be over expressed in osteoclasts, including large multinuclear giant cells. Accordingly, the invention provides a number of diagnostic methods and kits, based either on protein or nucleic acid detection, for the detection of DC STAMP's activity of enhancing osteoclastogenesis of a cell.
Confirmation of Specific Expression of the Human DC-STAMP Gene for Diagnosis
As a result of analyzing expression levels of the human DC-STAMP gene in various types of human cells, it was found that the gene is expressed at a significantly higher expression level in osteoclasts compared to other tissues. An analysis of a group of test samples from various human bone tissues for expression level of the DC-STAMP gene shows that the gene is expressed at a significantly higher level in giant cell tumor (GCT) which is a bone tumor abundant in osteoclastic multinuclear giant cells characterized by clinical findings of osteolytic bone destruction (Bullough et al., Atlas of Orthopedic Pathology 2nd edition, pp. 17.6-17.8, Lippincott Williams & Wilkins Publishers (1992)). It has been also found that DC-STAMP is expressed at a higher level when a monocyte-derived cell strain is differentiated into osteoclasts. From these findings, it can be concluded that human DC-STAMP may be involved in osteoclastogenesis of cells. This suggests that the state of osteoclastogenesis of cells caused by excessive expression of human DC-STAMP can be determined by measuring the level of expression of human DC-STAMP in individual cells and/or tissues.
Accordingly, DC-STAMP is believed to be associated with human conditions, such as GCT, which increase bone resorption. In other words, measurement of the expression level of DC-STAMP in different types of cells and/or different tissues enables determination of the state of metabolic bone disorders which may develop due to over-expression of DC-STAMP. Herein, metabolic bone disorders include, but are not limited to, osteoporosis (osteoporosis after menopause, senile osteoporosis, secondary osteoporosis due to the use of a steroid or immunosuppressant, and osteoporosis associated with rheumatoid arthritis), bone destruction caused by rheumatoid arthritis, cancerous hypercalcemia, bone destruction caused by multiple myeloma or metastasis of cancer to bone, loss of teeth due to dental periostitis, osteolysis around artificial joints, bone destruction due to chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy and osteogenesis imperfecta.
The nucleotide sequence of the cDNA for the human DC-STAMP is registered as Accession No: NM—030788 with GenBank, and shown in SEQ ID NO: 1 in the Sequence Listing; its amino acid sequence is shown in SEQ ID NO: 2 in the Sequence Listing. The long nucleotide sequence containing the third exon of the cDNA for the murine DC-STAMP is registered as Accession No: AB109560_ with GenBank, and shown in SEQ ID NO: 3 in the Sequence Listing; its amino acid sequence is shown in SEQ ID NO: 4 in the Sequence Listing. The nucleotide sequence having the short third exon of the splice variant cDNA for the murine DC-STAMP is registered as Accession No: AB109561 with GenBank, and shown in SEQ ID NO: 5 in the Sequence Listing; its amino acid sequence is shown in SEQ ID NO: 6 in the Sequence Listing. A cDNA for DC-STAMP can be produced, for example, by carrying out polymerase chain reaction (referred to as “PCR” hereinafter) using a cDNA library expressing DC-STAMP as the template and primers capable of specifically amplifying the DC-STAMP cDNA (Saiki, R. K., et al., Science, (1988) 239, 487-49), which is called the PCR technique.
5. B Diagnostic Assays using Nucleic Acids
As outlined herein, the present invention provides diagnostic assays and kits based on nucleic acids and/or proteins. Human DC-STAMP, since it is highly expressed in osteoclast cells is thought to be involved in osteoclastogenesis. Thus the present invention provides methods of detecting osteoclastogenesis, or a predisposition or propensity towards a metabolic bone disorder, in patient samples.
The term “sample” or “specimen” refers to a sample taken from a test subject or a clinical specimen, and includes samples of tissues, excrement or the like, such as samples of blood, body fluids, prostate gland, testes, penis, bladder, kidney, oral cavity, pharynx, lip, tongue, gingival, nasopharynx, esophagus, stomach, small intestine, large intestine, colon, liver, gall bladder, pancreas, nose, lung, bone, soft tissue, skin, breast, uterus, ovary, brain, thyroid, lymph node, muscle, and adipose tissue. In the present invention, blood and bone marrow are preferred tissue samples.
In one embodiment, the invention provides methods of detecting osteoclastogenesis using the level of expression of the human DC-STAMP gene.
In a first embodiment, the method utilizes the following steps 1) to 4):
1) a step of extracting a total RNA fraction from a specimen taken from a test subject;
2) a step of extracting a total RNA fraction from a specimen taken from a healthy person;
3) a step of measuring the level of expression of the human DC-STAMP gene in the total RNA fractions according to steps 1) and 2); and
4) a step of analyzing the difference in the level of expression of the gene between the total RNA fraction derived from steps 1) and 2), measured in step 3) and thereby detect a metabolic bone disorder in the subject of step 1).
In one embodiment, the steps are as follows. Step 1 comprises extracting a total RNA fraction from a specimen taken from a test subject.
In extracting the total RNA fraction from a specimen, human tissue is obtained by an appropriate method satisfying the ethical standards for experimentation. The tissue obtained is dissolved directly in an RNA extraction solvent (containing a ribonuclease inhibitor, such as phenol). Alternatively, cells of the tissue obtained are collected by abrading them using a scraper so as not to break the cells, or gently extracting them from the tissue using a proteolytic enzyme such as trypsin, and then immediately subjecting the cells to an RNA extraction step.
Examples of RNA extraction methods that may be used include: guanidine thiocyanate/cesium chloride ultracentrifugation methods, guanidine thiocyanate/hot phenol methods, guanidine hydrochloride methods, and acidic guanidine thiocyanate/phenol/chloroform methods (Chomczynski, P. and Sacci, N., Anal. Biochem. (1987), 162, 156-159). Of these, acidic guanidine thiocyanate/phenol/chloroform methods are particularly suitable. Alternatively, a commercially available RNA extraction reagent, such as ISOGEN (manufactured by Nippon Gene Co., Ltd.) or TRIZOL reagent (manufactured by Gibco BRL) may be used in accordance with the protocol provided with the reagent.
From the total RNA fraction obtained, if necessary, it is preferred that mRNA alone is purified and used. Any suitable purification method can be used. For example, mRNA can be purified by adsorbing mRNA onto a biotinylated oligo (dT) probe, attaching the mRNA to paramagnetic particles having streptavidin immobilized thereon via binding of biotin to streptavidin, washing the particles, and eluting mRNA. Alternatively, mRNA may be purified by adsorbing mRNA onto an oligo (dT) cellulose column and eluting the mRNA therefrom. However, an mRNA purification step is not essential in methods of the present invention. Provided that expression of a desired polynucleotide can be detected, a total RNA fraction may be used, as can be done in the later steps.
Step 2 comprises a control step, e.g. extracting a total RNA fraction from a specimen taken from a healthy person. In the present invention, a healthy person means a person who does not have a metabolic bone disorder. The determination as to whether or not a person is healthy can be made by measuring the concentration of human DC-STAMP and determining whether or not the concentration value measured falls within a predetermined range for a healthy person. Alternatively, the correlation between the expression level of human DC-STAMP and the level of formation of a metabolic bone disorder can be investigated in advance, and then, determination of whether or not a test subject is a healthy person can be made by measuring the expression level of human DC-STAMP in a specimen taken from the test subject. The preparation of a total RNA fraction from a healthy person can be performed in the same manner as described in Step 1) above.
It should be noted that in some instances, for example when the level of DC-STAMP expression from a particular tissue, patient or sample is already known, it is not necessary to determine the level of DC-STAMP expression from a “normal” or “healthy” sample.
Step 3 can be done by measuring the level of expression of the human DC-STAMP gene in a total RNA fraction according to steps 1) and 2).
The level of expression of the human DC-STAMP gene is represented by the expression level of a polynucleotide that can hybridize with a polynucleotide which comprises the nucleotide sequence represented by SEQ ID NO:1 of the sequence listing or a polynucleotide which comprises a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO:1 of the sequence listing, under stringent conditions.
It should be noted that in some cases, it is desirable to use the entire DC-STAMP gene in the expression analysis; in other embodiments, such as in the use of gene arrays, as described below, the nucleic acid probes used to test for the presence of DC-STAMP nucleic acid can be fragments of the full length gene. Thus, for example, fragments of DC-STAMP nucleic acid can be used as the probe to determine the expression levels. In general, the probes will range from about 8 nucleosides to about 100, with from about 10 to 50 being preferred, and from about 15 to 30 being particularly preferred. As will be appreciated by those in the art, the length of the probes used is generally sufficient to confer specificity. In addition, it should be appreciated that either the coding (“Watson”) strand or the non-coding strand (“Crick”) can be used, depending on the assay.
As will be appreciated by those in the art, diagnostic assays can be run either as solution phase assays (homogeneous assays) or as solid phase assays (heterogeneous assays).
In one embodiment, solution assays are run. In these cases, assays generally rely on probes that bind the DC-STAMP nucleic acids based on either the increase or decrease of fluorescence based on hybridization status, or on fluorescence resonance energy transfer (FRET) assays. For example, “molecular beacon” probes contain two labels and form hairpin loops that are quenched in the absence of target sequence; upon hybridization, the two labels are separated and a signal is generated. See for example U.S. Pat. Nos. 5,925,517, 6,103,476, 6,461,817 and 6,037,130, as well as other PHRI patents and applications, incorporated by reference herein. Similarly, “Hybeacon” probes are single-stranded probes that are labeled with a single fluorophore; upon binding to a complementary nucleic acid, the emission spectra of the label is altered and thus detected. FRET assays are done using probes and targets that contain two different labels; upon binding, the labels become spatially close so as to allow energy transfer.
In one embodiment, heterogeneous assays are done using nucleic acids attached to a solid support for testing specimens for binding and/or quantitation of the DC-STAMP nucleic acid. By “substrate” or “solid support” or other grammatical equivalents herein is meant any material appropriate for the attachment of capture probes and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates is very large. Possible solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, ceramics, and a variety of other polymers. In a some embodiments, the solid supports allow optical detection and do not themselves appreciably fluoresce. In addition, as is known the art, the solid support may be coated with any number of materials, including polymers, such as dextrans, acrylamides, gelatins, agarose, etc. Exemplary solid supports include silicon, glass, polystyrene and other plastics and acrylics.
Generally the solid support is flat (planar), although as will be appreciated by those in the art, other configurations of solid supports may be used as well, including the placement of the probes on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
In one embodiment, the support is a gene chip. A gene chip may be used on which there is immobilized either an anti-sense oligonucleotide, which is synthesized based on an EST (expressed sequence tag) sequence from a database, known sequences (e.g. DC-STAMP sequences, for example DC-STAMP probes) or an mRNA sequence. In some cases, full length genes or complements can be used. Examples of such gene chips include gene chips manufactured by Affymetrix (Lip Shutz, R. J. et al., Nature Genet. (1999), 21, supplement, 20-24), but are not limited thereto, and may be prepared based on any known method. When mRNA derived from a human cell is analyzed, a gene chip derived from human sequences is preferably used. For example, the human sequences U95 set or U133 set manufactured by Affymetrix may be used. However, suitable gene chips are not limited to these and a gene chip derived from, for example, an animal species closely related to a human may be used.
In an alternative embodiment, membrane filters on which there is immobilized a cDNA or RT-PCR product prepared from total human RNA or total RNA, taken from a specific tissue of a human subject, EST sequences, DC-STAMP sequences, etc. can be used.
The sample can be a number of things. The cDNA or RT-PCR product can be a clone obtained by performing a reverse transcription reaction and PCR using a primer prepared based from the DC-STAMP sequence. The cDNA or RT-PCR product may have been selected previously by use of a subtraction method (Diatchenki, L, et al., Proc. Natl, Acad. Sci, USA (1996) 93, 6025-6030) or a differential display method (Liang, P., et al., Nucleic Acids Res., (1992) 23, 3685-3690) based on total RNA in which the expression level differs between a person having a metabolic bone disorder and a person having no metabolic bone disorder. The array or filter may be one which is commercially available, such as those provided by IntelliGene (manufactured by Takara Bio). Alternatively, the cDNA or RT-PCR product may be immobilized using a commercially available spotter such as GMS417 arrayer (manufactured by Takara Bio) to make an array or a filter.
In one embodiment, not a specific mRNA clone but all of the expressed mRNA are labeled and used as a labeled sample that is put on the solid support. Crude mRNA (unpurified) may be used as a starting material for preparing a probe; however, preferably poly (A)+ RNA is used which has been purified by the aforementioned method. A method of preparing a labeled probe and a method of detecting and analyzing the probe using various types of immobilized sample are further described as follows.
A biotin-labeled cRNA probe is prepared in accordance with the protocol (Affymetrix's Expression Analysis Technical Manual) provided with the GeneChip manufactured by Affymetrix. Subsequently, hybridization and analysis is performed to detect and analyze light emitted from adipic acid using an Affymetrix analyzer (GeneChip Fluidics Station 400) in accordance with the protocol (Expression Analysis Technical Manual) provided with the GeneChip manufactured by Affymetrix.
In order to detect cDNA, a label must be attached to the cDNA when it is prepared from poly (A)+ RNA using a reverse transcriptase reaction. To obtain fluorescently labeled cDNA, d-UTP labeled with a fluorescent dye such as Cy3 or Cy5 may be included in the reaction mixture. If poly(A)+ RNA derived from a osteoclastogenic cell and poly (A)+ RNA derived from a cell used as a control are labeled with different dyes, then both types of poly (A)+ RNAs may be used simultaneously in a mixture. When a commercially available array is used, e.g. an array manufactured by Takara Bio Co., Ltd. hybridization and washing are performed in accordance with the protocol provided and then a fluorescent signal is detected using a fluorescent signal detector (for example, the GMS418 array scanner manufactured by Takara Bio Co., Ltd.) and thereafter subjected to analysis. The choice of array for use as described herein is not limited to those which are commercially available. A home-made array and an array specifically prepared in-house may be used.
In addition, as noted above, it is possible to use “sandwich” assays, wherein the capture probe on the surface of the solid support binds to a first domain of the target DC-STAMP sequence, and a label probe hybridizes to a second domain of the target sequence. The label probes can include “Molecular Beacons” and “Hybeacons”, or single stranded nucleic acids labeled with fluorophores or other labels as outlined herein.
When preparing cDNA from poly (A)+ RNA by reverse transcription, a labeled sample can be prepared by adding a radioisotope (for example, d-CTP) to the reaction. Hybridization is performed in accordance with customary methods. More specifically, hybridization can be performed using the Atlas system (manufactured by Clontech), which is a microarray formed using a commercially available filter, after hybridization the microarray is washed. Thereafter, detection and analysis are performed using an analyzer (for example, Atlas Image manufactured by Clontech).
In any of the methods, a sample derived from human tissue is hybridized with the immobilized samples of the same lot. The probe which is used can be charged, but the hybridization conditions used are kept the same. When fluorescently labeled probes are used, if the probes are labeled with different fluorescent dyes, then probes of different types can be added simultaneously in the form of a mixture and hybridized with the immobilized samples. Thereafter, fluorescent intensity can be read simultaneously (Brown, P. O. et al., Nature Genet., (1999) 21, supplement, p. 33-37).
In addition to the measurement methods mentioned above, there are subtraction cloning methods (see Experimental Medicine, Supplementary Volume, New Genetic Engineering Handbook, published by Yodosha Co., Ltd. (1996), p 32-35); differential display methods (Basic Biochemical Experimental Method 4, nucleic acid/gene experiment, II. Applied series, Tokyo Kagakudojin (2001), p 125-128); and methods using a reporter gene: chloramphenicol acetyltransferase (such as a pCAT3-Basic vector manufactured by Promega), β-galactosidase (such as a pβgal-Basic vector manufactured by Promega), secreted alkaline phosphotase (such as pSEAP2-Basic manufactured by Clontech); or green-fluorescent protein (such as pEGFP-1 manufactured by Clontech). However, the choice of measurement method is not limited to these methods.
Using any of the methods described above, The difference in the level of expression of human DC-STAMP between a specimen derived from a healthy person and a specimen derived from a test subject is analyzed. If a specimen shows a significantly high expression level of human DC-STAMP, it is determined that the possibility of having a metabolic bone disorder is high, that is, it can be detected. The term “significantly high expression level” refers to the case where, when analysis is performed by using GeneChip (manufactured by Affymetrix) and microarray Suite Ver. 3.0 (manufactured by Affymetrix), an average difference value of a gene derived from an osteoclast cell is significantly high compared to that of a normal non-osteoclast cell.
5 C Protein Diagnostic Assays
Alternatively, the level of expression of human DC-STAMP is measured, and then assessed to determine whether or not the measured concentration value falls within a predetermined range for a healthy person. If the value exceeds the range, the subject has a metabolic bone disorder. The diagnosis of a metabolic bone disorder can be made in this manner. Otherwise, the correlation between the level of expression of the human DC-STAMP gene and the degree of formation of a metabolic bone disorder in a healthy person is previously investigated, and then, the expression level of human DC-STAMP gene of a specimen taken from the test subject is measured. Also, in this manner, whether or not a test subject is a healthy person or not can be determined.
In addition to the nucleic acid diagnostic methods described herein, diagnosis can be done using protein expression assays as well. In general, this is done by measuring the level of expression of human DC-STAMP protein in a specimen taken from a subject and comparing the level to the level of expression in a healthy subject. Again, this can be done either as a solution assay, using the techniques outlined above or by immobilization on a surface. In the case of proteins, the use of beads coated with anti-DC-STAMP proteins find particular use.
The specimen may be prepared for protein analysis in a variety of ways as will be appreciated by those in the art. In one embodiment, the specimen is subjected to high-speed centrifugation as necessary to remove insoluble substances, and then prepared as a sample for ELISA/RIA and Western blot.
To prepare a sample for ELISA/RIA, blood or bone marrow taken from a subject is used directly, or diluted appropriately in a buffer solution before use. For Western blotting (electrophoresis), a solution extracted from blood or bone marrow can be used directly as the sample, or diluted appropriately with a buffer solution, and mixed with a sample buffer solution (manufactured by Sigma) containing 2-mercaptoethanol for SDS-polyacrylamide gel electrophoresis. For dot or slot blotting, a solution extracted from blood or bone marrow can be used undiluted or diluted appropriately in a buffer solution, the samples are directly adsorbed to a membrane using a blotting device.
A protein in the sample thus obtained can be specifically detected by precipitating the protein using a procedure such as immunoprecipitation or ligand binding, either without additional immobilization or after direct immobilization thereof. For immobilizing a protein, a membrane used can be one such as is used in Western blotting, dot blotting or slot blotting. Examples of such membranes include nitrocellulose membranes (for example, as manufactured by BioRad), nylon membranes such as Hybond-ECL (manufactured by Amersham Pharmacia), cotton membranes such as blot absorbent filters (for example, as manufactured by BioRad) and polyvinylidene difluoride (PVDF) membranes (for example, manufactured by BioRad). IN addition, a variety of protein chips can be used.
