Patent Application
20070025996
The present invention relates to compounds such as an antibodies useful in cancer treatment, a pharmaceutical composition for treating cancer characterized in that it contains the antibody as an active ingredient, a method of detecting cancer and a cancer detection kit.
Tumor cells are known to express antigenic proteins which are intrinsic to the particular type of tumor cells (hereinafter sometimes referred to as a “tumor-associated antigens”). Attempts have been made to develop new therapies for treating tumors by targeting tumor-associated antigens. Monoclonal antibodies that elicit an antigen-antibody response specific to such tumor-associated antigens are known to induce various types of in vivo immune responses (antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), etc.) to attack cancer cells, thereby inducing cell death. Monoclonal antibodies useful for tumor treatment have been developed.
However, the range of monoclonal antibodies useful for tumor treatment is limited. The monoclonal antibodies presently available are capable of treating only a few types of tumors including metastatic breast cancer, acute leukemic myelosis, intractable chronic lymphoma, non-Hodgkin's lymphoma, and multiple myeloma. Development of monoclonal antibodies applicable to treatment of other tumors is desirable.
To obtain a monoclonal antibody useful for tumor treatment, it is necessary to identify a protein specifically expressed in a tumor cell and obtain a monoclonal antibody against this protein antigen.
Human oculospanin protein was obtained as an Expressed Sequence Tag (EST) clone derived from a gene expressed on the retinal pigment epithelium and the ocular choroidal membrane (Molecular Vision (2002) 8, 25-220). The human oculospanin gene has an open reading frame of 1068 bp. Human oculospanin consists of 355 amino acids and is estimated to have a molecular weight of 36.4 kDa based on the DNA sequence. However, the relationship between human oculospanin and tumors is still unknown.
Accordingly, in one aspect, the present invention provides antibodies which specifically bind to a human oculospanin protein selected from at least one member of the group consisting of SEQ ID NO:2 and SEQ ID NO:4, and b) has cytotoxic activity against a cell expressing the oculospanin 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.
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 oculospanin comprising contacting a human oculospanin protein with a library of candidate agents and determining the presence or absence of binding of at least one candidate agent and the oculospanin 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 cytotoxicity induction in a population of cells expressing a human oculospanin protein comprising contacting the cells with a candidate agent to form a mixture and assaying for cytotoxicity. In some aspects, a library of candidate agents are tested, and in certain aspects, the candidate agents are antibodies. The method optionally includes adding effector cells to the mixture.
In an additional aspect, the invention provides methods of inducing cytotoxicity in a cell expressing human oculospanin comprising adding an agent that inhibits oculospanin activity such that cytotoxicity is induced.
In a further aspect, the invention provides methods of detecting cancer comprising measuring the amount of nucleic acid encoding oculospanin from a human test sample, measuring the amount of nucleic acid encoding oculospanin from a human healthy sample, and comparing the difference in the amounts to determine the presence of cancer in the test sample.
In yet another aspect, the invention provides methods of detecting cancer comprising measuring the amount of oculospanin protein from a human test sample, measuring the amount of oculospanin protein from a human healthy sample and comparing the difference in the amounts to determine the presence of cancer in the test sample.
1. General Overview of the Invention
The present invention is directed to the finding that the protein oculospanin is overexpressed in certain cancer cells, particularly melanoma, and that compounds that bind to oculospanin, such as antigen binding proteins including antibodies, induces cytotoxicity in cells expressing oculospanin. Thus the present invention provides compositions, including antibodies, which induce cytotoxicity in oculospanin-expressing cells, and methods of diagnosing cancer as well as methods of inducing cytotoxicity.
The identification of this correlation further allows a number of methods utilizing the oculospanin protein, including methods of screening for candidate agents that bind to and/or modulate the activity of oculospanin, including screening assays for candidate agents such as antibodies and/or other compounds for cytotoxicity of cells that express oculospanin. As is more fully described below, these assays can take on a number of formats, including the use of substantially pure oculospanin proteins (including fragments and derivatives thereof) in homogeneous and heterogeneous assays, biochemical assays, and cellular assays that utilize cells expressing human oculospanin proteins.
In addition, the invention provides for methods of inducing cytotoxicity through the use of agents that bind to oculospanin, including, but not limited to, antigen binding proteins such as antibodies.
Accordingly, in one embodiment, the present invention provides antigen binding proteins that bind to human oculospanin.
2. Oculospanin Proteins
By “human oculospanin protein” herein is meant the protein sequence depicted in SEQ ID NO:2 and/or SEQ ID NO:4, and allelic variations thereof. In some embodiments, for example when the oculospanin is used in screening, fragments or derivatives of oculospanin protein (and nucleic acids, as outlined below) can be used. Thus, variants of human oculospanin 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 fusion oculospanin 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 oculospanin proteins, such as rodent or other non-human mammalian proteins.
2A. Oculospanin Variants
In some embodiments, depending on the use of the oculospanin 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 oculospanin 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 oculospanin 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 oculospanin protein 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.
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 oculospanin 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 oculospanin 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.
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 oculospanin 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 oculospanin proteins as needed.
3. Oculospanin Antigen Binding Proteins
The invention provides antigen binding proteins that bind to oculospanin. 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 oculospanin.
The antigen binding proteins of the invention specifically bind to human oculospanin. “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 oculospanin having the amino acid sequence of SEQ ID NOs:2 and 4.
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 5β 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 embodiment, 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.
In one embodiment, the invention provides antibodies which have one or more CDRs from the murine antibody produced by the mouse hybridoma O3B8-2C9-4F3, deposited at the International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566, JAPAN, deposit number FERM BP-08627. That is, any combination of heavy and light chain CDRs, including CDR1; CDR2 and CDR3 from the light and heavy chains, from O3B8-2C9-4F3 can be used. In addition, amino acid variants of the murine CDRs that retain substantially the same activity as the parent antibody, e.g. binding to oculospanin and induction of cytoxicity, are also included, as is outlined above for variantst.
From studies of the structural features of 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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin antigen binding protein is an antibody fragment, that is a fragment of any of the antibodies outlined herein that retain binding specificity to oculospanin.
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 oculospanin 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 oculospanin 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 oculospanin 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 al., 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) h) Antibody Conjugates
In one embodiment, the oculospanin 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.
3. A ii) Covalent modifications of Antigen Binding Proteins such as Antibodies
Covalent modifications of antigen binding proteins, as well as the oculospanin 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.
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.
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. 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. 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 labelling 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.
4. Nucleic Acids Encoding Oculospanin Antigen Binding Proteins
The invention provides nucleic acids that encode oculospanin and oculospanin 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 oculospanin antigen binding proteins, nucleic acids used as candidate agents (e.g. antisense 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 NO:1 and SEQ ID NO:4) 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 oculospanin 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 oculospanin antigen binding proteins and oculospanin 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, oculospanin nucleic acids hybridize to the sequences depicted in SEQ ID NO:1 and SEQ ID NO:4 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 oculospanin 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. 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. The definition 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. 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 Oculospanin Proteins
There are a variety of techniques useful in obtaining oculospanin 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 and SEQ ID NO:4, may be used to clone expression vectors, as well as find other related oculospanin 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 oculospanin 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.
Once the oculospanin protein is amplified, it can be cloned and, if necessary, its constituent parts recombined to form the entire oculospanin 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 oculospanin nucleic acid can be expressed as outlined below.
4 B. Nucleic Acids Encoding Oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 Oculospanin 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 oculospanin proteins, oculospanin antigen binding proteins such as anti-oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 thymidine 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 oculospanin polypeptide. As a result, increased quantities of a polypeptide such as an oculospanin antigen binding protein are synthesized from the amplified DNA.
A ribosome-binding site is usually necessary for translation initiation of mRNA 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 oculospanin 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 oculospanin 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 al., 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 oculospanin 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 oculospanin antigen binding protein or oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 Oculospanin Antigen Binding Proteins for Diagnostic, Therapeutic and Screening Purposes
In the specification of the present invention, a compound having a cancer therapeutic effect is a compound having an activity in suppressing cancer growth and/or an activity of reducing cancer. In the specification of the present invention, the terms “cancer” and “tumor” have the same meaning. The term “canceration of a cell” used herein refers to the abnormal proliferation of cells, which is caused by their lack of sensitivity to contact inhibition and their scaffold independent-proliferation. A cell exhibiting such abnormal proliferation is referred to as a “cancer cell”. In the specification, a protein having the same function as that of human oculospanin, such as canceration activity, is also referred to as a “human oculospanin”. Note that the term “oncogene” as used in the present invention includes a precancerous gene and a proto-oncogene other than the oncogene.
The present invention provides agents that confer and/or induce cytotoxicity to cells expressing oculospanin. The term “cytotoxicity” used herein refers to both cell death and toxicity resulting in cell stasis, e.g. a loss of the ability to grow, as well as to apoptosis. Therefore, cytotoxicity includes not only externally inflicted direct damage, but also various structural and functional changes that may occur within a cell, which include DNA cleavage, dimerization of bases, chromosomal cleavage, malfunction of cellular mitotic apparatus, and a reduction in enzymatic activities. The term “cytotoxic activity” used herein refers to any activity that causes cytotoxicity, as mentioned above.
The correlation of the connection between oculospanin (including oculospanin levels in cancerous cells) allows a number of utilities, including diagnostic methods and kits, therapeutic uses, including in cancer, and screening technologies for modulation of oculospanin activity.
5 A. Diagnosis Utilizing Oculospanin and Oculospanin Antigen Binding Proteins
As is described herein, oculospanin has been shown to be over expressed in cancer cells, particularly melanoma cells. Accordingly, the invention provides a number of diagnostic methods and kits, based either on protein or nucleic acid detection, for the detection of cancer.