To detect and quantify a protein using an ELISA or RIA method, a sample or a diluted sample solution (for example, diluted with phosphate buffered saline (hereinafter referred to as “PBS”) containing 0.05% sodium azide) is dispensed into a 96-well plate, such as an Immunoplate, Maxisorp, (manufactured by Nunc) and incubated without agitation at a temperature in the range of 4° C. to room temperature overnight, or at 37° C. for 1 to 3 hours, thereby allowing the protein to adsorb the bottom surface of the wells to immobilize the protein.
Antibody against human DC-STAMP can be obtained using a customary method (see, for example, New Biochemical Experimental Course 1, Protein 1, p. 389-397, 1992), which comprises immunizing an animal with human DC-STAMP or a polypeptide arbitrarily selected from the amino acid sequences of human DC-STAMP, taking the antibody produced in the body and purifying it. Alternatively, a monoclonal antibody can be obtained in accordance with a method well known in the art (for example, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Antibody, p. 365-367, 1980, Prenum Press, N,Y.), which comprises fusing an antibody-producing cell producing an antibody against human DC-STAMP with a myeloma cell to form a hybridoma cell.
Human DC-STAMP protein for use as an antigen can be obtained by introducing a human DC-STAMP gene into a host cell by gene manipulation. To explain more specifically, human DC-STAMP protein may be obtained by preparing a vector capable of expressing the human DC-STAMP gene, introducing the vector into the host cell, expressing the gene, and purifying the expressed human DC-STAMP protein.
The level of expression of human DC-STAMP can be represented by the level of expression of a protein comprising the amino acid sequence represented by SEQ ID NO:2 of the sequence listing. The expression level can be measured by a method known in the art, such as a Western blotting or a dot/slot blotting method, using anti-human DC-STAMP antibody. Measurement of the level of expression of human DC-STAMP in a specimen taken from a healthy person can be performed in the same manner as described above. Then the difference between the level of expression of the protein measured in the specimen is compared to the level of expression in a healthy specimen and thereby detecting that a subject has a metabolic bone disorder.
The difference in the level of expression of human DC-STAMP between the specimens from a healthy person and a test subject is analyzed. As a result, if a specimen exhibits a significantly high expression level of human DC-STAMP, it can be determined that there is a high probability of a subject having a metabolic bone disorder. In this manner, the disorder can be detected.
Alternatively, a metabolic bone disorder can be detected by measuring the concentration of human DC-STAMP and analyzing whether or not the measured concentration value falls within the predetermined range for a healthy person. In this case, if the concentration value of a subject is higher than the range for a healthy person, it is determined that the subject has a bone disorder. Furthermore, by investigating the correlation between the level of expression of DC-STAMP and the degree of bone disorder formation in a healthy person, it is possible to determine whether or not a subject is healthy based on the level of expression of human DC-STAMP in a specimen taken from the subject.
5 D Specific Methods for Investigation of the Human DC-STAMP Gene and Human DC-STAMP
The human DC-STAMP gene and human DC-STAMP are expressed at a significantly high levels in osteoclasts in normal human tissues, and they are, expressed at a significantly higher level in giant cell tumors (GCT), which is a bone tumor abundant in osteoclastic multinuclear giant cells, as previously described herein.
In a method of examining the function of human DC-STAMP, full-length cDNA is first taken from a human cDNA library, derived from cells expressing human DC-STAMP, by a known method such as a colony hybridization method. Then, the full-length cDNA is introduced into a mouse or a human cell, highly-expressed therein, and assessment is carried out to investigate whether or not the cDNA affects the cell.
To express cDNA in an animal, a method may be used in which the full-length cDNA obtained is introduced into a virus vector and the vector is administered to the animal. Examples of gene transfection using a virus vector include methods of introducing cDNA by integrating it into a DNA virus or an RNA virus, such as a retrovirus, adeno virus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, or polio virus. Of these, methods using retrovirus, adeno virus, adeno-associated virus and vaccinia virus are preferred.
Examples of non-viral gene transfection include administering an expression plasmid directly into the muscle (DNA vaccination), liposome treatment, lipofection, micro-injection, calcium phosphate treatment, electroporation and the like. Of these, DNA vaccination and liposome treatment are preferred.
Furthermore, by transfecting full-length cDNA into cultured cells, such as muscle cells, liver cells, or adipose cells derived from human, mouse or rat; or into primary muscle cells, liver cells, adipose cells or skin cells, and expressing the cDNA therein at a high level, it is possible to examine the functions of a target cell, more specifically, production and intake of sugars and lipids, control of glycolipid metabolism such as glycogen accumulation, or to see if there is any effect on the morphology of a cell. Conversely, by introducing into a cell an antisense nucleic acid to the total RNA of a gene to be examined, it is possible to examine the effects produced on the function and morphology of the target cell.
To introduce a full-length cDNA into an animal or a cell, the cDNA is integrated into a vector containing appropriate promoter sequences and transformation is carried out to transform the host cell with the vector. The expression promoter for use with a vertebrate cell may have a promoter that is typically located upstream of the gene to be expressed, an RNA splicing site, a polyadenylation site, a transcription termination sequence, etc. Furthermore, if necessary, a replication initiation point may be present. Examples of such an expression vector include, but are not limited to, pSV2dhfr having an early promoter of simian virus 40 (SV40) (Subramani, S. et al., Mol. Cell. Biol., (1981), 1, p 854-864), retrovirus vectors pLNCX, pLNSX, pLXIN, pSIR (manufactured by Clontech), and cosmid vector pAxCw (manufactured by Takara Bio). These expression vectors can be integrated into a simian cell, such as a COS cell (Gluzman, Y. Cell (1981), 23, p. 175-182, ATCC: CRL-1650), a dihydrofolic acid reductase defective strain (Urlaub, G. and Chasin, L. A. Proc. Natl. Acad. Sci. USA (1980), 77, p. 4126-4220) of a Chinese hamster ovary cell (CHO cell, ATCC:CCL-61), human embryonic kidney derived 293 cell (ATCC: CRL-1573) and the like, by methods including a diethylaminoethyl (DEAE)-dextran method (Luthman, H and Magnusson, G., Nucleic Acids Res. (1983), 11, p. 1295-1308), a calcium phosphate-DNA co-precipitation method (Graham, F. L. and van der Eb, A. J. Virology (1973), 52, p. 456-457), and an electroporation method (Neumann, E. et al., EMBO J. (1982), 1, p. 841-845). However, the integration method and cell are not limited to those specifically described. In this manner, a desired transformant can be obtained.
Furthermore, using gene manipulation in a healthy animal, a transgenic animal can be obtained which highly expresses the desired gene. This can be used to examine the effects on cell phenotype, such as morphology. The function of DC-STAMP can be also analyzed by suppressing DC-STAMP expression and examining the effects of a lower level of expression on differentiation into, and maturation of, osteoclasts, or on the cell morphology. A suppressor for DC-STAMP expression may be an anti-sense nucleic acid, a siRNA or the like which acts against the DC-STAMP gene. An inhibitor for DC-STAMP function may be an antibody capable of specifically binding to DC-STAMP.
Suppression of DC-STAMP expression or inhibition of DC-STAMP function can be applied to examine of the effects thereof on the function of each type of cells, specifically, function related to bone metabolism, such as differentiation into, and maturation, of osteoclasts, or on cell morphology. In addition, knockout animals can be created from animals suffering from, or free of, a metabolic bone disorder to examine the resulting state of the cells or tissue. Alternatively, the state of cells may be examined by preparing a knockout animal by knocking out the target gene in an animal having a metabolic bone disorder.
5 E Human DC-STAMP Gene and/or Human DC-STAMP Detection Kit
The human DC-STAMP gene and/or human DC-STAMP can be detected using a kit containing one or more components as described herein. Generally, the kit includes nucleic acid primers and/or probes for the detection of DC-STAMP. For example, pairs of polymerase chain reaction (PCR) primers for amplifying DC-STAMP genes can be included in a kit. These generally comprise DC-STAMP specific sequences, such as primers having a continuous sequence of from about 10-15 to about 20-30 bases in length for specifically amplifying a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOS:1, 3, or 5 in the Sequence Listing.
In alternative embodiments, detection probes that hybridize specifically to DC-STAMP genes are included; in this context, “specificity” means that the DC-STAMP gene can be identified with little or no cross-hybridization to other genes. In general, the detection probes have a continuous sequence of at least 10 nucleotides capable of hybridizing with a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO:1, 3, or 5 of the sequence listing under stringent conditions, thereby enabling detection of the polynucleotide. Probes can be longer, with about 15, 20 and 25 and upwards nucleotides all being included. In addition, these detection probes can also be labeled, for example using biotin or fluorophores.
In addition, solid supports can be included in the kits, including planar arrays or beads, with immobilized probes in the case of nucleic acid detection, or DC-STAMP binding proteins such as antibodies, in the case of protein detection.
In the case of protein detection, DC-STAMP antigen binding proteins, such as DC-STAMP antibodies can also be included, and optionally, secondary antibodies capable of binding to an DC-STAMP antibody. Suitable antibodies are made as described below.
The primers described above in paragraph [00196] above can be easily constructed based on the nucleotide sequence of the human DC-STAMP gene (the nucleotide sequence represented by SEQ ID NO:1, 3, or 5 of the sequence listing) by a customary method, for example, by a method using commercially available primer construction software (e.g., Wisconsin GCG package Version 10.2) and subjected to amplification. The detection probe according to paragraph [00197] above is a polynucleotide capable of hybridizing specifically with human DC-STAMP and being 100 to 1500 bases in length, preferably 300 to 600 bases in length. These primers and probes may be tagged with an appropriate label (such as an enzyme label, radioactive label, biotin, or fluorescent label) or may have a linker added thereto.
A kit according to the present invention may contain a thermostable DNA polymerase, dNTPs (a mixture of dATP, dCTP, dGTP and dTTP) and a buffer solution. Examples of thermostable DNA polymerases include Taq DNA polymerase, LA Taq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.), Tth DNA polymerase, and Pfu DNA polymerase. The type of buffer solution can be selected in accordance with the DNA polymerase which is to be used and Mg2+ can be added, as needed.
A kit according to the present invention can be used for detection of a human DC-STAMP gene and/or human DC-STAMP protein, thereby determining the presence or absence of a metabolic bone disorder and for screening to identify a suppressor of the pathological progress of a metabolic bone disorder.
6. Therapeutic Methods
The present invention provides DC-STAMP binding proteins such as anti-DC-STAMP antibodies.
6. A. Preparation of Antigen
An antigen for preparing an anti-human DC-STAMP antibody can be a polypeptide comprising human DC-STAMP, a partial amino acid sequence thereof having a partial and continuous amino acid sequence comprising at least 6 bases, or derivatives thereof having an arbitrary amino acid sequence or a carrier added to these (fusion proteins).
Human DC-STAMP protein can be directly purified from blood cells or bone marrow cells, or a cell membrane fraction prepared therefrom, as well as synthesized in vitro, or produced in host cells by gene manipulation. More specifically, in producing human DC-STAMP by gene manipulation, a human DC-STAMP gene is integrated into an expression vector, and thereafter the human DC-STAMP is synthesized in a solution containing enzymes, substrates and energy substances required for its transcription and translation. Alternatively, a prokaryotic or eukaryotic host cell can be transformed with the expression vector and then human DC-STAMP can be isolated. The nucleotide sequence of human DC-STAMP cDNA is described herein. The human DC-STAMP cDNA can be obtained from a cDNA library expressing human DC-STAMP by using a primer for specifically amplifying human DC-STAMP cDNA from the cDNA library as a template through a polymerase chain reaction (hereinafter referred to as the “PCR”, (see Saiki, R. K., et al., (1988), Science 239, 487-49) herein termed a “PCR method”.
The in vitro synthesis for a polypeptide can be performed using, for example, the rapid translation system (RTS) manufactured by Roche Diagnostics; however, suitable synthesis methods are not limited to this particular method. In the case of RTS, the desired gene is cloned into an expression vector, under the control of a T7 promoter, and the expression vector is added to an in vitro reaction system. Consequently, mRNA is first transcribed from template DNA by T7 RNA polymerase and then translation is performed by ribosomes in a solution containing Escherichia coli lysate. In this manner, a target polypeptide can be synthesized in the reaction solution (Biochemica, 1, 20-23 (2001), Biochemica, 2, 28-29 (2001)).
Examples of suitable prokaryotic hosts include Escherichia coli and Bacillus subtilis. To transform a desired gene into these host cells, the host cells are transformed with a plasmid vector derived from a species compatible with the host, and containing a replicon, that is, a replication initiation point, and a regulatory sequence. Furthermore, it is preferred that the vector has a sequence capable of imparting a selectable phenotype to the cell to be transformed.
As a host cell an Escherichia coli strain, for example, a K12 strain can be used and pBR322 and pUC series plasmids can generally be used as vectors. However, the choice of host cell and vector is not limited thereto and any suitable known strain and vector may be used.
Promoters suitable for use in Escherichia coli, include the tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, and polypeptide chain extension factor Tu (tufB) promoter and the like. Any one of these promoters may be used for producing the desired polypeptide.
As a host cell, a Bacillus subtilis strain can be used, for example, the 207-25 strain is preferred. The vector pTUB 228 (Ohmura, K. et al., (1984), J. Biochem. 95, 87-93) can be used; however, the choice of Bacillus subtilis host and vector is not limited to this particular combination. By linking a DNA sequence encoding a signal peptide sequence for Bacillus subtilis α-amylase, the protein of interest can be expressed and secreted from the cell.
Eukaryotic host cells include cells from vertebrate animals, insects, yeasts and so forth, and vertebrate cells often used include, for example, but are not limited to, murine monocyte-derived RAW264.7 cells (ATCC Cat. No. TIB-71), RAW264 cells (ECACC Cat. No. 85062803), and RAW-D cells (Watanabe et al., J. Endocrinol., (2004) 180, 193-201); dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, 4126-4220) of simian COS cells (Gluzman, Y., Cell, (1981) 23, 175-182; ATCC CRL-1650), murine fibroblasts NIH3T3 (ATCC No. CRL-1658), and Chinese hamster ovarian cells (CHO cells; ATCC: CCL-61); and the like.
An expression promoter for use with a vertebrate cell, can be one having a promoter located upstream of the gene to be expressed, an RNA splicing site, a polyadenylation site, and a transcription termination sequence. Furthermore, a replication initiation site may be present. Examples of the suitable expression vectors include, but are not limited to, pCDNA3.1 (manufactured by Invitrogen) having an early promoter of a cytomegalo virus and pSV2dhfr (Subramani, S. et al., (1981), Mol. Cell. Biol. 1, 854-864) having an SV40 early promoter.
When using a COS cell or NIH3T3 cell as the host cell, suitable expression vectors have an SV40 replication initiation site, capable of self-proliferating in the COS cell or NIH3T3 cell and additionally may have a transcription promoter, transcription termination signal, and RNA splicing site. The expression vector may be integrated into the COS cell or NIH3T3 cell by DEAE-dextran treatment (Luthman, H and Magnusson, G. (1983), Nucleic Acids Res. 11, p. 1295-1308), calcium phosphate-DNA co-precipitation (Graham, F. L. and van der Eb, A. J. (1973), Virology, 52, p. 456-457), electroporation (Neumann, E. et al., (1982), EMBO J. 1, p. 841-845) or others. In this manner, a desired transformant cell can be obtained. Furthermore, when a CHO cell is used as a host cell, if a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker, such as pRSVneo (Sambrook, J. et al., (1989): Molecular Cloning A Laboratory Manual “Cold Spring Harbor Laboratory, NY) or pSV2neo (Southern, P. J., and Berg, P. (1982), J. Mol. Appl. Genet. 1, 327-341) is co-transfected with the expression vector, and then a G418 resistant colony is selected, a transformed cell stably producing the desired polypeptide can be obtained.
The transformant obtained in the manner mentioned above can be cultured in accordance with a customary method to obtain the desired polypeptide expressed within the cell or secreted outside the cell and thus present in the culture medium. As a culture medium, various types of media customarily used can be selected appropriately depending upon the type of host cell employed. More specifically, for COS cells, RPMI 1640 medium or Dulbecco's Modified Eagle's medium (hereinafter referred to as “DMEM”) may be used. If necessary, serum components such as fetal calf serum may be added to the medium.
A recombinant protein produced within a cell or secreted outside a transformant cell and present in the culture medium can be separated and purified by various known separation methods on the basis of the physical properties and chemical properties of the protein. Examples of such separation methods include treatment with a general protein precipitating agent, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, affinity chromatography, various types of liquid chromatographic methods such as high-performance liquid chromatography (HPLC), dialysis and combinations of these methods. If a hexa-his tag is fused to the recombination protein which is expressed, the recombinant protein can be efficiently purified by a nickel affinity column. If the aforementioned methods are used in combination, a large amount of a desired polypeptide can be obtained with a high purity and in a high yield.
Alternatively, the antigen used can be a membrane fraction prepared from a recombinant cell expressing human DC-STAMP or a recombinant cell expressing human DC-STAMP, or a chemically synthesized peptide fragment of a protein according to the present invention obtained by a method known to those skilled in the art.
Once the antigen is made, antibodies can be produced.
6. B. Production of Anti-Human DC-STAMP Monoclonal Antibody
An example of an antibody which specifically binds to human DC-STAMP, is a monoclonal antibody which specifically binds to human DC-STAMP. A method suitable for obtaining such monoclonal antibody is as follows:
To produce the monoclonal antibody, the steps necessary required include:
(a) purifying the biomacromolecule which is to be used as an antigen;
(b) immunizing an animal by injecting the antigen into the animal, taking a blood sample and checking the antibody titer to determine the time at which the spleen should be excised, and preparing antibody producing cells;
(c) preparing bone myeloma cells (hereinafter referred to as “myeloma”);
(d) fusing the antibody-producing cells and the myeloma;
(e) selecting hybridomas producing a desired antibody;
(f) segregating (cloning) them into single cell clones;
(g) optionally, culturing the hybridoma to produce a large amount of monoclonal antibody or raising an animal having the hybridoma transplanted therein; and
(h) analyzing the physiological activity and binding specificity of the monoclonal antibody thus produced, or characteristics of the monoclonal antibody as a labeling agent.
The method of producing a monoclonal antibody is described in more detail below in accordance with the steps mentioned above. However, methods of producing monoclonal antibody are not limited to the method described. For example, an antibody-producing cell other than a spleen cell and myeloma.