5 A i). Confirmation of Specific Expression of the Human Oculospanin Gene for Diagnosis
As a result of analyzing expression levels of the human oculospanin gene in various types of human cells, it was found that the gene is expressed at a significantly higher expression level in melanocytes compared to other tissues. Furthermore, the present inventors found that the level of expression of the human oculospanin gene in melanoma is significantly higher than in normal melanocytes. To explain more specifically, they found the following: when the level of expression of human oculospanin in melanocytes, lymphoblasts and glia cells, and epithelial cells is compared, the expression level in melanocytes is found to be significantly higher. Furthermore, when the level of expression of human oculospanin in normal skin cells is compared to that in melanoma, the expression level is significantly higher in the melanoma. From these findings, it can be concluded that human oculospanin may be involved in canceration of cells and/or in proliferation of cancer cells. This suggests that the canceration state and/or proliferation state of cancer cells caused by excessive expression of human oculospanin can be determined by measuring the level of expression of human oculospanin in individual cells and/or tissues. An example of such cancer is skin cancer, in particular, melanoma. However, this finding is applicable to cancers other than skin cancer, provided that human oculospanin is expressed in the cancer at a significantly higher level than in other tissues.
The nucleotide sequence of the open reading frame (ORF) of the human oculospanin gene is represented by Sequence ID No. 1 of the sequence listing and the amino acid sequence thereof is represented by Sequence ID No. 2. Furthermore, cDNA of the human oculospanin gene has been registered with GenBank as Homo sapiens oculospanin (OCSP) mRNA under Accession No. NM—031945. The cDNA nucleotide sequence registered at GenBank is represented by Sequence ID No. 3 of the sequence listing. The ORF is represented by nucleotide Nos. 65 to 1129 of Sequence ID No. 3. Furthermore, the amino acid sequence of human oculospanin registered at GenBank is represented by Sequence ID No. 4 of the sequence listing. There is a single amino acid difference between SEQ ID NO:2 and SEQ ID NO:4. A protein comprising an amino acid sequence having one or several amino acids replaced, deleted from or added to the amino acid sequence of human oculospanin and exhibiting the same biological activity as that of human oculospanin is also included herein as a human oculospanin.
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 oculospanin, since it is highly expressed in cancer cells, especially, melanoma, is thought to be involved in canceration of cells, particularly skin cells, and/or proliferation of cancer cells.
Thus the present invention provides methods of detecting cancer, or a predisposition or propensity to get cancer, 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, skin and lymph node are preferred tissue samples.
In one embodiment, the invention provides methods of detecting cancer using the level of expression of the human oculospanin 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 oculospanin 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 detecting cancer of the test 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 cancer. The determination as to whether or not a person is healthy can be made by measuring the concentration of human oculospanin 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 oculospanin and the degree of cancer formation 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 oculospanin 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 oculospanin expression from a particular tissue, patient or sample is already known, it is not necessary to determine the level of oculospanin expression from a “normal” or “healthy” sample.
Step 3 can be done by measuring the level of expression of the human oculospanin gene in a total RNA fraction according to steps 1) and 2).
The level of expression of the human oculospanin gene is represented by the expression level of a polynucleotide that can hybridize with a polynucleotide which comprises the nucleotide sequence represented by Sequence ID No. 1 of the sequence listing or a polynucleotide which comprises a nucleotide sequence complementary to the nucleotide sequence represented by Sequence 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 oculospanin 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 oculospanin nucleic acid can be fragments of the full length gene. Thus, for example, fragments of oculospanin 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 oculospanin 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 oculospanin 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. oculospanin sequences, for example oculospaninprobes) 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, oculospanin 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 oculospanin 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 tumor and a person having no tumor. 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 melanoma 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. An 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 oculospanin 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), p32-35); differential display methods (Basic Biochemical Experimental Method 4, nucleic acid/gene experiment, II. Applied series, Tokyo Kagakudojin (2001), p125-128); 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 oculospanin 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 oculospanin, it is determined that the possibility of having cancer, particularly skin cancer, and more particularly melanoma, is high, that is, cancer 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 a melanoma cell is significantly high compared to that of a normal melanocyte.
5 C Protein Diagnostic Assays
Alternatively, the level of expression of human oculospanin 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 cancer. The diagnosis of cancer can be made in this manner. Otherwise, the correlation between the level of expression of the human oculospanin gene and the degree of cancer formation in a healthy person is previously investigated, and then, the expression level of human oculospanin 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 oculospanin 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-oculospanin 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, skin or lymph node tissue taken from a subject is used directly, or diluted appropriately in a buffer solution before use. For Western blotting (electrophoresis), a solution extracted from skin or lymph node tissue 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 skin or lymph node tissue 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 oculospanin 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 oculospanin or a polypeptide arbitrarily selected from the amino acid sequences of human oculospanin, 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 oculospanin with a myeloma cell to form a hybridoma cell.
Human oculospanin protein for use as an antigen can be obtained by introducing a human oculospanin gene into a host cell by gene manipulation. To explain more specifically, human oculospanin protein may be obtained by preparing a vector capable of expressing the human oculospanin gene, introducing the vector into the host cell, expressing the gene, and purifying the expressed human oculospanin protein.
The level of expression of human oculospanin can be represented by the level of expression of a protein comprising the amino acid sequence represented by Sequence 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 oculospanin antibody.
Measurement of the level of expression of human oculospanin 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 cancer.
The difference in the level of expression of human oculospanin 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 oculospanin, it can be determined that there is a high probability of a subject having cancer, particularly, skin cancer, and more particularly, melanoma. In this manner, cancer can be detected.
Alternatively, cancer can be detected by measuring the concentration of human oculospanin 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 cancer. Furthermore, by investigating the correlation between the level of expression of human oculospanin and the degree of cancer 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 oculospanin in a specimen taken from the subject.
5 D Specific Methods for Investigation of the Human Oculospanin Gene and Human Oculospanin
The human oculospanin gene and human oculospanin are expressed at a significantly high level in melanocytes in normal human tissues, and they are, expressed at a significantly higher level in melanoma than in normal melanocytes.
In a method of examining the function of human oculospanin, full-length cDNA is first taken from a human cDNA library, derived from cells expressing human oculospanin, 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, p854-864), 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. Alternatively, the state of cells may be examined by preparing a knockout animal by knocking out the target gene in an animal having melanoma.
5 E Human Oculospanin Gene and/or Human Oculospanin Detection Kit
The human oculospanin gene and/or human oculospanin 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 oculospanin. For example, pairs of polymerase chain reaction (PCR) primers for amplifying oculospanin genes can be included in a kit. These generally comprise oculospanin 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 Sequence ID No. 1 of the sequence listing. In alternative embodiments, detection probes that hybridize specifically to oculospanin genes are included; in this context, “specificity” means that the oculospanin 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 Sequence ID No. 1 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 oculospanin binding proteins such as antibodies, in the case of protein detection.
In the case of protein detection, oculospanin antigen binding proteins, such as oculospanin antibodies can also be included, and optionally, secondary antibodies capable of binding to an oculospanin antibody. Suitable antibodies are made as described below.
The primer according to section 1) above can be easily constructed based on the nucleotide sequence of the human oculospanin gene (the nucleotide sequence represented by Sequence ID No. 1 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. As an example of such a primer, more specifically, a primer for amplifying a polynucleotide comprising the nucleotide sequence represented by Sequence ID No. 1 of the sequence listing, use can be made of the combination of an oligonucleotide comprising the nucleotide sequence represented by Sequence ID No. 5 of the sequence listing and an oligonucleotide comprising the nucleotide sequence represented by Sequence ID No. 6 of the sequence listing. The probe according to section 2) above is a polynucleotide capable of hybridizing specifically with human oculospanin 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 oculospanin gene and/or human oculospanin protein, thereby determining the presence or absence of cancer and screening for a substance capable of suppressing cancer growth.
6. Therapeutic Methods
The present invention provides oculospanin binding proteins such as anti-oculospanin antibodies.
6. A. Preparation of Antigen
An antigen for preparing an anti-human oculospanin antibody can be a polypeptide comprising human oculospanin, 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 oculospanin protein can be directly purified from human tumor tissues or cells, synthesized in vitro, or produced in host cells by gene manipulation. More specifically, in producing human oculospanin by gene manipulation, a human oculospanin gene is integrated into an expression vector, and thereafter the human oculospanin 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 oculospanin can be isolated. The nucleotide sequence of human oculospanin cDNA is described in: Graeme Wistow, Steven L. Bernstein, M. Keith Wyatt, Robert N. Fariss, Amita Behal, Jeffrey W. Touchman, Gerard Bouffard, Don Smith, and Katherine Peterson (2002), Expressed sequence tag analysis of human RPE/choroids for the NEIBank Project: Over 6000 non-redundant transcripts, novel genes and splice variants, Molecular Vision 8:205-220, and registered in the GenBank under Accession No. NM—031945. The ORF of the cDNA is shown in Sequence ID No. 1 of the sequence listing. The human oculospanin cDNA can be obtained from a cDNA library expressing human oculospanin by using a primer for specifically amplifying human oculospanin 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.
Examples of eukaryotic host cells include vertebrate, insect and yeast cells. Examples of vertebrate cells include, but are not limited to, a simian cell, COS cell (Gluzman, Y. (1981), Cell 23, 175-182, (ATCC CRL-1650)), mouse fibroblast cell NIH3T3 (ATCC No. CRL-1658), and a dihydrofolic acid reductase defective strain (Urlaub, G. and Chasin, L. A. (1980), Proc., Natl. Acad. Sci, USA 77, 4126-4220) of Chinese hamster ovary cell (CHO cell, (ATCC CCL-61)).
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 oculospanin or a recombinant cell expressing human oculospanin, 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 Oculospanin Monoclonal Antibody
An example of an antibody which specifically binds to human oculospanin, is a monoclonal antibody which specifically binds to human oculospanin. 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 oculospanin 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 Ill. (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 colour 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), P3×63-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), GM1500.GTG-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. I. 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 oculospanin 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 oculospanin or cells expressing human oculospanin 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 oculospanin 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 oculospanin.
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 oculospanin antibody. As the template DNA used herein, use may be made of cDNA synthesized from mRNA of a hybridoma producing the anti-human oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin antibody molecule, a binding activity capable of binding to human oculospanin 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 per se. 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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 2/3 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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin antibody can be manufactured easily with high purity and in high yield.
6. D A Pharmaceutical Composition Containing an Anti-Human Oculospanin Antibody
From the anti-human oculospanin antibodies obtained by a method described in Section “5. Preparation of anti-human oculospanin antibody”, antibody neutralizing the biological activity of human oculospanin or an antibody specifically damaging a cancer cell expressing human oculospanin can be obtained. These antibodies can inhibit the biological activity of human oculospanin in the living body, in other words, canceration of a cell. Therefore, they can be used as a medicament, in particular, as a therapeutic agent for cancer. The activity of an anti-human oculospanin antibody in neutralizing a biological activity of human oculospanin in vitro can be determined by the ability to inhibit canceration of a cell in which human oculospanin is overexpressed. To explain more specifically, the inhibitory activity can be determined by culturing mouse fibroblast cell strain, NIH3T3, which overexpresses human oculospanin, adding an anti-human oculospanin antibody to the culture system in various concentrations. In this way, the inhibitory activities against focus formation, colony formation and spheroid growth can be determined. The cytotoxic activity of an anti-human oculospanin antibody against a cancer cell in vitro can be determined by antibody-dependent cytotoxic activity, complement-dependent cytotoxicity or complement-dependent cell-mediated cytotoxicity exhibited by the anti-human oculospanin antibody against a cell overexpressing human oculospanin. To be more specific, 293T cells overexpressing human oculospanin are cultured; then, an anti-human oculospanin antibody is added at various concentrations to the culture system. Mouse spleen cells are further added to the culture system and cultured for an appropriate time. Thereafter, the ratio of induction of cell death for the cells overexpressing human oculospanin is determined. The effect of an anti-human oculospanin antibody in cancer treatment can be determined in vivo by using an experimental animal, more specifically, by administering the anti-human oculospanin antibody to a transgenic animal overexpressing human oculospanin and determining a change in the cancer cells.
An antibody thus obtained for neutralizing the biological activity of human oculospanin or an antibody specifically damaging cancer cells expressing human oculospanin is useful as a medicament, especially as a pharmaceutical composition for use in cancer treatment or as an antibody for use in immunological diagnosis of such a disease. As the type of cancer, skin cancer and melanoma, a kind of skin cancer, may be mentioned; but cancers that can be treated or diagnosed in accordance with the invention are not limited to these examples.
The present invention provides a pharmaceutical composition containing an anti-human oculospanin 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 preferably 0.01 to 100 times, more preferably 0.1 to 10 times the weight of the anti-human oculospanin 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 oculospanin 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 oculospanin antibody contained in a pharmaceutical composition according to the present invention exhibits higher affinity for human oculospanin; in other words, the higher the affinity of anti-human oculospanin antibody for human oculospanin, 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 oculospanin antibody may be administered to a person as a single dose at an interval of 1 to 30 days in an amount of about 0.1 to 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.
7. Screening Methods
The present invention provides methods for screening for candidate agents that bind to oculospanin proteins. In some cases, screens are done for agents that induce 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 inducement of cytotoxicity) oculospanin.
“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 oculospanin and therefore potential disease states.
Candidate agents encompass numerous chemical classes. In one embodiment, the candidate agent is an organic molecule, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Particularly preferred are small organic compounds having a molecular weight of more than 100 and less than about 2,000 daltons, more preferably less than about 1500 daltons, more preferably less than about 1000 daltons, more preferably 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 occuring proteins or fragments of naturally occuring 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 oculospanin 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. By “nucleic acid” or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, for example in the use of nucleic acids as candidate agents in screening assays, 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) pp169-176). 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 ETMs, 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 occuring 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.
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 oculospanin 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 oculospanin 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 oculospanin 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 oculospanin proteins are then added to cytotoxicity assays, as described herein. Alternatively, cytotoxicity assays are run with libraries of candidate agents without a binding assay done first.
Cytotoxicity assays are generally done as outlined below in the Examples; this generally is done by adding the candidate agent, a cell expressing oculospanin, and effector cells. The oculospanin-expressing cells can be selected from a number of different cells. In one embodiment, the oculospanin-expressing cells are naturally occurring cells, such as primary melanoma cells. In some embodiments, oculospanin-expressing cells are cells or cell lines that have been transformed to produce oculospanin, particularly human oculospanin.
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 oculospanin 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 oculospanin 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 oculospanin, 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 oculospanin by directly binding to the human oculospanin or by inhibiting the interaction between the human oculospanin and other factors.
Furthermore, according to another aspect, the present invention relates to a polypeptide associated with human oculospanin of the present invention, in other words, a partner protein for controlling the activity of human oculospanin. More specifically, the present invention relates to a screening method for such a partner protein for controlling the activity of human oculospanin.
One aspect of such a screening method comprises a step of bringing a test protein sample into contact with human oculospanin, thereby selecting a protein binding to the human oculospanin. Such a method includes purification of a protein by making use of its affinity for purified human oculospanin. To describe more specifically, first, a sequence formed of 6 histidines is bound to human oculospanin as an affinity tag. The resultant human oculospanin 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 oculospanin 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 oculospanin and a protein forming a complex as a subunit with a protein which binds directly to human oculospanin, but having no binding activity for human oculospanin, thus binding indirectly to human oculospanin [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, pp76-81, 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, pp66-75, 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 oculospanin in this manner is available, it can be used in functional screening of a substance inhibiting the interaction between human oculospanin and the partner protein. More specifically, a fusion protein of human oculospanin with glutathione-5-transferase can be prepared. The fusion protein is allowed to bind to a microplate covered with anti-glutathione-5-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 oculospanin, it is possible to screen an anti-cancer agent, for example, a useful candidate substance as a therapeutic agent for prostate cancer, in accordance with a test method using an expression vector comprising the human oculospanin gene, as described above. Furthermore, when the obtained partner protein has the activity of suppressing the function of human oculospanin, a polynucleotide having a nucleotide sequence encoding such a suppressor can be used in gene therapy for cancer.
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 oculospanin 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 anti-cancer factor is produced and functions to suppress proliferation of cancer cells. In this manner, it is possible to treat cancer.
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 Sequence 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 Sequence ID No. 1 of the sequence listing and formed generally of 15 to 30 mer. 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 Sequence ID No. 1 of the sequence listing.
A composition containing the antisense oligonucleotide 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.
Expression profile analysis was performed, using an EST probe (Affymetrix GeneChip HG-133 probe 223795_at: manufactured by Affymetrix) having a nucleotide sequence partially overlapping with the sequence represented by Sequence ID No. 1 of the sequence listing, by use of the data base (GeneExpress Software System Release 1.4.2) provided by Genelogic company.
Expression of the human oculospanin gene in various cells was quantitatively compared by considering its transcription. As a result, the expression levels in 8 melanocyte samples were to found to be significantly high, compared to the levels in other cells samples, including 12 blood-cell samples, 6 glia cell samples, 62 epithelial cell samples (P values thereof were <0.0001, =0.0007, and <0.0001 sequentially in order,
Next, the expression levels of the human oculospanin gene were compared in samples derived from tissue. More specifically, the amount of transcription was compared with respect to 66 skin samples from healthy individuals and 33 melanoma samples. As a result, the amount of transcription in the melanoma samples was found to be significantly high (P value=0.0001,
When 66 healthy person's skin samples were compared to 12 melanoma samples derived from lymph node tissue, the amount of transcription in the melanoma samples was found to be significantly higher (P value=0.0003,
Furthermore, when 13 healthy person's samples derived from lymph node were compared to 12 melanoma samples derived from lymph node tissue, the amount of transcription in the melanoma samples was found to be significantly higher (P value=0.0011, the panel of
a) PCR Reaction
As a primer for amplifying human oculospanin cDNA by PCR, oligonucleotides having the following sequences were synthesized in accordance with a customary method.
Note that Primer 1 is an oligonucleotide constructed by adding 4 bases, CACC, as a KOZAK sequence, upstream of the initiation codon of the human oculospanin gene, in other words, an oligonucleotide constructed by adding the 4 bases, the CACC sequence, to the 5′ side of the nucleotide sequence consisting of nucleotides No. 1 to 23 of the Sequence ID No. 1 of the sequence listing. The CACC sequence, since it forms a chain complementary to the 3′ terminus of the vector when it is integrated into the cloning vector pENTR/D-TOPO, makes it possible to integrate the gene into the vector whilst maintaining the orientation of the gene. Primer 2 is an oligonucleotide composed of a chain complementary to a nucleotide sequence consisting of nucleotides No. 1043 to 1065 of the sequence ID No. 1 of the sequence listing.