6. C Preparation of an Antibody Producing Cell
An antigen obtained as above is mixed with Freund's complete or incomplete adjuvant or an auxiliary agent such as potassium aluminum sulfate. The mixture is used as an immunogen and is injected into an animal. A suitable experimental animal would be an animal known to be suitable for use in a hybridoma preparation method. Specific examples of such animals include mice, rats, goats, sheep, cows and horses. However, in view of the availability of myeloma cells which are to be fused with the antibody-producing cells taken from the animal, mice or rats are preferred as the animals to be immunized. The choice of strains of mice or rats used in practice is not particularly limited. Examples of suitable mouse strains include A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BR, C57L, DBA, FL HTH, HTI, LP, NZB, NZW, RF, R III, SJL, SWR, WB, and 129. Examples of rat strains include Low, Lewis, Spraque, Daweley, ACI, BN, and Fischer. These mice and rats are available from experimental animal-raising distributors such as Clea Japan Inc., Charles River Japan Inc., Japan SLC Inc., and The Jackson Laboratories. In view of fusion compatibility with myeloma cells as discussed later, “BALB/c” as a mouse line and “Low” as a rat line are particularly preferred as the immunized animal. In consideration of homology of an antigen between a human and a mouse, a mouse having a reduced biological function for removing autoantibody, in other words, a mouse suffering from autoimmune disease is preferably used. Note that a mouse or a rat which is to be immunized is preferably 5 to 12 weeks old, more preferably 6 to 8 weeks old.
An animal can be immunized with human DC-STAMP or a recombinantly produced version thereof by known methods, such as the methods specifically described in, for example, Weir, D. M. Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C Thomas Publisher Springfield Illinois (1964), etc. Of these immunization methods, a method preferably used in the present invention is, for example, performed as follows. First, an antigen, that is, a membrane protein fraction, or a cell expressing an antigen, is injected into an animal intradermally or intraperitoneally. To improve immunization efficiency, both injection methods can be used together. More specifically, when the intradermal injection is performed in the first half of the injections and the intraperitoneal injection is performed in the second half of the injections or only the last time, the immunization efficiency can be particularly increased. The dosing regimen of the antigen differs depending upon the type and individual differences, etc. of the animal body to be immunized. However, the antigen is preferably injected 3 to 6 times at intervals of 2 to 6 weeks, and more preferably 3 to 4 at intervals of 2 to 4 weeks. It is preferred not to excessively increase the number of dosings, because then the antigen may be wasted. Also, it is preferred not to overly extend the length of the dosing interval, because the activity of the cells decreases due to aging of the animal. The dose of the antigen differs depending upon the type and individual differences, etc. of the animal body; however, the dose generally falls within the range of about 0.05 to 5 ml, preferably about 0.1 to 0.5 ml. Booster immunization is performed 1 to 6 weeks after the antigen is administered, preferably after 2 to 4 weeks, more preferably after 2 to 3 weeks. If the booster immunization is performed after more than 6th weeks or within 1 week, the booster immunization will be less effective. Note that the dose of the antigen to be injected as a booster differs depending upon the type and size of the animal body; however, for example, for mice, it generally falls within the range of about 0.05 to 5 ml, preferably about 0.1 to 0.5 ml, and more preferably about 0.1 to 0.2 ml. It is preferable not to administer an unnecessarily large amount of antigen because then the immunization effect decreases and it is unfavorable to the animal to be immunized.
One to 10 days, preferably, 2 to 5 days, more preferably 2 to 3 days after the booster immunization, spleen cells or lymphocytes containing antibody-producing cells are removed from the immunized animal under aseptic conditions. At this time, an antibody titer is determined. If an animal having a sufficiently high antibody titer is used as the supply source for the antibody-producing cells, the efficiency of the following operations can be enhanced. As a method of determining the antibody titer to be used herein, various types of known technologies are appropriate, such as RIA methods, ELISA methods, fluorescent antibody methods, and passive blood cell agglutination reaction methods. In view of detection sensitivity, speed, accuracy, and the possibility of automatic operation, RIA methods and ELISA methods are preferred.
The determination of an antibody titer according to the present invention can be performed by an ELISA method as follows. First, the purified or partially purified antigen is adsorbed onto a solid surface such as 96-well plate for ELISA. Then, solid surface having no antigen adsorbed thereon is covered with a protein unrelated to the antigen, such as bovine serum albumin (hereinafter referred to as “BSA”). After washing the surface, the surface is brought into contact with a serially-diluted sample (e.g., mouse serum) serving as a primary antibody, thereby allowing a monoclonal antibody contained in the sample to bind to the antigen. Furthermore, a secondary antibody, that is, an enzyme-labeled antibody against a mouse antibody, is added to bind to the mouse antibody. After washing the resultant complex, a substrate for the enzyme is added and the change in absorbance, which occurs due to a color change induced by degradation of the substrate, is measured to calculate the antibody titer.
Antibody-producing cells are separated from the spleen cells or lymphocytes in accordance with known methods (for example, described in Kohler et al., Nature, 256, 495, 1975; Kohler et al., Eur J. Immunol., 6, 511, 1977; Milstein et al., Nature, 266, 550, 1977; Walsh, Nature, 266, 495, 1977). More specifically, in the case of spleen cells, the antibody-producing cells can be separated by a general method which comprises homogenizing tissue, filtering the homogenized through a stainless steel mesh, and suspending the cells obtained in Eagle's Minimum Essential Medium (MEM).
6. C i) Preparation of Bone Myeloma Cells (hereinafter Referred to as “Myeloma”)
The choice of myeloma cells which are to be used for cell fusion is not particularly limited and suitable cells can be selected from known cell strains. For convenience when hybridoma are selected from fused cells, it is preferable to use a HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) defective strain whose selection procedure has been established. More specifically, examples of HGPRT defective strains include X63-Ag8(X63), NSI-Ag4/1(NS1), P3X63-Ag8.U1(P3U1), X63-Ag8.653(X63.653), P2/0-Ag14(SP2/0), MPC11-45.6TG1.7(45.6TG), F0, S149/5XXO and BU.1 derived from mice, 210.RSY3.Ag.1.2.3 (Y3) derived from rat; and U266AR(SKO-007), GM1500GTG-A12(GM1500), UC729-6, LICR-LOW-HMy2(HMy2), and 8226AR/NIP4-1(NP41) derived from humans. These HGPRT defective strains are available from the American Type Culture Collection (ATCC), etc.
These strains are subcultured in an appropriate medium such as 8-azaguanine medium [RPMI-1640 supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as “FCS”) and further 8-azaguanine is added thereto]; Iscove's Modified Dulbecco's Medium (hereinafter referred to as “IMDM”), or Dulbecco's Modified Eagle Medium (hereinafter referred to as “DMEM”). In this case, 3 to 4 days before performing the cell fusion operation, the cells are transferred to a regular medium [for example, ASF104 medium (manufactured by Ajinomoto Co. Inc.) containing 10% FCS] and subcultured therein to obtain not less than 2×107 cells by the day of cell fusion.
6 C ii) Cell Fusion
Fusion between antibody-producing cells and myeloma cells is appropriately performed in accordance with known methods (including: Weir, D. M. Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, E. A., and Mayer, M. M. Experimental Immunochemistry, Charles C Thomas Publisher, Springfield, Ill. (1964)), under conditions such that the survival rate of cells is not excessively reduced. Examples of such methods include chemical methods in which antibody-producing cells and myeloma cells are mixed in a high concentration polymer solution, for example, polyethylene glycol; and physical methods using electric stimulation. Of these methods, the chemical method is more specifically explained as follows. When polyethylene glycol is used as the high concentration polymer solution, antibody-producing cells and myeloma cells are mixed in a solution of polyethylene glycol having a molecular weight of 1,500 to 6,000, more preferably, 2,000 to 4,000, at a temperature of 30 to 40° C., preferably 35 to 38° C., for 1 to 10 minutes, more preferably 5 to 8 minutes.
6. C. iii) Selection of Hybridoma Populations
The method of selecting hybridoma obtained by cell fusion is not particularly restricted. Usually, use is made of the HAT (hypoxanthine, aminopterin, thymidine) selection method [Kohler et al., Nature, 256, 495 (1975); Milstein at al., Nature 266, 550 (1977)]. This is an effective method when hybridoma are obtained using myeloma cells of a HGPRT defective strain incapable of surviving in the presence of aminopterin. More specifically, by culturing unfused cells and hybridoma in HAT medium, only hybridoma having aminopterin resistance are selected and allowed to remain and proliferate.
6. C iv) Segregation to Single Cell Clone (Cloning)
As a cloning method for hybridoma, known methods such as a methylcellulose method, soft agarose method, or limiting dilution method can be used [see, for example, Barbara, B. M. and Stanley, M. S.: Selected Methods in Cellular Immunology, W. H. Freeman and Company, San Francisco (1980)]. Examples of a cloning method include a limiting dilution method in which hybridoma cells are diluted so as to contain a single hybridoma cell per well of a plate and cultured; a soft agarose method in which hybridoma cells are cultured in a soft agarose medium and colonies are recovered; a method of taking individual hybridoma cells by means of a micro manipulator and culturing them; and a so-called “clone sorter method” in which hybridoma cells are separated one by one by means of a cell sorter. Of these methods, the limiting dilution method is preferred. In this method, a fibroblast cell strain derived from a rat fetus or feeder cells such as healthy mouse spleen cells, thymus gland cells, or ascites cells are seeded. Hybridoma cells are diluted in medium to provide a dilution ratio of 0.2 to 0.5 cells per 0.2 ml. The diluted hybridoma suspension solution is transferred into wells to provide 0.1 ml per well and continuously cultured for about 2 weeks with changes of about ⅓ of the medium with fresh medium at predetermined time intervals (for example, every 3 days). In this manner, hybridoma clones can be proliferated.
The hybridoma cells in the well for which antibody titer has been confirmed are subjected to repeat cloning by the limiting dilution method, 2 to 4 times. Hybridoma cells, with an antibody titer which is confirmed to be stable, are selected as anti-human DC-STAMP monoclonal antibody producing hybridoma strains. One of the cloned hybridoma strains thus obtained is designated as “O3B8-2C9-4F3” and this has been deposited at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science Technology (located at Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as of Feb. 17, 2004 under deposition No. FERM BP-08627.
6 C v) Preparation of Monoclonal Antibody by Culturing Hybridoma Cells
The hybridoma cells thus selected are cultured to efficiently obtain monoclonal antibody. However, prior to culturing, it is desirable that a hybridoma cell producing a desired monoclonal antibody is screened. The screening is performed by a known method.
The determination of antibody titer can be performed in the present invention by, for example, an ELISA method in accordance with the following procedure. First, purified or partially purified human DC-STAMP or cells expressing human DC-STAMP are adsorbed onto a solid surface of a 96-well plate for ELISA. Then, the solid surface having no antigen adsorbed thereon is covered with a protein unrelated to the antigen, for example, bovine serum albumin (hereinafter referred to as “BSA”). After washing the surface, the surface is brought into contact with a serially-diluted sample (for example, mouse serum) as a first antibody, thereby allowing binding of an anti-human DC-STAMP antibody in the sample to the antigen. Furthermore, an antibody against the mouse antibody and labeled with an enzyme, serving as a secondary antibody, is added to bind to the mouse antibody. After washing the resultant complex, a substrate for the enzyme is added and the change of absorbance, which occurs due to the colour change induced by degradation of the substrate, is determined to calculate the antibody titer. In this way, the antibody titer is calculated. Note that such a screening operation can be performed after or before cloning of the hybridoma cell as mentioned above.
A hybridoma obtained by the aforementioned method can be stored in a frozen state in liquid nitrogen or in a refrigerator at 80° C. or less.
After completion of cloning, hybridoma are transferred from HT medium to a general medium and cultured. Large-scale culture is performed by rotation culture using a large culture bottle or by spinner culture. The supernatant obtained from the large-scale culture is purified by a known method to those skilled in the art, such as gel filtration, to obtain a monoclonal antibody which specifically binds to a protein according to the present invention. The hybridoma can be injected into the abdominal cavity of a mouse of the same line as the hybridoma (for example, BALB/c) or a Nu/Nu mouse to proliferate the hybridoma. In this way, ascites fluid containing a large amount of the monoclonal antibody according to the present invention can be obtained. When hybridoma cells are injected into the abdominal cavity, if a mineral oil such as 2,6,10,14-tetramethyl pentadecane (pristine) has (3 to 7 days before) been administered previously, the ascites fluid can be obtained in a larger amount. To explain more specifically, an immunosuppressive agent is previously injected into the abdominal cavity of a mouse of the same strain as the hybridoma. Twenty days after inactivation of the T cells, 106 to 107 of hybridoma clone cells are suspended in a serum-free medium (0.5 ml) and the suspension is injected into the abdominal cavity. When the abdomen is expanded and filled with the ascites fluid, the ascites fluid is taken. By virtue of this method, the monoclonal antibody can be obtained at a concentration 100-fold higher than that of the culture medium.
A monoclonal antibody obtained in the aforementioned method can be purified by the methods described in, for example, Weir, D. M.: Handbook of Experimental Immunology Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978). To explain more specifically, examples of such methods include ammonium sulfate precipitation methods, gel-filtration methods, ion exchange chromatographic methods, and affinity chromatographic methods. Of these, the ammonium sulfate precipitation method, if it is repeated 3 to 4 times, preferably 3 to 6 times, successfully purifies the monoclonal antibody. However, in this method, the yield of the purified monoclonal antibody is extremely low. Therefore, the monoclonal antibody is crudely purified by performing the ammonium sulfate precipitation method once or twice and then subjected to at least one method, and preferably two methods, selected from gel filtration, ion exchange chromatography, and affinity chromatography and the like. In this way, highly purified monoclonal antibody can be obtained in a high yield. The ammonium sulfate precipitation method may be performed in the following combination and in the following order: a) ammonium sulfate precipitation method—ion exchange chromatographic method-gel filtration method; b) ammonium sulfate precipitation method—ion exchange chromatographic method—affinity chromatographic method; and c) ammonium sulfate precipitation method—gel filtration method—affinity chromatographic method, etc. Of these combinations, to obtain the monoclonal antibody with a high purity in a high yield, combination c) is particularly preferable.
As a simple purification method, a commercially available antibody purification kit (for example, MAbTrap GII kit manufactured by Pharmacia) and the like can be used.
The monoclonal antibody thus obtained has high antigen specificity for human DC-STAMP.
6 C vi) Analysis of Monoclonal Antibody
The monoclonal antibody thus obtained is checked for isotype and subclass thereof as follows. Suitable identification methods include the Ouchterlony method, ELISA methods and RIA methods. The Ouchterlony method is simple; although, if monoclonal antibody is obtained at low concentration it must be concentrated. Alternatively, when an ELISA method or RIA method is used, the culture supernatant can be directly reacted with an antigen adsorption solid phase. In addition, if various types of antibodies corresponding to immunoglobulin isotypes and subclasses are used as secondary antibodies, the isotype and subclass of the monoclonal antibody can be identified. As a further simple method, a commercially available identification kit (for example, Mouse Typer kit manufactured by BioRad) and the like can be used.
The quantification of a protein can be performed by the Folin Lowry assay based on the adsorption at 280 nm [1.4 (OD280)=Immunoglobulin 1 mg/ml].
6 C vii) Method using a Polymerase Chain Reaction
When the amino acid sequence of a desired protein has been elucidated in its entirety or in part, oligonucleotide primers of a sense strand and an antisense strand corresponding to a part of the amino acid sequence are synthesized. Then, the polymerase chain reaction [Saiki, R. K., et al. (1988) Science 239, 487-49] is performed by using these primers in combination to amplify a DNA fragment encoding heavy chain and light chain subunits of a desired anti-human DC-STAMP antibody. As the template DNA used herein, use may be made of cDNA synthesized from mRNA of a hybridoma producing the anti-human DC-STAMP monoclonal antibody by a reverse transcriptase reaction.
The DNA fragment thus prepared can be directly integrated into a plasmid vector by use of a commercially available kit, etc. Alternatively, the DNA fragment may be used for selecting a desired clone by labeling the fragment with 32P, 35S, or biotin, and performing colony hybridization or plaque hybridization by using it as a probe.
For example, a method of examining a partial amino acid sequence of each subunit of the anti-human DC-STAMP monoclonal antibody of the present invention is preferably performed by isolating each subunit by use of a known method such as electrophoresis or column chromatography and then analyzing the N-terminal amino acid sequence of each subunit using an automatic protein sequencer (for example, PPSQ-10, manufactured by Shimadzu Corporation).
A method of isolating cDNA encoding each subunit of the anti-human DC-STAMP monoclonal antibody protein from the desired transformant obtained as mentioned above is performed in accordance with a known method [see Maniatis, T., et al. (1982) in “Molecular Cloning A Laboratory Manual” Cold Spring Harbor Laboratory, NY.], and more specifically, can be performed by separating fractions corresponding to vector DNA from a cell and excising a DNA region encoding a desired subunit from the vector DNA (plasmid DNA).
(b) Screening method using a synthesized oligonucleotide probe **TT
When the whole or part of the amino acid sequence of a desired protein is elucidated (any sequence is taken from any region of the desired protein as long as it is a specific sequence having a plurality of contiguous amino acids), an oligonucleotide is synthesized (in this case, use may be made of either a nucleotide sequence presumed based on the degree of frequency of codons in use or a plurality of nucleotide sequences of conceivable nucleotide sequences in combination; in the latter case, the number of types of nucleotide sequences can be reduced by integrating inosine) so as to correspond to the amino acid sequence, used as a probe (labeled with 32P, 35S or biotin); hybridized with a nitrocellulose filter on which the DNA of a transformant strain is denatured and immobilized, and then the positive strain obtained is isolated.
The sequence of the DNA thus obtained can be determined by the Maxam-Gilbert chemical modification method [see Maxam, A. M. and Gilbert, W. (1980) in “Methods in Enzymology” 65, 499-576] and the dideoxynucleotide chain termination method [Messing, J. and Vieira, J. (1982) Gene 19, 269-276].
Recently, an automatic base sequence determination system using a fluorescent dye has been widely used (for example, sequence robots “CATALYST 800” and model 373ADNA sequencer, etc. manufactured by PerkinElmer Japan Co., Ltd.)
Using such a system also makes it possible to efficiently and safely determine a DNA nucleotide sequence. Based on the data of the present invention thus determined including the nucleotide sequence of DNA and the N-terminal amino acid sequences of the heavy chain and light chain, it is possible to determine the entire amino acid sequence of the heavy chain and light chain of the monoclonal antibody of the present invention.
The heavy chain and light chain of immunoglobulin each constitute a variable region and a constant region. The variable region further constitutes complementarity-determining regions (hereinafter referred to as “CDR”, there are 3 sites in each of the heavy chain and light chain) and framework regions adjacent to these CDRs (4 sites in each of the heavy chain and light chain).