The PCR reaction was performed using PLATINUM Pfx DNA polymerase (manufactured by Invitrogen) in accordance with the protocol provided. More specifically, to 0.1 μl of the first strand cDNA obtained, 1.5 μl of each of 10 pmol/μl synthetic primer 1 and synthetic primer 2, 5 μl of 10× Pfx Amplification Buffer, 1.5 μl of 10 mM dNTP Mix, 1 μl of 50 mM MgSO4, 0.5 μg of PLATINUM Pfx DNA polymerase, 10 μl of 10× PCRx Enhancer Solution, and 28.9 μl of sterilized water were added to prepare 50 μl of a PCR reaction solution. The PCR reaction was performed using a Peltier Thermal Cycler TPC-200 DNA Engine (manufactured by MJ Research), first by heating the PCR solution at 94° C. for 2 minutes, repeating 5 times a thermal cycle consisting of reactions at 94° C. for 30 seconds and 65° C. for 2 minutes; 5 times a thermal cycle consisting of reactions at 94° C. for 30 seconds 60° C. for 40 seconds, and 68° C. for one minute and 20 seconds; 5 times a thermal cycle consisting of reactions at 94° C. for 30 seconds, 55° C. for 40 seconds, and 68° C. for one minute and 20 seconds; 35 times a thermal cycle consisting of reactions at 94° C. for 30 seconds, 50° C. for 40 seconds, and 68° C. for one minute and 20 seconds and finally maintaining the PCR solution at 68° C. for 10 minutes, and then storing the solution at 4° C. A desired cDNA was obtained by subjecting the reaction product to 1.5% agarose gel electrophoresis, confirming amplification of the NM—031945 cDNA (1069 bp), and purifying the DNA from the agarose gel using the S.N.A.P. UV-Free Gel Purification Kit (manufactured by Invitrogen) in accordance with the protocol provided. The concentration of cDNA thus purified was determined by use of 1 D Image Analysis Software Version 3.5 (Kodak Digital Science EDAS290: manufactured by Kodak) with reference to a 1 kb DNA Ladder which was used as a concentration reference.
b) Cloning of the Human Oculospanin cDNA into the pENTR/D-TOPO Vector
The NM—031945 cDNA obtained in Example 2a) was cloned into the pENTR/D-TOPO vector using the pENTR Directional TOPO Cloning Kit (manufactured by Invitrogen) in accordance with the protocol provided. More specifically, NM—031945 cDNA was mixed with the pENTR/D-TOPO vector, having Topoisomerase bound thereto, in the reaction buffer supplied with the kit and incubated at room temperature for 30 minutes. OneShot TOP10 Chemically Competent E. coli (manufactured by Invitrogen) was transformed using the reaction product obtained and cultured on an LB agar medium containing 50 μg/ml kanamycin. The resultant E. coli colonies, which exhibited resistance to kanamycin, were selected and cultured, in a liquid TB medium containing 1 ml of 50 μg/ml kanamycin, at 37° C. overnight. Plasmid DNA was isolated and purified by using a Montage Plasmid Miniprep96 Kit (manufactured by Millipore). Then, the plasmid DNA thus obtained was subjected to a reaction using the BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit in accordance with the protocol provided, the nucleotide sequence was analyzed using an ABI PRISM 3100 DNA Analyzer (manufactured by Applied Biosystems). As a result, it was confirmed that cDNA (Sequence ID No. 1 of the sequence listing) having an open reading frame of the nucleotide represented by GenBank ACCESSION NO.NM—031945 was integrated into the pENTR/D-TOPO vector.
Next, the gene was transferred to an expression vector, pcDNA3.1/DEST40 (manufactured by Invitrogen), using the GATEWAY™ system. To explain more specifically, 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix (manufactured by Invitrogen), 4 μl of LR Reaction Buffer, 0.3 μg of pENTR/D-TOPO-NM—031945, and 0.3 μg of pcDNA3.1/DEST40 were mixed and made up to a 20 μl reaction solution in TE buffer. The reaction solution was allowed to react at 25° C. for one hour. After the reaction, 2 μl of proteinase K was added and a reaction was performed at 37° C. for 10 minutes. Using the resulting reaction product, OneShot TOP10 Chemically Competent E. coli (manufactured by Invitrogen) were transformed and cultured in a LB agar medium containing 50 μg/ml of ampicillin. The resulting E. coli colonies, exhibiting ampicillin resistance, were selected and cultured in 100 ml of liquid LB medium containing 50 μg/ml ampicillin at 37° C. overnight, and plasmid DNA (pcDNA3.1-DEST40-NM—031945) was isolated and purified by use of Plasmid MAXI Kit (manufactured by QIAGEN).
a) Transfection of NIH3T3 cells with the plasmid pcDNA3.1-DEST40-NM—031945
NIH3T3 cells were transfected with plasmid pcDNA3.1-DEST40-NM—031945 obtained in Example 2 as follows. The transfection of the NIH3T3 cells was performed by lipofection using the Lipofectamine 2000 Reagent manufactured by Invitrogen. To explain more specifically, first, NIH3T3 cells were grown in a 6 well plate up to a semi-confluent state. Next, the cells were washed once with antibiotic-free DMEM containing 10% fetal calf serum, then 200 μl of antibiotic-free DMEM containing 10% fetal calf serum was added to the cells. Then, to a 1.5 ml Eppendorf tube, 100 μl of serum-free medium (DMEM) and 2 μg of plasmid DNA (pcDNA3.1-DEST40-NM—031945) recovered in the aforementioned manner were added and mixed. To another 1.5 ml Eppendorf tube, 96 μl of serum-free medium (DMEM) and 4 μl of Lipofectamine 2000 Reagent were added and mixed. The DNA solution and the Lipofectamine solution were mixed and allowed to stand still at room temperature for 20 minutes. Thereafter, the DNA-Lipofectamine solution mixture was added to the cells and cultured at 37° C. in 5% CO2. After 4 hours, 1 ml of DMEM containing 10% fetal calf serum was added to the cells which were cultured at 37° C. overnight in 5% CO2.
b) Confirmation of Expression of the Plasmid pcDNA3.1-DEST40-NM—031945 in NIH3T3 Cells
The cell culture product thus obtained was recovered. The negative control containing no cDNA or NIH3T3 cells transfected with the pcDNA3.1-DEST40-NM—031945 obtained were washed with a PBS (−) buffer solution (manufactured by Invitrogen). The cells were dispersed in a sample buffer solution (manufactured by BioRad) containing 2-mercaptoethanol for use in SDS polyacrylamide electrophoresis (SDS-PAGE). SDS-PAGE was performed using 12.5% polyacrylamide gel (e PAGEL E-T12.5L; manufactured by ATTO corporation) under reducing conditions.
After electrophoresis, bands were transferred from the polyacrylamide gel to a Polyvinylidene Difluoride(PVDF) membrane (manufactured by Millipore) by use of a gel-membrane transfer apparatus (NP7513 manufactured by Marysol) in a transfer buffer solution (192 mM glycine, 20% methanol, 25 mM Tris) under the following conditions: 4° C., 120 minutes and 200 mA.
After transfer, the PVDF membrane was subjected to Western blot analysis using an anti-V5-tag antibody (manufactured by Invitrogen). To explain more specifically, first, the PVDF membrane was blocked using blockace (manufactured by Yukijirushi Co.,) once at room temperature for 30 minutes, and put in a plastic bag (trade name: Hybribag manufactured by Cosmo Bio). To the bag, the anti-V5-tag antibody (1000-fold dilution) and 5 ml of blockace were added and the bag was shaken at room temperature for one hour. After one hour, the membrane was removed and washed with PBS containing 0.05% Tween 20 (hereinafter referred to as “0.05% Tween 20-PBS) once at room temperature for 15 minutes and twice for 5 minutes. Thereafter, the membrane was transferred to a new plastic bag. To the bag, 30 ml of a solution containing a horseradish peroxidase labeled anti-rabbit IgG antibody (manufactured by Amersham Pharmacia) diluted 5000 fold with 0.05% Tween 20-PBS, was added and shaken at room temperature for one hour. After one hour, the membrane was taken out and washed with 0.05% Tween 20-PBS once for 15 minutes and four times for 5 minutes. After washing, the membrane was placed on a wrapping film and a band having the anti-V5-tag antibody bound thereto was detected by use of ECL Western blotting detection solution (manufactured by Amersham Pharmacia). The membrane was placed on the wrapping film and soaked in the ECL Western blotting detection solution for one minute and then exposed to an X-ray film (one minute). As a result, a band specific to the NIH3T3 cells having plasmid pcDNA3.1-DEST40-NM—031945 DNA introduced therein was detected due to the presence of the anti-V5-tag antibody (
c) Transfection of BALB-3T3 Cells with the Plasmid pcDNA3.1-DEST40-NM—031945
BALB-3T3 cells (American Type Culture Collection No. CCL-163) were cultured in three Cell Trays (culturing area: 500 cm2 manufactured by Sumitomo Bakelite Co., Ltd.) for cell culture in Dulbecco's Modified Eagle Medium (hereinafter referred to as “DMEM”) manufactured by Nissui Pharmaceutical Co., Ltd., containing 10% bovine serum (hereinafter referred to as “BS”) manufactured by Gibco), at 37° C. in 5% CO2 gas up to a semi-confluent state. Thereafter, the BALB-3T3 cells were transfected with the plasmid pcDNA3.1-DEST40-NM—031945. The transfection of the BALB-3T3 cells was performed by lipofection using Geneporter™ 2 Transfection Reagent (manufactured by Gene Therapy Systems). To explain more specifically, the cells were washed once using a serum-free medium, DMEM. To the cells, 500 ml of the serum-free medium (DMEM) was added. Then, to a 50 ml Falcon tube, 6 ml of New DNA diluent and 240 μg of plasmid DNA (pcDNA3.1-DEST40-NM—031945) recovered by the aforementioned method were added and mixed. To another 50 ml Falcon tube, 4.8 ml of serum-free medium (DMEM) and 1200 μl of Geneporter™ 2 Reagent were added and mixed. The DNA solution and the Geneporter™ 2 solution were mixed and allowed to stand still at room temperature for 20 minutes. Thereafter, the solution mixture with DNA-Geneporter™ 2 was added to the cells (4 ml/tray) and cultured at 37° C. in the presence of 5% CO2. After 4 hours, DMEM containing 20% bovine serum was added in an amount of 50 ml/tray and cultured at 37° C. in 5% CO2 overnight.
d) Preparation of the Cell Membrane Fraction
The cells cultured by the aforementioned method were washed with PBS (−) buffer solution (manufactured by Invitrogen). The cells were collected using a cell scraper (manufactured by Sumitomo bakelite Co., Ltd.), and suspended in 7 ml of 5 mM Tris buffer at pH 8.0. The resulting cell solution was allowed to stand still at 4° C. for 30 minutes. The cells were crushed using a Dounce Type B homogenizer (30 strokes) and centrifuged at 1000 G for 10 minutes. The supernatant was recovered and centrifuged at 78,000 G for 100 minutes using an ultracentrifugation apparatus (manufactured by Hitachi) and the precipitate was recovered. The precipitate was subjected to a sugar density gradient to concentrate the membrane fragments. More specifically, the precipitate was dissolved in 3 ml of a solution of 57% sugar and 0.25M Tris buffer, pH 8.0. The resulting solution was transferred to an ultracentrifuge tube. An aliquot of 3 ml of a solution of 57% sugar and 0.25M Tris buffer, pH 8.0 and 1.5 ml of a solution of 37.5% sugar and 0.25M Tris buffer pH 8.0 were layered sequentially onto the cell precipitate solution. Then, centrifugation was performed using an ultracentrifugation apparatus at 75,500 G for 16 hours. An aliquot of 1 ml was taken from the top of each tube. To each aliquot (fraction), 10 mL of 5 mM Tris buffer pH 8.0 was added and this was subjected to ultracentrifugation at 78,000 G for one hour to recover the precipitate. To the precipitate 500 μl of 5 mM Tris buffer, pH 8.0 was added and the cell solution was homogenized using a Dounce type B homogenizer (10 strokes). The cell membrane fraction was identified by Western Blotting method described in the Section “Confirmation of Expression” and used as an immunogen.
(4-1) Immunization
1 ml (total protein amount: 100 μg) of the membrane fraction solution of the human oculospanin expressing cells obtained in Example 3 was injected intraperitoneally into BALB/c mice which were 4 to 10 weeks old (purchased from Japan SLC Inc.) After two weeks, the same membrane fraction solution (20 μg protein/mouse) was injected into the abdominal cavity as a booster immunization.
(4-2) Cell Fusion
The spleen was excised from a mouse at three days after the booster immunization and added to 10 ml of a serum-free RPMI 1640 medium (10.4 g/l, RPMI 1640 “Nissui” (1): manufactured by Nissui Pharmaceutical Co., Ltd., hereinafter referred to as “serum-free RPMI medium”) containing 20 mM HEPES buffer (pH 7.3), 350 mg/ml sodium hydrogen carbonate, 0.05 mM β-mercaptoethanol, 50 units/ml penicillin, 50 μg/ml streptomycin, and 300 μg/ml L glutamic acid, and the spleen was crushed on the mesh of a cell strainer (cell strainer; manufactured by Falcon) using a spatula. The cell suspension solution passed through the mesh was centrifuged to collect the spleen cells. The spleen cells were washed twice with serum-free RPMI medium, suspended in serum-free RPMI medium and the number of cells was counted.
Myeloma cells NSI (American Type Culture Collection TIB-18) were cultured in ASF 104 medium (manufactured by Ajinomoto; hereinafter referred to as the “serum-containing ASF medium”) containing 10% FCS (manufactured by Gibco BRL) at 37° C. in 5% CO2 gas such that the cell density did not exceed 1×108 cells/ml. The myeloma cells thus prepared were washed with serum-free RPMI medium in the same manner as above and suspended in serum-free RPMI medium and the number of cells was counted.
The NSI cell suspension solution containing about 3×107 cells and the spleen cell suspension solution containing about 3×108 cells were mixed and subjected to centrifugation, and thereafter the supernatant was completely removed. The cell fusion operation below was performed whilst maintaining the plastic centrifuge tube containing the pellet in a beaker containing hot water at 37° C. To the pellet, 1 ml of 50% (w/v) polyethylene glycol 1500 (manufactured by Boehringer Mannheim) was slowly added by pipette whilst agitating the pellet using the tip. Thereafter, 1 ml of the serum-free RPMI medium, previously warmed to 37° C., was gently added in twice and a further 7 ml of serum-free RPMI medium was added. After centrifugation, the supernatant was removed and 10 ml of hypoxanthine aminopterin thymidine medium (hereinafter referred to as “HAT medium”; manufactured by Boehringer Mannheim) containing 10% FCS was added by pipette whilst gently agitating using the tip. After 20 ml of the HAT medium containing 10% FCS was added, the resulting solution was dispensed to a 96-well cell culture microplate at an amount of 100 μl/well and cultured at 37° C. in 5% CO2 gas. Seven to eight days later, to wells containing medium with a tinge of yellow, fresh HAT medium was added in an amount of 100 μl/well. The fused cells thus obtained were subjected to screening by limiting dilution analysis as mentioned below.
(4-3) Limiting Dilution
The thymus gland was excised from female BALB/c mouse which were 4 to 10 weeks old (purchased from Japan SLC Inc.) and crushed on the mesh of a cell strainer (Cell Strainer, manufactured by Falcon) using a spatula. The cells passed through the mesh were washed twice with hypoxanthine thymidine medium (hereinafter referred to as the “HT medium”, manufactured by Boehringer Mannheim) containing 10% FCS. The thymus gland cells of the mouse were suspended in 30 ml of the HT medium containing 10% FCS. The suspension solution thus obtained was used as a feeder cell solution. The culture solution containing the fused cells obtained in Section (4-2) was diluted 10 to 100 fold with the feeder cell solution depending upon the cell density and further serially diluted with the feeder cell solution until the density of the fused cells was 5 cells/ml, 1 cell/ml and 0.5 cells/ml. Each of the samples thus prepared was dispensed into a 96-well cell culture microplate in an amount of 100 μl per well and cultured at 37° C. in 5% CO2 gas for 5 days.
(4-4) Screening
(4-4-1) Cell ELISA
Human oculospanin expressing cells were maintained by culturing them in RPMI 1640 medium (manufactured by Invitrogen) supplemented with 10% fetal calf serum (manufactured by Moregate Biotech), 20 mM HEPES (manufactured by Sigma) and 55 μM 2-mercaptoethanol (manufactured by Invitrogen) at 37° C. in 5% CO2 gas. Human oculospanin expressing cells in the logarithmic growth phase were seeded into a cell culture flask at a density of 2×104 cells/cm2 and cultured for 3 days. The human oculospanin expressing cells thus prepared were transferred to a 50 ml tube and centrifuged using a HITACHI himac CF8DL at 1,000 rpm for 5 minutes (Centrifugation condition 1). The supernatant was removed and the human oculospanin expressing cells were suspended in a medium. Thereafter, the number of living cells was counted using 0.4% tryphan blue solution (manufactured by Sigma). The density of the live human oculospanin expressing cells was adjusted using the medium to be 107 cells per ml and the resultant medium was dispensed to a 96-well U-bottom plate in an amount of 100 μl/well. The 96-well U-bottom plate was centrifuged using a HITACHI himac CF8DL at 15,000 rpm for one minute (Centrifugation condition 2). The supernatant was removed using a 200 μl tip. The 96-well U-bottom plate was tapped on the side surface to suspend the human oculospanin expressing cells. To the suspension, hybridoma culture supernatant solutions whose concentrations were adjusted to 10 μg/ml, 5 μg/ml, 2.5 μg/ml with a medium cooled on ice, were added in an amount of 100 μl/well. Whilst the 96-well U-bottom plate was stirred using a plate mixer (manufactured by Fujirebio Inc.) at intervals of 15 minutes, a reaction was performed at 4° C. for 1.5 hours. After completion of the reaction, the 96-well U-bottom plate was centrifuged under Centrifugation condition 2, and the supernatant was removed using a 200 μl tip. A solution (PBS-5% FBS) prepared by adding 5% fetal calf serum to PBS(−)(manufactured by Nissui Pharmaceutical Co., Ltd.) was added to the wells in an amount of 200 μl per well. After stirring using a plate mixer, centrifugation was performed under Centrifugation condition 2 and the supernatant was removed using a 200 μl tip. Thereafter, the aforementioned operation was repeated twice. The 96-well U-bottom plate was tapped on the side surface to suspend the human oculospanin expressing cells. To the suspension, peroxidase-labeled anti-human IgG antibody (manufactured by Kirkegaad & Perry Laboratories) diluted 500 fold with PBS-5% FBS cooled in ice was added in an amount of 100 μl/well. While the 96-well U-bottom plate was stirred using a plate mixer at intervals of 15 minutes, a reaction was performed at 4° C. for 1.5 hours. After completion of the reaction, the 96-well U-bottom plate was centrifuged under Centrifugation condition 2 and the supernatant was removed using a 200 μl tip. Then, PBS-5% FBS was added in an amount of 200 μl/well and stirred using a plate mixer, centrifuged under Centrifugation condition 2, and then the supernatant was removed using a 200 μl tip. Thereafter, the aforementioned operation was repeated twice. The 96-well U-bottom plate was tapped on the side surface to suspend the human oculospanin expressing cells. To the suspension, a color development substrate for peroxidase (manufactured by Nacalai Tesque Inc.) adjusted to room temperature was added in an amount of 100 μl/well and stirred using a plate mixer for 10 minutes. After centrifugation was performed under Centrifugation condition 2, the supernatant was transferred to 96-well flat-bottomed plate in an amount of 50 μl/well and absorbance was measured at 405 nm using a plate reader (1420 ARVO multilabel counter, manufactured by PerkinElmer Inc.)