The amino acid sequence of the constant region is common to antibodies belonging to the same immunoglobulin class regardless of the type of antigen. In the variable region, the amino acid sequence of a CDR is intrinsic to each antibody. However, according to a study comparing amino acid sequence data of numerous antibodies, it is known that the position of the CDR and the length of a framework sequence are similar between the subunits of different antibodies as long as they belong to the same subgroup [see Kabat, E. A., et al. (1991) in “Sequence of Proteins of Immunological Interest Vol. II”: U.S. Department of Health and Human Services]. Therefore, it is possible to determine the position of the CDRs and framework regions and further the constant region in each amino acid sequence, by comparing the amino acid sequences of the heavy chain and the light chain of the anti-human DC-STAMP monoclonal antibody of the present invention with the known amino acid sequence data. Note that the chain length of FRH1, that is, the framework region located at the side proximal to the N terminus, is sometimes shorter than the general length of 30 amino acids. In some cases, the framework region is known to have a minimum of 18 amino acids [see Kabat et al. cited above]. From this, in the antibody of the present invention, the chain length of the framework region at the N-terminus of the heavy chain is set at 18 to 30 amino acids, preferably 30 amino acids, as long as the function of the anti human DC-STAMP antibody is not impaired.
In summary, only by artificially modifying a peptide having the same amino acid sequence as each of the CDRs of light chains or heavy chains or a partial contiguous amino acid sequence thereof, as determined above, thereby approximating the structure to the tertiary structure of the CDR actually taken from within the anti-human DC-STAMP antibody molecule, a binding activity capable of binding to human DC-STAMP can be imparted to the CDR [see, for example, U.S. Pat. No. 5,331,573]. Hence, a peptide containing the same amino acid sequence as that of a CDR or a partial amino acid sequence thereof is also included as being a molecule of the present invention.
A modified amino acid sequence can be prepared by deleting at least one or more amino acids from its original amino acid sequence in accordance with cassette mutagenesis [see Toshimitu Kishimoto, “New Biochemical Experimental Lecture 2, Nucleic acid III, Recombinant DNA technique”, p 242-251].
Such various types of DNA sequences can be produced in accordance with a customary method for chemically synthesizing a nucleic acid, for example, the phosphite triester method [see Hunkapiller, M., et al. (1984) Nature 310, 105-111]. Note that codons corresponding to a desired amino acid are already known perse. Any codon may be selected. Alternatively, which codon is used can be determined in accordance with a customary method by considering the frequency with which codons are used by the host cell. The partial modification of the nucleotide sequences of codons, may be performed in accordance with a customary method, more specifically, in accordance with a site-specific mutagenesis method [see Mark, D. F., et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666] using a synthetic oligonucleotide primer encoding a desired modification.
Furthermore, it is possible to check whether a certain type of DNA can hybridize with DNA encoding a heavy chain or light chain of an anti-human DC-STAMP monoclonal antibody of the present invention by subjecting the DNA to the following experiment performed using a probe DNA labeled with [α-32P]dCTP, in accordance with the random primer method [see Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13] or the nick translation method [see Maniatis, T., et al. (1982) in “Molecular Cloning A laboratory Manual” Cold Spring Harbor Laboratory, NY.].
To explain more specifically, the DNA to be checked is adsorbed onto, for example, a nitrocellulose or nylon membrane. After it is denatured with alkali, if necessary, the membrane is heated or UV-irradiated, thereby immobilizing the DNA onto the membrane. The membrane is soaked in a pre-hybridization solution containing 6×SSC (1×SSC contains 0.15M sodium chloride, 0.015 trisodium citrate solution) and 5% Denhardt's solution, and 0.1% sodium dodecylsulfate (SDS), and maintained at 55° C. for 4 hours or more. Subsequently, the probe prepared in advance is added to the pre-hybridization solution so as to have a final specific activity of 1×106 cpm/ml and the temperature is maintained at 60° C. overnight. Thereafter, the membrane is washed with 6×SSC at room temperature for 5 minutes several times, further washed with 2×SSC for 20 minutes and subjected to autoradiography.
Using the aforementioned methods, DNA which hybridizes with the DNA encoding a heavy chain or light chain of the humanized anti-human DC-STAMP antibody of the present invention can be isolated from a random cDNA library or a genomic library [see Maniatis, T., et al. (1982) in “Molecular Cloning A Laboratory Manual” Cold Spring Harbor Laboratory, NY.].
Each of the DNA sequences obtained in the aforementioned manner can be integrated into an expression vector, which can be then introduced into a prokaryotic or eukaryotic host cell. In this way, the gene (having the DNA) can be expressed in the host cell as described herein.
A fraction containing an anti-human DC-STAMP antibody protein produced within or outside the transformant cell can be treated by various known protein isolation procedures based on the use of physical and/or chemical properties to isolate and purify the protein. Examples of these methods include treatment with a protein precipitation agent generally used, ultrafiltration, chromatography, such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, and affinity chromatography, or high performance liquid chromatography (HPLC), dialysis, and combinations thereof.
To humanize the anti-human DC-STAMP monoclonal antibody, the amino acid sequence of a variable region must be designed such that the entire CDR sequence and a partial amino acid sequence of the FR sequence determined are transplanted into a human antibody framework, as follows:
Conventionally, in designing a humanized antibody, an acceptor subgroup is selected based on the following guidelines.
a) the natural combination of a heavy chain and light chain of a known human antibody having a naturally occurring amino acid sequence is used as it is;
b) although the combination of a heavy chain and a light chain as a subgroup is maintained; the heavy chain and the light chain may be derived from different human antibodies. The heavy chain and the light chain which are to be used may be selected from amino acid sequences with high identity to those of the heavy chain and light chain of the donor, respectively, and the consensus sequences. In the present invention, the aforementioned guidelines may be employed. However, there are alternative methods as follows:
c) regardless of consideration of the combination of the subgroup, a method may be employed for selecting FRs of the heavy chain and light chain with high identity to those of a donor from the library of primary sequences of a human antibody. In these selection methods, the degree of identity of the amino acids of the FR region between a donor and an acceptor can be set at 70% or more. By employing such a method, it is possible to reduce the number of amino acid residues of an antibody to be transplanted from a donor, thereby inducing less HAMA response.
There is an operation (hereinafter referred to as “molecular modeling”) for predicting the tertiary structure of an antibody molecule from its primary sequence; however, the accuracy of prediction of this operation is limited. Therefore, the role of an amino acid residue appearing only rarely in the subgroup to which the donor belongs cannot be sufficiently specified. It is generally difficult to determine which amino acid residue of a donor or an acceptor should be selected for such a position of the amino acid residue in accordance with the method described above by Queen and co-workers. However, in accordance with the selection method (c), it is possible to reduce the frequency with which such determination must be made.
The present inventors have further improved such humanization methods by providing a novel method of identifying an amino acid derived from the FR of a donor and important for maintaining the structure and function of a CDR of the donor.
After a human acceptor molecule for each of a light chain and heavy chain is selected, the amino acid residue to be transferred from the FR of a donor is selected by the method mentioned below.
In the amino acid sequences of the donor and the acceptor, when the corresponding amino acid residues of their FRs differ from each other, it must be determined which amino acid residue should be selected. When making such a selection, care must be taken so as not to damage the tertiary structure of the CDR derived from the donor.
Queen et al. have proposed, in the Japanese National Publication of International Patent Application No. 4-502408, a method of transplanting an amino acid residue on the FR into an acceptor together with a CDR sequence, if it satisfies at least one of the following conditions:
1) The amino acid is rarely present at the position within a human FR region of an acceptor, whereas the corresponding amino acid of a donor is usually present at the equivalent position;
2) the amino acid is located extremely close to one of the CDRs;
3) it is predicted that the amino acid has a side chain atom within about 3 angstroms from the CDR in its three dimensional immunoglobulin model and the side chain atom can interact with an antigen or the CDR of a humanized antibody.
In the above, a residue satisfying requirement 2) above often exhibits the property of requirement 3). Therefore, in the present invention, requirement 2) is omitted and two requirements are newly set. More specifically, in the present invention, if the amino acid residue on the donor's FR to be transferred together with the CDR satisfies the following:
a) the amino acid is rarely present at the position within an FR region of an acceptor, whereas the corresponding amino acid of a donor is usually present at the equivalent position;
b) in the tertiary structure model, the amino acid presumably interacts with a constituent amino acid atom of the CDR and an antigen or the CDR loop to be transplanted;
c) the position mentioned above is that of a canonical class determination residue; or
d) the position is that which forms a contact surface between a heavy chain and a light chain,
then the amino acid residue is transplanted from the FR of the donor.
In requirement a), in accordance with the Kabat list mentioned above, an amino acid found at a frequency of 90% or more at a position in the same subclass of antibody is defined as “usually present”, whereas an amino acid found at a frequency of less than 10% is defined as “rarely present”.
In requirement c), as to whether or not “the position mentioned above is a canonical class determining residue”, the determination can be made uniquely in accordance with Chothia's list as mentioned above.
In requirements b) and d), molecular modeling of the antibody's variable region must be performed in advance. As software for molecular modeling, any commercially available software may be used; however, preferably AbM (manufactured by Oxford Molecular Limited Company) is used.
The accuracy of prediction by molecular modeling is somewhat limited. Therefore, in the present invention, by considering X-ray crystallographic data for variable regions of various antibodies, the reliability of the structure predicted by molecular modeling can be evaluated in two steps.
In the tertiary structure of the variable region constructed by the molecular modeling software, such as AbM, if the distance between two atoms is shorter than a value of the sum of the van der Waals radius of two atoms plus 0.5 angstroms, the two molecules are assumed to be in van der Waals contact. On the other hand, if the distance between atoms having polarity, such as amide nitrogen or carbonyl oxygen, of the main and side chains, is shorter than a distance of an average hydrogen binding distance, 2.9 angstroms plus 0.5 angstroms, it is assumed that hydrogen bonding may exist between the atoms. Furthermore, if the distance between the oppositely charged atoms is shorter than a distance of 2.85 angstroms plus 0.5 angstroms, it is assumed that an ionic bond is formed between the atoms.
On the other hand, from X-ray crystallographic experimental results for variable regions of various antibodies, as the position on the FR at which contact with the CDR can be found with a high frequency regardless of the subgroup, the following positions can be specified: in the light chain, the 1, 2, 3, 4, 5, 23, 35, 36, 46, 48, 49, 58, 69, 71, and 88th positions, and in the heavy chain, 2, 4, 27, 28, 29, 30, 36, 38, 46, 47, 48, 49, 66, 67, 69, 71, 73, 78, 92, 93, 94, and 103rd positions (numerals all represent amino acid numbers defined in the documents described by Kabat et al. The same definition will be also applied below). When the same standard as that of the molecular modeling is applied, the amino acid residues of these positions are confirmed to be in contact with the amino acid residues of the CDR in the ⅔ portion of the known antibody's variable region. Based on the findings, the sentence: “b) In the tertiary structure model, the amino acid presumably interacts with a constituent amino acid atom of the CDR and an antigen or the CDR loop to be transplanted” means as follows.
In molecular modeling, if a position in the FR which is expected to be in contact with the CDR agrees with any one of the positions at which the contact between the FR and the CDR is reported to frequently occur according to experimental detection by X-ray crystallography, selection of the amino acid residue from the donor is preferred. In other cases, requirement b) is not taken into consideration.
The sentence: “d) the position is that which forms a contact surface between the heavy chain and the light chain” means the following requirement. From the X-ray crystallographic experimental results for the variable regions of various antibodies, it is confirmed that heavy chain-light chain contact is frequently observed at the 36, 38, 43, 44, 46, 49, 87, 98th amino acid residues in the light chain and at the 37, 39, 45, 47, 91, 103, and 104th amino acid residues in the heavy chain. In cases where the possibility of heavy chain-light chain contact is predicted in the molecule modeling and the contact position agrees with any one of the aforementioned positions, transplantation of the amino acid residue from the donor is preferably performed. In other cases, requirement d) is not taken into consideration.
The DNA encoding variable regions of the heavy chain and light chain of a humanized anti-human DC-STAMP antibody of the present invention can be produced by the methods described below.
For example, a plurality of polynucleotide fragments comprising a partial nucleotide sequence of the DNA, of 60 to 70 nucleotides in length, are chemically synthesized alternately from the sense and antisense strands. Thereafter, individual polynucleotide fragments are annealed and ligated using DNA ligase. In this way, it is possible to obtain a DNA having DNA encoding variable regions of the heavy chain and light chain of a desired humanized anti-human DC-STAMP antibody.
In another method, DNA encoding the total amino acid sequence of the variable region of an acceptor is extracted from human lymphocytes, replacement of nucleotides is performed in the region encoding a CDR by a method known to those skilled in the art to introduce a restriction enzyme cleavage sequence. After the region is cleaved with the corresponding restriction enzyme, the nucleotide sequence encoding a CDR of the donor is synthesized and ligated using DNA ligase. In this way, it is possible to obtain the DNA encoding variable regions of the heavy chain and light chain of a desired humanized anti-human DC-STAMP antibody.
Furthermore, in the present invention, it is possible to obtain DNA comprising DNA encoding variable regions of the heavy chain and light chain of a desired humanized anti-human DC-STAMP antibody, preferably in accordance with the overlap extension PCR method (Horton et al., Gene, 77, 61-68, (1989)) described below.
To explain more specifically, two different DNA sequences, which encode two different amino acid sequences, respectively and which are desired to be ligated to each other, are designated as (A) and (B), for the sake of convenience. A sense primer of 20 to 40 nucleotides (hereinafter referred to as a “primer (C)”) to be annealed to the 5′ side of the DNA sequence (A) and an antisense primer of 20 to 40 nucleotides (hereinafter referred to as a “primer (D)) to be annealed to the 3′ side of the DNA sequence (B) are chemically synthesized. Furthermore, a chimeric-type sense primer (hereinafter referred to as “primer (E)) is formed by ligating a nucleotide sequence of 20 to 30 nucleotides to the 3′ side of the DNA sequence (A) and a nucleotide sequence of 20 to 30 nucleotides is ligated to the 5′ side of the DNA sequence (B). An antisense primer (hereinafter referred to as “primer (F)) complementary to the primer (E) is synthesized. When a PCR is performed by using appropriate vector DNA containing DNA (A) as a substrate, sense primer (C) and the chimeric-type antisense primer (F), DNA in which the 20 to 30 nucleotides of the 5′ end of DNA (B) is attached to the 3′ end of the DNA (A) can be obtained (the DNA newly formed is designated as DNA (G)). Similarly, when a PCR is performed by using appropriate vector DNA containing DNA (B) as a substrate, antisense primer (D) and the chimeric-type sense primer (E), DNA in which 20 to 30 nucleotides of the 3′ end of DNA (A) is attached to the 5′ end of the DNA (B) can be obtained (the DNA newly formed is designated as DNA (H)). In the DNAs (G) and (H), the 40 to 60 nucleotides on the 3′ side of the DNA (G) form a sequence complementary to that formed by the 40 to 60 nucleotides on the 5′ side of the DNA (H). The amplified DNA (G) and (H) are mixed and subjected to PCR, DNA (G) and (H) are formed into a single strand in a first denaturation reaction. Although most chains of DNA revert to their original states following an annealing reaction, a part of DNA forms into a hetero-double-stranded DNA by the annealing of the complementary nucleotide sequence region. A protruding single stranded part is filled in by a subsequent extension reaction to obtain a chimeric type DNA (hereinafter referred to as DNA (I)) formed of DNA (A) and DNA (B) ligated to each other. DNA (I) can be amplified by performing PCR using DNA (I) as a substrate, the sense primer (C) and the antisense primer (D). In the present invention, DNA encoding a CDR region of a heavy chain and light chain of an anti-human DC-STAMP mouse monoclonal antibody, DNA encoding an FR region of human immunoglobulin IgG, furthermore DNA encoding a secretion signal of human immunoglobulin IgG may be used as DNA (A) and (B), on a case-by-case basis, and subjected to the ligation reaction mentioned above.
Note that codons corresponding to a desired amino acid are known per se and can be arbitrarily chosen. More specifically, the codons can be determined in accordance with a customary method in consideration of the frequency with which the codon is used by a host. A part of nucleotide sequence of the codons may be modified in accordance with a customary method such as site-specific mutagenesis (see, Mark, D. F., et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666) using a synthetic oligonucleotide primer encoding a desired modification. Therefore, if each primer is designed so as to introduce a point mutation and thereafter chemically synthesized, it is possible to obtain DNA encoding variable regions of a heavy chain and light chain of a desired anti-human DC-STAMP antibody.
By integrating each of the DNAs of the present invention thus obtained into an expression vector, a prokaryotic or eukaryotic host cell can be transformed. Furthermore, by introducing an appropriate promoter and a sequence related to phenotypic expression into these vectors, each gene can be expressed in the corresponding host cell.
By virtue of the method mentioned above, a recombinant anti-human DC-STAMP antibody can be manufactured easily with high purity and in high yield.
6. D A Pharmaceutical Composition Containing an Anti-Human DC-STAMP Antibody
From the anti-human DC-STAMP antibodies obtained by a method described in Section “3. DC-STAMP Antigen Binding Proteins”, antibody suppressing the biological activity of DC-STAMP can be obtained. An antibody capable of neutralizing the bioactivity of DC-STAMP may be obtained from anti-DC-STAMP antibodies produced by the methods described herein. An antibody capable of neutralizing the bioactivity of DC-STAMP may be used as therapeutic agent for metabolic bone disorders caused by abnormal differentiation into osteoclasts, because the antibody inhibits the in vivo bioactivity of DC-STAMP, i.e., differentiation into and/or maturation of osteoclasts.
The neutralizing activity of the anti-DC-STAMP antibody for the in vitro bioactivity of DC-STAMP may be assayed, for example, by its ability to suppress osteoclastic differentiation of cells overexpressing DC-STAMP. For instance, a murine monocyte-derived cell strain, such as RAW264.7 cells, RAW264 cells, or RAW-D cells, overexpressing DC-STAMP may be cultured, then supplied with different levels of the anti-DC-STAMP antibody, and stimulated with RANKL and TNF-α to measure suppression of differentiation of the cells into osteoclasts. Also, a primary cell culture from bone marrow may be supplied with different levels of the anti-DC-STAMP antibody, and stimulated with RANKL and TNF-α to measure suppression of differentiation of the cells into osteoclasts. Furthermore, in a pit assay experiment (Takada et al., Bone and Mineral, (1992) 17, 347-359) using cells from femur and/or tibia, the cells from femur and/or tibia may be supplied with different levels of the anti-DC-STAMP antibody and the formation of pits on ivory pieces observed to measure suppression of osteoclast bone resorption. The therapeutic effect in vivo of the anti-DC-STAMP antibody on metabolic bone disorder in laboratory animals may be examined, for example, by administering the anti-DC-STAMP antibody to transgenic animals overexpressing DC-STAMP and measuring a change in the osteoclasts.