(4-4-2) Flow Cytometry
The human oculospanin expressing cells obtained in Example 3 were cultured and grown in RPMI 1640 medium containing 10% FCS at 37° C. in 5% CO2 gas. A cell suspension solution, prepared so as to contain 1×107 cells/ml, was dispensed into 96-well U-bottom microplate (manufactured by Nunk) in an amount of 50 μl/well and centrifuged (at 90×g, 4° C. for 10 minutes). The supernatant was removed and the supernatant of the fused cells cultured in Section (4-3) above was added in an amount of 50 μl/well and stirred. The plate was allowed to stand for one hour on ice, subjected to centrifugation (at 90×9, 4° C. for 10 minutes) and the supernatant was removed. The pellet was washed twice with a flow cytometric buffer solution (PBS containing 5% FCS and 0.04% (w/v) sodium azide) in an amount of 100 μl/well and 50 μl of 500-fold diluted goat anti-mouse IgG antibody IgG fraction (manufactured by Organon Technica) labeled with fluorescein-5-isothiocyanate (hereinafter referred to as “FITC”) was added as a secondary antibody and allowed to stand still on ice for one hour. After centrifugation (at 90×9, 4° C. for 10 minutes), the supernatant was removed. The pellet was washed twice with 100 μl of the flow cytometric buffer solution per well, and thereafter 50 μl of a 3.7% formalin solution was added and the resulting solution mixture was allowed to stand for 10 minutes on ice. In this manner, the cells were immobilized. After centrifugation (at 90×g, 4° C. for 10 minutes), the supernatant was removed. The pellet was washed again with 100 μl of the flow cytometric buffer solution per well and suspended in 100 μl of the flow cytometric buffer per well. This was used as a sample for flow cytometry. The intensity of FITC fluorescence emitted from the cells in each sample was measured using a flow cytometer (Epics Elite manufactured by Coulter) at an excitation wavelength of 488 nm and a detection wavelength of 530 nm. When the FITC fluorescence intensity of the human oculospanin expressing cells exposed to supernatant from the fusion cell culture was much higher (about 100 to 1,000) than that (about 0.3) of the human oculospanin expressing cells unexposed to the supernatant from the fusion cell culture, the corresponding fusion cells were selected.
(4-5) Cloning
The cells selected in Section (4-4) above were subjected to a series of steps (4-3) to (4-4), five times. In this way, several hybridoma clones were obtained which were capable of producing a single antibody capable of binding to human oculospanin expressing cells but incapable of binding to the non-transfected parent cells.
Mouse-mouse hybridoma cells constructed in Example 4 were cultured in 1 litre of ASF medium containing 10% FCS at 37° C. in 5% CO2 gas until the cell density reached 1×106 cells/ml. The culture solution was centrifuged (at 1,000 rpm for 2 minutes), the supernatant was discarded, and the cells collected were washed once using serum-free ASF medium. Thereafter, the cells were resuspended in 1 litre of serum-free ASF medium and cultured at 37° C. in 5% CO2 gas for 48 hours. The culture solution was centrifuged (at 1,000 rpm for 2 minutes) and the supernatant was recovered and transferred into a dialysis tube (exclusion limit molecular weight: 12,000 to 14,000, manufactured by Gibco BRL). Dialysis was performed against a 10-fold amount of 10 mM sodium phosphate buffer solution (pH 8.0). The IgG contained in the solution within the dialysis tube was crudely purified using high performance liquid chromatographic apparatus (FPLC system, manufactured by Pharmacia) under the conditions described below:
Column: DEAE Sepharose CL-6B column (Column size 10 ml, manufactured by Pharmacia)
Solvent: 10 mM sodium phosphate buffer solution (pH 8.0)
Flow rate: 1 ml/minute
Elution: 1M sodium chloride linear concentration gradient (0-50%, 180 minutes)
The eluate was fractionated into 5 ml samples. The antibody titer of the anti-human oculospanin antibody in each fraction was checked by the ELISA method using human oculospanin protein. First, a membrane fraction solution prepared from human oculospanin expressing cells prepared in Example 3 was added to a 96-well microplate for ELISA in an amount of 100 μl/well and kept warm at 37° C. for one hour. Then the membrane fraction solution was discarded and each well was washed three times with 100 μl of PBS-Tween per well. Then, 100 μl of PBS containing 2% bovine serum albumin was added per well and kept warm at 37° C. for one hour. After washing three times with 100 μl of PBS-Tween per well, 100 μl of the elution fraction was added and kept warm at 37° C. for one hour. Furthermore, after wells were washed three times with 100 μl of PBS-Tween per well, horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by Amersham) diluted 2000 fold in PBS-Tween was added in an amount of 100 μl/well and allowed to react at 37° C. for one hour, and then washed three times with 100 μl of PBS-Tween per well. Subsequently, a substrate for horseradish peroxidase (manufactured by BioRad) was added in an amount of 100 μl/well and allowed to stand still for 5 minutes, and thereafter, the absorbance of each well at 415 nm was measured using a microplate reader.
Consequently, the fractions exhibiting high absorbance were collected and loaded onto two antibody affinity purification columns (Hitrap Protein G column, column volume: 5 ml, manufactured by Pharmacia). After washing the inside of the columns with 25 ml of equilibrium buffer (20 mM, sodium phosphate buffer (pH 7.0) per column, the antibody was eluted using 15 ml of an elution buffer (0.1M glycine-hydrochloride (pH 2.7)) per column. Each eluate was collected in a test tube containing 1.125 ml of 1M Tris-hydrochloride (pH 9.0). Immediately after completion of the elution, the eluate was loaded onto the upper portion of an ultrafilter of centrifugation-tube form (Centriprep 10 manufactured by Grace Japan) and centrifuged at 3000×g at 4° C. for 2 hours. After the filtrate collected in the lower portion of the filter was removed, 15 ml of PBS was added to the upper portion and again centrifuged at 3000×g, and 4° C. for 2 hours. In all, this operation was repeated five times. At the 5th time of operation, the centrifugation operation was performed until the liquid amount in the upper portion of the filter reached 0.5 ml. The liquid left in the upper portion of the filter was used as a sample of the anti-human oculospanin antibody.
Antibody-dependent cytotoxic activity was measured as an index of bioactivity.
The number of human oculospanin expressing cells (Example 3) was counted by the tryphan blue staining method, the concentration of the cells was adjusted to 1×106 cells/ml with RPMI 1640 medium (manufactured by Invitrogen, hereinafter referred to as the “RPMI medium”) containing 10% fetal bovine serum (manufactured by Moregate). To the cells, 2.5 μl of bis(acetoxymethyl)2,2′:6′2″-terpyridine-6,6″-dicarboxylic acid (BATDA labeling agent, manufactured by PerkinElmer) was added, stirred well and incubated at 37° C. in 5% carbon dioxide for 30 minutes while mixing at intervals of 15 minutes by inverting the culture. To the culture medium, 10 ml of the RPMI medium was added, stirred and centrifuged at 1,500 rpm for 5 minutes. This washing operation was repeated a further two times. The BATDA labeled human oculospanin expressing cells thus obtained were resuspended in 10 ml of RPMI 1640 medium. An aliquot of 50 μl (5×103 cells) of the suspension solution was seeded in each well of a 96-well round bottom microplate, which was previously prepared by adding a purified mouse anti-human oculospanin antibody previously adjusted with RPMI 1640 medium to a concentration of 1 μg/ml, or the supernatant of the hybridoma culture medium, and leaving it stand still at 4° C. for 30 minutes. The microplate was allowed to stand still at 4° C. for a further 30 minutes. To a negative control well there was added either the purified mouse anti-human oculospanin antibody or RPMI 1640 medium in place of the hybridoma supernatant.
Effector cells were prepared as follows. J774A.1 cells (available from Dainippon Pharmaceutical Co., Ltd.) were cultured in the presence of 100 ng/ml macrophage colony stimulating factor (manufactured by Sigma) for 3 days. The number of J774A.1 cells was counted by the tryphan blue staining method and then adjusted with RPMI medium to a concentration of 1×106 cells/ml. To each well of the 96-well round-bottom microplate mentioned above, an 100 μl aliquot (1×105 cells) of the cells was seeded. The microplate was centrifuged at 1,500 rpm for 5 minutes and incubated at 37° C. in 5% CO2 gas for 4 hours. To a positive control well, 1% Triton-X-100 was added in place of the effector cells, in order to completely kill the BATDA-labeled human oculospanin expressing cells. After a 4 hour incubation, 20 μl of the culture supernatant was taken from each well and transferred to 96-well white plate. To the plate, 200 μl of a europium solution (manufactured by PerkinElmer) was added. The plate was shaken at room temperature for 15 minutes and the decomposition of fluorescence with time was measured.
The rate of cell death induction in each well was calculated based on the equation below:
Cell death induction rate (%)=(fluorescent count for each test well−background count for the negative control well)/(the fluorescent count for the positive control well−background count for the negative control well)×100.
By comparison with a control containing only RPMI 1640 medium, it was confirmed that cell death of the human oculospanin expressing cells was induced by addition of the purified mouse anti-human oculospanin antibody or the hybridoma supernatant.
a) Construction of Plasmid pEF/DEST51-NM—031945
The NM—031945 cDNA obtained in Example 2a) was cloned into the pENTR/D-TOPO vector by using the pENTR Directional TOPO cloning kit (manufactured by Invitrogen) in accordance with the protocol provided. The NM—031945 cDNA was mixed with pENTR/D-TOPO vector having Topoisomerase bound thereto, in a reaction buffer provided with the kit and incubated at room temperature for 30 minutes. Using the reaction product obtained, Oneshot TOP10 chemically competent E. coli. (manufactured by Invitrogen) were transformed and cultured in LB agar medium containing 50 μg/ml kanamycin. The resulting E. coli colonies, resistant to kanamycin, were selected and cultured in 1 ml of liquid TB medium containing 50 μg/ml of kanamycin at 37° C. overnight. The plasmid DNA was isolated and purified using a Montage Plasmid Miniprep96 Kit (manufactured by Millipore). Next, the plasmid DNA thus obtained was subjected to a sequencing reaction performed using a BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit in accordance with the protocol provided, the nucleotide sequence was analyzed using an ABI PRISM 3100 DNA Analyzer (manufactured by Applied Biosystem). As a result, it was confirmed that the cDNA (Sequence ID No. 1 of the sequence listing) having an open reading frame of the nucleotide sequence represented by Accession No. NM—031945 was integrated in the pENTR/D-TOPO vector.