An antibody capable of neutralizing the bioactivity of DC-STAMP thus obtained is useful as a pharmaceutical agent, especially in a pharmaceutical composition to treat diseases such as osteoporosis, rheumatoid arthritis and cancerous hypercalcemia, which are attributable to metabolic bone disorder; or as an antibody for immunological diagnosis of these diseases.
An anti-DC-STAMP antibody may be given, for example, alone or in combination with at least one therapeutic agent for treating bone diseases in order to treat metabolic bone disorder. As an example, an anti-DC-STAMP antibody may be also given together with a therapeutically effective amount of a therapeutic agent against a metabolic bone disorder. The therapeutic agent suitably administered together with the anti-DC-STAMP antibody may include, but is not limited to, bisphosphonates, activated vitamin D3, calcitonin and its derivatives, hormone preparations such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K2 (menatetrenone), and calcium preparations. Depending on the condition of the metabolic bone disorder and/or the extent of therapy required, two, three or more different agents may be given, or supplied as a formulation having these agents combined therein. These agents and the anti-DC-STAMP antibody may be also supplied as a combined formulation. Further, these agents may be supplied as a therapeutic kit, the agents being contained therein. Also, these agents may be supplied separately from the anti-DC-STAMP antibody. When the therapy is in the form of gene therapy, the gene for the anti-DC-STAMP antibody may be inserted downstream of the same promoter together with, or separately from, a gene for a proteinaceous therapeutic agent of bone disease, and they may be integrated into different vectors or into the same vector.
The present invention also provides a pharmaceutical composition containing a therapeutically effective amount of an anti-DC-STAMP antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjunct.
The present invention also provides a pharmaceutical composition containing a therapeutically effective amount of an anti-DC-STAMP antibody, a therapeutically effective amount of at least one therapeutic agent for bone disease and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjunct. The therapeutic agent for bone disease may include, but is not limited to, bisphosphonates, activated vitamin D3, calcitonin and its derivatives, hormone preparations such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K2 (menatetrenone), calcium preparations, PTH (parathyroid hormone) preparations, non-steroidal anti-inflammatory agents, anti-TNFα antibodies, anti-PTHrP (parathyroid hormone-related protein) antibodies, IL-1 receptor antagonists, anti-RANKL antibodies and OCIF (osteoclastogenesis inhibitory factor).
The antibodies of the present invention can inhibit the biological activity of human DC-STAMP in the living body, in other words, osteoclastogenesis of a cell. Therefore, they can be used as a medicament, in particular, as a therapeutic agent for metabolic bone disorders. The activity of an anti-human DC-STAMP antibody in neutralizing a biological activity of human DC-STAMP in vitro can be determined by the ability to inhibit osteoclastogenesis of a cell in which human DC-STAMP is overexpressed.
To explain more specifically, the inhibitory activity can be determined by culturing murine monocyte-derived RAW-D cells (Watanabe et al., J. Endocrinol., (2004) 180, 193-201), which, overexpresses human DC-STAMP, adding an anti-human DC-STAMP antibody to the culture system in various concentrations. In this way, the inhibitory activities against osteoclast formation can be determined, such as for example, the number of tartrate resistant acid phosphatase (TRAP) positive multinuclear osteoclasts, which is indicative of the level of multinucleation or fusion of mononuclear osteoclast precursor cells of monocyte or macrophage lineage to form osteoclasts.
An antibody thus obtained for neutralizing the biological activity of human DC-STAMP is useful as a medicament, especially as a pharmaceutical composition for use in the treatment of metabolic bone disorders or as an antibody for use in immunological diagnosis of such a disorder. The types of metabolic bone disorders that may be treated and/or diagnosed in accordance with the invention are not limited to the examples disclosed herein.
The present invention provides a pharmaceutical composition containing an anti-human DC-STAMP antibody in an amount useful for treatment, a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or an auxiliary agent.
A substance to be used as a pharmaceutically acceptable preparation in a pharmaceutical composition according to the present invention is preferably non-toxic to a patient to which the pharmaceutical composition is to be administered, in view of the dose and concentration.
A pharmaceutical composition according to the present invention can contain substances, suitable for inclusion in a preparation, which are capable of changing, maintaining, and stabilizing pH, osmotic pressure, viscosity, transparency, isotonic condition, aseptic condition, stability, solubility, release rate, absorbtion rate, and permeability. Examples of such substances for inclusion in a preparation include, but are not limited to, amino acids such as glycine, alanine, glutamine, asparagine, arginine, and lysine; anti-oxidant agents such as anti-bacterial agents, ascorbic acid, sodium sulfate and sodium hydrogen sulfite; buffering agents such as phosphate, citrate, borate buffers, hydrocarbonate, Tris-HCl solution; fillers such as mannitol and glycine; chelating agents such as ethylenediamine tetraacetate (EDTA); complex forming agents such as caffeine, polyvinylpyrrolidine, β-cyclodextrin and hydroxypropyl-β-cyclodextrin; thickening agents such as glucose, mannose, and dextrin; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, dextrin; hydrophilic polymers such as colorants, flavors, diluents, emulsifiers, 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; polysorbates such as suspending agents, PEG, sorbitan ester, polysorbate 20, and polysorbate 80; surfactants such as Triton, tromethamine, lecithin, cholesterol; stability-enhancing agents such as sucrose, and sorbitol; elasticity-enhancing agents; transport agents, diluents; excipients; and/or pharmaceutical auxiliary agents such as sodium chloride, potassium chloride, mannitol/sorbitol. The amount of these substances added to a preparation is about 0.01 to about 100 times or about 0.1 to about 10 times the weight of the anti-human DC-STAMP antibody. Those skilled in the art can appropriately determine the formulation suitable for preparation of a pharmaceutical composition depending upon the disease and administration route.
The excipient and carrier used in a pharmaceutical composition may be a liquid or solid substance. Examples of a suitable excipient and carrier may include injectable solutions, saline, artificial cerebral spinal fluid and other substances usually used for parenteral administration. Furthermore, neutral saline and saline containing serum albumin may be used as a carrier. A pharmaceutical composition may contain a Tris buffer of pH 7.0 to 8.5 and an acetate buffer of pH 4.0 to 5.5, which may be supplemented with sorbitol and other compounds. A pharmaceutical composition according to the present invention having a selected composition is prepared with a requisite purity in appropriate drug form, or as a lyophilized product or a liquid product. To describe this more specifically, a pharmaceutical composition containing the anti-human DC-STAMP antibody can be formed into a lyophilized product using an appropriate excipient such as sucrose.
A pharmaceutical composition according to the present invention can be prepared for parenteral use or for oral use for gastrointestinal absorption. The composition and concentration of a preparation can be chosen depending upon the administration method. As an anti-human DC-STAMP antibody contained in a pharmaceutical composition according to the present invention exhibits higher affinity for human DC-STAMP; in other words, the higher the affinity of anti-human DC-STAMP antibody for human DC-STAMP, as expressed by the dissociation constant (Kd value), that is, the lower the Kd value, the higher the efficacy of the pharmaceutical composition of the present invention at a lower dose. Therefore, based on this, the dose amount of the pharmaceutical composition of the present invention to a person can be determined. The humanized anti-human DC-STAMP antibody may be administered to a person as a single dose at an interval of about 1 to about 30 days in an amount of about 0.1 to about 100 mg/kg.
Examples of forms of a pharmaceutical composition of the present invention may include injections such as drip infusions, suppository agents, pernasal agents, sublingual agents, and percutaneous absorption agents, depot agents, transdermal patch agents, topical agents, oral agents such as a tablet, a capsule, a granule, a powder, a syrup and the like.
7. Screening Methods
The present invention provides methods for screening for candidate agents that bind to DC-STAMP proteins. In some cases, screens are done for agents that induce suppression of osteoclastogenesis and/or cytotoxicity as described herein.
Screening methods can be homogeneous or heterogeneous, with the latter being preferred.
Thus, the present invention provides methods of screening candidate agents for agents that bind to and/or modulate the activity of (in particular, the suppression of osteoclastogenesis) DC-STAMP, including the inhibition of enhanced differentiation of a cell expressing DC-STAMP into an osteoclast.
“Candidate agent” or “candidate drug” as used herein describes any molecule, e.g., proteins including biotherapeutics including antibodies and enzymes, small organic molecules including known drugs and drug candidates, polysaccharides, fatty acids, vaccines, nucleic acids, etc. that can be screened for activity as outlined herein. Candidate agents are evaluated in the present invention for discovering potential therapeutic agents that affect DC-STAMP and therefore potential disease states.
Candidate agents encompass numerous chemical classes. In one embodiment, the candidate agent is an organic molecule and may be small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. In other embodiments, the candidate agents are small organic compounds having a molecular weight of more than 100 and less than about 2,000 daltons, less than about 1500 daltons, less than about 1000 daltons, or less than 500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least one of an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression and/or synthesis of randomized oligonucleotides and peptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. By “protein,” as used herein, is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. In some embodiments, the two or more covalently attached amino acids are attached by a peptide bond. The protein may be made up of naturally occurring amino acids and peptide bonds, for example when the protein is made recombinantly using expression systems and host cells, as outlined below. Alternatively, proteins (for example when used as candidate agents in screening assays, as outlined below) may include synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine), or peptidomimetic structures, i.e., “peptide or protein analogs”, such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:9367, incorporated by reference herein), which can be resistant to proteases or other physiological and/or storage conditions. Such synthetic amino acids may be incorporated in particular when fragments of DC-STAMP or antigen binding proteins are synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptidomimetic, synthetic and naturally occurring residues/structures can be used. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The amino acid “R group” or “side chain” may be in either the (L)- or the (S)-configuration. In a specific embodiment, the amino acids are in the (L)- or (S)-configuration.
Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of procaryotic and eucaryotic proteins may be made for screening in the systems described herein. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occuring nucleic acids, random and/or synthetic nucleic acids.
For example, digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins. In addition, RNAis are included herein.
7. A. Screens
The screens may take on a variety of formats. In general, either the candidate agents or the DC-STAMP protein (including fragments thereof are attached to solid supports as described herein. This is generally done using any immobilization techniques, including those described herein, for example through the use of absorbtion to the solid support or covalent attachment using functional groups.
In one embodiment, the DC-STAMP protein is attached to the solid support and labeled candidate agents are added, unbound agents are washed away, and detection of binding of the candidate agent to the DC-STAMP protein is done. The contacting step is done under reaction conditions that favor agent-target interactions. Generally, this will be physiological conditions. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away.
A variety of other reagents may be included in the assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
Once a binding event has been detected, the candidate agent may be identified.
In some embodiments, candidate agents that are identified as binding to the DC-STAMP proteins are then added to suppressor activity assays, as described herein. Alternatively, suppressor activity assays are run with libraries of candidate agents without a binding assay done first.
Suppressor assays are generally done as outlined below in the Examples; this generally is done by adding the candidate agent, a cell expressing DC-STAMP, and in some cases, effector cells. The DC-STAMP-expressing cells can be selected from a number of different cells. In one embodiment, the DC-STAMP-expressing cells are naturally occurring cells, such as primary bone-marrow derived cells. In some embodiments, DC-STAMP-expressing cells are cells or cell lines that have been transformed to produce DC-STAMP, particularly human DC-STAMP.
The cultured cells used in a screening method of the present invention may be healthy mammalian cells, or cells capable of abnormal proliferation, such as cancer cells, as long as they can express DC-STAMP, including, for example, but not limited to, murine monocyte-derived RAW264.7 cells (ATCC Cat. No. TIB-71), RAW264 cells (ECACC Cat. No. 85062803), and RAW-D cells (Watanabe et al., J. Endocrinol., (2004) 180, 193-201); and murine bone marrow-derived primary culture cells. The cultured cells may originate from mammalian species. preferably including, but not being limited to, human, mouse or other animals (e.g., guinea pig, rat, chicken, rabbit, pig, sheep, cow, monkey, etc.). It is more preferable that the cultured cells are mammalian cells overexpressing DC-STAMP, such as RAW264.7 cells, RAW264 cells and RAW-D cells, which all have the DC-STAMP gene introduced therein together with a promoter region for over-expression of DC-STAMP.
The screening method of the invention also includes a method of administering a candidate agent or test substance to individual mammalian animals, rather than culturing cells with the test substance, removing an organ or tissue from the animals, and detecting expression of the DC-STAMP gene in the animal's cells. The organ or tissue from which the gene expression is to be detected has only to express DC-STAMP, but it is preferably a tissue developing a metabolic bone disorder, more preferably bone marrow. A mammalian species used here may be a non-human animal, preferably mouse, rat or guinea pig, more preferably mouse or rat. An animal model having a metabolic bone disorder may be an animal having had an ovary removed, an animal having had a testis removed, a cancer-bearing animal having tumor cells implanted into hypoderm, intraderm, left ventricle, bone marrow, vein, peritoneum or elsewhere, an animal having had a sciatic nerve removed, an animal model for adjuvant arthritis, an animal model for collagen-induced arthritis, an animal model for glucocorticoid-induced osteoporosis, senescence accelerated mice (SAM P6 mice, Matsushita et al., Am. J. Pathol. 125, 276-283 (1986)), an animal having had the thyroid/parathyroid removed, an animal receiving a continuous infusion of parathyroid hormone-related peptides (PTHrP), knockout mice having lost osteoclast inhibitory factor (OCIF) (Mizuno et al., Biochem. Biophys. Res. Commun., (1998) 247, 610-615) or the like. Additionally, an animal model having lost teeth due to periodontal disease or an animal created to over-express DC-STAMP may be used. Test substances selected by screening may be administered to the above-described animal models to measure the parameters that vary with metabolic bone disorder, such as the number of osteoclasts, bone density and bone strength in bone tissue, or blood Ca2+ level, and thereby evaluate their therapeutic and/or preventive effect on metabolic bone disorder.
A test substance can be given to animal subjects that over-express DC-STAMP to measure, over time, the incidence of metabolic bone disorder, severity thereof and/or survival rate etc. and to compare those with the counterparts for animals that over-express DC-STAMP but to which the test substance has not been given. If the animals that over-express DC-STAMP and have been given the test substance have a significantly lower incidence, a significantly lower severity and/or a survival rate that is higher by about 10%, preferably about 30% or more, and more preferably about 50% or more, the test substance may be selected as a compound effective to treat and/or prevent metabolic bone disorder.
The cells for culture used in the present invention may be cultured under any conditions so long as they can express DC-STAMP by addition of RANKL and TNF-α in the absence of a test substance. For instance, the cells may be cultured under known conditions provided that they can express DC-STAMP. The animals used to detect DC-STAMP expression in an organ or a tissue removed therefrom may be raised also under any conditions whereby the organ or tissue can express DC-STAMP in the absence of a test substance.
The effect of a test substance on DC-STAMP expression may be studied either by measuring the level of expression of the DC-STAMP gene or by measuring the level of expression of DC-STAMP which is the translation product of the DC-STAMP gene. A test substance capable of suppressing the expression of the DC-STAMP gene and/or DC-STAMP may be considered to be a substance having a therapeutic and/or preventive effect on metabolic bone disorders, preferably osteoporosis, rheumatoid arthritis and/or cancerous hypercalcemia.
The extraction of the total RNA from the cultured cells, measurement of the expression level of the DC-STAMP gene or measurement of the expression level of DC-STAMP may be carried out in accordance with a method described in the section of “5.B. Diagnostic Assays using Nucleic Acids”. In culturing mammalian cells, appropriate levels of RANKL and TNF-α may be added optionally to the medium with the test substance, or even without the substance in the case of a control culture.
In a preferred embodiment, the methods of the invention utilize a robotic system. Many systems are generally directed to the use of 96 (or more) well microtiter plates, but as will be appreciated by those in the art, any number of different plates or configurations may be used. In addition, any or all of the steps outlined herein may be automated; thus, for example, the systems may be completely or partially automated.
As will be appreciated by those in the art, there are a wide variety of components which may be used, including, but not limited to, one or more robotic arms; plate handlers for the positioning of microplates; automated lid handlers to remove and replace lids for wells on non-cross contamination plates; tip assemblies for sample distribution with disposable tips; washable tip assemblies for sample distribution; 96 well loading blocks; cooled reagent racks; microtitler plate pipette positions (optionally cooled); stacking towers for plates and tips; and computer systems.
Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications. This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration. These manipulations are cross-contamination-free liquid, particle, cell, and organism transfers. This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.
In a preferred embodiment, chemically derivatized particles, plates, tubes, magnetic particle, or other solid phase matrix with specificity to the assay components are used. The binding surfaces of microplates, tubes or any solid phase matrices include non-polar surfaces, highly polar surfaces, modified dextran coating to promote covalent binding, antibody coating, affinity media to bind fusion proteins or peptides, surface-fixed proteins such as recombinant protein A or G, nucleotide resins or coatings, and other affinity matrix are useful in this invention.
In a preferred embodiment, platforms for multi-well plates, multi-tubes, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity. This modular platform includes a variable speed orbital shaker, electroporator, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
In a preferred embodiment, thermocycler and thermoregulating systems are used for stabilizing the temperature of the heat exchangers such as controlled blocks or platforms to provide accurate temperature control of incubating samples from 4° C. to 100° C.
In some preferred embodiments, the instrumentation will include a detector, which may be a wide variety of different detectors, depending on the labels and assay. In a preferred embodiment, useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluroescence resonance energy transfer (FRET), SPR systems, luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation. These will enable the monitoring of the size, growth and phenotypic expression of specific markers on cells, tissues, and organisms; target validation; lead optimization; data analysis, mining, organization, and integration of the high-throughput screens with the public and proprietary databases.
These instruments can fit in a sterile laminar flow or fume hood, or are enclosed, self-contained systems, for cell culture growth and transformation in multi-well plates or tubes and for hazardous operations. The living cells will be grown under controlled growth conditions, with controls for temperature, humidity, and gas for time series of the live cell assays. Automated transformation of cells and automated colony pickers will facilitate rapid screening of desired cells.
Flow cytometry or capillary electrophoresis formats may be used for individual capture of magnetic and other beads, particles, cells, and organisms.
The flexible hardware and software allow instrument adaptability for multiple applications. The software program modules allow creation, modification, and running of methods. The system diagnostic modules allow instrument alignment, correct connections, and motor operations. The customized tools, labware, and liquid, particle, cell and organism transfer patterns allow different applications to be performed. The database allows method and parameter storage. Robotic and computer interfaces allow communication between instruments.
In a preferred embodiment, the robotic workstation includes one or more heating or cooling components. Depending on the reactions and reagents, either cooling or heating may be required, which may be done using any number of known heating and cooling systems, including Peltier systems.
In a preferred embodiment, the robotic apparatus includes a central processing unit that communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. The general interaction between a central processing unit, a memory, input/output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.
7. B. Rational Drug Design
According to another aspect, the present invention is directed to a drug design approach for obtaining a substance capable of inhibiting the activity of human DC-STAMP based on the tertiary structure of the protein. This approach is known as a rational drug design method and is used to search for a compound capable of efficiently inhibiting or activating a function, such as enzymatic activity or binding to a ligand, cofactor or DNA. As an example of such a compound, a protease inhibitor serving as anti-HIV agent presently marketed is well known. In analyzing the three-dimensional structure of human DC-STAMP according to the present invention, a generally well known method such as X-ray crystallography or nuclear magnetic resonance conceivably can be used. Furthermore, in searching for or designing a substance for inhibiting the function of human DC-STAMP, a computer-aided drug design method (CADD) can be used. As an example of this case, a low molecular weight compound (International Publication WO 99/58515) inhibiting the action of AP-1 is known which is expected to act as a novel genomic drug for treating chronic rheumatoid arthritis. By virtue of such a method, it is possible to obtain a substance inhibiting the function of human DC-STAMP by directly binding to the human DC-STAMP or by inhibiting the interaction between the human DC-STAMP and other factors.
Furthermore, according to another aspect, the present invention relates to a polypeptide associated with human DC-STAMP of the present invention, in other words, a partner protein for controlling the activity of human DC-STAMP. More specifically, the present invention relates to a screening method for such a partner protein for controlling the activity of human DC-STAMP.
One aspect of such a screening method comprises a step of bringing a test protein sample into contact with human DC-STAMP, thereby selecting a protein binding to the human DC-STAMP. Such a method includes purification of a protein by making use of its affinity for purified human DC-STAMP. To describe more specifically, first, a sequence formed of 6 histidines is bound to human DC-STAMP as an affinity tag. The resultant human DC-STAMP is incubated in a cell extract solution (that is, a fraction passed through a column charged with nickel-agarose) at 4° C. for 12 hours. Then, a nickel-agarose carrier is separately added to the mixture and the mixture is incubated at 4° C. for one hour. After the nickel-agarose carrier is sufficiently washed with a washing buffer, 100 mM imidazole is added to the mixture to elute a protein specifically binding to human DC-STAMP and contained in the cell extract solution. The purified protein is analyzed to determine its structure. A protein that can be purified as described above includes a protein which binds directly to human DC-STAMP and a protein forming a complex as a subunit with a protein which binds directly to human DC-STAMP, but having no binding activity for human DC-STAMP, thus binding indirectly to human DC-STAMP [see Experimental Medicine, Supplementary volume, Biomanual series 5, “Transcriptional factor investigation method” pp 215-219 (published by Yodosha Co., Ltd.)].
As alternative methods, there is a cloning method in accordance with Far-Western blot (Experimental Medicine, Supplementary volume, New Genetic Engineering Handbook, pp 76-81, published by Yodosha Co., Ltd.), and a two-hybrid system using a yeast or a mammalian cell (Experimental Medicine, Supplementary volume, New Genetic Engineering Handbook, pp 66-75, published by Yodosha Co., Ltd.), and “Checkmate mammalian two hybrid system” (manufactured by Promega). However, the present invention is not limited to use of these methods.
If cDNA of a partner protein directly or indirectly interacting with human DC-STAMP in this manner is available, it can be used in functional screening of a substance inhibiting the interaction between human DC-STAMP and the partner protein. More specifically, a fusion protein of human DC-STAMP with glutathione-S-transferase can be prepared. The fusion protein is allowed to bind to a microplate covered with anti-glutathione-S-transferase antibody and a biotinylated partner protein is brought into contact with the fusion protein. The binding of the partner protein with the fusion protein can be detected using alkaline phosphatase conjugated with streptavidin. When the biotinylated partner protein is added, test substances are added at the same time to select a substance which promotes or inhibits the binding of the fusion protein and the partner protein. By this method, a substrate directly acting on the fused protein or a substance directly acting on the partner protein can be obtained.
When the fused protein binds indirectly to the partner protein via another factor, the assay is performed in the presence of a cell extraction solution containing this factor. In this case, a substance, which may act upon the factor, may be selected.
When the partner protein obtained has the activity of suppressing the function of human DC-STAMP, it is possible to screen an osteoclastogensis suppressive agent, for example, a useful candidate substance as a therapeutic agent for osteoporosis, in accordance with a test method using an expression vector comprising the human DC-STAMP gene, as described above. Furthermore, when the obtained partner protein has the activity of suppressing the function of human DC-STAMP, a polynucleotide having a nucleotide sequence encoding such a suppressor can be used in gene therapy for metabolic bone disorders.
Such a polynucleotide can be obtained by analyzing the amino acid sequence of the identified inhibitor, synthesizing an oligonucleotide probe comprising a nucleotide sequence encoding the amino acid sequence and screening a cDNA library or genomic library. Furthermore, in the case where a peptide having inhibitory activity against a function of human DC-STAMP is derived from an artificial peptide library synthesized at random, DNA comprising a nucleotide sequence encoding the amino acid sequence of the peptide can be chemically synthesized.
In gene therapy, a gene encoding such an inhibitor is integrated, for instance, into a virus vector and a patient can be infected with a virus (attenuated) comprising the resultant recombinant virus vector. In the body of the patient, an osteoclastogenesis suppressive factor is produced and functions to suppress osteoclast formation. In this manner, it is possible to treat metabolic bone disorders.
As a method of introducing a gene therapeutic agent into a cell, both a gene transfection using a virus vector and a non-viral gene transfection can be used [Nikkei Science, 4, (1994), p. 20-45; Experimental Medicine, Extra number, 12 (15) (1994); Experimental Medicine, Supplementary volume, “Basic Technology of Gene Therapy” Yodosha, Co., Ltd. (1996)].
Examples of gene transfection using a virus vector include methods of integrating DNA encoding an inhibitor or a mutated version of the DNA into DNA virus or using a RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, or sindbis virus and introducing the virus vector into a body. Of these, methods using retrovirus, adenovirus, adeno-associated virus, and vaccinia virus are particularly preferred. Examples of non-viral gene transfection include a method of administering an expression plasmid directly into the muscle (DNA vaccination method), liposome treatment, lipofection, microinjection, calcium phosphate treatment, and an electroporation method. Of these, DNA vaccination and liposome treatment are preferred.
To use a gene therapeutic agent as a medicine in practice, there is an in vivo method for introducing DNA directly into the body, and an ex vivo method which comprises taking a certain type of cells out of the body, introducing DNA into the cells, and returning the cells into the body [Nikkei Science, 4, (1994), p. 20-45; The Pharmaceutical Monthly, 36(1), 23-48 (1994); Experimental Medicine, Extra number 12 (15) (1994)].
When the gene therapeutic agent is administered in accordance with the in vivo method, it is administered through an appropriate administration route, such as a vein, artery, subcutaneous tissue, intradermal tissue, or muscle, which differs depending upon the type of disease and symptoms. When the agent is administered in accordance with an in vivo method, the gene therapeutic agent is generally prepared in the form of an injection; however if necessary, a customarily used carrier may be added. Furthermore, when the agent is prepared in the form of a liposome or membrane-fused liposome (Sendai virus-liposome, etc.), the liposome agent may be supplied as a suspension agent, lyophilized agent, or centrifugally concentrated and lyophilized agent.
A complementary sequence to the nucleotide sequence represented by SEQ ID NO:1 or a complementary sequence to a partial sequence of this nucleotide sequence can be used as a so-called antisense therapy. As an antisense molecule, use may be made of DNA partially complementary to the nucleotide sequence represented by SEQ ID NO:1 of the sequence listing and formed generally of 15 to 30 mers complementary to a portion of a nucleotide sequence, which may include, without limitation, SEQ ID NOS: 1, 3 and 5. Also, use may be made of a stable DNA derivative such as a phosphorothioate derivative, methyphosphonate derivative, or morpholino derivative of the DNA, or a stable RNA derivative such as 2′-O-alkyl RNA. Such an antisense molecule can be introduced into a cell by a method known in the art of the present invention, for example by injecting an extremely small amount of the antisense molecule, by forming a liposome capsule, or by expressing it by use of a vector having an antisense sequence. Such an antisense therapy is useful for treating a disease caused by excessive activity of a protein encoded by the nucleotide sequence represented by SEQ ID NO:1 of the sequence listing.
Also, double-stranded short RNA (siRNA) can be used (Genes and Developments, 15th/Jan./2001, 15, 2, pp. 188-200). For instance, siRNA against the DC-STAMP gene may be prepared and introduced into cells, according to the method described in the aforementioned document, to treat disease due to metabolic bone disorder caused by over-expression of DC-STAMP.
A composition containing the antisense oligonucleotide and/or siRNA useful as a medicine can be prepared by a known method including mixing a pharmaceutically acceptable carrier. Examples of such a carrier and the preparation method are described in Applied Antisense Oligonucleotide Technology (1998 Wiley-Liss, Inc.). A preparation containing an antisense oligonucleotide can be administered orally by mixing with a pharmaceutically acceptable appropriate excipient or diluent, in the form of tablets, capsules, granules, powder or syrup, or administered parenterally in the form of an injection, suppository, patch, or external preparation. These preparations can be prepared by a known method using additives, excipients including organic excipients such as sugar derivatives (e.g., lactose, white sugar (sucrose), glucose, mannitol, and sorbitol); starch derivatives (e.g., corn starch, potato starch, α starch, and dextrin); cellulose derivatives (e.g., crystalline cellulose); Arabic gum; dextran; and pullulan; and inorganic excipients such as silicate derivatives (e.g., soft anhydrous silicic acid, synthesized aluminium silicate, calcium silicate, and magnesium aluminate metasilicate); phosphates (e.g., calcium hydrogen phosphate); carbonates (e.g., calcium carbonate), and sulfates (e.g., calcium sulfate); lubricant agents including metal stearates (e.g., stearic acid, calcium stearate, and magnesium stearate); talc; colloidal silica; waxes (e.g., beeswax and spermaceti wax), boric acid; adipic acid; sulfates (e.g., sodium sulfate), glycol; fumaric acid; sodium benzoate; DL leucine; lauryl sulfates (e.g., sodium lauryl sulfate and magnesium lauryl sulfate); silicates (e.g., anhydrous silicate, silicate hydrate); and starch derivatives mentioned above; binding agents including hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, macrogol, and the same compounds as mentioned as excipients; disintegrating agents including cellulose derivatives (e.g., low substitution degree hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, inner-cross-linked carboxymethylcellulose sodium; and chemically modified starch celluloses (e.g., carboxymethylstarch, carboxymethylstarch sodium, and cross-linked polyvinyl pyrrolidone); emulsifying agents including colloid silica (bentnite and bee gum), metal hydroxides (e.g., magnesium hydroxide and aluminium hydroxide), anionic surfactants (e.g., sodium lauryl sulfate and calcium stearate); cationic surfactants (e.g., benzalkonium chloride) and non-ionic surfactants (e.g., polyoxyethylene alkylether, polyoxyethylene sorbitan fatty acid ether, and sucrose fatty acid ester); stabilizing agents including paraoxy benzoates (e.g., methyl paraben, propyl paraben); alcohols (e.g., chloro butanol, benzyl alcohol, and phenylethyl alcohol); benzalkonium chloride; phenols (e.g., phenol and cresol); thimerosal; dehydro acetate; and sorbic acid; flavoring agents including sweeteners, acidic flavors and flavors generally used; and diluents.
As a method of introducing a compound of the present invention into a patient, a colloidal dispersion system may be used in addition to the aforementioned methods. The colloidal dispersion system is expected to contribute to increasing the stability of the compound in the body and efficiently transporting the compound to a specific organ, tissue or cell. The choice of colloidal dispersion system is not particularly limited as long as it is generally used, and for example, a lipid-based dispersion system may be used which includes polymer complexes, nanocapsules, microspheres, beads, or oil-in-water emulsifiers, micelles, micelle mixtures, or liposomes. A preferable colloidal dispersion system consists of multiple liposomes or vesicles of an artificial membrane, which is effective in efficiently transferring a compound to a specific organ, tissue or cell (Mannino et al., Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem. et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech., 1995, 6, 698).
A unilamellar liposome ranging from 0.2 to 0.4 μm in size is capable of encapsulating a large proportion of macromolecules contained in an aqueous buffer. A compound can be encapsulated in such an aqueous inner membrane and transported to the brain cells in biological active form (Fraley et al., Trends Biochem. Sci., 1981, 6, 77). The liposome is generally composed of a mixture of a lipid, particularly a phospholipid, more particularly a phospholipid having a high phase transition temperature, with one or more types of steroid, in particular, cholesterol. Examples of a lipid useful for producing a liposome include phosphatidyl compounds such as phosphatidyl glycerol, phosphatidyl choline, phosphatidylserine, sphingolipid, phosphatidylethanolamine, cerebroside, and ganglioside. Of these, particularly useful is diacylphosphatidyl glycerol in which a lipid moiety has 14 to 18 carbon atoms, in particular, 16 to 18 carbon atoms and is saturated (that is, no double bond is present within the C14-C18 carbon atom chain). Typical phospholipids include phosphatidyl choline, dipalmitoyl phosphatidyl choline and distearoyl phosphatidyl choline.
The colloidal dispersion system containing liposomes can be used for passive or active targeting. Passive targeting can be attained using a tendency inherent to liposomes, which tend to distribute in the reticuloendothelial system of an organ containing sinusoids. Alternatively, active targeting can be attained by modifying a liposome, for example, by binding a specific ligand thereto, such as viral protein coat (Morishita et al., Proc. Natl. Acad. Sci. (U.S.A.), 1993, 90, 8474), a monoclonal antibody (or its appropriate binding portion), sugar, glycolipid, or protein (or its appropriate oligopeptide fragment); or alternatively, by modifying the composition of the liposome in order to distribute it in organs or cell types other than those where liposomes are naturally localized. The surface of the colloidal dispersion system can be modified in various methods for targeting. In a delivery system using a liposome as a targeting means, to maintain a ligand for use in targeting by keeping tight association with a lipid bilayer, a lipid group is integrated into the lipid bilayer of the liposome. To bind a lipid chain to the targeting ligand, various linking groups can be used. Examples of such a targeting ligand binding to a specific cell surface molecule predominantly found on the cell to which an oligonucleotide according to the present invention is desired to be delivered include (1) hormone, growth factor or an appropriate oligopeptide fragment thereof binding to a specific cellular receptor predominantly expressed by a cell to which delivery is desired; and (2) a polyclonal antibody, monoclonal antibody, or an appropriate fragment thereof (e.g., Fab; F (ab)′2) specifically binding to an antigenic epitope predominantly found on a target cell. Two or more bio activators can be formed into a complex within a single liposome and administered. A medicinal agent for improving intracellular stability and/or targeting ability of the contents can be added to the colloidal dispersion system.
Although a therapeutic gene of the present invention can be used in an amount varying with symptom intensity, age, etc. In the case of peroral administration, the lowermost limit per dose is 1 mg (preferably 30 mg) and the uppermost limit per dose is 2,000 mg (preferably 1,500 mg). In the case of injection, the lowermost limit per dose is 0.1 mg (preferably 5 mg) and the uppermost limit per dose is 1,000 mg (preferably 500 mg). Such a dose can be administered subcutaneously, intramuscularly or intravenously.
Now, the present invention will be more specifically described in detail by way of Examples, which should not be construed as limiting the present invention. Note that individual operations regarding gene manipulation in the following Examples are performed in accordance with the methods described in “Molecular Cloning” (by Sambrook, J., Fritsch, E. F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press 1989), or performed using commercially available reagents or kits in accordance with the protocols thereof.
a) Isolation of RAW-D cells and RAW-N cells by limiting dilution culture. Stimulation of murine monocyte-derived cell strain RAW264.7 with soluble RANKL is known to strongly induce gene expressions for markers of differentiation into osteoclasts, such as tartrate resistant acid phosphatase (hereinafter referred to as “TRAP”) and cathepsin K (Hsu et al., Proc. Natl. Acad. Sci. USA, (1999) 96, 3540-3545). Consequently, stimulation of RAW264.7 cells with RANKL is believed to induce their differentiation into osteoclasts. It was thus attempted to obtain cells subcloned from the parent strain RAW264 cells, designated RAW264.7 cells, which would be more sensitive to RANKL and TNF-α, or more differentiation by these stimuli (Watanabe et al., J. Endocrinol., (2004) 180, 193-201). RAW264 cells can be purchased from The European Collection of Cell Cultures (Catalog No. 85062803). The RAW264 cells were subjected to limiting dilution in the normal manner, using α-MEM medium containing 10% fetal calf serum and plated on a 96-well plate in 100 μl aliquots. They were cultured for 10-14 days and the colonies formed were harvested. Each colony was prepared at 4.5×104 cells/ml in α-MEM medium containing 10% fetal calf serum. The preparation was plated on a 96-well plate at 150 μl/well, and the following were added: human RANKL (from PeproTech Inc.) to a final concentration of 20 ng/ml and human TNF-α (from PeproTech Inc.) to a final concentration of 1 ng/ml. The cells were cultured for 3 days and then stained for TRAP with a Leukocyte Acid Phosphatase kit (from Sigma Co.) according to the protocol provided therewith to check for formation of TRAP positive multinuclear osteoclasts. This series of cloning procedures through limiting dilution culture was repeated twice for each colony. As a result, RAW-D cells capable of efficiently differentiating to osteoclasts following stimulation with RANKL and TNF-α were obtained, as well as RAW-N cells totally incapable of differentiating to osteoclasts following stimulation with RANKL and TNF-α.
b) Study by TRAP staining on the tendencies of RAW-D cells and RAW-N cells to differentiate to osteoclasts. The RAW-D cells and RAW-N cells were examined for their responses to stimulation with osteoclast-inducing substances such as RANKL and TNF-α. The RAW-D, RAW-N and RAW264 cells were prepared at 4.5×104 cells/ml in α-MEM medium containing 10% fetal calf serum, respectively. Each preparation was plated on a 96-well plate at 150 μl/well, and the following were added: human TNF-α (from PeproTech Inc.) to a final concentration of 1 ng/ml and human RANKL (from PeproTech Inc.) to a final concentration of 10, 20, 40 or 80 ng/ml. The cells were cultured for 3 days and then stained for TRAP with a Leukocyte Acid Phosphatase kit (from Sigma Co.) according to the protocol provided therewith to count the number of TRAP positive osteoclasts formed. As a result, the RAW-D cells formed TRAP positive multinuclear osteoclasts depending on the concentration of the RANKL added (
a) Extraction of total RNA. RAW-D and RAW-N were prepared at 7×104 cells/ml in α-MEM medium containing 10% fetal calf serum, respectively. Each preparation was plated on a 24-well plate at 500 μl/well, and the following were added: human RANKL (from PeproTech Inc.) to a final concentration of 20 ng/ml, human TNF-α (from PeproTech Inc.) to a final concentration of 2 ng/ml and murine MIP-1α (from PeproTech Inc.) to a final concentration of 1 ng/ml, and cultured for 3 days. In parallel, each preparation was also cultured in the absence of human RANKL (from PeproTech Inc.), human TNF-α and murine MIP-1α.