Then, the gene was transferred into expression vector pcDNA3.1/DEST40 (manufactured by Invitrogen) by use of the GATAWAY™ system. More specifically, 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix (manufactured by Invitrogen), 4 μl of LR Reaction Buffer, 0.3 μg of pENTR/D-TOPO-NM—031945, 0.3 μg of pcDNA3.1/DEST40, were mixed with TE buffer to prepare a 20 μl solution, which was allowed to react at 25° C. for one hour. After completion of the reaction, 2 μl of Proteinase K was added and a reaction was performed at 37° C. for 10 minutes. OneShot TOP10 Chemically Competent E. coli (manufactured by Invitrogen) were transfected with the reaction product and cultured on LB agar medium containing 50 μg/ml of ampicillin. The resulting E. coli colonies, resistant to ampicillin, were selected and incubated in 100 ml of liquid LB medium containing 50 μg/ml of ampicillin at 37° C. overnight. As a result, plasmid DNA (pcDNA3.1-DEST40-NM—031945) was isolated and purified using the Plasmid MAXI Kit (manufactured by Qiagen).
Similarly, the gene was transferred to the expression vector pEF/DEST51 (manufactured by Invitrogen) by use of the Gateway™ system. To explain more specifically, 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix (manufactured by Invitrogen), 4 μl of LR Reaction Buffer, 0.3 μg of pENTR/D-TOPO-NM—031945 and 0.3 μg of pEF/DEST51 were mixed with TE buffer to prepare a 20 μl solution and allowed to react at 25° C. for one hour. After the reaction, 2 μl of proteinase K was added and allowed to react at 37° C. for 10 minutes. OneShot TOP10 Chemically Competent E. coli (manufactured by Invitrogen) were transformed with the reaction product obtained and cultured on LB agar medium containing 50 μg/ml ampicillin. The resulting E. coli colonies, resistant to ampicillin, were selected and cultured in 100 ml of liquid LB medium, containing 50 μg/ml ampicillin, at 37° C. overnight. As a result, plasmid DNA (pEF-DEST51-NM—031945) was isolated and purified using the Plasmid MAXI Kit (manufactured by Qiagen).
b) Transfection of BALB-3T3 Cells and 293T Cells with the Plasmid pEF-DEST51-NM—031945
BALB-3T3 cells (available from RIKEN, clone A31) were cultured in 330 150 mm cell-culture dishes (culturing area: 148 cm2, manufactured by IWAKI) containing Dulbecco's Modified Eagle's medium (hereinafter referred to as the “DMEM”, manufactured by SIGMA) supplemented with 10% bovine serum (manufactured by GIBCO; hereinafter referred to as “BS”) at 37° C. in 5% CO2 gas up to a semi-confluent state. Thereafter, the BALB-3T3 cells were transfected with plasmid pEF-DEST51-NM—031945. The Transfection of BALB-3T3 cells was performed by lipofection using the Geneporter™ 2 transfection reagent manufactured by Gene Therapy Systems. More specifically, the cells were washed once with serum-free medium (DMEM) and 20 ml of the serum-free medium (DMEM) was added. Then, to a 50 ml Falcon tube, 0.6 ml of New DNA diluent and 24 μg of plasmid DNA (pEF-DEST51-NM—031945) recovered by the aforementioned method were added and mixed. To another 50 ml Falcon tube, 0.35 ml of a serum free medium (Opti-MEM I, manufactured by GIBCO) and 84 μl of Geneporter™ 2 Reagent were added and mixed. The DNA solution and the Geneporter™ 2 solution were mixed and allowed to stand still at room temperature for 20 minutes. Thereafter, the solution mixture of DNA-Geneporter™ 2 was added to the cells (1 ml/dish) and cultured at 37° C. in 5% CO2. After 3 hours, the medium was replaced with 20 ml of DMEM containing 10% bovine serum per dish and cultured at 37° C. overnight in 5% CO2.
Furthermore, plasmid pEF-DEST51-NM—031945 was introduced in 293T cells as follows. Introduction into the 293T cells was performed by using LIPOFECTAMINE 2000 reagent (manufactured by Invitrogen). The 293T cells were seeded at a density of 2.5×105 cells/9.2 cm2 and cultured at 37° C. overnight in 5% CO2. In a 5 ml polypropylene tube, 10 μl of LIPOFECTAMINE 2000 reagent and 250 μl of OPTI-MEM I Reduced Serum Medium (manufactured by Invitrogen) were mixed and allowed to react with each other at room temperature for 5 minutes. In another 5 ml polyethylene tube, 4 μg of plasmid (pEF-DEST51-NM—031945) and 250 μl of OPTI-MEM I Reduced Serum Medium were mixed. The LIPOFECTAMINE solution and the DNA solution were mixed and allowed to react with each other at room temperature for 20 minutes. The supernatant was removed from the 293T cells cultured overnight and an antibiotic-free Dulbecco's Modified Eagle medium (manufactured by Gibco) containing 10% fetal calf serum (manufactured by Moregate) was added to the cells in an amount of 2 ml/9.2 cm2. The LIPOFECTAMINE-DNA solution mixture was added to the 293T cells and incubated at 37° C. in 5% CO2 gas for 2 days.
c) Preparation of the Cell Membrane Fraction (10 Liter)
The cells cultured by the aforementioned method were washed with a PBS (−) buffer solution (manufactured by Dainippon Pharmaceutical Co., Ltd). The cells were collected using a cell scraper (manufactured by IWAKI) and suspended in 230 ml of a 5 mM Tris buffer solution, pH 7.4. The resulting cell solution was allowed to stand still at 4° C. for 30 minutes. The cells were crushed using a Dounce Type B homogenizer (50 strokes) and centrifuged at 1000 G for 10 minutes. The supernatant was recovered and centrifuged at 1,000 G for 10 minutes using an ultracentrifugation apparatus (manufactured by KUBOTA) and the supernatant was recovered.
The supernatant was centrifuged at 78,000 G for 100 minutes using an ultracentrifugation apparatus (manufactured by BECKMAN) and the precipitate was recovered. To the precipitate, 14 ml of 57% sucrose in Tris buffer was superposed and subjected to sugar density gradient at 78,000 G for 16 hours at 4° C. As a result, the membrane fragment of the upper layer was recovered. To the membrane fraction, 55 ml of 5 mM Tris buffer, pH 7.4, was added and centrifuged at 78,000 G for 60 minutes at 4° C. The precipitate was recovered. To the precipitate, 1500 μl of 5 mM Tris buffer, pH 7.4, was added and then the cell solution was homogenized by the Dounce type B homogenizer (10 strokes). The membrane fraction was identified using a Western blotting method described in the Section “Confirmation of expression”.
a) Immunization
1×107 cells of the human oculospanin gene expressing cells obtained in Example 7 were injected intraperitoneally into BALB/c female mice which were 5 weeks old (purchased from Japan SLC Inc.) After 2, 4, 6 and 8 weeks, the human oculospanin gene expressing cells (1×107 cells/mouse) were injected intraperitoneally as a booster in the same manner.
B) Cell Fusion
The spleen was excised out from a mouse on the fourth day after the booster immunization and added to 10 ml of a serum-free MEM medium (9.4 g/L, Eagle MEM medium “Nissui” (1): manufactured by Nissui Pharmaceutical Co., Ltd., hereinafter referred to as “serum-free MEM medium”) containing 10 mM HEPES buffer (pH 7.4), 0.02 mg/l sodium hydrogen carbonate, and 300 μg/ml L-glutamic acid, and then the spleen cells were withdrawn using a 21 G′ syringe and tweezers. The cell suspension solution was centrifuged to precipitate the spleen cells. The spleen cells were washed twice with the serum-free MEM medium and suspended in serum-free MEM medium and the number of cells was counted.
Myeloma cells SP2/0 were cultured in myeloma growth medium (hereinafter referred to as the “ME medium”) containing 15% FBS (manufactured by GIBCO), 306 μg/ml glutamic acid, and 0.05 mM β-mercaptoethanol at 37° C. in the presence of 7% carbon dioxide gas such that the cell density did not exceed 1×106 cells/ml. The myeloma cells SP2/0 thus cultured were washed with the serum-free MEM medium and suspended in serum-free MEM medium and the number of cells was counted.
The SP2/0 cell suspension solution containing cells, the number of which corresponded to about ⅕ of the spleen cells, and the suspension solution for the whole spleen cells were mixed. After centrifugation, the supernatant was completely removed. The cell fusion operation below was performed while keeping a plastic centrifuge tube containing the pellet at room temperature. To the pellet, 1 ml of 40% (w/v) polyethylene glycol 4000 (manufactured by Merck) was slowly added while shaking the centrifuge tube. Thereafter, 9 ml of serum-free MEM medium previously warmed at 37° C. was gently added in three times. After centrifugation, the supernatant was removed and hypoxanthine aminopterin thymidine medium (hereinafter referred to as the “HAT medium”; manufactured by SIGMA) containing 20% FBS was added using a pipette while gently stirring with the pipette tip such that the cell density became 2.5×106 cells/ml. The HAT medium was dispensed to a 96-well cell-culture microplate in an amount of 100 μl/well and cultured at 37° C. in the presence of 7% carbon dioxide gas. After one day, fresh HAT medium was added to all the wells in an amount of 100 μl/well and thereafter, the medium was replaced with fresh medium at intervals of 2 to 3 days. The fused cells thus obtained were subjected to screening by limiting dilution analysis as mentioned below.
c) Limiting Dilution
The culture solution containing the fused cells obtained in Section (b) above was serially diluted such that the density of fused cells in the HT medium (HY medium in the case of 2nd cloning or later) became 1 cell/well (10 cells/ml), and 5 cells/well (50 cells/ml). Each of the samples thus prepared was dispensed in an amount of 100 μl per well, in a 96-well microplate already containing 100 μl of the HY medium, and the microplates were cultured at 37° C. in the presence of 7% carbon dioxide gas for 10 days.