Afterwards, the total RNA was extracted from RAW-D or RAW-N, cultured under the conditions described above, using a total RNA extraction reagent (TRIZol reagent from Invitrogen Corporation) according to the protocol provided therewith. The total RNA recovered was stored at −80° C.
b) Electrophoresis and blotting of total RNA. The recovered total RNA was prepared at 0.5 μg/μl in RNA sample buffer (1×MOPS buffer-containing 20 mM MOPS, 8 mM sodium acetate and 1 mM EDTA), 50% formamide, 18 μg/ml bromophenol blue, 5.8% formaldehyde, 5% glycerol), kept at 65° C. for 15 minutes, and cooled rapidly on ice for 5 minutes. A 20 μl aliquot of the sample solution was dispensed into a well on a 1% agarose gel including formaldehyde for electrophoresis (1×MOPS buffer, 1.2% agarose (from Sigma Co.), 6% formaldehyde) and was subjected to electrophoresis. Electrophoresis was carried out by applying electric current through a submarine electrophoretic bed containing 1×MOPS buffer at 100 V for about 3 hours.
After electrophoresis, the RNA in the agarose gel was transferred to a nylon membrane (Hibond N+ from Amersham Pharmacia Biotech) overnight by the capillary transfer technique (Maniatis, T. et al., in “Molecular Cloning A Laboratory Manual”, Cold Spring Harbor Laboratory, NY, (1982))(a solution for transfer was 20×SSC). The membrane was washed with 2×SSC for 5 minutes, air-dried, and exposed to UV light (300 mJ/cm2) on a crosslinking black light (Stratalinker 2400 from Stratagene Corporation) to immobilize the RNA.
c) Preparation of probes. A plasmid DNA, which was prepared by inserting a nucleotide sequence represented by the nucleotide sequence at positions 457 to 1208 of the murine DC-STAMP ΔT7 cDNA (SEQ ID NO: 5 in the Sequence Listing; GenBank Accession No: AB109561) into the TA cloned site of pGEM-T Easy vector (from Promega Corporation), was digested with NcoI (from Takara Shuzo Co., Ltd.) at the NcoI site near the TA cloned site to make a linear DNA. An antisense RNA probe labeled with DIG (digoxigenin) was prepared by using DIG RNA labeling mix (from Roche Diagnostics K.K.) and SP6 RNA polymerase (from Roche Diagnostics K.K.) according to the protocols provided therewith. This liquid probe preparation was mixed with 20 units of RNase-free DNase I (from Roche Diagnostics K.K.) to digest the template DNA. The RNA probe thus prepared can detect mRNAs for both DC-STAMP and DC-STAMP ΔT7, since the probe corresponds to the nucleotide sequences represented by positions 457 to 1078 and 1247 to 1376 in the SEQ ID NO: 3 (murine DC-STAMP cDNA) in the Sequence Listing.
d) Hybridization. The membrane prepared in b) was placed in a 6 ml hybridization solution (a solution of DIG Easy Hyb Granules from Roche Diagnostics K.K. in redistilled water prepared according to the protocol provided therewith), incubated at 65° C. for 15 minutes (pre-hybridization), and then incubated at 65° C. for 16 hours in a 6 ml hybridization solution containing the DIG-labeled RNA probe. Thereafter, the membrane was washed twice in a solution of 2×SSC containing 0.1% SDS at ambient temperature for 5 minutes, and further washed twice in a solution of 0.5×SSC containing 0.1% SDS at 65° C. for 30 minutes. Next, the membrane was treated with a blocking solution (a solution of a blocking reagent from Roche Diagnostics K.K. in a maleate buffer prepared according to the protocol provided therewith) for 30 minutes and with a blocking solution containing alkaline phosphatase-labeled anti-digoxigenin Fab fragments (0.075 units/ml) (from Roche Diagnostics K.K.) for 30 minutes. Further, the membrane was washed three times with a wash buffer (5 mM maleate buffer, pH 7.5, 150 mM NaCl, 0.3% Tween 20) for 15 minutes, CDP-Star (from Roche Diagnostics K.K.) was added as the luminescent substrate, and analyzed with a Luminoimage Analyzer (LAS-1000 plus from Fuji Photo Film Co., Ltd.).
As a result, RAW-D was shown to have little expression of the murine DC-STAMP in the absence of RANKL and TNF-α, but a significantly increased expression of the murine DC-STAMP in the presence of RANKL and TNF-α (
On the other hand, RAW-N had little or no expression of the DC-STAMP either in the absence or in the presence of RANKL and TNF-α. It should be added that the expression of the murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also determined as a control.
RAW-D was prepared at 7×104 cells/ml in α-MEM medium containing 10% fetal calf serum. The preparation was plated on a 24-well plate at 500 μl/well, and the following were added: human RANKL (from PeproTech Inc.) to a final concentration of 20 ng/ml, human TNF-α (from PeproTech Inc.) to a final concentration of 2 ng/ml and murine MIP-1α (from PeproTech Inc.) to a final concentration of 1 ng/ml, and cultured for 0, 4, 8, 16, 32, 48 or 72 hours.
Thereafter, at each time point of culture total RNA was extracted from RAW-D using a total RNA extraction reagent (TRIZol reagent from Invitrogen Corporation) according to the protocol provided therewith. The total RNA recovered was stored at −80° C. until it was used. The total RNA (1 μg) and 1 μl of oligo(dT) 18 primer (0.5 μg/μl) were added to H2O to make a 11 μl solution, which was then heated at 70° C. for 10 minutes and then stored at 4° C. To the solution were added: 4 μl of 5×1st Strand Buffer (from Invitrogen Corporation), 1 μl of 10 mM dNTPs, 2 μl of 0.1 M dithiothreitol, 1 μl of Superscript II reverse transcriptase (200 U/μl from Invitrogen Corporation), and 1 μl of H2O to make a total 20 μl solution, which underwent a reaction at 42° C. for 1 hour, and was then heated at 70° C. for 10 minutes and stored at 4° C.
The resultant single-stranded cDNA was amplified with each pair of primers as described below.
PCR conditions: The primers for amplifying murine DC-STAMP and murine DC-STAMP ΔT7 include: 5′-aaaacccttg ggctgttctt-3′ (mDC-STAMP-F: SEQ ID NO: 7 in the Sequence Listing) and 5′-cttcgcatgc aggtattcaa-3′ (mDC-STAMP-R: SEQ ID NO: 8 in the Sequence Listing); Primers for amplifying murine cathepsin K: 5′-gagggccaac tcaagaagaa-3′ (mcatK-F: SEQ ID NO: 9 in the Sequence Listing) and 5′-gccgtggcgt tatacataca-3′ (mcatK-R: SEQ ID NO: 10 in the Sequence Listing). The primers for amplifying murine TRAP include: 5′-cagctgtcct ggctcaaaa-3′ (mTRAP-F: SEQ ID NO: 11 in the Sequence Listing) and 5′-acatagccca caccgttctc-3′ (mTRAP-R: SEQ ID NO: 12 in the Sequence Listing). The primers for amplifying murine GAPDH include: 5′-aaacccatca ccatcttcca-3′ (mGAPDH-F: SEQ ID NO: 13 in the Sequence Listing) and 5′-gtggttcaca cccatcacaa-3′ (mGAPDH-R: SEQ ID NO: 14 in the Sequence Listing).
PCR was conducted under the conditions described below using a thermal cycler (GeneAmp PCR System 9700 from Applied Biosystems Division, Perkin Elmer Japan Co., Ltd.). Platinum Taq DNA Polymerase (from Invitrogen Corporation) was used for the reaction. To distilled water were added 8 pmol of each primer, 20 ng of the single-stranded cDNA, 0.5 μl of 10× reaction buffer, 0.2 μl of 50 mM MgCl2, 0.4 μl of each 2.5 mM dNTP, and 0.05 μl of Taq DNA polymerase (5 units/μl) to make 5 μl of a reaction solution. The reaction solution was heated at 94° C. for 2 minutes, treated repeatedly 30 times with a temperature cycle of 94° C. for 0.5 minutes, 65° C. for 1 minute, and 72° C. for 1 minute, then heated at 72° C. for 10 minutes and kept at 4° C. The whole reaction solution was subjected to electrophoresis on a 2.0% agarose gel.
The DC-STAMP gene began to be expressed 8 hours after RANKL, TNF-α and MIP-1α were added, and its expression level continued to increase until 72 hours (
When murine bone marrow-derived primary culture cells are cultured in the presence of activated vitamin D3, a large number of TRAP-positive multinuclear osteoclasts appear (Takahashi et al., Endocrinology, (1988) 122, 1373-1382). A male DDY mouse aged 6 weeks was euthanized by cervical dislocation under ether anesthesia to remove femur and tibia. The femur and tibia were stripped of soft tissues and cut on both ends, respectively. A serum-free α-MEM medium was infused into the bone marrow using a syringe with a 25-gauge needle to collect bone marrow cells. The cell number was counted, and the cells were prepared at 2×106 cells/ml in α-MEM medium containing 15% fetal calf serum. The preparation was plated on a 24-well plate at 500 μl/well, and activated vitamin D3 was added (Biomol International LP) to a final concentration of 1×10−8 M. The cells were cultured for 1, 3, 5 or 6 days.
Thereafter, at each time point of culture total RNA was extracted from the cells using a total RNA extraction reagent (TRIZol reagent from Invitrogen Corporation) according to the protocol provided therewith. The total RNA recovered was stored at −80° C. until it was used.
An RT-PCR reaction was conducted using a RNA LA PCR kit (AMV) Ver1.1 (Takara Biochemicals Inc.). First, the following were mixed to make a 10 μl reaction solution: 2 μl of 25 mM MgCl2, 1 μl of 10× RNA PCR Buffer, 1 μl of dNTP Mix (10 mM each), 0.25 μl of RNase Inhibitor (40 U/μl), 0.5 μl of reverse transcriptase (5 U/μl), 0.5 μl of Oligo dT-Adapter primer (2.5 pmol/μl), 1 μg of the total RNA and RNase-free dH2O. Then, the reaction solution was heated at 50° C. for 25 minutes, then heated at 99° C. for 5 minutes and then stored at 4° C. The resulting single-stranded cDNA was amplified with each pair of the primers described in Example 2.
RT-PCR was conducted under the conditions described below using a thermal cycler (GeneAmp PCR System 9700). To 5 μl of the reaction solution containing cDNA the following were added: 1.5 μl of 25 mM MgCl2, 2 μl of 10× LA PCR Buffer II (Mg2+ free), 0.125 μl of Takara LA Taq (5 U/μl), a primer set (1 μM each of final concentration), and redistilled water to make a 25 μl reaction solution. The reaction solution was heated at 94° C. for 2 minutes, treated repeatedly 25 times with a temperature cycle of 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds, and then stored at 4° C. A 9 μl aliquot of the reaction solution was subjected to electrophoresis on a 2.0% agarose gel.
As a result, the DC-STAMP gene was expressed slightly 1 day after activated vitamin D3 was added, but expressed pronouncedly after 3 days when mononuclear osteoclastic precursor cells were formed, and still expressed pronouncedly after 5 and 6 days when multinucleation occurred actively (
DC-STAMP ΔT7 was expressed at a lower level than DC-STAMP, but the time course of its expression was similar to that of DC-STAMP. In
From the murine DC-STAMP amino acid sequence (SEQ ID NO: 4 in the Sequence Listing; GenBank Accession No: AB109560), preparation of a partial peptide of the murine DC-STAMP protein was attempted based on a peptide comprising an amino acid sequence which is located between the 6th and 7th transmembrane domains and represented by amino acids of positions 330 to 343. A partial peptide having the above-described sequence plus a cysteine residue bound on its N-terminus: Cys Ser Leu Pro Gly Leu Glu Val His Leu Lys Leu Arg Gly Glu (SEQ ID NO: 15 in the Sequence Listing) was synthesized. This peptide was conjugated to KLH (keyhole limpet hemocyanin), which is an antigen-stimulating carrier protein, by the MBS (maleimidebenzoyloxysuccinimide) process. A rabbit was immunized with the conjugate to obtain rabbit antiserum as usual. The antiserum was purified by passing it through a peptide affinity column onto which the partial peptide used for immunization was immobilized to provide rabbit anti-mouse DC-STAMP polyclonal antibody. Since DC-STAMP ΔT7 (GenBank Accession No: AB109561) also contains this peptide sequence, the antibody was considered to bind to both DC-STAMP and DC-STAMP ΔT7. This peptide sequence was further compared with a sequence represented by amino acids of positions 330 to 343 in the human DC-STAMP amino acid sequence (SEQ ID NO: 2 in the Sequence Listing; GenBank Accession No: NM—030788). As a result, both sequences were found to be identical except that Leu (mouse) at position 334 was replaced by Phe (human) and Arg (mouse) at position 341 was replaced by His (human), and thus the antibody was very likely to bind also to the human DC-STAMP.
a) Sampling of neonatal mouse tibia-derived osteoclasts. Tibia was removed from a DDY mouse aged 1 day and stripped of the soft tissue. The tibia was minced with postmortem scissors in α-MEM medium containing 15% fetal calf serum and then pipetted a little harder to disperse and suspend the cells. The cell suspension was plated on a chamber slide (from Nalge Nunc International) and cultured for 1 hour to provide multinuclear cells adhering to the slide as osteoclasts.
b) Development of DC-STAMP protein with immunostaining. The osteoclasts obtained in a) were fixed with a 4% paraformaldehyde solution at ambient temperature for 20 minutes, washed four times with phosphate-buffered saline (pH 7.4), and blocked with phosphate-buffered saline (pH 7.4) containing 3% goat serum at ambient temperature for 30 minutes. After the blocking solution was removed, the following solution was added to the osteoclasts phosphate-buffered saline (with 1% horse serum) containing the rabbit anti-mouse DC-STAMP polyclonal antibody (10 μg/ml), which was prepared in Example 4, and allowed to react at ambient temperature for 30 minutes. As a negative control, an IgG antibody (from DAKO Japan Co., Ltd.) from a rabbit which was not immunized was provided and subjected to the same procedure. The osteoclast sample was then washed four times with phosphate-buffered saline containing 1% horse serum, and reacted with a biotinylated goat anti-rabbit IgG antibody (from Vector Laboratories Inc.) used as a secondary antibody at ambient temperature for 30 minutes. It was washed four times with phosphate-buffered saline, and underwent a staining reaction using an ABC-AP kit (from Vector Laboratories Inc.) according to the protocol provided. As a result, the osteoclasts exposed to the anti-DC-STAMP antibody were observed to be stained intensely, which demonstrated that the DC-STAMP was expressed in the neonatal mouse tibia-derived osteoclasts. In contrast, the osteoclasts were not stained at all for the antibody of the negative control.
a) Preparation of test sample from neonatal mouse mandibular tissue. A DDY mouse aged 1 day was anesthetized with ether and injected with phosphate-buffered saline (pH 7.4) containing 4% paraformaldehyde into the left ventricle to fix it under perfusion. The mandibule was removed, soaked in phosphate-buffered saline (pH 7.4) containing 4% paraformaldehyde as described above, fixed therein at 4° C. for 12 hours, washed three times with phosphate-buffered saline, and washed further in phosphate-buffered saline at 4° C. overnight. Thereafter, it was decalcified with 10% EDTA (ethylenediaminetetraacetic acid) at 4° C. for a week. It was washed in phosphate-buffered saline containing 30% sucrose at 4° C. overnight, embedded in an OCT compound (from Sakura Finetek Japan Co., Ltd.) and frozen in isopentane containing dry ice. The resultant embedded block was sliced at a 10 μm thickness with a cryomicrotome (Leica Microsystems GmbH) to prepare a mandibular tissue section.
b) Development of DC-STAMP protein with immunostaining. The mandibular tissue section prepared in a) was dried in air to remove moisture, and reacted with methanol containing 0.3% hydrogen peroxide at ambient temperature for 30 minutes to eliminate endogenous peroxidase activity. The section was washed three times with phosphate-buffered saline (at ambient temperature for 5 minutes each), and blocked with phosphate-buffered saline containing 10% donkey serum at ambient temperature for 30 minutes. After the blocking solution was removed, the section was immersed in phosphate-buffered saline (with 2% donkey serum) containing the rabbit anti-mouse DC-STAMP polyclonal antibody (10 μg/ml), which was prepared in Example 4, and reacted in a wet chamber at 4° C. overnight. As a negative control, an IgG antibody (from DAKO Japan Co., Ltd.) from a rabbit which was not immunized was provided and subjected to the same procedure. The section was then washed three times with phosphate-buffered saline (at ambient temperature for 5 minutes each), and reacted with a 200-fold dilution with phosphate-buffered saline of a biotinylated donkey anti-rabbit IgG antibody (from Jackson ImmunoResearch Laboratories Inc.) used as a secondary antibody at ambient temperature for 1 hour. It was washed three times with phosphate-buffered saline (at ambient temperature for 5 minutes each), and reacted with a 300-fold dilution with distilled water of a peroxidase-labeled streptavidin conjugate (from DAKO Japan Co., Ltd.) at ambient temperature for 30 minutes. It was washed three times with phosphate-buffered saline (at ambient temperature for 5 minutes each), and underwent a staining reaction by using a DAB substrate kit (from Vector Laboratories Inc.) according to the protocol provided therewith. As a result, the mandibular tissue section that was reacted with the anti-DC-STAMP antibody was observed to be intensely stained only on the osteoclasts, which demonstrated that the DC-STAMP was expressed in the neonatal mouse mandibular-derived osteoclasts. In contrast, none of the cells were stained at all when the antibody of the negative control was used.
a) Preparation of siRNAs against the murine DC-STAMP gene. Murine DC-STAMP siRNAs with two uridine units (UU) added on the respective 3′-terminus of sense and antisense chains were prepared by transcription using a Silencer siRNA Construction kit (from Ambion Inc.) according to the protocol provided therewith. Sets of template oligoDNAs needed for preparation of siRNA are described below.
Firstly, a siRNA for the 5′ side (corresponding to the 7th transmembrane region in the amino acid sequence predicted from the human DC-STAMP cDNA sequence) of the third exon and a variant siRNA thereof were prepared using the following respective combinations of template oligoDNAs.
The siRNA #135 templates include: 5′-aatactagga ttgttgtctt ccctgtctc-3′ (mDC-STAMP-#135-AS; SEQ ID NO: 16 in the Sequence Listing) and 5′-aagaagacaa caatcctagt acctgtctc-3′ (mDC-STAMP-#135-S; SEQ ID NO: 17 in the Sequence Listing).
The variant siRNA #135 templates include: 5′-aatactagga gcgttgtctt ccctgtctc-3′ (mDC-STAMP-#135-Mut-AS; SEQ ID NO: 18 in the Sequence Listing, where t is mutated to g at nucleotide position 11, and t is mutated to c at nucleotide position 12) and 5′-aagaagacaa cgctcctagt acctgtctc-3′ (mDC-STAMP-#135-Mut-S; SEQ ID NO: 19 in the Sequence Listing, where a is mutated to g at nucleotide position 12, and a is mutated to c at nucleotide position 13)
Secondly, a siRNA for the cDNA sequence portion characteristic of the murine DC-STAMP, which is located on the 3′ side of the above siRNA (#135) portion in the third exon, and a variant siRNA thereof were prepared using the following respective combinations of template oligoDNAs.
The siRNA *6 templates include: 5′-aattctcgtg tcagtctcct tcctgtctc-3′ (mDC-STAMP-*6-AS; SEQ ID NO: 20 in the Sequence Listing) and 5′-aaaaggagac tgacacgaga acctgtctc-3′ (mDC-STAMP-*6-S; SEQ ID NO: 21 in the Sequence Listing)
The variant siRNA *6 templates include: 5′-aattctcgta ccagtctcct tcctgtctc-3′ (mDC-STAMP-*6-Mut-AS; SEQ ID NO: 22 in the Sequence Listing, where g is mutated to a at nucleotide position 9, and t is mutated to c at nucleotide position 10) and 5′-aaaaggagac tggtacgaga acctgtctc-3′ (mDC-STAMP-*6-Mut-S; SEQ ID NO: 23 in the Sequence Listing, where a is mutated to g at nucleotide position 13, and c is mutated to t at nucleotide position 14)
b) Suppression of differentiation of RAW-D cells into osteoclasts using siRNA. RAW-D was prepared at 4.5×104 cells/ml in α-MEM medium containing 10% fetal calf serum. The preparation was plated on a 96-well plate at 80 μl/well. On the next day, the medium was replaced with 80 μl of OPTI-MEM I medium (from Invitrogen Corporation), to which was added the DC-STAMP siRNA or variant siRNA prepared in a) to a final concentration of 0.1, 1 or 5 nM, and the cells were transfected using a transfection reagent, siPORT Lipid (from Ambion Inc.) according to the protocol provided therewith (20 μl added). A control (mock) free of siRNA but containing the transfection reagent was also provided. The cells were transfected in a CO2 incubator for 4 hours, and then the following were added: 100 μl of α-MEM medium containing the human RANKL (from PeproTech Inc.) at 40 ng/ml, the human TNF-α (from PeproTech Inc.) at 2 ng/ml and 20% fetal calf serum. The cells were cultured for 3 days and then stained for TRAP with a Leukocyte Acid Phosphatase kit (from Sigma Co.) according to the protocol provided to count the number of TRAP positive multinuclear osteoclasts formed from the cells. The DC-STAMP siRNA #135 at a concentrations of 0.1, 1 or 5 nM was observed to suppress significantly the formation of osteoclasts, this was not the case when the variant siRNA #135 was added. The variant siRNA at a concentration of 5 nM was observed to suppress slightly the formation of osteoclasts, but no suppression of osteoclastic formation was observed at a concentration of 0.1 or 1 nM. From the results, the DC-STAMP siRNA suppressed formation of TRAP positive multinuclear osteoclasts which may be induced in RAW-D by RANKL and TNF-α depending on its concentration, in contrast to the mock control (siRNA level=0 nM) and a negative control, i.e., the variant siRNA (
a) Extraction of total RNA from RAW-D. RAW-D was prepared at 7×104 cells/ml in α-MEM medium containing 10% fetal calf serum. The preparation was plated on a 24-well plate at 500 μl/well, and the following were added: human RANKL (from PeproTech Inc.) to a final concentration of 20 ng/ml, human TNF-α (from PeproTech Inc.) to a final concentration of 2 ng/ml and murine MIP-1α (from PeproTech Inc.) to a final concentration of 1 ng/ml, and then cultured for 3 days.
Then, the total RNA was extracted from RAW-D using a total RNA extraction reagent (TRIZol reagent from Invitrogen Corporation) according to the protocol provided therewith. The total RNA recovered was stored at −80° C.
b) Synthesis of the first strand cDNA. The total RNA (1 μg) and 1 μl of oligo(dT) 18 primer (0.5 μg/μl) were added to H2O to make a 11 μl solution, which was then heated at 70° C. for 10 minutes and stored at 4° C. To the solution were added: 4 μl of 5×1st Strand Buffer (from Invitrogen Corporation), 1 μl of 10 mM dNTPs, 2 μl of 0.1 M dithiothreitol, 1 μl of Superscript II reverse transcriptase (200 U/μl from Invitrogen Corporation), and 1 μl of H2O to make a total 20 μl solution, which underwent the reaction at 42° C. for 1 hour, was then heated at 70° C. for 10 minutes and stored at 4° C.
c) PCR reaction. Oligonucleotides, as primers for amplifying the ORF cDNAs of murine DC-STAMP and murine DC-STAMP ΔT7 by PCR, and having the sequences: 5′-tttgtcgaca tgaggctctg gaccttgggc accagtattt t-3′ (mDC-STAMP-cDNA-F: SEQ ID NO: 24 in the Sequence Listing) and 5′-tttgcggccg ctcatagatc atcttcattt gcagggattg t-3′ (mDC-STAMP-cDNA-R: SEQ ID NO: 25 in the Sequence Listing) were synthesized as usual. This combination of the primers was used for PCR, which was conducted under the conditions described below using a thermal cycler (GeneAmp PCR System 9700). To redistilled water were added primers (final concentration of 1.0 μM each), 5 μl of 10× Pyrobest PCR buffer (from Takara Shuzo Co., Ltd.), 4 μl of 2.5 mM dNTPs, and 1 μl of cDNA (prepared in b)) to make a 50 μl solution. Further, 0.5 μl of a Pyrobest DNA polymerase (5 U/μl) (from Takara Shuzo Co., Ltd.) was added to make the reaction solution. The reaction solution was heated at 94° C. for 2 minutes, treated repeatedly 30 times with a temperature cycle of 94° C. for 0.5 minutes, 60° C. for 0.5 minutes, and 72° C. for 5 minutes, then heated at 72° C. for 10 minutes and stored at 4° C.
d) Cloning into the pCl-neo vector. The whole PCR reaction solution obtained in c) was purified using a QIAquick PCR Purification Kit (from Qiagen Inc.) according to the protocol provided therewith. The resultant fragment was digested with restriction enzymes SalI and NotI, ligated to pCl-neo (from Promega Corporation) preliminarily digested also with SalI and NotI using a DNA Ligation Kit Ver. 1 (from Takara Shuzo Co., Ltd.), and transformed into E. coli XL1-Blue MRF' (from Stratagene Corporation). Transformed E. coli having the plasmid pCl-neo-murine DC-STAMP were isolated from the E. coli colonies thus obtained.
Analysis of the entire nucleotide sequence of the ORF cDNA inserted in the resulting plasmid using a DNA sequencer (ABI Prism 310 DNA sequencer from Applied Biosystems Division, PerkinElmer Japan Co., Ltd.) revealed that the sequence was a sequence shown in SEQ ID NO: 26 in the Sequence Listing. This nucleotide sequence was identical to the ORF coding region in the sequence registered as “murine DC-STAMP” (Accession No. AB109560) with the NCBI GeneBank data base, and the amino acid sequence (SEQ ID NO: 27 in the Sequence Listing) encoded by the nucleotide sequence was 100% identical to the amino acid sequence of the murine DC-STAMP.
In the pCl-neo-murine DC-STAMP plasmid obtained in Example 8, the open reading frame sequence for the murine DC-STAMP is integrated under the control of the CMV promoter derived from pCl-neo. Therefore, transfer of the plasmid into a host may induce the expression of the murine DC-STAMP protein. The gene transfer (transient transfection) of this expression plasmid into RAW-D was carried out by the DEAE-dextran process. This pCl-neo-murine DC-STAMP vector (3 μg) or a pCl-neo vector (3 μg) without any the DNA insert was combined with a mixture of 50 μl of a DEAE-dextran solution (from Promega Corporation) at 10 mg/ml and 950 μl of OPTI-MEMI (from Invitrogen Corporation) to make a transfection solution.
RAW-D (3.0×106 cells) were washed (centrifuged at 200×g for 5 minutes) twice with serum-free α-MEM (10 ml) and suspended in the transfection solution (1 ml) described above. The suspension was kept at a constant temperature in a CO2 incubator (at 37° C.) for 30 minutes, washed (centrifuged under 200×g for 5 minutes) once with serum-free α-MEM (10 ml), and further washed once with α-MEM containing 5% fetal calf serum (10 ml). The suspended wash was centrifuged at 200×g for 10 minutes to precipitate the cells, which were then re-suspended in α-MEM containing 10% fetal calf serum (2 ml). The cell density was measured with a hemocytometer and adjusted to 4.5×104 cells/ml. The resultant suspension was plated on a 96-well plate at 0.15 ml/well, and the following were added: human RANKL (from PeproTech Inc.) to a final concentration of 20 ng/ml and human TNF-α (from PeproTech Inc.) to a final concentration of 1 ng/ml, or, alternatively, neither of these were added. The cells were cultured for 3 days and then stained for TRAP with a Leukocyte Acid Phosphatase kit (from Sigma Co.) according to the protocol provided herewith to count the number of TRAP positive multinuclear osteoclasts derived therefrom. As a result, in the absence of RANKL and TNF-α, no formation of TRAP positive multinuclear osteoclasts was induced, even when the murine DC-STAMP protein was over-expressed, but, in the presence of RANKL and TNF-α, over-expression of the murine DC-STAMP protein enhanced significantly formation of the TRAP positive multinuclear osteoclasts, compared with the control, i.e., when RAW-D was transfected with the pCl-neo vector without the DNA insert (
The rabbit anti-murine DC-STAMP polyclonal antibody prepared in Example 4 was used to examine its effect on differentiation of RAW-D cells into osteoclasts.
RAW-D was prepared at 4.5×104 cells/ml in α-MEM medium containing 10% fetal calf serum. The suspension was plated on a 96-well plate at 150 μl/well, and the following were added: human RANKL (from PeproTech Inc.) to a final concentration of 20 ng/ml and human TNF-α (from PeproTech Inc.) to a final concentration of 1 ng/ml. To the cell culture supernatant, the rabbit anti-murine DC-STAMP polyclonal antibody prepared in Example 4 was added to a final concentration of 0, 5, 10 or 20 μg/ml. The cells were cultured for 3 days and then stained for TRAP with a Leukocyte Acid Phosphatase kit (from Sigma Co.) according to the protocol provided therewith to count the number of TRAP positive multinuclear osteoclasts derived therefrom. As a result, formation of the TRAP positive multinuclear osteoclasts was suppressed by addition of anti-murine DC-STAMP polyclonal antibody in a dose-dependent manner (
The results indicate that the antibody potentially capable of specifically binding to DC-STAMP and DC-STAMP ΔT7 suppressed formation of the TRAP positive multinuclear osteoclasts from RAW-D, and thus DC-STAMP and DC-STAMP ΔT7 were suggested to be very important in differentiation into osteoclasts.
A male DDY mouse aged 6 weeks was euthanized by cervical dislocation under ether anesthesia to remove the femur and tibia. The femur and tibia were stripped of soft tissues and cut on both ends, respectively. A serum-free α-MEM medium was infused into the bone marrow using a syringe with a 25-gauge needle to collect bone marrow cells. The cell number was counted, and the cells were prepared at 2×106 cells/ml in α-MEM medium containing 15% fetal calf serum. The cell suspension was plated on a 24-well plate at 500 μl/well, and activated vitamin D3 was added (from Biomol Corporation) to a final concentration of 1×10−8 M. To the cell culture supernatant, the rabbit anti-murine DC-STAMP polyclonal antibody prepared in Example 4 was added to a final concentration of 0, 5, 10 or 20 μg/ml. The cells were cultured for 6 days and then stained for TRAP with a Leukocyte Acid Phosphatase kit (from Sigma Co.) according to the protocol provided therewith to count the number of TRAP positive multinuclear osteoclasts derived therefrom. As a result, formation of the TRAP positive multinuclear osteoclasts was suppressed by addition of the anti-murine DC-STAMP polyclonal antibody in a dose-dependent manner (
When cells derived from murine femur and tibia are cultured on an ivory section in the presence of activated vitamin D3, osteoclasts are observed to erode the ivory surface with many pits of bone resorption widely distributed (Takada et al., Bone and Mineral, 17, 347-359 (1992)).
An ICR mouse aged 14 days (either sex may be used) was euthanized by cervical dislocation under ether anesthesia to remove femur and tibia. The femur and tibia were stripped of soft tissues and minced finely using scissors in a dish 60 mm in diameter containing 1 ml of DMEM medium with 10% fetal calf serum. The minced sample was transferred into a 15-ml centrifuge tube, to which was added 10 ml of DMEM medium containing 10% fetal calf serum, agitated for 30 seconds in a vortex mixer (from M & S Instruments Inc.) and left to stand for 2 minutes. The supernatant was recovered and the cell number was counted. A cell suspension was prepared at 1×107 cells/ml in DMEM medium containing 10% fetal calf serum. The suspension was plated on a 96-well plate at 100 μl/well on which ivory sections of 150-200 μm in thickness and 6 mm in diameter (prepared in Kureha Chemical Industry Co., Ltd.) were laid, and cultured for 4 hours in a CO2 incubator. Thereafter, the medium was replaced by 200 μl of DMEM medium containing 10% fetal calf serum, to which activated vitamin D3 was added to a final concentration of 1×10−8 M (a group without the vitamin was also provided). To the cell culture supernatant, the rabbit anti-murine DC-STAMP polyclonal antibody prepared in Example 4 was added to a final concentration of 0, 2, 6 or 20 μg/ml. The cells were cultured for 4 days. After the culture was finished, the culture supernatant was removed from the plate containing the ivories. The plate was washed once with distilled water, and another portion of distilled water was added. The cells attached on each ivory section were removed with a polishing brush (from Tagaya Seisakusho Co., Ltd.) connected to a hand motor (from Tokyo Nakai Co., Ltd.). The ivory section was washed twice with distilled water, stained with acid hematoxylin solution (from Sigma Co.) for 13 minutes on the pits formed on its surface, and washed twice with distilled water. The ivory section was reversed and the area of the pits was measured microscopically. To measure the total area of the pits, a micrometer (10×10 squares) attached to the eye lens of the microscope was used to count the total number of squares (meshes) where pits were located and convert the number to the pit area. As a result, addition of activated vitamin D3 induced formation of many pits on the ivory section, but when the anti-murine DC-STAMP polyclonal antibody was added at the same time, pit formation was suppressed by the antibody in a dose-dependent manner (
Giant cell tumor (GCT) is a bone tumor, characterized by osteolytic bone destruction as a clinical symptom, in which a large number of multinuclear giant cells occur that are osteoclastic histologically, (Bullough et al., Atlas of Orthopedic Pathology 2nd edition, 17.6-17.8, Lippincott Williams & Wilkins Publishers (1992)). The EST probe (Affymetrix Genechip HG-U133 probe 221266_s_at, made by Affymetrix), which has a nucleotide sequence partially overlapping with the human DC-STAMP gene, was analyzed for expression profile in GCT tissues using the data base (Genesis 2003 Release 2.0) made by GeneLogic. EST probes for RANK (Affymetrix Genechip HG-U133 probe 207037_at, made by Affymetrix) and RANKL (Affymetrix Genechip HG-U133 probe 210643_at, made by Affymetrix) which play a key role in differentiation into osteoclasts, and for cathepsin K (Affymetrix Genechip HG-U133 probe 202450_s_at, made by Affymetrix) and TRAP (Affymetrix Genechip HG-U133 probe 204638_at, made by Affymetrix) which are markers for differentiation into osteoclasts, were also analyzed for expression profile in GCT tissues.
A comparison of the expression levels was made among 9 cases of healthy bone tissues, 14 cases of GCT tissues and 10 cases of bone tumor tissues other than GCT: this revealed that the GCT tissues specifically had a higher level of transcription of RANK and RANKL than the healthy bone tissues (
When human peripheral blood mononuclear cells (HPBMC) are stimulated with RANKL in the presence of M-CSF and dexamethasone, TRAP positive multinuclear osteoclasts are formed (Matsuzaki et al., Biochem. Biophys. Res. Commun., (1998) 246, 199-204). HPBMCs purchased from Takara Bio Inc. were prepared at 5×106 cells/ml in α-MEM medium containing 10% fetal calf serum. The cells were plated on a 96-well plate at 100 μl/well, and to which was added a medium containing human M-CSF (from R & D Systems Inc.) to a final concentration of 200 ng/ml, dexamethasone (from Wako Pure Chemical Industries, Ltd.) to a final concentration of 1×10−7 M and human RANKL (from PeproTech Inc.) to a final concentration of 100 ng/ml to 200 μl/well. A non-RANKL group of the cell suspensions was also provided. To the cell culture supernatant, the rabbit anti-murine DC-STAMP polyclonal antibody prepared in Example 4 was added to a final concentration of 0, 2 or 6 μg/ml. After the culture was initiated, on days 4, 7 and 11, the medium was replaced and a test sample was added, and on day 13, the cells were stained for TRAP with a Leukocyte Acid Phosphatase kit (from Sigma Co.) according to the protocol provided therewith to count the number of TRAP positive multinuclear osteoclasts formed. As a result, a large number of osteoclasts were formed by stimulation with RANKL, but addition of the anti-murine DC-STAMP polyclonal antibody suppressed formation of the TRAP positive multinuclear osteoclasts in a dose-dependent manner (
By virtue of the present invention, it was found that the expression level of human DC-STAMP in osteoclast cells is significantly high. According to the present invention, there are provided a method of detecting a metabolic bone disorder using the human DC-STAMP gene and a bone disorder detection kit, and further provided an antibody having a suppressive activity towards osteoclast formation and a pharmaceutical composition for treating a bone disorder containing the antibody.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-035216 | Feb 2004 | JP | national |
This application is a Continuation-in-Part of International Application No. PCT/JP2005/002373, filed Feb. 9, 2005, which claims the benefit under 35 U.S.C. 365(c) of Japanese Application No. 2004-035216, filed Feb. 12, 2004. The entire disclosure of the above-listed prior applications is considered to be part of the disclosure of the instant application and is hereby incorporated by reference therein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP05/02373 | Feb 2005 | US |
| Child | 11502767 | Aug 2006 | US |