d) Screening
d-1) ELISA
The cell membrane fraction obtained in Example 7 was prepared in a solution of 1 μg/ml dispensed into a 96-well EIA plate (manufactured by COSTAR) in an amount of 50 μl/well. After the plate was allowed to stand at 4° C. for one day, the antigen solution within the plate was discarded by shaking well and 80 μl of a solution containing 1% BSA in PBS(−) was added per well. The plate was sealed and stored at 4° C. until use. When used, the plate was returned to room temperature and washed three times using a Serawasher (manufactured by Bio-Tec) through which PBS (PBS-T) containing 0.1% Tween 20 was supplied. As a primary antibody, 50 μl of cell culture supernatant obtained after 10 to 12 days of cell fusion was added to each well and allowed to stand at room temperature for one hour. After completion of the reaction with the primary antibody, the plate was washed three times with PBS-T and alkaline phosphatase labeled anti-mouse IgG antibody (manufactured by BIO SOURCE), diluted 5000 fold with a solution (antigen dilution solution) containing 0.5% BSA added to PBS-T, was added to the wells in an amount of 50 μl/well, and allowed to stand still at room temperature for one hour. After completion of the reaction with the secondary antibody, a color-emitting substrate for alkaline phosphatase, p-nitrophenyl phosphate, 2Na6H2O (pNPP, manufactured by Wako Pure Chemical Industries Ltd.) returned to room temperature was dissolved to a concentration of 1 mg/ml in pNPP Buffer (97 ml/l diethanolamine, 0.1 g/l MgCl2.6H2O, pH 9.8) and added to the wells in an amount of 100 μl/well. The absorbance was measured at 405 nm and 630 nm using a plate reader (manufactured by Nalgene Nunc International)
d-2) Flow Cytometry
HEK293 culture cells obtained in Example 7 were cultured in DMEM medium containing 10% FBS at 37° C. in 5% CO2 gas. After transfection, the cells were cultured for 24 hours and a cell suspension solution was prepared so as to contain 2×107 cells/ml. The cell suspension solution was dispensed into 96-well V-shape bottom microplates (manufactured by Corning) in an amount of 50 μl/well and subjected to centrifugation (1000×g, 20° C. for 5 minutes). The supernatant was removed and the supernatant of the fused cells cultured in step (c) above was added at an amount of 50 μl/well, stirred, allowed to stand still on ice for 0.75 hours, centrifuged (1000×g, 20° C. for 5 minutes), and then the supernatant was removed. The pellet was washed twice with a flow cytometry buffer solution (MEM containing 5% FBS) in an amount of 150 μl/well. Thereafter, to the pellet, 100 μl of 33-fold diluted rabbit anti-mouse IgG antibody (manufactured by Wako Pure Chemical Industries Ltd.) labeled with fluorescein-5-isothiocyanate (hereinafter referred to as “FITC”) was added as a secondary antibody, allowed to stand still on ice for 0.75 hours, and subjected to centrifugation (1000×g, 20° C. for 5 minutes). The supernatant was removed, the pellet was washed twice with flow cytometry buffer using 150 μl/well and suspended in the flow cytometry buffer in an amount of 500 μl/well. This was used as a sample for flow cytometry. In each sample, the intensity of FITC fluorescence emitted from cells was measured by flow cytometry (FC500, manufactured by BECKMAN) at an excitation wavelength of 488 nm and a detection wavelength of 530 nm. As a result, the fused cells were selected from the sample exhibiting higher FITC fluorescent intensity than those of HEK293 transient expressing cells to which the supernatant of the fusion cell culture was not added.
e) Cloning
The cells selected in the step (d) above were subjected twice to the operations of a series of steps c) to d). As a result, several hybridoma clones were obtained which produced a monoclonal antibody which binds to HEK293 transient expressing cells, but does not bind to cells into which the anti-human oculospanin expression plasmid has not been introduced. One of the hybridoma strains thus cloned was designated as O3B8-2C9-4F3 and deposited at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science Technology as of Feb. 17, 2004 under deposition No. FERM BP-08627.
The mouse-mouse hybridoma prepared in Example 8 was suspended in HY medium at a concentration of 1×106 cells/ml and allowed to stand still at 37° C. in the presence of 7% carbon dioxide for 3 days. The culture solution thus obtained was centrifuged at 1,600 rpm for 5 minutes. The supernatant was recovered and IgG was roughly purified as follows:
Binding buffer: pH 7.0 (20 mM Na2HPO4.12H2O, 20 mM Na2HPO4.2H2O)
Elution buffer: pH 3.0, 100 mM glycine-HCl
Neutralization buffer: pH 9.0, 1M Tris-HCl
A requisite aliquot of Protein G carrier (manufactured by Amersham Biosciences) was taken. After ethanol was removed, the protein G carrier aliquot was washed twice with ultra pure water and washed once with the binding buffer. The binding buffer was added to the protein G aliquot carrier to make a 50% gel slurry. The protein G gel slurry was added to the supernatant of the hybridoma. The resulting mixture was rotated at 4° C. for 24 hours and washed three times with the binding buffer. After washing, the elution buffer was added to allow antibody to elute. The eluate was received by a tube containing neutralization buffer in an amount of 1/10 of the elution buffer. The eluate was loaded onto the upper portion of an ultrafilter of a sample tube (Amicon Ultrafree-MC: manufactured by Millipore) and centrifuged at 5000×g, 4° C. for 20 minutes. While the filtrate collected in the lower portion of the filter was removed, the eluate was added such that the liquid amount in the upper portion of the filter was at least 50 μl. After the whole amount of eluate was added, PBS (−) was added in the volume 3 times as large as the eluate. In this manner, buffer exchange was performed. The liquid left in the upper portion of the filter was treated as the anti-human oculospanin antibody sample.
As an index of biological activity, antibody-dependent cytotoxic activity was measured. The number of the human oculospanin expressing cells prepared in Example 7 was counted by the tryphan blue staining method and thereafter the concentration of the cells was adjusted with RPMI 1640 medium (manufactured by Invitrogen, hereinafter referred to as “RPMI medium”) containing 10% fetal bovine serum (manufactured by Moregate) to 8×105 cells/0.4 ml. Then 40 μl of Chromium-51 (sodium chromate manufactured by Amersham Bioscience) was added to the cells, the cells were incubated at 37° C. in 5% CO2 for 2 hours. To the cells, 8 ml of RPMI medium was added, stirred and then centrifuged at 1,500 rpm for 5 minutes. This washing operation was repeated further twice. The Chromium-51 labeled human oculospanin expressing cells thus obtained were resuspended in 4 ml of RPMI medium and seeded in a 96-well round bottom plate, in which 50 μl of 5 μm/ml purified mouse anti-human oculospanin antibody adjusted with RPMI medium was already present, in an amount of 50 μl (1×104 cells) per well and allowed to stand still at 4° C. for 30 minutes. In a negative control well or background measurement well, RPMI medium was added in place of the purified mouse anti-human oculospanin antibody.
Effector cells were prepared as mentioned below. The spleen cells were taken from BALB/c-nu/nu mouse (female, 7 weeks old) in accordance with the customary method. Then, the cell number was counted by the tryphan blue staining method, the concentration of the cells was adjusted with RPMI medium to 1.5×107 cells/ml. The cells were seeded into 96-well round bottom microplates in an amount of 100 μl (1.5×106 cells/ml) per well, centrifuged at 1,500 rpm for 5 minutes and incubated at 37° C. in 5% CO2 for 4 hours. To the positive control well, 2% Triton-X-100 was added in place of the effector cells in order to completely kill the Chromium-51 labeled human oculospanin expressing cells. To the background measurement well, the RPMI medium was added in place of the effector cells. Next, incubation was performed for 4 hours, 50 μl of the culture supernatant was taken from each of the wells and transferred to a 96-well Luma Plate (manufactured by PerkinElmer). The plate was dehydrated at 50° C. overnight, the amount of Chromium-51 in each well was measured by a microplate scintillation counter (TopCourt NTX, manufactured by PerkinElmer).
The rate at which cell death was induced in each well was calculated in accordance with the following formula:
Cell death induction rate (%)=(radioactivity count for each test well−background count for the negative control well)/(the radioactivity count for the positive control well−background count for the negative control well)×100.
Compared to the negative control, it was confirmed that addition of the purified mouse anti-human oculospanin antibody (
By virtue of the present invention, it was found that the expression level of human oculospanin in melanoma is significantly high. According to the present invention, there are provided a method of detecting cancer using the human oculospanin gene and a cancer detection kit, and further provided an antibody having cytotoxic activity against oculospanin and a pharmaceutical composition for treating cancer containing the antibody.
Sequence list free text
Sequence ID No. 5: PCR sense primer for human oculospanin amplification.
| Number | Date | Country | Kind |
|---|---|---|---|
| JP 2003-063648 | Mar 2003 | JP | national |
This application is a continuation-in-part application of U.S. application Ser. No. 11/223,812, filed Sep. 9, 2005, which is a continuation of International Application No. PCT/JP2004/003048, filed Mar. 9, 2004, which claims the benefit under 35 U.S.C. 365(c) of Japanese Application No. 2003-063648, filed Mar. 10, 2003; and of U.S. Application No. 10/548,688, filed with the U.S. Patent Office on Sep. 9, 2005, under 35 U.S.C. 371 from International Application No. PCT/JP2004/003048, filed Mar. 9, 2004, which claims the benefit under 35 U.S.C. 365(c) of Japanese Application No. 2003-063648. 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/JP04/03048 | Mar 2004 | US |
| Child | 11223812 | Sep 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11223812 | Sep 2005 | US |
| Child | 11345651 | Jan 2006 | US |
| Parent | 10548688 | US | |
| Child | 11345651 | Jan 2006 | US |
